U.S. patent application number 16/909521 was filed with the patent office on 2021-01-14 for flt3l-fc fusion proteins and methods of use.
The applicant listed for this patent is Gilead Sciences, Inc.. Invention is credited to Alexandre Ambrogelly, Manuel Baca, Brian A. Carr, Hon Man Hamlet Chu, Magdeleine S. Hung, Manu Kanwar, Michelle R. Kuhne, Douglas S. Rehder, Matthew R. Schenauer, Nicholas S. Wilson.
Application Number | 20210009718 16/909521 |
Document ID | / |
Family ID | 1000005164985 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210009718 |
Kind Code |
A1 |
Ambrogelly; Alexandre ; et
al. |
January 14, 2021 |
FLT3L-Fc FUSION PROTEINS AND METHODS OF USE
Abstract
Provided are FLT3L-Fc fusion proteins, polynucleotides encoding
such fusion proteins, expression cassettes, vectors, cells and kits
comprising such fusion proteins, and methods of using.
Inventors: |
Ambrogelly; Alexandre;
(Carlsbad, CA) ; Baca; Manuel; (Lexington, MA)
; Carr; Brian A.; (Foster City, CA) ; Chu; Hon Man
Hamlet; (El Cerrito, CA) ; Hung; Magdeleine S.;
(Mountain View, CA) ; Kanwar; Manu; (San Marcos,
CA) ; Kuhne; Michelle R.; (San Francisco, CA)
; Rehder; Douglas S.; (Bonsall, CA) ; Schenauer;
Matthew R.; (Dana Point, CA) ; Wilson; Nicholas
S.; (San Carlos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gilead Sciences, Inc. |
Foster City |
CA |
US |
|
|
Family ID: |
1000005164985 |
Appl. No.: |
16/909521 |
Filed: |
June 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62866584 |
Jun 25, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/94 20130101;
A61K 45/05 20130101; C07K 2319/30 20130101; C07K 2317/53 20130101;
C07K 19/00 20130101; C12N 15/64 20130101; A61K 2035/122 20130101;
C07K 14/4705 20130101 |
International
Class: |
C07K 19/00 20060101
C07K019/00; A61K 45/00 20060101 A61K045/00; C07K 14/47 20060101
C07K014/47; C12N 15/64 20060101 C12N015/64 |
Claims
1.-350. (canceled)
351. A fusion protein comprising: a human fms related tyrosine
kinase 3 ligand (FLT3L) extracellular domain operably linked to an
immunoglobulin fragment crystallizable region (Fc region), wherein
the fusion protein comprises an amino acid sequence that is at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1-18 and
21-27, and wherein i. at least 5 amino acids are truncated from the
C-terminus of the FLT3L extracellular domain; and/or ii. the Fc
region does not comprise a hinge region.
352. The fusion protein of claim 351, comprising an amino acid
sequence of SEQ ID NO:1.
353. The fusion protein of claim 351, comprising an amino acid
sequence of SEQ ID NO:9.
354. The fusion protein of claim 351, comprising an amino acid
sequence of SEQ ID NO:6.
355. The fusion protein of claim 351, comprising an amino acid
sequence of SEQ ID NO:14.
356. The fusion protein of claim 351, wherein the Fc region is from
a human IgG1 and does not comprise a hinge region.
357. The fusion protein of claim 356, wherein the C-terminus of the
FLT3L extracellular domain is not truncated.
358. The fusion protein of claim 351, wherein the Fc region is from
a human IgG4 and at least 5 amino acids are truncated from the
C-terminus of the FLT3L extracellular domain.
359. The fusion protein of claim 358, wherein the Fc region
comprises a hinge region.
360. The fusion protein of claim 351, wherein the Fc region
comprises the following amino acids at the indicated positions (EU
index numbering): i. Tyrosine at position 252, threonine at
position 254 and glutamic acid at position 256 (YTE); or ii.
Leucine at position 428 and serine at position 434 (LS).
361. The fusion protein of claim 351, wherein the fusion protein
has a serum half-life of at least about 7 days.
362. A homodimer comprising two identical fusion proteins of claim
351.
363. A heterodimer comprising two non-identical fusion proteins of
claim 351.
364. A polynucleotide encoding a fusion protein of claim 351.
365. An expression cassette comprising one or more regulatory
sequences operably linked to the polynucleotide of claim 364.
366. A vector comprising the polynucleotide of claim 364.
367. A lipid nanoparticle (LNP) comprising the polynucleotide of
claim 364.
368. A cell or population of cells comprising the polynucleotide of
claim 364, wherein the cell expresses or the population of cells
express the fusion protein of claim 351.
369. A pharmaceutical composition comprising the fusion protein of
claim 351 and a pharmaceutically acceptable carrier.
370. A method of promoting, inducing and/or increasing the
expansion and/or proliferation of a cell or a population of cells
that express fms related tyrosine kinase 3 (FLT3, CD135),
comprising contacting the cell or population of cells in vitro with
an effective amount of the fusion protein of claim 351.
371. A method of preventing, reducing and/or inhibiting the
recurrence, growth, proliferation, migration and/or metastasis of a
cancer cell or population of cancer cells in a subject in need
thereof, comprising administering to the subject an effective
amount of the fusion protein of claim 351.
372. A method of enhancing, promoting, and/or increasing the tumor
infiltration of T-cells and/or NK cells in a subject in need
thereof, comprising administering to the subject an effective
amount of the fusion protein of claim 351.
373. A method of enhancing, improving, and/or increasing the
response to an anticancer therapy in a subject in need thereof,
comprising co-administering to the subject (1) an effective amount
of the fusion protein of claim 351; and (2) an effective amount of
an anticancer agent.
374. A method of enhancing, improving, and/or increasing the
response to an immune checkpoint protein in a subject in need
thereof, comprising co-administering to the subject (1) an
effective amount of the fusion protein of claim 351; and (2) an
effective amount of the immune checkpoint protein.
375. A method of treating or preventing a virus infection
comprising administering an effective amount of the fusion protein
of claim 351 to a subject in need thereof.
376. A kit comprising one or more unitary doses of the fusion
protein of claim 351.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. provisional application No. 62/866,584, filed on
Jun. 25, 2019, which is hereby incorporated herein by reference in
its entirety for all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 27, 2020, is named 1290_PC_SL.txt and is 195,679 bytes in
size.
BACKGROUND
[0003] Dendritic cells (DCs) are the most potent antigen-presenting
cell in the body. DCs function to process antigen material and
present it on the cell surface to the T cells. DCs act as
messengers between the innate and the adaptive immune systems. Fms
related tyrosine kinase 3 ligand (FLT3LG, FLT3L, NCBI Gene ID:
2323) selectively expands DCs from bone marrow precursors, as well
as promotes proliferation of terminally differentiated DCs in
lymphoid and tumor tissues.
[0004] Soluble recombinant human protein forms of FLT3L have a
serum half-life in humans of about 12-28 hours after five
consecutive subcutaneous (SC) doses, requiring daily administration
to the patient over a 28-day therapeutic cycle. Daily
administration is undesirable, for both the patient and clinician,
and is dose scheduling that does not align with other approved
immune-oncology therapeutic agents, which is usually once every 2
to 3 weeks. Longer acting antiviral therapeutic agents are also
considered desirable.
SUMMARY
[0005] Provided are FLT3L-Fc fusion proteins that have an extended
serum half-life in a human subject, relative to soluble FLT3L.
[0006] Accordingly, in one aspect, provided are fusion proteins
comprising: a human fms related tyrosine kinase 3 ligand (FLT3L)
extracellular domain operably linked to an immunoglobulin fragment
crystallizable region (Fc region), wherein: at least 5 amino acids
are truncated from the C-terminus of the FLT3L extracellular
domain; and/or the Fc region does not comprise a hinge region. In
some embodiments, the FLT3L extracellular domain is derived from a
human FLT3L extracellular domain. In some embodiments, the FLT3L
extracellular domain is a human FLT3L extracellular domain. In some
embodiments, the fusion protein is capable of binding to human
FLT3. In some embodiments, the FLT3L extracellular domain is from
FLT3L isoform 1 or from FLT3L isoform 2. In some embodiments, at
least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids are
truncated from the C-terminus of the FLT3L extracellular domain. In
some embodiments, the FLT3L extracellular domain does not comprise
(e.g., is deleted, removed or excluded) the amino acid sequence
PTAPQ (SEQ ID NO:85), APTAPQ (SEQ ID NO:86), TAPTAPQ (SEQ ID
NO:87), ATAPTAPQ (SEQ ID NO:88), EATAPTAPQ (SEQ ID NO:89), or
LEATAPTAPQ (SEQ ID NO:90), PTAPQPP (SEQ ID NO:91), APTAPQPP (SEQ ID
NO:92), TAPTAPQPP (SEQ ID NO:93), ATAPTAPQPP (SEQ ID NO:94),
EATAPTAPQPP (SEQ ID NO:95), or LEATAPTAPQPP (SEQ ID NO:96). In some
embodiments, the FLT3L extracellular domain comprises an N-terminal
signal peptide. In some embodiments, the FLT3L extracellular domain
comprises one or more of the following amino acid substitutions:
H8Y; K84E; S102A; and/or S125A; wherein the amino acid residue
positions are with reference to SEQ ID NOs: 1-18, 21-27 or 71-81.
In some embodiments, one or both of serine residues at positions
102 and 125 are substituted to alanine, wherein the amino acid
residue positions are with reference to SEQ ID NOs: 1-18, 21-27 or
71-81. In some embodiments, the Fc region is from a human IgG1,
IgG2, IgG3 or IgG4. In some embodiments, the Fc region is from a
human IgG1 or IgG4. In some embodiments, the Fc region comprises a
human IgG1 isotype and comprises one or more amino acid
substitutions in the Fc region at a residue position selected from
the group consisting of: N297A, N297G, N297Q, N297G, D265A, L234A,
L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G,
P329A, P329G, K322A, L234F, L235E, P331S, T394D, A330L, M252Y,
S254T, T256E, M428L, N434S, T366W, T366S, L368A, Y407V, and any
combination thereof, wherein the numbering of the residues is
according to EU numbering. In some embodiments, the Fc region
comprises a human IgG1 isotype and comprises one or more amino acid
substitutions in the Fc region at a residue position selected from
the group consisting of: L234A, L234V, L234F, L235A, L235E, P331S,
and any combination thereof, wherein the numbering of the residues
is according to EU numbering. In some embodiments, the Fc region
comprises a human IgG4 isotype and comprises one or more amino acid
substitutions in the Fc region at a residue position selected from
the group consisting of: E233P, F234V, F234A, L235A, G237A, E318A,
S228P, L235E, T394D, M252Y, S254T, T256E, N297A, N297G, N297Q,
T366W, T366S, L368A, Y407V, M428L, N434S, and any combination
thereof, wherein the numbering of the residues is according to EU
numbering. In some embodiments, the Fc region comprises a human
IgG4 isotype and comprises one or more amino acid substitutions in
the Fc region at a residue position selected from the group
consisting of: F234V, F234A, L235A, L235E, S228P, and any
combination thereof, wherein the numbering of the residues is
according to EU numbering. In some embodiments, the Fc region
comprises the following amino acids at the indicated positions (EU
index numbering): (i) Tyrosine at position 252, threonine at
position 254 and glutamic acid at position 256 (YTE); or (ii)
Leucine at position 428 and serine at position 434 (LS). In some
embodiments, the FLT3L extracellular domain comprises an amino acid
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%,
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 71-81. In some embodiments, the Fc region
comprises an amino acid sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or 100%, identical to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 103-107. In some
embodiments, the fusion protein comprises an amino acid sequence
that is at least 80%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100%, identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 1-18 and 21-27. In some embodiments, the Fc region is from a
human IgG1 and does not comprise a hinge region. In some
embodiments, the C-terminus of the FLT3L extracellular domain is
not truncated. In some embodiments, the fusion protein comprises or
consists of an amino acid sequence of SEQ ID NO:1. In some
embodiments, the fusion protein comprises or consists of an amino
acid sequence of SEQ ID NO:9. In some embodiments, the fusion
protein comprises or consists of an amino acid sequence selected
from the group consisting of SEQ ID NOs: 1, 2, 5, 7, 9, 10, 13, 15,
22, 23 and 24, or comprises or consists of an amino acid sequence
that is at least 80%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 1,
2, 5, 7, 9, 10, 13, 15, 22, 23 and 24, wherein the Fc region is
derived from a human IgG1 isotype and does not comprise a hinge
region, e.g., does not the amino acid sequence EPKSCDKTHTCPPCP (SEQ
ID NO:101) or EPKSCDKTHTCPPCPAPELL (SEQ ID NO:110). In some
embodiments, the Fc region is from a human IgG4 and at least 5
amino acids are truncated from the C-terminus of the FLT3L
extracellular domain. In some embodiments, the Fc region comprises
a hinge region. In some embodiments, the fusion protein comprises
or consists of an amino acid sequence of SEQ ID NO:6. In some
embodiments, the fusion protein comprises or consists of an amino
acid sequence of SEQ ID NO:14. In some embodiments, the fusion
protein comprises or consists of an amino acid sequence selected
from the group consisting of SEQ ID NOs: 3, 4, 6, 8, 11, 12, 14,
16, 17, 18, 25 and 26, or comprises or consists of an amino acid
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 3, 4, 6, 8, 11, 12, 14, 16, 17, 18, 25 and 26, wherein the Fc
region is derived from a human IgG4 isotype and wherein at least 5
amino acids are truncated from the C-terminus of the FLT3L
extracellular domain, e.g., wherein the FLT3L extracellular domain
does not comprise the amino acid sequence PTAPQ (SEQ ID NO:85).
[0007] In a further aspect, provided is a fusion protein comprising
an amino acid sequence that is at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100%, identical to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 19-20.
[0008] In a further aspect, provided are fusion proteins
comprising: (i) a FLT3L-Fc fusion protein comprising an amino acid
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%,
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-27; and (ii) a second polypeptide. In
some embodiments, the second polypeptide comprises a targeting
moiety or domain, a growth factor, a cytokine, a chemokine or a TNF
superfamily (TNFSF) member. In some embodiments, the second
polypeptide is N-terminal to the FLT3L extracellular domain. In
some embodiments, the second polypeptide is C-terminal to the Fc
region. In some embodiments, the second polypeptide is between the
FLT3L extracellular domain and the Fc region. In some embodiments,
the targeting moiety domain binds to a target protein or antigen
identified in Table B, below, such as without limitation CD19,
MS4A1 (CD20), CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37,
CD38, CD40, CD44, CD48, CD52, CD70, NTSE (CD73), ENTPD1 (CD39),
CD74, CD79b, CD80, CD86, IL3RA (CD123), PROM1 (CD133), CD137, SDC1
(CD138), alpha fetoprotein (AFP), c-Met; c-Kit; C-type lectin
domain family 12 member A (CLEC12A, CLL1, CD371); C-type lectin
domain containing 9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin,
PCAD); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9,
CAIX); carcinoembryonic antigen related cell adhesion molecule 3
(CEACAM3); carcinoembryonic antigen related cell adhesion molecule
5 (CEACAM5); carcinoembryonic antigen related cell adhesion
molecule 6 (CEACAM6, CD66c); chorionic somatomammotropin hormone 1
(CSH1, CS1); coagulation factor III, tissue factor (F3, TF);
collectin subfamily member 10 (COLEC10); delta like canonical Notch
ligand 3 (DLL3); ectonucleotide pyrophosphatase/phosphodiesterase 3
(ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor
(EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2);
epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine
kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP);
fibroblast growth factor receptor 2 (FGFR2); fibroblast growth
factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate
receptor 1 (FOLR1, FRa); GD2 ganglioside; glycoprotein NMB (GPNMB,
osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human
papillomavirus (HPV) E6; HPV E7; major histocompatibility complex
(MHC) class I-presented neoantigens; major histocompatibility
complex (MHC) class II-presented neoantigens; major
histocompatibility complex, class I, E (HLA-E); major
histocompatibility complex, class I, F (HLA-F); major
histocompatibility complex, class I, G (HLA-G, MHC-G); integrin
subunit beta 7 (ITGB7); leukocyte immunoglobulin like receptor B1
(LILRB1, ILT2); leukocyte immunoglobulin like receptor B2 (LILRB2,
ILT4); LY6/PLAUR domain containing 3 (LYPD3, C4.4A); glypican 3
(GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family member A1
(MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4
(MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1
(MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1
(MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin
1 (MUC1) and splice variants thereof (e.g., MUC1/C, D, and Z);
mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4
(NECTIN4); SLIT and NTRK like family member 6 (SLITRK6);
promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7
(inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM
family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid
binding Ig like lectin 7 (SIGLEC7); sialic acid binding Ig like
lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate),
member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6;
LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily
member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9
(TNFSF9; 4-1BB-L, CD137L); TNF receptor superfamily member 10a
(TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily member
10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily
member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor
superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor
superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF);
transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein
(TPBG, 5T4); trophinin (TRO, MAGED3); tumor associated calcium
signal transducer 2 (TACSTD2, TROP2, EGP1); Fucosyl GM1; sialyl
Lewis adhesion molecule (sLe); and Lewis Y antigen.
[0009] In a related aspect, provided is a homodimer comprising two
(i.e., first and second) identical FLT3L-Fc fusion proteins, as
described above and herein. In a related aspect, provided is a
heterodimer comprising two (i.e., first and second) non-identical
FLT3L-Fc fusion proteins, as described above and herein. In various
embodiments of such heterodimers, the FLT3L domain is the same and
the Fc region is different between the first and second FLT3L-Fc
fusion proteins.
[0010] In a further aspect, provided are heterodimers comprising a
FLT3L-Fc fusion protein, as described above and herein, and a
second fusion protein comprising a targeting moiety domain fused to
a second Fc region. In some embodiments, the targeting moiety
domain binds to a target protein or antigen identified in Table B,
below, such as without limitation CD19, MS4A1 (CD20), CD22, IL2RA
(CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40, CD44, CD48,
CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b, CD80, CD86,
IL3RA (CD123), PROM1 (CD133), CD137, SDC1 (CD138), alpha
fetoprotein (AFP), c-Met; c-Kit; C-type lectin domain family 12
member A (CLEC12A, CLL1, CD371); C-type lectin domain containing 9A
(CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic
anhydrase 6 (CA6); carbonic anhydrase 9 (CA9, CAIX);
carcinoembryonic antigen related cell adhesion molecule 3
(CEACAM3); carcinoembryonic antigen related cell adhesion molecule
5 (CEACAM5); carcinoembryonic antigen related cell adhesion
molecule 6 (CEACAM6, CD66c); chorionic somatomammotropin hormone 1
(CSH1, CS1); coagulation factor III, tissue factor (F3, TF);
collectin subfamily member 10 (COLEC10); delta like canonical Notch
ligand 3 (DLL3); ectonucleotide pyrophosphatase/phosphodiesterase 3
(ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor
(EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2);
epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine
kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP);
fibroblast growth factor receptor 2 (FGFR2); fibroblast growth
factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate
receptor 1 (FOLR1, FRa); GD2 ganglioside; glycoprotein NMB (GPNMB,
osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human
papillomavirus (HPV) E6; HPV E7; major histocompatibility complex
(MHC) class I-presented neoantigens, major histocompatibility
complex (MHC) class II-presented neoantigens, major
histocompatibility complex, class I, E (HLA-E); major
histocompatibility complex, class I, F (HLA-F); major
histocompatibility complex, class I, G (HLA-G, MHC-G); integrin
subunit beta 7 (ITGB7); leukocyte immunoglobulin like receptor B1
(LILRB1, ILT2); leukocyte immunoglobulin like receptor B2 (LILRB2,
ILT4); LY6/PLAUR domain containing 3 (LYPD3, C4.4A); glypican 3
(GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family member A1
(MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4
(MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1
(MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1
(MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin
1 (MUC1) and splice variants thereof (e.g., MUC1/C, D, and Z);
mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4
(NECTIN4); SLIT and NTRK like family member 6 (SLITRK6);
promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7
(inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM
family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid
binding Ig like lectin 7 (SIGLEC7); sialic acid binding Ig like
lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate),
member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6;
LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily
member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9
(TNFSF9; 4-1BB-L, CD137L); TNF receptor superfamily member 10a
(TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily member
10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily
member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor
superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor
superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF);
transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein
(TPBG, 5T4); trophinin (TRO, MAGED3); tumor associated calcium
signal transducer 2 (TACSTD2, TROP2, EGP1); Fucosyl GM; sialyl
Lewis adhesion molecule (sLe); and Lewis Y antigen. In some
embodiments, the targeting moiety domain comprises an
antigen-binding antibody fragment. In some embodiments, the
antibody fragment comprises a Fab or a single-chain variable
fragment (scFv). In some embodiments, the targeting moiety domain
comprises a non-immunoglobulin binding moiety or an antibody
mimetic protein. In some embodiments, the non-immunoglobulin
antigen-binding domain or antibody mimetic protein is selected from
the group consisting of adnectins, affibody molecules, affilins,
affimers, affitins, alphabodies, anticalins, peptide aptamers,
armadillo repeat proteins (ARMs), atrimers, avimers, designed
ankyrin repeat proteins (DARPins.RTM.), fynomers, knottins, Kunitz
domain peptides, monobodies, and nanoCLAMPs. In some embodiments,
both the first Fc region and the second Fc region do not comprise a
hinge region. In some embodiments, the heterodimer is stabilized by
an interaction between the first Fc region and the second Fc
region. In some embodiments, the heterodimer comprises a
heterodimeric human IgG1 or human IgG4. In some embodiments, the
heterodimeric human IgG1 or human IgG4 comprises a first Fc region
and a second Fc region, comprising the following amino acids at the
indicated positions (EU numbering): (i) the first Fc region
comprises a tryptophan at position 366 (T366W); and the second Fc
region comprises a serine at position 366 (T366S), an alanine at
position 368 (L368A) and a valine at position 407 (Y407V); (ii) the
first Fc region comprises a serine at position 366 (T366S), an
alanine at position 368 (L368A) and a valine at position 407
(Y407V); and the second Fc region comprises a tryptophan at
position 366 (T366W); (iii) the first Fc region comprises a
cysteine at position 354 (S354C), a tryptophan at position 366
(T366W); and the second Fc region comprises a cysteine at position
349 (Y349C), a serine at position 366 (T366S), an alanine at
position 368 (L368A) and a valine at position 407 (Y407V); or (iv)
the first Fc region comprises cysteine at position 349 (Y349C), a
serine at position 366 (T366S), an alanine at position 368 (L368A)
and a valine at position 407 (Y407V); and the second Fc region
comprises a cysteine at position 354 (S354C), a tryptophan at
position 366 (T366W).
[0011] In a further aspect, provided is a conjugate comprising: (i)
a FLT3L-Fc fusion protein, as described above and herein, or a
homodimer or heterodimer of such FLT3L-Fc protein; attached to a
therapeutic agent or a detectable label. In some embodiments, the
therapeutic agent is covalently linked. In some embodiments, the
therapeutic agent is a small organic compound. In some embodiments,
the therapeutic agent is an agonist or activator of a toll-like
receptor (TLR) or a stimulator of interferon genes (STING)
receptor. In some embodiments, the TLR agonist or activator is
selected from the group consisting of a TLR2 agonist, a TLR3
agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8
agonist and a TLR9 agonist. In some embodiments, the TLR7 agonist
is selected from the group consisting of GS 9620, DS-0509, LHC-165
and TMX-101 (imiquimod), and/or wherein the TLR8 agonist is
selected from the group consisting of GS-9688 and NKTR-262 (dual
TLR7/TLR8 agonist). In some embodiments, the STING receptor agonist
or activator is selected from the group consisting of ADU-S100
(MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532,
SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthenone-4-acetic acid
(DMXAA), cyclic-GAMP (cGAMP) and cyclic-di-AMP. In some
embodiments, the therapeutic agents is an immune checkpoint
inhibitor. In some embodiments, the immune checkpoint inhibitor is
a small molecule inhibitor of CD274 (PDL1, PD-L1), programmed cell
death 1 (PDCD1, PD1, PD-1) or CTLA4. In some embodiments, the small
molecule inhibitor of CD274 or PDCD1 is selected from the group
consisting of GS-4224, GS-4416, INCB086550 and MAX10181. In some
embodiments, the small molecule inhibitor of CTLA4 is BPI-002.
[0012] In a further aspect, provided are polynucleotides encoding a
FLT3L-Fc fusion protein, as described above and herein. In some
embodiments, the polynucleotide is selected from the group
consisting of DNA, cDNA, RNA or mRNA. In some embodiments, the
polynucleotide comprises a nucleic acid sequence that is at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100%, identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 28-70.
Further provided are expression cassettes comprising one or more
regulatory sequences operably linked to a FLT3L-Fc-encoding
polynucleotide, as described herein.
[0013] In a further aspect, provided are vectors comprising a
polynucleotide encoding the FLT3L-Fc fusion proteins described
herein, or an expression cassette comprising such FLT3L-Fc-encoding
polynucleotide. In some embodiments, the vector is a plasmid vector
or a viral vector. In some embodiments, the viral vector comprises
an oncolytic viral vector. In some embodiments, the viral vector
comprises a DNA virus or a RNA virus. In some embodiments, the
viral vector is from a viral family selected from the group
consisting of Adenoviridae (e.g., Adenovirus), Arenaviridae (e.g.,
lymphocytic choriomeningitis mammarenavirus, Cali mammarenavirus
(a.k.a., Pichinde mammarenavirus), Poxviridae (e.g., Vaccinia
virus), Herpesviridae (e.g., Herpesvirus, e.g., HSV-1),
Parvoviridae (e.g., Parvovirus H1), Reoviridae (e.g., Reovirus),
Picornaviridae (e.g., Coxsackievirus, Seneca Valley Virus,
Poliovirus), Paramyxoviridae (e.g., Measles virus, Newcastle
disease virus (NDV)), Rhabdoviridae (e.g., Vesicular stomatitis
virus (VSV)), Togaviridae (e.g., Alphavirus, Sindbis virus),
Enteroviridae (e.g., Echovirus).
[0014] In a further aspect, provided is a lipoplex, such as a lipid
nanoparticle (LNP), comprising a polynucleotide encoding the
FLT3L-Fc fusion proteins described herein, or an expression
cassette or vector comprising such FLT3L-Fc-encoding
polynucleotide.
[0015] In a further aspect, provided is a cell or population of
cells comprising a polynucleotide encoding the FLT3L-Fc fusion
proteins described herein, or an expression cassette or vector
comprising such FLT3L-Fc-encoding polynucleotide, wherein the cell
expresses the FLT3L-Fc fusion protein, or homodimer or heterodimer
comprising such FLT3L-Fc fusion protein. In some embodiments, the
cell or population of cells is a eukaryotic cell. In some
embodiments, the cell or population of cells comprises a mammalian
cell, an insect cell, a plant cell or a yeast cell. In some
embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO)
cell or a human cell. In some embodiments, the human cell is a
human embryonic kidney cell. In some embodiments, the cell
predominantly sialylates N-linked and/or O-linked glycosylation
sites in the fusion protein. In some embodiments, at least 50%, at
least 60%, at least 70%, least 80%, at least 85%, at least 90%, or
more, N-linked and/or O-linked glycosylation sites in the fusion
protein are sialylated. In some embodiments, the sialylated
N-linked and/or O-linked glycosylation sites in the fusion protein
comprise from 2 to 7 sialic acid residues, e.g., from 3 to 6 sialic
acid residues, e.g., from 4 to 5 sialic acid residues.
[0016] In a further aspect, provided are pharmaceutical
compositions comprising a FLT3L-Fc fusion protein, as described
herein, or a fusion protein, homodimer, heterodimer or conjugate
comprising such FLT3L-Fc fusion protein; a polynucleotide encoding
the FLT3L-Fc fusion proteins described herein, or an expression
cassette, vector or lipoplex, such as an LNP, comprising such
FLT3L-Fc-encoding polynucleotide, and a pharmaceutically acceptable
carrier. In some embodiments, the composition comprises an aqueous
formulation. In some embodiments, the composition comprises the
FLT3L-Fc fusion protein, or a fusion protein, homodimer,
heterodimer or conjugate comprising such FLT3L-Fc fusion protein at
a concentration in the range of about 1 mg/ml to about 2 mg/ml, 3
mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10
mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml,
17 mg/ml, 18 mg/ml, 19 mg/ml or 20 mg/ml. In some embodiments, the
composition is lyophilized. In some embodiment, the composition
further comprises one or more additional therapeutic agents, e.g.,
second, third or fourth therapeutic agents.
[0017] Further provided are methods of promoting, inducing and/or
increasing the expansion and/or proliferation of a cell or a
population of cells that express fms related tyrosine kinase 3
(FLT3, CD135). In some embodiments, the methods comprise contacting
the cell or population of cells with an effective amount of a
FLT3L-Fc fusion protein, as described herein, or a fusion protein,
homodimer, heterodimer, conjugate or pharmaceutical composition
comprising such FLT3L-Fc fusion protein; a polynucleotide encoding
a FLT3L-Fc fusion protein described herein, or an expression
cassette, vector, lipoplex, such as an LNP, or pharmaceutical
composition comprising such FLT3L-Fc-encoding polynucleotide. In
some embodiments, the cell or population of cells that express FLT3
comprise dendritic cells (e.g., cDC1 cells and/or cDC2 cells),
monocyte-derived dendritic cells (moDCs), and/or progenitor cells
thereof. In some embodiments, the cell or population of cells that
express FLT3 comprise hematopoietic progenitor cells. In some
embodiments, the hematopoietic progenitor cells are selected from
the group consisting of: Common Lymphoid Progenitors (CLPs), Early
Progenitors with Lymphoid and Myeloid potential (EPLMs),
granulocyte-monocyte (GM) progenitors (GMP), monocyte-derived
dendritic cells (moDCs) progenitors, and early multi-potent
progenitors (MPP) within the Lineage-kit+Sca1 (LSK) compartment. In
various embodiments, the cell or population of cells is contacted
in vitro or in vivo. In some embodiments, the cell or population of
cells proliferate or are expanded within a solid tumor. In some
embodiments, conventional dendritic cells (e.g., cDC1 and/or cDC2)
are expanded or induced to proliferate. In some embodiments, cDC1
dendritic cells (e.g., positive for surface expression of X-C motif
chemokine receptor 1 (XCR1), thrombomodulin (THBD, CD141), and
C-type lectin domain containing 9A (CLEC9A)) are expanded or
induced to proliferate. In some embodiments, dendritic cells
expressing C-C motif chemokine receptor 5 (CCR5, CD195) and/or X-C
motif chemokine receptor 1 (XCR1) on their cell surface are
expanded or induced to proliferate. In some embodiments, dendritic
cells expressing one or more cell surface proteins selected from
the group consisting of XCR1, cell adhesion molecule 1 (CADM1),
C-type lectin domain containing 9A (CLEC9A, CD370), and
thrombomodulin (THBD) on their cell surface are expanded or induced
to proliferate. In some embodiments, dendritic cells expressing one
or more cell surface proteins selected from the group consisting of
CD1A, CD1C, CD1E, signal regulatory protein alpha (SIRPA; CD172A),
CD207 and Fc fragment of IgE receptor Ia (FCER1A) on their cell
surface are expanded or induced to proliferate. In some
embodiments, dendritic cells expressing one or more proteins
selected from the group consisting of basic leucine zipper ATF-like
transcription factor 3 (BATF3) and interferon regulatory factor 8
(IRF8) on their cell surface are expanded or induced to
proliferate. In some embodiments, dendritic cells expressing one or
more proteins selected from the group consisting of BATF3, IRF8,
THBD, CLEC9A and XCR1 on their cell surface are expanded or induced
to proliferate. In some embodiments, cDC2 dendritic cells (e.g.,
positive for surface expression of CDlc molecule (BDCA) are
expanded or induced to proliferate. In some embodiments, the
FLT3L-Fc fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide or the pharmaceutical composition is
administered to a subject or patient, e.g., a mammal, e.g., a
human.
[0018] Further provided are methods of expanding hematopoietic stem
cells (HSCs) ex vivo, comprising culturing HSCs in vitro in the
presence of mesenchymal lineage precursor or stem cells (MLPSCs)
and an effective amount of a FLT3L-Fc fusion protein, as described
herein, or a fusion protein, homodimer, heterodimer, conjugate or
pharmaceutical composition comprising such FLT3L-Fc fusion protein;
a polynucleotide encoding a FLT3L-Fc fusion protein described
herein, or an expression cassette, vector, or lipoplex, such as an
LNP, comprising such FLT3L-Fc-encoding polynucleotide such that
HSCs having the phenotype CD34+ are expanded. In some embodiments
the HSCs are further cultured in the presence of at least one
histone deacetylase inhibitor (HDACi). In some embodiments, the
HDACi is selected from the group consisting of valproic acid (VPA),
trichostatin A (TSA), DLS3, MS275, and SAHA. In some embodiments,
the HSCs have the phenotype CD34+, CD90+ or CD34+, CD45RA-, CD49f+
and are expanded at least 5-fold, at least 10-fold, at least
20-fold, or at least 40-fold. In some embodiments, the methods
further comprise isolating cells having the phenotype the phenotype
CD34+, CD90+ or CD34+, CD45RA-, CD49f+ to provide an enriched
population of cells having the phenotype CD34+, CD90+ or CD34+,
CD45RA-, CD49f+.
[0019] In a related aspect, provided are methods of preventing,
reducing and/or inhibiting the recurrence, growth, proliferation,
migration and/or metastasis of a cancer cell or population of
cancer cells in a subject in need thereof. In another aspect,
provided are methods of enhancing, promoting, and/or increasing the
tumor infiltration of T-cells and/or NK cells in a subject in need
thereof. Also provided are methods of enhancing, promoting, and/or
accelerating the recovery from or reversing the effects of
lymphopenia in a subject in need thereof. Further provided are
methods of treating or preventing a viral infection. Also provided
are methods for inhibiting the replication of a virus, treating a
viral infection or delaying the onset of symptoms of a viral
infection in a subject in need thereof. Further provided are
methods of enhancing, improving, and/or increasing the response to
an anticancer therapy or an antiviral therapy in a subject in need
thereof. Further provided are methods of promoting, increasing,
supplementing and/or boosting the immune response induced by the
vaccine. Further provided are methods of enhancing, improving,
and/or increasing the response to an immune checkpoint protein in a
subject in need thereof. In some embodiments, the methods comprise
administering to the subject effective amount of a FLT3L-Fc fusion
protein, as described herein, or a fusion protein, homodimer,
heterodimer, conjugate or pharmaceutical composition comprising
such FLT3L-Fc fusion protein; a polynucleotide encoding a FLT3L-Fc
fusion protein described herein, or an expression cassette, vector,
lipoplex, such as an LNP, or pharmaceutical composition comprising
such FLT3L-Fc-encoding polynucleotide. In some embodiments, one or
more additional therapeutic agents, e.g., second, third and/or
fourth therapeutic agents, are co-administered. In some
embodiments, the one or more additional therapeutic agents
comprises one or more of AGEN1884 (zalifrelimab), AGEN1181,
AGEN2034 (balstilimab), AGEN1307, AGEN2373, AGEN1223 and GS-1423
(AGEN1423; see WO2019/173692). In some embodiments, the one or more
additional therapeutic agents comprises a vaccine. In some
embodiments, the vaccine is selected from the group consisting of
an antiviral vaccine, an antibacterial vaccine and an anticancer
vaccine (e.g., a neoantigen vaccine). In some embodiments, the
vaccine comprises an antiviral vaccine against a virus selected
from the group consisting of hepatitis A virus (HAV), hepatitis B
virus (HBV), human immunodeficiency virus (HIV), cytomegalovirus
(CMV), a herpes simplex virus (HSV), Epstein-Barr virus (EBV),
human orthopneumovirus or human respiratory syncytial virus (RSV),
human papillomavirus (HPV), varicella-zoster virus, measles virus,
mumps virus, poliovirus vaccine, influenza virus, paramyxovirus,
rotavirus, Zika virus, Dengue virus, Ebola virus and coronavirus.
In some embodiments, the vaccine comprises an antibacterial vaccine
against a bacterium selected from the group consisting of
Mycobacterium tuberculosis, pertussis, tetanus, diphtheria,
meningococcus, pneumococcus, Haemophilus influenza, cholera,
typhoid, and anthrax. In some embodiments, the one or more
additional therapeutic agents comprises an oncolytic viral vector.
In some embodiments, the oncolytic viral vector comprises a DNA
virus or a RNA virus. In some embodiments, the viral vector is from
a viral family selected from the group consisting of. Adenoviridae
(e.g., Adenovirus), Arenaviridae (e.g., lymphocytic
choriomeningitis mammarenavirus, Cali mammarenavirus (a.k.a.,
Pichinde mammarenavirus), Poxviridae (e.g., Vaccinia virus),
Herpesviridae (e.g., Herpesvirus, e.g., HSV-1), Parvoviridae (e.g.,
Parvovirus H1), Reoviridae (e.g., Reovirus), Picornaviridae (e.g.,
Coxsackievirus, Seneca Valley Virus, Poliovirus), Paramyxoviridae
(e.g., Measles virus, Newcastle disease virus (NDV)), Rhabdoviridae
(e.g., Vesicular stomatitis virus (VSV)), Togaviridae (e.g.,
Alphavirus, Sindbis virus), Enteroviridae (e.g., Echovirus). In
some embodiments, the one or more additional therapeutic agents
comprises an immunotherapy, an immunostimulatory therapy, a
cytokine therapy, a chemokine therapy, a cellular therapy, a gene
therapy, and combinations thereof. In some embodiments, the
immunotherapy comprises co-administering one or more antibodies or
antigen-binding antibody fragments thereof, or antibody-drug
conjugates thereof, CD3-targeting multi-specific molecules,
CD16-targeting multi-specific molecules, or non-immunoglobulin
antigen-binding domains or antibody mimetic proteins directed
against one or more targets or tumor associated antigens (TAAs)
selected from the group consisting of: CD19, MS4A1 (CD20), CD22,
IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40, CD44,
CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b, CD80,
CD86, IL3RA (CD123), PROM1 (CD133), CD137, SDC1 (CD138), alpha
fetoprotein (AFP), c-Met; c-Kit; C-type lectin domain family 12
member A (CLEC12A, CLL1, CD371); C-type lectin domain containing 9A
(CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic
anhydrase 6 (CA6); carbonic anhydrase 9 (CA9, CAIX);
carcinoembryonic antigen related cell adhesion molecule 3
(CEACAM3); carcinoembryonic antigen related cell adhesion molecule
5 (CEACAM5); carcinoembryonic antigen related cell adhesion
molecule 6 (CEACAM6, CD66c); chorionic somatomammotropin hormone 1
(CSH1, CS1); coagulation factor III, tissue factor (F3, TF);
collectin subfamily member 10 (COLEC10); delta like canonical Notch
ligand 3 (DLL3); ectonucleotide pyrophosphatase/phosphodiesterase 3
(ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor
(EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2);
epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine
kinase 2 (ERBB2, HER2); fibroblast activation protein alpha (FAP);
fibroblast growth factor receptor 2 (FGFR2); fibroblast growth
factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate
receptor 1 (FOLR1, FRa); GD2 ganglioside; glycoprotein NMB (GPNMB,
osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human
papillomavirus (HPV) E6; HPV E7; major histocompatibility complex
(MHC) class I-presented neoantigens, major histocompatibility
complex (MHC) class II-presented neoantigens, major
histocompatibility complex, class I, E (HLA-E); major
histocompatibility complex, class I, F (HLA-F); major
histocompatibility complex, class I, G (HLA-G, MHC-G); integrin
subunit beta 7 (ITGB7); leukocyte immunoglobulin like receptor B1
(LILRB1, ILT2); leukocyte immunoglobulin like receptor B2 (LILRB2,
ILT4); LY6/PLAUR domain containing 3 (LYPD3, C4.4A); glypican 3
(GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family member A1
(MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4
(MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1
(MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1
(MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin
1 (MUC1) and splice variants thereof (e.g., MUC1/C, D, and Z);
mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4
(NECTIN4); SLIT and NTRK like family member 6 (SLITRK6);
promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7
(inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM
family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid
binding Ig like lectin 7 (SIGLEC7); sialic acid binding Ig like
lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate),
member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6,
LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily
member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9
(TNFSF9, 4-1BB-L, CD137L); TNF receptor superfamily member 10a
(TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily member
10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily
member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor
superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor
superfamily member 18 (TNFRSF18, GITR, CD357); transferrin (TF);
transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein
(TPBG, 5T4); trophinin (TRO, MAGED3); tumor associated calcium
signal transducer 2 (TACSTD2, TROP2, EGP1); Fucosyl GM1; sialyl
Lewis adhesion molecule (sLe); and Lewis Y antigen. In some
embodiments, the one or more antibodies or antigen-binding antibody
fragments thereof, or antibody-drug conjugates thereof,
CD3-targeting multi-specific molecules, CD16-targeting
multi-specific molecules, or non-immunoglobulin antigen-binding
domains or antibody mimetic proteins binds to an epitope of a
target or tumor associated antigen (TAA) presented in a major
histocompatibility complex (MHC) molecule (e.g., a neoantigen). In
some embodiments, the TAA is a cancer testis antigen. In some
embodiments, the cancer testis antigen is selected from the group
consisting of acrosin binding protein (ACRBP, CT23, OY-TES-1,
SP32), alpha fetoprotein (AFP, AFPD, FETA, HPAFP); A-kinase
anchoring protein 4 (AKAP4, AKAP 82, AKAP-4, AKAP82, CT99, FSC1,
HI, PRKA4, hAKAP82, p82), ATPase family AAA domain containing 2
(ATAD2, ANCCA, CT137, PRO2000), kinetochore scaffold 1 (KNL1,
AF15Q14, CASC5, CT29, D40, MCPH4, PPP1R55, Spc7, hKNL-1, hSpc105),
centrosomal protein 55 (CEP55, C10orf3, CT111, MARCH, URCC6),
cancer/testis antigen 1A (CTAG1A, ESO1, CT6.1, LAGE-2, LAGE2A,
NY-ESO-1), cancer/testis antigen 1B (CTAG1B, CT6.1, CTAG, CTAG1,
ESO1, LAGE-2, LAGE2B, NY-ESO-1), cancer/testis antigen 2 (CTAG2,
CAMEL, CT2, CT6.2, CT6.2a, CT6.2b, ESO2, LAGE-1, LAGE2B),
CCCTC-binding factor like (CTCFL, BORIS, CT27, CTCF-T, HMGB1L1,
dJ579F20.2), catenin alpha 2 (CTNNA2, CAP-R, CAPR, CDCBM9, CT114,
CTNR), cancer/testis antigen 83 (CT83, CXorf61, KK-LC-1, KKLC1),
cyclin A1 (CCNA1, CT146), DEAD-box helicase 43 (DDX43, CT13, HAGE),
developmental pluripotency associated 2 (DPPA2, CT100, ECAT15-2,
PESCRG1), fetal and adult testis expressed 1 (FATE1, CT43, FATE),
FMR1 neighbor (FMR1NB, CT37, NY-SAR-35, NYSAR35), HORMA domain
containing 1 (HORMAD1, CT46, NOHMA), insulin like growth factor 2
mRNA binding protein 3 (IGF2BP3, CT98, IMP-3, IMP3, KOC, KOC1,
VICKZ3), leucine zipper protein 4 (LUZP4, CT-28, CT-8, CT28,
HOM-TES-85), lymphocyte antigen 6 family member K (LY6K, CT97,
HSJ001348, URLC10, ly-6K), maelstrom spermatogenic transposon
silencer (MAEL, CT128, SPATA35), MAGE family member A1 (MAGEA1,
CT1.1, MAGE1); MAGE family member A3 (MAGEA3, CT1.3, HIP8, HYPD,
MAGE3, MAGEA6); MAGE family member A4 (MAGEA4, CT1.4, MAGE-41,
MAGE-X2, MAGE4, MAGE4A, MAGE4B); MAGE family member A11 (MAGEA11,
CT1.11, MAGE-11, MAGE11, MAGEA-11); MAGE family member C1 (MAGEC1,
CT7, CT7.1); MAGE family member C2 (MAGEC2, CT10, HCA587, MAGEE1);
MAGE family member D1 (MAGED1, DLXIN-1, NRAGE); MAGE family member
D2 (MAGED2, 11B6, BARTS5, BCG-1, BCG1, HCA10, MAGE-D2); kinesin
family member 20B (KIF20B, CT90, KRMP1, MPHOSPH1, MPP-1, MPP1);
NUF2 component of NDC80 kinetochore complex (NUF2, CDCA1, CT106,
NUF2R), nuclear RNA export factor 2 (NXF2, CT39, TAPL-2, TCP11X2);
PAS domain containing repressor 1 (PASD1, CT63, CT64, OXTES1), PDZ
binding kinase (PBK, CT84, HEL164, Nori-3, SPK, TOPK); piwi like
RNA-mediated gene silencing 2 (PIWIL2, CT80, HILI, PIWIL1L, mili);
preferentially expressed antigen in melanoma (PRAME, CT130, MAPE,
OIP-4, OIP4); sperm associated antigen 9 (SPAG9, CT89, HLC-6, HLC4,
HLC6, JIP-4, JIP4, JLP, PHET, PIG6), sperm protein associated with
the nucleus, X-linked, family member A1 (SPANXA1, CT11.1, CT11.3,
NAP-X, SPAN-X, SPAN-Xa, SPAN-Xb, SPANX, SPANX-A), SPANX family
member A2 (SPANXA2, CT11.1, CT11.3, SPANX, SPANX-A, SPANX-C,
SPANXA, SPANXC), SPANX family member C (SPANXC, CT11.3, CTp11,
SPANX-C, SPANX-E, SPANXE), SPANX family member D (SPANXD, CT11.3,
CT11.4, SPANX-C, SPANX-D, SPANX-E, SPANXC, SPANXE, dJ171K16.1), SSX
family member 1 (SSX1, CT5.1, SSRC), SSX family member 2 (SSX2,
CT5.2, CT5.2A, HD21, HOM-MEL-40, SSX), synaptonemal complex protein
3 (SYCP3, COR1, RPRGL4, SCP3, SPGF4), testis expressed 14,
intercellular bridge forming factor (TEX14, CT113, SPGF23),
transcription factor Dp family member 3 (TFDP3, CT30, DP4, HCA661),
serine protease 50 (PRSS50, CT20, TSP50), TTK protein kinase (TTK,
CT96, ESK, MPH1, MPS1, MPS1L1, PYT), and zinc finger protein 165
(ZNF165, CT53, LD65, ZSCAN7). In some embodiments, the
non-immunoglobulin antigen-binding domains or antibody mimetic
proteins are selected from the group consisting of adnectins,
affibody molecules, affilins, affimers, affitins, alphabodies,
anticalins, peptide aptamers, armadillo repeat proteins (ARMs),
atrimers, avimers, designed ankyrin repeat proteins (DARPins.RTM.),
fynomers, knottins, Kunitz domain peptides, monobodies, and
nanoCLAMPs. In some embodiments, the immunotherapy comprises
co-administering one or more antagonists or inhibitors of an
inhibitory immune checkpoint protein or receptor and/or one or more
activators or agonists of a stimulatory immune checkpoint protein
or receptor. In some embodiments, the one or more immune checkpoint
proteins or receptors are selected from the group consisting of:
CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and
immunoglobulin domain containing 2 (TMIGD2, CD28H), CD84 (LY9B,
SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3);
V-set domain containing T cell activation inhibitor 1 (VTCN1,
B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA);
immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural
killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6);
HERV-H LTR-associating 2 (HHLA2, B7H7); inducible T cell
co-stimulator (ICOS, CD278); inducible T cell costimulator ligand
(ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40);
TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8
(CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9
(CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL);
TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte
associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF);
TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related
sequence A (MICA); MHC class I polypeptide-related sequence B
(MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1,
PD1, PD-1); cytotoxic T-lymphocyte associated protein 4 (CTLA4,
CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2
(NECTIN2, CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell
adhesion molecule (PVR, CD155); PVR related immunoglobulin domain
containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM
domains (TIGIT); T cell immunoglobulin and mucin domain containing
4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2,
TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activating 3 (LAG3,
CD223); signaling lymphocytic activation molecule family member 1
(SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3);
SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7,
CD319); UL16 binding protein 1 (ULBP1); UL16 binding protein 2
(ULBP2); UL16 binding protein 3 (ULBP3); retinoic acid early
transcript 1E (RAETIE; ULBP4); retinoic acid early transcript 1G
(RAETIG; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6);
killer cell immunoglobulin like receptor, three Ig domains and long
cytoplasmic tail 1 (KIR, CD158E1); killer cell lectin like receptor
C1 (KLRC1, NKG2A, CD159A); killer cell lectin like receptor K1
(KLRK1, NKG2D, CD314); killer cell lectin like receptor C2 (KLRC2,
CD159c, NKG2C); killer cell lectin like receptor C3 (KLRC3, NKG2E);
killer cell lectin like receptor C4 (KLRC4, NKG2F); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig
domains and long cytoplasmic tail 2 (KIR2DL2); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 3 (KIR2DL3); killer cell immunoglobulin like receptor, three
Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell
lectin like receptor D1 (KLRD1); killer cell lectin like receptor
G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid binding Ig like lectin
7 (SIGLEC7); and sialic acid binding Ig like lectin 9 (SIGLEC9). In
some embodiments, the immunotherapy comprises co-administering one
or more blockers or inhibitors of one or more T-cell inhibitory
immune checkpoint proteins or receptors. In some embodiments, the
T-cell inhibitory immune checkpoint proteins or receptors are
selected from the group consisting of CD274 (CD274, PDL1, PD-L1);
programmed cell death 1 ligand 2 (PDCDLG2, PD-L2, CD273);
programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T-lymphocyte
associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain
containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set
immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin
superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270),
TNFSF14 (HVEML); CD272 (B and T lymphocyte associated (BTLA)); PVR
related immunoglobulin domain containing (PVRIG, CD112R); T cell
immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte
activating 3 (LAG3, CD223); hepatitis A virus cellular receptor 2
(HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell
immunoglobulin like receptor, three Ig domains and long cytoplasmic
tail 1 (KIR, CD158E1); killer cell immunoglobulin like receptor,
two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 2 (KIR2DL2); killer
cell immunoglobulin like receptor, two Ig domains and long
cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin like
receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1).
In some embodiments, the immunotherapy comprises co-administering
one or more agonists or activators of one or more T-cell
stimulatory immune checkpoint proteins or receptors. In some
embodiments, the T-cell stimulatory immune checkpoint proteins or
receptors are selected from the group consisting of CD27, CD70;
CD40, CD40LG; inducible T cell costimulator (ICOS, CD278);
inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor
superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4
(TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR),
TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2
(NECTIN2, CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell
adhesion molecule (PVR, CD155). In some embodiments, the
immunotherapy comprises co-administering one or more blockers or
inhibitors of one or more NK-cell inhibitory immune checkpoint
proteins or receptors. In some embodiments, the NK-cell inhibitory
immune checkpoint proteins or receptors are selected from the group
consisting of killer cell immunoglobulin like receptor, three Ig
domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig
domains and long cytoplasmic tail 2 (KIR2DL2); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 3 (KIR2DL3); killer cell immunoglobulin like receptor, three
Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell
lectin like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin
like receptor D1 (KLRD1, CD94); killer cell lectin like receptor G1
(KLRG1; CLEC15A, MAFA, 2F1); sialic acid binding Ig like lectin 7
(SIGLEC7); and sialic acid binding Ig like lectin 9 (SIGLEC9). In
some embodiments, the immunotherapy comprises co-administering one
or more agonists or activators of one or more NK-cell stimulatory
immune checkpoint proteins or receptors. In some embodiments, the
NK-cell stimulatory immune checkpoint proteins or receptors include
without limitation CD16, CD226 (DNAM-1); killer cell lectin like
receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7
(SLAMF7). In some embodiments, the one or more immune checkpoint
inhibitors comprises a proteinaceous inhibitor (e.g., an antibody
or antigen binding fragment thereof, or a non-immunoglobulin
antibody mimetic protein) of PD-L1 (CD274), PD-1 (PDCD1) or CTLA4.
In some embodiments, the proteinaceous inhibitor of CTLA4 is
selected from the group consisting of ipilimumab, tremelimumab,
BMS-986218, AGEN1181, AGEN1884 (zalifrelimab), BMS-986249, MK-1308,
REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071,
ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144,
PBI-5D3H5, FPT-155 (CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4),
MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1),
XmAb-20717 (PD-1/CTLA4) and AK-104 (CTLA4/PD-1). In some
embodiments, the proteinaceous inhibitor of PD-L1 (CD274) or PD-1
(PDCD1) is selected from the group consisting of pembrolizumab,
nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI680 (AMP-514),
spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559,
CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055),
AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091,
AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genolimzumab
(CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210
(camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306,
(MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311,
JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308
(sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001),
BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4/PD-L1/CD28),
PF-06936308 (PD-1/CTLA4), MGD-013 (PD-1/LAG-3), FS-118
(LAG-3/PD-L1) MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752
(CTLA4/PD-1), RO-7121661 (PD-1/TIM-3), XmAb-20717 (PD-1/CTLA4),
AK-104 (CTLA4/PD-1), M7824 (PD-L1/TGFO-EC domain), CA-170
(PD-L1/VISTA), CDX-527 (CD27/PD-L1), LY-3415244 (TIM3/PDL1), and
INBRX-105 (4-1BB/PDL1). In some embodiments, the one or more immune
checkpoint inhibitors comprises a small molecule inhibitor of CD274
(PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1) or CTLA4.
In some embodiments, the small molecule inhibitor of CD274 or PDCD1
is selected from the group consisting of GS-4224, GS-4416,
INCB086550 and MAX10181. In some embodiments, the small molecule
inhibitor of CTLA4 comprises BPI-002. In some embodiments, the
immunotherapy comprises co-administering one or more cellular
therapies selected from the group consisting of: natural killer
(NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK)
cells, macrophage (MAC) cells, tumor infiltrating lymphocytes
(TILs) and dendritic cells (DCs). In some embodiments, the one or
more cellular therapies comprise a T cell therapy selected from the
group consisting of: alpha/beta TCR T cells, gamma/delta TCR T
cells, regulatory T (Treg) cells and TRuC.TM. T cells. In some
embodiments, the one or more cellular therapies comprise a NK cell
therapy comprising NK-92 cells. In some embodiments, the one or
more cellular therapies comprise cells that are autologous,
syngeneic or allogeneic to the subject. In some embodiments, the
one or more cellular therapies comprise cells comprising chimeric
antigen receptors (CARs). In some embodiments, the cells in the
cellular therapy bind to a target or tumor associated antigen (TAA)
(e.g., via a chimeric antigen receptor (CAR)) selected from the
group consisting of selected from the group consisting of: CD19,
MS4A1 (CD20), CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37,
CD38, CD40, CD44, CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39),
CD74, CD79b, CD80, CD86, IL3RA (CD123), PROM1 (CD133), CD137, SDC1
(CD138), alpha fetoprotein (AFP), c-Met; c-Kit; C-type lectin
domain family 12 member A (CLECI2A, CLL1, CD371); C-type lectin
domain containing 9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin,
PCAD); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9,
CAIX); carcinoembryonic antigen related cell adhesion molecule 3
(CEACAM3); carcinoembryonic antigen related cell adhesion molecule
5 (CEACAM5); carcinoembryonic antigen related cell adhesion
molecule 6 (CEACAM6, CD66c); chorionic somatomammotropin hormone 1
(CSH1, CS1); coagulation factor III, tissue factor (F3, TF);
collectin subfamily member 10 (COLEC10); delta like canonical Notch
ligand 3 (DLL3); ectonucleotide pyrophosphatase/phosphodiesterase 3
(ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor
(EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2);
epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine
kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP);
fibroblast growth factor receptor 2 (FGFR2); fibroblast growth
factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate
receptor 1 (FOLR1, FRa); GD2 ganglioside; glycoprotein NMB (GPNMB,
osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human
papillomavirus (HPV) E6; HPV E7; major histocompatibility complex
(MHC) class I-presented neoantigens, major histocompatibility
complex (MHC) class II-presented neoantigens, major
histocompatibility complex, class I, E (HLA-E); major
histocompatibility complex, class I, F (HLA-F); major
histocompatibility complex, class I, G (HLA-G, MHC-G); integrin
subunit beta 7 (ITGB7); leukocyte immunoglobulin like receptor B1
(LILRB1, ILT2); leukocyte immunoglobulin like receptor B2 (LILRB2,
ILT4); LY6/PLAUR domain containing 3 (LYPD3, C4.4A); glypican 3
(GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family member A1
(MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4
(MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1
(MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1
(MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin
1 (MUC1) and splice variants thereof (e.g., MUC1/C, D, and Z);
mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4
(NECTIN4); SLIT and NTRK like family member 6 (SLITRK6);
promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7
(inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM
family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid
binding Ig like lectin 7 (SIGLEC7); sialic acid binding Ig like
lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate),
member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6;
LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily
member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9
(TNFSF9; 4-1BB-L, CD137L); TNF receptor superfamily member 10a
(TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily member
10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily
member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor
superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor
superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF);
transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein
(TPBG, 5T4); trophinin (TRO, MAGED3); tumor associated calcium
signal transducer 2 (TACSTD2, TROP2, EGP1); Fucosyl GM1; sialyl
Lewis adhesion molecule (sLe); and Lewis Y antigen. In some
embodiments, the cells in the cellular therapy bind to an epitope
of a target or tumor associated antigen (TAA) presented in a major
histocompatibility complex (MHC) molecule (e.g., a neoantigen). In
some embodiments, the TAA is a cancer testis antigen. In some
embodiments, the cancer testis antigen is selected from the group
consisting of acrosin binding protein (ACRBP, CT23, OY-TES-1,
SP32), alpha fetoprotein (AFP, AFPD, FETA, HPAFP); A-kinase
anchoring protein 4 (AKAP4, AKAP 82, AKAP-4, AKAP82, CT99, FSC1,
HI, PRKA4, hAKAP82, p82), ATPase family AAA domain containing 2
(ATAD2, ANCCA, CT137, PRO2000), kinetochore scaffold 1 (KNL1,
AF15Q14, CASC5, CT29, D40, MCPH4, PPP1R55, Spc7, hKNL-1, hSpc105),
centrosomal protein 55 (CEP55, COorf3, CT111, MARCH, URCC6),
cancer/testis antigen 1A (CTAG1A, ESO1, CT6.1, LAGE-2, LAGE2A,
NY-ESO-1), cancer/testis antigen 1B (CTAG1B, CT6.1, CTAG, CTAG1,
ESO1, LAGE-2, LAGE2B, NY-ESO-1), cancer/testis antigen 2 (CTAG2,
CAMEL, CT2, CT6.2, CT6.2a, CT6.2b, ESO2, LAGE-1, LAGE2B),
CCCTC-binding factor like (CTCFL, BORIS, CT27, CTCF-T, HMGB1L1,
dJ579F20.2), catenin alpha 2 (CTNNA2, CAP-R, CAPR, CDCBM9, CT114,
CTNR), cancer/testis antigen 83 (CT83, CXorf61, KK-LC-1, KKLC1),
cyclin A1 (CCNA1, CT146), DEAD-box helicase 43 (DDX43, CT13, HAGE),
developmental pluripotency associated 2 (DPPA2, CT100, ECAT15-2,
PESCRG1), fetal and adult testis expressed 1 (FATE1, CT43, FATE),
FMR1 neighbor (FMR1NB, CT37, NY-SAR-35, NYSAR35), HORMA domain
containing 1 (HORMAD1, CT46, NOHMA), insulin like growth factor 2
mRNA binding protein 3 (IGF2BP3, CT98, IMP-3, EIP3, KOC, KOC1,
VICKZ3), leucine zipper protein 4 (LUZP4, CT-28, CT-8, CT28,
HOM-TES-85), lymphocyte antigen 6 family member K (LY6K, CT97,
HSJ001348, URLC10, ly-6K), maelstrom spermatogenic transposon
silencer (MAEL, CT128, SPATA35), MAGE family member A1 (MAGEA,
CT1.1, MAGE1); MAGE family member A3 (MAGEA3, CT1.3, HIP8, HYPD,
MAGE3, MAGEA6); MAGE family member A4 (MAGEA4, CT1.4, MAGE-41,
MAGE-X2, MAGE4, MAGE4A, MAGE4B); MAGE family member A11 (MAGEA11,
CT1.11, MAGE-11, MAGE11, MAGEA-11); MAGE family member C1 (MAGEC1,
CT7, CT7.1); MAGE family member C2 (MAGEC2, CT10, HCA587, MAGEE1);
MAGE family member D1 (MAGED1, DLXIN-1, NRAGE); MAGE family member
D2 (MAGED2, 11B6, BARTS5, BCG-1, BCG1, HCA10, MAGE-D2); kinesin
family member 20B (KIF20B, CT90, KRMP1, MPHOSPH1, MPP-1, MPP1);
NUF2 component of NDC80 kinetochore complex (NUF2, CDCA1, CT106,
NUF2R), nuclear RNA export factor 2 (NXF2, CT39, TAPL-2, TCP11X2);
PAS domain containing repressor 1 (PASD1, CT63, CT64, OXTES1), PDZ
binding kinase (PBK, CT84, HEL164, Nori-3, SPK, TOPK); piwi like
RNA-mediated gene silencing 2 (PIWIL2, CT80, HILI, PIWIL1L, mili);
preferentially expressed antigen in melanoma (PRAME, CT130, MAPE,
OIP-4, OIP4); sperm associated antigen 9 (SPAG9, CT89, HLC-6, HLC4,
HLC6, JIP-4, JIP4, JLP, PHET, PIG6), sperm protein associated with
the nucleus, X-linked, family member A1 (SPANXA1, CT11.1, CT11.3,
NAP-X, SPAN-X, SPAN-Xa, SPAN-Xb, SPANX, SPANX-A), SPANX family
member A2 (SPANXA2, CT11.1, CT11.3, SPANX, SPANX-A, SPANX-C,
SPANXA, SPANXC), SPANX family member C (SPANXC, CT11.3, CTp11,
SPANX-C, SPANX-E, SPANXE), SPANX family member D (SPANXD, CT11.3,
CT11.4, SPANX-C, SPANX-D, SPANX-E, SPANXC, SPANXE, dJ171K16.1), SSX
family member 1 (SSX1, CT5.1, SSRC), SSX family member 2 (SSX2,
CT5.2, CT5.2A, HD21, HOM-MEL-40, SSX), synaptonemal complex protein
3 (SYCP3, COR1, RPRGL4, SCP3, SPGF4), testis expressed 14,
intercellular bridge forming factor (TEX14, CT113, SPGF23),
transcription factor Dp family member 3 (TFDP3, CT30, DP4, HCA661),
serine protease 50 (PRSS50, CT20, TSP50), TTK protein kinase (TTK,
CT96, ESK, MPH1, MPS1, MPS1L1, PYT), and zinc finger protein 165
(ZNF165, CT53, LD65, ZSCAN7). In some embodiments, the cytokine or
chemokine therapy comprises co-administering one or more
immunostimulatory cytokines or chemokines that promote or increase
the proliferation or activation of T cells (including alpha/beta
TCR T cells and gamma/delta TCR T cells), NK-T cells, NK cells,
and/or dendritic cells. In some embodiments, the one or more
immunostimulatory cytokines or chemokines are selected from the
group consisting of: IL 2, IL-12, IL-15, IL-18, IL-21, interferon
(IFN)-.alpha., IFN-.beta., IFN-.gamma., CXCL9/Mig (monokine induced
by interferon-y), CXCL10/IP10 (interferon-y-inducible 10 kDa
protein) and CXCL11/I-TAC (interferon-inducible T cell
a-chemoattractant), CXCL4/PF4 (platelet factor 4), monocyte
chemoattractant protein 2 (MCP-2), macrophage inflammatory protein
1 alpha (MIP-1.alpha.), macrophage inflammatory protein 1 beta
(MIP-10) and regulated on activation normal T expressed and
secreted protein (RANTES). In some embodiments, the one or more
additional therapeutic agents comprises an activator or agonist of:
a toll-like receptor (TLR); a stimulator of interferon genes
(STING) receptor; inducible T cell costimulator (ICOS, CD278);
and/or a TNF receptor superfamily (TNFRSF) member. In some
embodiments, the TNF receptor superfamily (TNFRSF) member is
selected from the group consisting of: TNFRSF1A, TNFRSF1B, TNFRSF4
(OX40), TNFRSF5 (CD40), TNFRSF6 (FAS), TNFRSF7 (CD27), TNFRSF8
(CD30), TNFRSF9 (4-1BB, CD137), TNFRSF10A (CD261, DR4, TRAILR1),
TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10C (CD263, TRAILR3),
TNFRSF10D (CD264, TRAILR4), TNFRSF11A (CD265, RANK), TNFRSF11B,
TNFRSF12A (CD266), TNFRSF13B (CD267), TNFRSF13C (CD268), TNFRSF16
(NGFR, CD271), TNFRSF17 (BCMA, CD269), TNFRSF18 (GITR, CD357),
TNFRSF19, TNFRSF21 (CD358, DR6), and TNFRSF25 (DR3). In some
embodiments, the TNFRSF4 (OX40 or CD134) activator or agonist
comprises INCAGN1949, tavolimab (MEDI0562), pogalizumab
(MOXR0916/RG7888), MED16469, BMS 986178, PF-04518600, GSK3174998,
IBI101, ATOR-1015, ABBV-368 or SL-279252. In some embodiments, the
TNFRSF9 (4-1BB or CD137) activator or agonist comprises urelumab,
BMS-663513, utomilumab (PF-05082566), CTX-471, MP-0310, ADG-106,
ATOR-1017 or AGEN2373. In some embodiments, the TNFRSF18 (GITR or
CD357) activator or agonist comprises GWN323, MEDI1873, MK-1248,
MK-4166, TRX518, INCAGN1876, BMS-986156, BMS-986256, AMG-228,
ASP1951 (PTZ 522), FPA-154 or OMP-336B11. In some embodiments, the
one or more additional therapeutic agents comprises a molecule that
concurrently binds to TNF receptor superfamily member 4 (TNFRSF4,
OX40 or CD134) and TNF receptor superfamily member 18 (TNFRSF18,
GITR or CD357). In some embodiments, the TLR agonist or activator
is selected from the group consisting of a TLR2 agonist, a TLR3
agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8
agonist and a TLR9 agonist. In some embodiments, the TLR7 agonist
is selected from the group consisting of GS 9620, DS-0509, LHC-165
and TMX-101 (imiquimod), and/or wherein the TLR8 agonist is
selected from the group consisting of GS-9688 and NKTR-262 (dual
TLR7/TLR8 agonist). In some embodiments, the STING receptor agonist
or activator is selected from the group consisting of ADU-S100
(MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532,
SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthenone-4-acetic acid
(DMXAA), cyclic-GAMP (cGAMP) and cyclic-di-AMP. In some
embodiments, the one or more additional therapeutic agents
comprises an anti-CD47 antibody. In some embodiments, the anti-CD47
antibody is magrolimab. In some embodiments, the one or more
additional therapeutic agents comprises an inhibitor of SIRPalpha.
In some embodiments, the SIRPalpha inhibitor is selected from the
group consisting of AL-008, RRx-001, CTX-5861, FSI-189 (GS-0189),
ES-004, BI765063, ADU1805, and CC-95251. In some embodiments, the
one or more additional therapeutic agents comprises an inhibitor or
antagonist of. protein tyrosine phosphatase, non-receptor type 11
(PTPN11 or SHP2), myeloid cell leukemia sequence 1 (MCL1) apoptosis
regulator, mitogen-activated protein kinase kinase kinase kinase 1
(MAP4K1) (also called Hematopoietic Progenitor Kinase 1 (HPK1)),
phosphatidylinositol-4,5-bisphosphate 3-kinase, including
catalytic
subunit alpha (PIK3CA), catalytic subunit beta (PIK3CB), catalytic
subunit gamma (PIK3CG) and catalytic subunit delta (PIK3CD),
diacylglycerol kinase alpha (DGKA, DAGK, DAGK1 or DGK-alpha),
5'-nucleotidase ecto (NT5E or CD73), ectonucleoside triphosphate
diphosphohydrolase 1 (ENTPD1 or CD39), transforming growth factor
beta 1 (TGFB1 or TGFO), heme oxygenase 1 (HMOX1, HO-1 or HO1), heme
oxygenase 2 (HMOX2, HO-2 or H02), vascular endothelial growth
factor A (VEGFA or VEGF), erb-b2 receptor tyrosine kinase 2 (ERBB2,
HER2, HER2/neu or CD340), epidermal growth factor receptor (EGFR,
ERBB, ERBB1 or HER1), ALK receptor tyrosine kinase (ALK, CD246),
poly(ADP-ribose) polymerase 1 (PARP1), poly(ADP-ribose) polymerase
2 (PARP2), TCDD inducible poly(ADP-ribose) polymerase (TIPARP,
PARP7), cyclin dependent kinase 4 (CDK4), cyclin dependent kinase 6
(CDK6), TNF receptor superfamily member 14 (TNFRSF14, HVEM, CD270),
T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked
inhibitor of apoptosis (XIAP, BIRC4, IAP-3), baculoviral IAP repeat
containing 2 (BIRC2, cIAPI), baculoviral IAP repeat containing 3
(BIRC3, cIAP2), baculoviral IAP repeat containing 5 (BIRC5,
survivin), C-C motif chemokine receptor 2 (CCR2, CD192), C-C motif
chemokine receptor 5 (CCR5, CD195), C-C motif chemokine receptor 8
(CCR8, CDw198), C-X-C motif chemokine receptor 2 (CXCR2, CD182),
C-X-C motif chemokine receptor 3 (CXCR3, CD182, CD183), C-X-C motif
chemokine receptor 4 (CXCR4, CD184), cytokine inducible SH2
containing protein (CISH), arginase (ARG1, ARG2), carbonic
anhydrase (CA1, CA2, CA3, CA4, CA5A, CA5B, CA6, CA7, CA8, CA9,
CA10, CA11, CA12, CA13, CA14), prostaglandin-endoperoxide synthase
1 (PTGS1, COX-1), prostaglandin-endoperoxide synthase 2 (PTGS2,
COX-2), secreted phospholipase A2, prostaglandin E synthase (PTGES,
PGES), arachidonate 5-lipoxygenase (ALOX5, 5-LOX), soluble epoxide
hydrolase 2 (EPHX2), indoleamine 2,3-dioxygenase 1 (IDO1),
indoleamine 2,3-dioxygenase 2 (IDO2), hypoxia inducible factor 1
subunit alpha (HIF1A), angiopoietin 1 (ANGPT1), Endothelial TEK
tyrosine kinase (TIE-2, TEK), Janus kinase 1 (JAK1), catenin beta 1
(CTNNB1), histone deacetylase 9 (HDAC9), 5'-3' exoribonuclease 1
(XRN1) and/or WRN RecQ like helicase (WRN). In some embodiments,
the inhibitor comprises an antibody or an antigen-binding fragment
thereof, or antibody-drug conjugate thereof, CD3-targeting
multi-specific molecule, CD16-targeting multi-specific molecule,
non-immunoglobulin antigen binding molecule or antibody mimetic
protein. In some embodiments, the inhibitor comprises an inhibitory
nucleic acid (e.g., an siRNA). In some embodiments, the inhibitor
comprises a small organic molecule. In some embodiments, the
inhibitor of 5'-nucleotidase ecto (NT5E or CD73) is selected from
the group consisting of MEDI9447 (oleclumab), CPI-006, BMS-986179,
IPH5301, TJ4309 (TJD5), NZV-930, AB-680, PSB-12379, PSB-12441,
PSB-12425, CB-708, GS-1423 (AGEN1423) and PBF-1662. In some
embodiments, the inhibitor of CCR2 and/or CCR5 is selected from the
group consisting of BMS-813160, PF-04136309 and CCX-872. In some
embodiments, the inhibitor of MCL1 is selected from the group
consisting of AMG-176, AMG-397, S-64315, AZD-5991, 483-LM, A
1210477, UMI-77 and JKY-5-037. In some embodiments, the inhibitor
of PTPN11 or SHP2 is selected from the group consisting of TNO155
(SHP-099), RMC-4550, JAB-3068 and RMC-4630. In some embodiments,
the inhibitor of Janus kinase 1 (JAK1) is selected from the group
consisting of filgotinib, tofacitinib, baricitinib and ABT-494. In
some embodiments, the one or more additional therapeutic agents
comprises a regulatory T cell (Treg) inhibitor. In some
embodiments, the subject further receives radiation therapy. In
some embodiments, the radiation therapy comprises stereotactic body
radiation therapy (SBRT). In some embodiments, the one or more
additional therapeutic agents comprises one or more anti-neoplastic
or chemotherapeutic agents. In some embodiments, the one or more
anti-neoplastic or chemotherapeutic agents are selected from the
group consisting of a nucleoside analog (e.g., 5-fluorouracil,
gemcitabine, cytarabine, cladribine, pentostatin, fludarabine), a
taxane (e.g., paclitaxel, nab-paclitaxel, docetaxel, cabazitaxel),
a platinum coordination complex (cisplatin, carboplatin,
oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin,
picoplatin, satraplatin, dicycloplatin, eptaplatin, lobaplatin,
miriplatin), a dihydrofolate reductase (DHFR) inhibitor (e.g.,
methotrexate, trimetrexate, pemetrexed), a topoisomerase inhibitor
(e.g., doxorubicin, daunorubicin, dactinomycin, eniposide,
epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone,
pixantrone, sobuzoxane, topotecan, irinotecan, MM-398 (liposomal
irinotecan), vosaroxin and GPX-150, aldoxorubicin, AR-67,
mavelertinib, AST-2818, avitinib (ACEA-0010), irofulven (MGI-114)),
an alkylating agent (e.g., a nitrogen mustard (e.g.,
cyclophosphamide, chlormethine, uramustine or uracil mustard,
melphalan, chlorambucil, ifosfamide, bendamustine, temozolomide,
carmustine), a nitrosourea (e.g., carmustine, lomustine,
streptozocin), an alkyl sulfonate (e.g., busulfan)), and mixtures
thereof. In some embodiments, the one or more additional
therapeutic agents comprises a FOLFOX regimen, a FOLFIRI regimen, a
FOLFOXIRI regimen or a FOLFIRINOX regimen. In some embodiments, the
one or more additional therapeutic agents comprises an antiviral
therapy. In some embodiments, the antiviral therapy comprises
co-administering a hepatitis B virus (HBV) therapeutic agent. In
some embodiments, the HBV therapeutic agent is selected from an HBV
vaccine, HBV polymerase inhibitor, immunomodulator, interferon
alpha receptor ligand, hyaluronidase inhibitor, Hepatitis B Surface
Antigen (HBsAg) inhibitor, cyclophilin inhibitor, antisense
oligonucleotide, short interfering RNA (siRNA), or DNA-directed RNA
interference (ddRNAi) targeting HBV viral mRNA, endonuclease
modulator (e.g., PGN-514), ribonucleotide reductase inhibitor
(e.g., Trimidox), HBV replication inhibitor, non-canonical RNA
polymerase PAPD5 and PAPD7 inhibitor (e.g., siRNA), covalently
closed circular DNA inhibitor (cccDNA), caspase 9 stimulator (e.g.,
ENOB-B-01), CD3 modulator (e.g., IMC-I109V), Ffar2 and Ffar3
agonist (e.g., SFA-001), additional HBV antibody, CCR2 chemokine
(e.g., propagermanium), FXR agonist, thymosine antagonist,
nucleoprotein modulator, retinoic acid-inducible gene stimulator 1,
arginase inhibitor (e.g., astodrimer, CB-1158, C-201, resminostat),
endonuclease inhibitor (e.g., PGN-154), ribonuclease reductase
inhibitor (e.g., Trimidox), non-nucleoside reverse transcriptase
inhibitor (NNRTI), HBV replication inhibitor, capsid inhibitor,
transcript inhibitor, CAR-T cell therapy, TCR-T cell therapy, and
inhibitor of an HCV nonstructural protein (e.g., NS5A inhibitor
(e.g., ledipasvir, velpatasvir), a NS5B inhibitor (e.g.,
sofosbuvir, mericitabine), a NS3 inhibitor (e.g., voxilaprevir)).
In some embodiments, the HBV vaccine is selected from the group
consisting of HBsAG-HBIG complex, ARB-1598, Bio-Hep-B, NASVAC,
abi-HB (intravenous), ABX-203, Tetrabhay, GX-110E, GS-4774, peptide
vaccine (epsilonPA-44), Hepatrol-07, NASVAC (NASTERAP), MP-321,
BEVAC, Revac B mcf, Revac B+, MGN-1333, KW-2, CVI-HBV-002,
AltraHepB, VGX-6200, FP-02, FP-02.2 (HepTeell), NU-500, HBVax,
im/TriGrid/antigen vaccine, Mega-CD40L-adjuvanted vaccine, HepB-v,
RG7944 (INO-1800), recombinant VLP-based therapeutic vaccine (HBV
infection, VLP Biotech), hepatitis B therapeutic DNA vaccine,
AdTG-17909, AdTG-17910 AdTG-18202, ChronVac-B, TG-1050, VVX-001,
GSK-3528869A (ChAd155-hli-HBV+MVA-HBV +Hbc-HBs/AS01B-4), VBI-2601,
VTP-300 (ChAdOx1-Si--HBV-CPmut-TPA-Ssh prime and
MVA-SIi-HBV-CPmut-TPA-Ssh boost), MVA-BN, AVA-2100, HBV-ADV311,
YS-HBV-002, and Lm HBV. In some embodiments, the HBV polymerase
inhibitor is selected from the group consisting of adefovir
(HEPSERA.RTM.), emtricitabine (EMTRIVA.RTM.), tenofovir disoproxil
fumarate (VIREAD.RTM.), tenofovir alafenamide, tenofovir, tenofovir
disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide
hemifumarate, tenofovir dipivoxil, tenofovir dipivoxil fumarate,
tenofovir octadecyloxyethyl ester, CMX-157, tenofovir exalidex,
besifovir, entecavir (BARACLUDE.RTM.), entecavir maleate,
telbivudine (TYZEKA.RTM.), filocilovir, pradefovir, clevudine,
ribavirin, lamivudine (EPIVIR-HBV.RTM.), phosphazide, famciclovir,
fusolin, metacavir, ATI-2173, SNC-019754, FMCA, AGX-1009,
AR-II-04-26, HIP-1302, tenofovir disoproxil aspartate, tenofovir
disoproxil orotate, and HS-10234. In some embodiments, the
immunomodulator is selected from the group consisting of
rintatolimod, imidol hydrochloride, ingaron, dermaVir, plaquenil
(hydroxychloroquine), proleukin, hydroxyurea, mycophenolate mofetil
(MPA) and its ester derivative mycophenolate mofetil (MMF),
JNJ-440, WF-10, AB-452, ribavirin, IL-12, INO-9112, polymer
polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ-0535,
TG-1050, ABI-H2158, BMS-936559, GS-9688, RO-7011785, RG-7854,
RO-6871765, AIC-649, and IR-103. In some embodiments, the
interferon alpha receptor ligand is selected from the group
consisting of interferon alpha-2b (INTRON A.RTM.), pegylated
interferon alpha-2a (PEGASYS.RTM.), PEGylated interferon alpha-1b,
interferon alpha 1b (HAPGEN.RTM.), Veldona, Infradure, Roferon-A,
YPEG-interferon alfa-2a (YPEG-rhIFNalpha-2a), P-1101, Algeron,
Alfarona, Ingaron (interferon gamma), rSIFN-co (recombinant super
compound interferon), Ypeginterferon alfa-2b (YPEG-rhIFNalpha-2b),
MOR-22, peginterferon alfa-2b (PEG-INTRON.RTM.), Bioferon,
Novaferon, Inmutag (Inferon), MULTIFERON.RTM., interferon
alfa-n1(HUMOFERON), interferon beta-ia (AVONEX.RTM.), Shaferon,
interferon alfa-2b (Axxo), Alfaferone, interferon alfa-2b
(BioGeneric Pharma), interferon-alpha 2 (CJ), Laferonum, VIPEG,
BLAUFERON-A, BLAUFERON-B, Intermax Alpha, Realdiron, Lanstion,
Pegaferon, PDferon-B PDferon-B, interferon alfa-2b (IFN,
Laboratorios Bioprofarma), alfainterferona 2b, Kalferon, Pegnano,
Feronsure, PegiHep, interferon alfa 2b (Zydus-Cadila), interferon
alfa 2a, Optipeg A, Realfa 2B, Reliferon, interferon alfa-2b
(Amega), interferon alfa-2b (Virchow), ropeginterferon alfa-2b,
rHSA-IFN alpha-2a (recombinant human serum albumin intereferon
alpha 2a fusion protein), PEG-IFN-alpha, rHSA-IFN alpha 2b,
recombinant human interferon alpha-(lb, 2a, 2b), peginterferon
alfa-2b (Amega), peginterferon alfa-2a, Reaferon-EC, Proquiferon,
Uniferon, Urifron, interferon alfa-2b (Changchun Institute of
Biological Products), Anterferon, Shanferon, Layfferon, Shang Sheng
Lei Tai, INTEFEN, SINOGEN, Fukangtai, Pegstat, rHSA-IFN alpha-2b,
SFR-9216, and Interapo (Interapa). In some embodiments, the
hyaluronidase inhibitor is astodrimer. In some embodiments, the
Hepatitis B Surface Antigen (HBsAg) inhibitor is selected from the
group consisting of AK-074, HBF-0259, PBHBV-001, PBHBV-2-15,
PBHBV-2-1, REP-9AC, REP-9C, REP-9, REP-2139, REP-2139-Ca, REP-2055,
REP-2163, REP-2165, REP-2053, REP-2031, REP-006, and REP-9AC'. In
some embodiments, the HBsAg inhibitor is an HBsAg secretion
inhibitor selected from the group consisting of BM601, GST-HG-131,
and AB-452. In some embodiments, the cyclophilin inhibitor is
selected from the group consisting of CPI-431-32, EDP-494, OCB-030,
SCY-635, NVP-015, NVP-018, NVP-019, and STG-175. In some
embodiments, the antisense oligonucleotide targeting viral mRNA is
selected from the group consisting of ISIS-HBVRx, IONIS-HBVRx,
IONIS-HBV-LRx, IONIS-GSK6-LRx, GSK-3389404, and RG-6004. In some
embodiments, the short interfering RNA (siRNA) or DNA-directed RNA
interference (ddRNAi) is selected from the group consisting of
TKM-HBV (TKM-HepB), ALN-HBV (e.g., ALN-HBV02), SR-008, HepB-nRNA,
ARC-520, ARC-521, ARB-1740, ARB-1467, AB-729, DCR-HBVS, RG-6084
(PD-L1), RG-6217, ALN-HBV-02, JNJ-3989 (ARO-HBV), STSG-0002,
ALG-010133, ALG-ASO, LUNAR-HBV and DCR-HBVS (DCR-219). In some
embodiments, the ddRNAi is BB-HB-331. In some embodiments, the HBV
replication inhibitor is selected from the group consisting of
GP-31502, isothiafludine, IQP-HBV, RM-5038, and Xingantie. In some
embodiments, the cccDNA is selected from the group consisting of
BSBI-25, ccc-R08, and CHR-101. In some embodiments, the additional
HBV antibody targets a surface antigen of hepatitis B virus. In
some embodiments, the additional HBV antibody is selected from
lenvervimab (GC-1102), XTL-17, XTL-19, KN-003, IV Hepabulin SN,
VIR-3434, Omri-Hep-B, Nabi-HB, Hepatect CP, HepaGam B, igantibe,
Niuliva, CT-P24, Fovepta (BT-088), and HBC-34. In some embodiments,
the FXR agonist is selected from the group consisting of EYP-001,
GS-9674, EDP-305, MET-409, Tropifexor, AKN-083, RDX-023, BWD-100,
LMB-763, INV-3, NTX-023-1, EP-024297 and GS-8670. In some
embodiments, the thymosine antagonist is selected from Thymalfasin,
recombinant thymosin alpha 1 (GeneScience), NL-004 and PEGylated
thymosin alpha-1. In some embodiments, the nucleoprotein modulator
is selected from GS-4882, AB-423, AB-836, AT-130, ALG-001075,
ALG-001024, ALG-000184, EDP-514, GLS4, NVR-1221, NVR-3778, AL-3778,
BAY 41-4109, morphothiadine mesilate, ARB-168786, ARB-880,
ARB-1820, GST-HG-141, JNJ-379, JNJ-632, RG-7907, GST-HG-141,
HEC-72702, KL-060332, AB-506, ABI-H0731, ABI-H3733, JNJ-440,
ABI-H2158, CB-HBV-001, AK-0605, SOC-10, SOC-11 and DVR-23. In some
embodiments, the retinoic acid-inducible gene stimulator 1 is
selected from the group consisting of inarigivir soproxil
(SB-9200), SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537, ORI-9020,
ORI-9198, ORI-7170, and RGT-100. In some embodiments, the arginase
inhibitor is selected from the group consisting of CB-1158, C-201,
and resminostat. In some embodiments, the CAR-T cell therapy
directed to HBV therapy includes a population of immune effector
cells engineered to express a chimeric antigen receptor (CAR),
wherein the CAR includes an HBV antigen-binding domain (e.g.,
HbsAg-CART). In some embodiments, the TCR-T cell therapy includes T
cells expressing HBV-specific T cell receptors (e.g.,
(HBsAg)-specific TCR). In some embodiments, the HBV therapeutic
agent is selected from alpha-hydroxytropolones, amdoxovir,
antroquinonol, beta-hydroxycytosine nucleosides, ARB-199, CCC-0975,
ccc-R08, elvucitabine, ezetimibe, cyclosporin A, gentiopicrin
(gentiopicroside), HH-003, hepalatide, JNJ-56136379, nitazoxanide,
birinapant, NJK14047, NOV-205 (molixan, BAM-205), oligotide,
mivotilate, feron, GST-HG-131, levamisole, Ka Shu Ning, alloferon,
WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3,
BP-Inter-014, oleanolic acid, HepB-nRNA, cTP-5 (rTP-5), HSK-II-2,
HEISCO-106-1, HEISCO-106, Hepbarna, IBPB-006IA, Hepuyinfen,
DasKloster 0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210,
OB-AI-004, PF-06, picroside, DasKloster-0039, hepulantai, IMB-2613,
NCO-48 Fumarate, TCM-800B, reduced glutathione, RO-6864018,
RG-7834, QL-007sofosbuvir, ledipasvir, UB-551, PA-1010, HPN-BV1,
STSG-0002, and ZH-2N. In some embodiments, the antiviral therapy
comprises co-administering a hepatitis C virus (HCV) therapeutic
agent. In some embodiments, the HCV therapeutic agent is selected
from daclatasvir, ledipasvir, ombitasvir, elbasvir, sofosbuvir,
dasabuvir, ribavirin, asunaprevir, simeprevir, paritaprevir,
ritonavir, elbasvir, and grazoprevir. In some embodiments, the
antiviral therapy comprises co-administering a human
immunodeficiency virus (HIV) therapeutic agent. In some
embodiments, the HIV therapeutic agent comprises an HIV protease
inhibitor, HIV ribonuclease H inhibitor, HIV Nef inhibitor, HIV
reverse transcriptase inhibitor, HIV integrase inhibitor, HIV entry
inhibitor, HIV maturation inhibitor, a latency reversing agent, HIV
capsid inhibitor, HIV targeting antibody, HIV vaccine, or a birth
control or contraceptive regimen. In some embodiments, the HIV
protease inhibitor is selected from the group consisting of
amprenavir, atazanavir, brecanavir, darunavir, fosamprenavir,
fosamprenavir calcium, indinavir, indinavir sulfate, lopinavir,
nelfinavir, nelfinavir mesylate, ritonavir, saquinavir, saquinavir
mesylate, tipranavir, AEBL-2, DG-17, GS-1156, TMB-657 (PPL-100),
T-169, BL-008, MK-8122, TMB-607, GRL-02031, and TMC-310911. In some
embodiments, the HIV ribonuclease H inhibitor is NSC-727447. In
some embodiments, the HIV Nef inhibitor is FP-1. In some
embodiments, the HIV reverse transcriptase inhibitor is a
non-nucleoside/non-nucleotide reverse transcriptase inhibitor. In
some embodiments, the non-nucleoside/non-nucleotide inhibitor is
select from the group consisting of dapivirine, delavirdine,
delavirdine mesylate, doravirine, efavirenz, etravirine, lentinan,
nevirapine, rilpivirine, ACC-007, ACC-008, AIC-292, F-18, KM-023,
PC-1005, VM-1500A-LAI, PF-3450074, elsulfavirine (sustained release
oral, HIV infection), elsulfavirine (long-acting injectable
nanosuspension, HIV infection), and elsulfavirine (VM-1500). In
some embodiments, the HIV reverse transcriptase inhibitor is a
nucleoside or nucleotide inhibitor. In some embodiments the
nucleoside or nucleotide inhibitor is selected from the group
consisting of adefovir, adefovir dipivoxil, azvudine,
emtricitabine, tenofovir, tenofovir alafenamide, tenofovir
alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir
disoproxil, tenofovir disoproxil
fumarate, tenofovir octadecyloxyethyl ester (AGX-1009), tenofovir
disoproxil hemifumarate, VIDEX.RTM. and VIDEX EC.RTM. (didanosine,
ddl), abacavir, abacavir sulfate, alovudine, apricitabine,
censavudine, didanosine, elvucitabine, festinavir, fosalvudine
tidoxil, CMX-157, dapivirine, doravirine, etravirine, OCR-5753,
tenofovir disoproxil orotate, fozivudine tidoxil, lamivudine,
phosphazid, stavudine, zalcitabine, zidovudine, rovafovir et
alafenamide (GS-9131), GS-9148, MK-8504, MK-8591, MK-8583, VM-2500
and KP-1461. In some embodiments, the HIV integrase inhibitor is
selected from the group consisting of elvitegravir, elvitegravir
(extended-release microcapsules), curcumin, derivatives of
curcumin, chicoric acid, derivatives of chicoric acid,
3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic
acid, aurintricarboxylic acid, derivatives of aurintricarboxylic
acid, caffeic acid phenethyl ester, derivatives of caffeic acid
phenethyl ester, tyrphostin, derivatives of tyrphostin, quercetin,
derivatives of quercetin, raltegravir, PEGylated raltegravir,
dolutegravir, JTK-351, bictegravir, AVX-15567, cabotegravir
(long-acting injectable), diketo quinolin-4-1 derivatives,
integrase-LEDGF inhibitor, ledgins, M-522, M-532, MK-0536,
NSC-310217, NSC-371056, NSC-48240, NSC-642710, NSC-699171,
NSC-699172, NSC-699173, NSC-699174, stilbenedisulfonic acid, T-169,
STP-0404, VM-3500 and cabotegravir. In some embodiments, the HIV
integrase inhibitor is an HIV non-catalytic site, or allosteric,
integrase inhibitor (NCINI). In some embodiments, the NCINI is
selected from the group consisting of CX-05045, CX-05168, and
CX-14442. In some embodiments, the HIV an entry inhibitor is
AAR-501, LBT-5001, cenicriviroc, a CCR5 inhibitor, a gp41
inhibitor, a CD4 attachment inhibitor, a gpl20 inhibitor, a gp160
inhibitor a, and a CXCR4 inhibitor. In some embodiments, the CCR5
inhibitor is selected from the group consisting of aplaviroc,
vicriviroc, maraviroc, maraviroc (long-acting injectable
nanoemulsion), cenicriviroc, leronlimab (PRO-140), adaptavir
(RAP-101), nifeviroc (TD-0232), anti-GP120/CD4 or CCR5 bispecific
antibodies, B-07, MB-66, polypeptide C25P, TD-0680, thioraviroc and
vMIP (Haimipu). In some embodiments, the gp41 inhibitor is selected
from the group consisting of albuvirtide, enfuvirtide, griffithsin
(gp41/gp120/gp160 inhibitor), BMS-986197, enfuvirtide biobetter,
enfuvirtide biosimilar, HIV-1 fusion inhibitors (P26-Bapc), ITV-1,
ITV-2, ITV-3, ITV-4, CPT-31, Cl3hmAb, PIE-12 trimer and
sifuvirtide. In some embodiments, the a CD4 attachment inhibitor is
ibalizumab or a CADA analog. In some embodiments, the gp120
inhibitor selected from the group consisting of anti-HIV
microbicide, Radha-108 (receptol) 3B3-PE38, BanLec, bentonite-based
nanomedicine, fostemsavir tromethamine, IQP-0831, VVX-004, and
BMS-663068. In some embodiments, gp160 inhibitor is fangchinoline.
In some embodiments, the CXCR4 inhibitor selected from the group
consisting of plerixafor, ALT-1188, N15 peptide, and vMIP
(Haimipu). In some embodiments, the HIV entry inhibitor is selected
from docosanol, enfuvirtide, maraviroc, palivizumab, respiratory
syncytial virus immune globulin, intravenous [RSV-IGIV],
varicella-zoster immunoglobulin [VariZIG], and varicella-zoster
immune globulin [VZIG]). In some embodiments, the HIV maturation
inhibitor is selected from the group consisting of BMS-955176,
GSK-3640254 and GSK-2838232. In some embodiments, the latency
reversing agent is selected from the group consisting of toll-like
receptor (TLR) agonists (including TLR7 agonists, e.g., GS-9620
(vesatolimod), vesatolimod analogs), histone deacetylase (HDAC)
inhibitors, proteasome inhibitors (e.g., velcade), protein kinase C
(PKC) activators (e.g., indolactam, prostratin, ingenol B,
DAG-lactones), Smyd2 inhibitors, BET-bromodomain 4 (BRD4)
inhibitors, ionomycin, IAP antagonists (inhibitor of apoptosis
proteins; e.g., APG-1387, LBW-242), SMAC mimetics (including
TL32711, LCL161, GDC-0917, HGS1029, AT-406, Debio-1143), PMA, SAHA
(suberanilohydroxamic acid, or suberoyl, anilide, and hydroxamic
acid), NIZ-985, IL-15 modulating antibodies, IL-15, IL-15 fusion
proteins, IL-15 receptor agonists, JQ1, disulfiram, amphotericin B,
and ubiquitin inhibitors (e.g., largazole analogs, APH-0812,
GSK-343). In some embodiments, the HIV capsid inhibitor is selected
from the group consisting of capsid polymerization inhibitors,
capsid disrupting compounds, HIV nucleocapsid p7 (NCp7) inhibitors
(e.g., azodicarbonamide), and HIV p24 capsid protein inhibitors
(e.g., GS-6207, GS-CA1, AVI-621, AVI-101, AVI-201, AVI-301,
AVI-CAN1-15 series, and PF-3450074). In some embodiments, the HIV
targeting antibody is selected from bNAbs (broadly neutralizing
HIV-1 antibodies), TMB-360, antibodies targeting HIV gp120 or gp41,
antibody-recruiting molecules targeting HIV, anti-CD63 monoclonal
antibodies, anti-GB virus C antibodies, anti-GP120/CD4, gp120
bispecific monoclonal antibodies, CCR5 bispecific antibodies,
anti-Nef single domain antibodies, anti-Rev antibody, camelid
derived anti-CD18 antibodies, camelid-derived anti-ICAM-1
antibodies, DCVax-001, gp140 targeted antibodies, gp41-based HIV
therapeutic antibodies, human recombinant mAbs (PGT-121),
PGT121.414.LS, ibalizumab, Immuglo, MB-66, and VRC-HIVMAB091-00-AB.
In some embodiments, the HIV targeting antibody is selected from
the group consisting of UB-421, BF520.1, CHO1, CH59, C2F5, C4E10,
C2F5+C2G12+C4E10, 3BNC117, 3BNC117-LS, 3BNC60, DH270.1, DH270.6,
D1D2, 10-1074-LS, Cl3hmAb, GS-9722 (elipovimab), DH411-2, BG18,
GS-9721, PGT145, PGT121, PGT-121.60, PGT-121.66, PGT122, PGT-123,
PGT-124, PGT-125, PGT-126, PGT-151, PGT-130, PGT-133, PGT-134,
PGT-135, PGT-128, PGT-136, PGT-137, PGT-138, PGT-139, MDXO10
(ipilimumab), DH511, DH511-2, N6, N6LS, N49P6, N49P7, N49P7.1,
N49P9, N49P11, N60P1.1, N60P25.1, N60P2.1, N60P31.1, N60P22, NIH
45-46, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGDM1400, PGDM12,
PGDM21, PCDN-33A, 2Dm2m, 4Dm2m, 6Dm2m, PGDM1400, VRCO1, VRC-01-LS,
A32, 7B2, 10E8, VRC-07-523, VRC07-523LS, VRC24, VRC41.01, 10E8VLS,
3810109, 10E8v4, IMC-HIV, iMabm36, eCD4-Ig, IOMA, CAP256-VRC26.25,
DRVIA7, VRC-HIVMAB080-00-AB, VRC-HIVMAB060-00-AB, P2G12, VRC07,
354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411,
354BG426, VRC29.03, CAP256, CAP256-VRC26.08, CAP256-VRC26.09,
CAP256-VRC26.25, PCT64-24E and VRC38.01, PGT-151, CAP248-2B, 35022,
ACS202, VRC34 and VRC34.01, 10E8, 10E8v4, 10E8-5R-100cF, 4E10,
DH511.11P, 2F5, 7b2, and LN01. In some embodiments, the HIV
targeting antibody is a bispecific or trispecific antibody selected
from the group consisting of MGD014, B12BiTe, BiIA-SG,
TMB-bispecific, SAR-441236, VRC-01/PGDM-1400/10E8v4, 10E8.4/iMab,
and 10E8v4/PGT121-VRCO1. In some embodiments, the HIV targeting
antibody is an in vivo delivered bNAbs (e.g., AAV8-VRC07; mRNA
encoding anti-HIV antibody VRCO1; or engineered B-cells encoding
3BNC117). In some embodiments, the HIV vaccine is selected from the
group consisting of peptide vaccines, recombinant subunit protein
vaccines, live vector vaccines, DNA vaccines, HIV MAG DNA vaccine,
CD4-derived peptide vaccines, vaccine combinations, adenoviral
vector vaccines (an adenoviral vector such as Ad5, Ad26 or Ad35),
simian adenovirus (chimpanzee, gorilla, rhesus i.e., rhAd),
adeno-associated virus vector vaccines, chimpanzee adenoviral
vaccines (e.g., ChAdOXI, ChAd68, ChAd3, ChAd63, ChAd83, ChAd155,
ChAd157, Pan5, Pan6, Pan7, Pan9), Coxsackieviruses based vaccines,
enteric virus based vaccines, Gorilla adenovirus vaccines,
lentiviral vector based vaccine, arenavirus vaccines (e.g., LCMV,
Pichinde), bi-segmented or tri-segmented arenavirus based vaccine,
trimer-based HIV-1 vaccine, measles virus based vaccine, flavivirus
vector based vaccines, tobacco mosaic virus vector based vaccine,
Varicella-zoster virus based vaccine, human parainfluenza virus 3
(PIV3) based vaccines, poxvirus based vaccine (modified vaccinia
virus Ankara (MVA), orthopoxvirus-derived NYVAC, and
avipoxvirus-derived ALVAC (canarypox virus) strains); fowlpox virus
based vaccine, rhabdovirus-based vaccines, such as VSV and
marabavirus; recombinant human CMV (rhCMV) based vaccine,
alphavirus-based vaccines, such as semliki forest virus, venezuelan
equine encephalitis virus and sindbis virus; lipoplex (e.g., LNP)
formulated mRNA based therapeutic vaccines; and lipoplex (e.g.,
LNP)-formulated self-replicating RNA/self-amplifying RNA vaccines.
In some embodiments, the HIV vaccine is selected from the group
consisting of anti-CD40.Env-gp140 vaccine, Ad4-EnvC150, BG505
SOSIP.664 gp140 adjuvanted vaccine, BG505 SOSIP.GT1.1 gp140
adjuvanted vaccine, Chimigen HIV vaccine, ConM SOSIP.v7 gp140,
rgpl20 (AIDSVAX), ALVAC HIV (vCP1521)/AIDSVAX B/E (gp120) (RV144),
monomeric gp120 HIV-1 subtype C vaccine, MPER-656 liposome subunit
vaccine, Remune, ITV-1, Contre Vir, Ad5-ENVA-48, DCVax-001
(CDX-2401), Vacc-4x, Vacc-C5, VAC-3S, multiclade DNA recombinant
adenovirus-5 (rAd5), rAd5 gag-pol env A/B/C vaccine, Pennvax-G,
Pennvax-GP, Pennvax-G/MVA-CMDR, HIV-TriMix-mRNA vaccine,
HIV-LAMP-vax, Ad35, Ad35-GRIN, NAcGM3/VSSP ISA-51, poly-ICLC
adjuvanted vaccines, TatImmune, GTU-multiHIV (FIT-06),
ChAdV63.HIVconsv, gp140[delta]V2.TV1+MF-59, rVSVIN HIV-1 gag
vaccine, SeV-EnvF, SeV-Gag vaccine, AT-20, DNK-4, ad35-Grin/ENV,
TBC-M4, HIVAX, HIVAX-2, N123-VRC-34.01 inducing epitope-based HIV
vaccine, NYVAC-HIV-PT1, NYVAC-HIV-PT4, DNA-HIV-PT123, rAAV1-PG9DP,
GOVX-B11, GOVX-B21, GOVX-C55, TVI-HIV-1, Ad-4 (Ad4-env Clade
C+Ad4-mGag), Paxvax, EN41-UGR7C, EN41-FPA2, ENOB-HV-11, PreVaxTat,
AE-H, MYM-V101, CombiHIVvac, ADVAX, MYM-V201, MVA-CMDR, MagaVax,
DNA-Ad5 gag/pol/nef/nev (HVTN505), MVATG-17401, ETV-01, CDX-1401,
DNA and Sev vectors vaccine expressing SCaVII, rcAD26.MOS1.HIV-Env,
Ad26.Mod.HIV vaccine, Ad26.Mod.HIV+MVA mosaic vaccine+gp140,
AGS-004, AVX-101, AVX-201, PEP-6409, SAV-001, ThV-01, TL-01,
TUTI-16, VGX-3300, VIR-1111, IHV-001, and virus-like particle
vaccines such as pseudovirion vaccine, CombiVICHvac, LFn-p24 B/C
fusion vaccine, GTU-based DNA vaccine, HIV gag/pol/nef/env DNA
vaccine, anti-TAT HIV vaccine, conjugate polypeptides vaccine,
dendritic-cell vaccines (such as DermaVir), gag-based DNA vaccine,
GI-2010, gp41 HIV-1 vaccine, HIV vaccine (PIKA adjuvant), i-key/MHC
class II epitope hybrid peptide vaccines, ITV-2, ITV-3, ITV-4,
LIPO-5, multiclade Env vaccine, MVA vaccine, Pennvax-GP,
pp71-deficient HCMV vector HIV gag vaccine, rgp160 HIV vaccine,
RNActive HIV vaccine, SCB-703, Tat Oyi vaccine, TBC-M4, UBI HIV
gp120, Vacc-4x+romidepsin, variant gp120 polypeptide vaccine, rAd5
gag-pol env A/B/C vaccine, DNA.HTI and MVA.HTI,
VRC-HIVDNA016-00-VP+VRC-HIVADV014-00-VP, INO-6145, JNJ-9220, gp145
C.6980; eOD-GT8 60mer based vaccine, PD-201401, env (A, B, C,
A/E)/gag (C) DNA Vaccine, gp120 (A,B,C,A/E) protein vaccine,
PDPHV-201401, Ad4-EnvCN54, EnvSeq-1 Envs HIV-1 vaccine (GLA-SE
adjuvanted), HIV p24gag prime-boost plasmid DNA vaccine, HIV-1
iglbl2 neutralizing VRC-01 antibody-stimulating anti-CD4 vaccine,
arenavirus vector-based vaccines (Vaxwave, TheraT), MVA-BN HIV-1
vaccine regimen, UBI HIV gpl20, mRNA based prophylactic vaccines,
VPI-211, and TBL-1203HI. In some embodiments, the birth control or
contraceptive regimen is selected from the group consisting of
cyproterone acetate, desogestrel, dienogest, drospirenone,
estradiol valerate, ethinyl estradiol, ethynodiol, etonogestrel,
levomefolate, levonorgestrel, lynestrenol, medroxyprogesterone
acetate, mestranol, mifepristone, misoprostol, nomegestrol acetate,
norelgestromin, norethindrone, noretynodrel, norgestimate,
ormeloxifene, segestersone acetate, ulipristal acetate, and any
combinations thereof. In some embodiments, the antiviral therapy
comprises co-administering an influenza therapeutic agent. In some
embodiments, the influenza therapeutic agent is selected from a
matrix 2 inhibitor (e.g., amantadine, rimantadine), neuraminidase
inhibitor (e.g., zanamivir, oseltamivir, peramivir, laninamivir
octanoate), and polymerase inhibitor (e.g., ribavirin,
favipiravir). In some embodiments, the influenza virus inhibitor is
selected from amantadine, rimantadine, arbidol (umifenovir),
baloxavir marboxil, oseltamivir, peramivir, ingavirin, laninamivir
octanoate, zanamivir, favipiravir, ribavirin, and combinations
thereof. In some embodiments, the influenza virus inhibitor is
selected from amantadine, rimantadine, zanamivir, oseltamivir,
peramivir, laninamivir octanoate, ribavirin, and favipiravir. In
some embodiments, the antiviral therapy comprises co-administering
a respiratory syncytial virus (RSV) therapeutic agent. In some
embodiments, the RSV therapeutic agent is selected from ribavirin,
ALS-8112, and presatovir. In some embodiments, the antiviral
therapy comprises co-administering a picornavirus therapeutic
agent. In some embodiments, the picorna therapeutic agent is
selected from hydantoin, guanidine hydrochloride, L-buthionine
sulfoximine, Py-11, and rupintrivir. In some embodiments, the
antiviral therapy comprises co-administering an Ebola virus
therapeutic agent. In some embodiments, the Ebola virus therapeutic
agent is selected from ribavirin, palivizumab, motavizumab,
RSV-IGIV (RespiGam.RTM.), MEDI-557, A-60444, MDT-637, BMS-433771,
amiodarone, dronedarone, verapamil, Ebola Convalescent Plasma
(ECP), TKM-100201, BCX4430
((2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hyd-
roxymethyl)pyrrolidine-3,4-diol), favipiravir (also known as T-705
or Avigan), T-705 monophosphate, T-705 diphosphate, T-705
triphosphate, FGI-106
(1-N,7-N-bis[3-(dimethylamino)propyl]-3,9-dimethylquinolino[8,7-h-
]quinolone-1,7-diamine), JK-05, TKM-Ebola, ZMapp, rNAPc2,
VRC-EBOADC076-00-VP, OS-2966, MVA-BN filo, brincidofovir, Vaxart
adenovirus vector 5-based ebola vaccine, Ad26-ZEBOV, FiloVax
vaccine, GOVX-E301, GOVX-E302, Ebola virus entry inhibitors (NPC1
inhibitors), rVSV-EBOV. In some embodiments, the Ebola virus
therapeutic agent is selected from ZMapp, mAB114, and REGEN-EB3. In
some embodiments, the antiviral therapy comprises co-administering
a coronavirus therapeutic agent. In some embodiments, the
coronavirus is a Severe Acute Respiratory Syndrome
(SARS)-associated coronavirus. In some embodiments, the coronavirus
is a Middle-East Respiratory Syndrome (MERS)-associated
coronavirus. In some embodiments, the coronavirus is SARS-CoV-2. In
some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is a
RNA polymerase inhibitor (e.g., remdesivir, galidesivir). In some
embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is
remdesivir (GS-5734). In some embodiments, the SARS-CoV-2
(COVID-19) therapeutic agent is an anti-SARS-CoV-2 hyperimmune
globulin therapy (plasma from convalescent COVID-19 patients, e.g.,
processed into a hyperimmune globulin) (e.g., TAK-888). In some
embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is
selected from a COVID-19 vaccine (e.g., BN162, Ad5-nCoV, INO-4800,
mRN1273), anti-IL6 receptor antibody (e.g., tocilizumab, sarilumab,
TZLS-501), an anti-IL6 antibody (e.g., siltuximab), RNA dependent
RNA polymerase (RdRp) inhibitor (e.g., favipravir, remdesivir),
anti-CCR5 antibody (e.g., leronlimab (PRO 140)), broadly
neutralizing antibody (e.g., an anti-ACE2 receptor antibody,
SAB-185, COVID-HIG, COVID-EIG), ACE2 (angiotensin-converting enzyme
2)-Fc fusion protein (COVIDTRAP) or recombinant human ACE2 protein
(APN1), ACE-MAB.TM. bi-specific fusion protein designed to bind to
the spike protein of coronaviruses-including SARS-CoV-2 and
SARS-CoV (e.g., STI-4920, CMAB020), Janus kinase (JAK1/JAK2)
inhibitor (e.g., ruxolitinib, baricitinib), an siRNA (e.g.,
targeting angiotensin converting enzyme-2 (ACE2) or transmembrane
protease, serine 2 (TMPRSS2)), HIV-1 protease inhibitor (e.g.,
lopinavir/ritonavir; darunavir alone or in combination with
cobicistat), complement inhibitor (e.g., eculizumab), HCV protease
inhibitor (e.g., danoprevir), stem cell therapy (e.g.,
MultiStem.RTM., Remestemcel-L, CYNK-001), NK cell therapy
(NKG2D-ACE2 CAR-N cells), a neutralizing antibody against human
granulocyte-macrophage colony stimulating factor (GM-CSF) (e.g.,
IZN-101, gimsilumab), a vasoconstrictor (e.g., angiotensin II),
selective inhibitor of nuclear export (SINE), such as XPO1
inhibitor (e.g., selinexor), NSAID, including COX inhibitors (e.g.,
ibuprofen, aspirin, diclofenac, naxopren) and selective COX2
inhibitors (e.g., celecoxib, rofecoxib, etoricoxib, lumiracoxib,
valecoxib), and other antiviral agents (e.g., ENU200,
lopinavir/ritonavir combination). In some embodiments, the COVID-19
vaccine is an mRNA vaccine (e.g., BN162), including a lipoplex
(e.g., lipid-nanoparticle (LNP)) encapsulated vaccine (e.g.,
mRNA1273). In some embodiments, the COVID-19 vaccine is a DNA
vaccine (e.g., INO-4800). In some embodiments, the COVID-19 vaccine
encodes for a prefusion stabilized form of the Spike (S) protein
(e.g., mRNA1273). In some embodiments, the COVID-19 vaccine is a
recombinant protein-based vaccine consisting of the receptor
binding domain (RBI) of the spike protein of the coronavirus. In
some embodiments, the COVID-19 vaccine uses a Ligand Antigen
Epitope Presentation System (LEAPS) peptide including conserved
regions of coronavirus proteins to stimulate protective cell
mediated T cell responses and reduce viral load. In some
embodiments, the COVID-19 vaccine is a microneedle array
(MNA)-delivered vaccine. In some embodiments, the vaccine is based
on a flu vector expressing the surface antigen of SARS-CoV-2. In
some embodiments, the COVID-19 vaccine is an intranasal vaccine
(e.g., AdCOVID). In some embodiments, the COVID-19 vaccine is
NVX-CoV2373, NO4800, or BNT-162. In some embodiments, the
SARS-CoV-2 (COVID-19) therapeutic agent is selected from a PIKfyve
kinase inhibitor (e.g., apilimod), immunomodulator (e.g.,
rintatolimod), T-cell immunotherapy, recombinant sialidase (e.g.,
DAS181), CRAC channel inhibitor (e.g., CM-4620-IE), cardiac cell
therapy using allogeneic cardiosphere-derived cells (e.g.,
CAP-1002), cardioprotective drug (e.g., aspirin, plavix, lipitor,
opremazole), SiP receptor antagonist (e.g., fingolimod), a
cyclooxygenase-2 (COX-2) inhibitor (e.g., celecoxib),
phosphodiesterase-5 (PDE5) inhibitor (e.g., sildenafil citrate),
serine protease TMPRSS2 inhibitor (camostat mesylate), anti-human
complement 5a antibody (e.g., IFX-1), macrophage migration
inhibitory factor (MIF) inhibitor, phosphodiesterase (PDE)-4 and
-10 inhibitor (e.g., ibudilast), an eEFA2 inhibitor (e.g.,
plitidepsin), sphingosine kinase 2 (SK2) inhibitor (e.g.,
ABC294640, RHB-107), galectin inhibitor (e.g., BXT-10), membrane
fusion inhibitor (e.g., umifenovir), anti-PD1 antibody, thymosine,
antimalarial (e.g., chloroquine, hydroxychloroquine), and other
antiviral therapeutics (e.g., HTCC
(N-(2-hydroxypropyl)-3-trimethylammonium 47 chitosan chloride,
OYA1). In some embodiments, the subject has cancer. In some
embodiments, the subject is in cancer remission. In some
embodiment, the subject has a hematological cancer, e.g., a
leukemia (e.g., Acute Myelogenous Leukemia (AML), Acute
Lymphoblastic Leukemia (ALL), B-cell ALL, Myelodysplastic Syndrome
(MDS), myeloproliferative disease (MPD), Chronic Myelogenous
Leukemia (CML), Chronic Lymphocytic Leukemia (CLL),
undifferentiated leukemia), a lymphoma (e.g., small lymphocytic
lymphoma (SLL), mantle cell lymphoma (MCL), follicular lymphoma
(FL), T-cell lymphoma, B-cell lymphoma, diffuse large B-cell
lymphoma (DLBCL), marginal zone lymphoma (MZL), Waldestrom's
macroglobulinemia (WM)) and/or a myeloma (e.g., multiple myeloma
(MM)). In some embodiments, the subject has a solid tumor. In some
embodiments, the tumor or cancer is malignant or metastatic. In
some embodiments, the subject has a tumor infiltrated with
conventional dendritic cells (cDC1). In some embodiments, the tumor
infiltrating dendritic cells express C-C motif chemokine receptor 5
(CCR5, CD195) and/or X-C motif chemokine receptor 1 (XCR1) on their
cell surface. In some embodiments, the tumor infiltrating dendritic
cells express one or more cell surface proteins selected from the
group consisting of XCR1, cell adhesion molecule 1 (CADM1), C-type
lectin domain containing 9A (CLEC9A, CD370), and thrombomodulin
(THBD). In some embodiments, the tumor infiltrating dendritic cells
express one or more cell surface proteins selected from the group
consisting of CD1A, CD1C, CD1E, signal regulatory protein alpha
(SIRPA; CD172A), CD207 and Fc fragment of IgE receptor Ia (FCERIA).
In some embodiments, the tumor infiltrating dendritic cells express
one or more proteins selected from the group consisting of basic
leucine zipper ATF-like transcription factor 3 (BATF3) and
interferon regulatory factor 8 (IRF8). In some embodiments, the
tumor infiltrating dendritic cells express one or more proteins
selected from the group consisting of BATF3, IRF8, THBD, CLEC9A and
XCR1. In some embodiments, the subject has a cancer that detectably
expresses or overexpresses one or more cell surface immune
checkpoint receptors. In some embodiments, the one or more cell
surface immune checkpoint receptors are selected from the group
consisting of: CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2),
transmembrane and immunoglobulin domain containing 2 (TMIGD2,
CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244
(SLAMF4); CD276 (B7H3); V-set domain containing T cell activation
inhibitor 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR,
B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3);
natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1,
B7H6); HERV-H LTR-associating 2 (HHLA2, B7H7); inducible T cell
co-stimulator (ICOS, CD278); inducible T cell costimulator ligand
(ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40);
TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8
(CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9
(CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL);
TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte
associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF);
TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related
sequence A (MICA); MHC class I polypeptide-related sequence B
(MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1,
PD1, PD-1); cytotoxic T-lymphocyte associated protein 4 (CTLA4,
CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2
(NECTIN2, CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell
adhesion molecule (PVR, CD155); PVR related immunoglobulin domain
containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM
domains (TIGIT); T cell immunoglobulin and mucin domain containing
4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2,
TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activating 3 (LAG3,
CD223); signaling lymphocytic activation molecule family member 1
(SLAMFI, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3);
SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7,
CD319); UL16 binding protein 1 (ULBP1); UL16 binding protein 2
(ULBP2); UL16 binding protein 3 (ULBP3); retinoic acid early
transcript 1E (RAETIE; ULBP4); retinoic acid early transcript 1G
(RAETIG; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6);
killer cell immunoglobulin like receptor, three Ig domains and long
cytoplasmic tail 1 (KIR, CD158E1); killer cell lectin like receptor
C1 (KLRC1, NKG2A, CD159A); killer cell lectin like receptor K1
(KLRK1, NKG2D, CD314); killer cell lectin like receptor C2 (KLRC2,
CD159c, NKG2C); killer cell lectin like receptor C3 (KLRC3, NKG2E);
killer cell lectin like receptor C4 (KLRC4, NKG2F); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig
domains and long cytoplasmic tail 2 (KIR2DL2); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 3 (KIR2DL3); killer cell immunoglobulin like receptor, three
Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell
lectin like receptor D1 (KLRD1); killer cell lectin like receptor
G1 (KLRG1; CLECI5A, MAFA, 2F1); sialic acid binding Ig like lectin
7 (SIGLEC7); and sialic acid binding Ig like lectin 9 (SIGLEC9). In
some embodiments, greater than about 50% of the cancer or tumor
cells detectably express one or more cell surface immune checkpoint
receptor proteins (e.g., PD1 or PD-L1; a so-called "hot" cancer or
tumor). In some embodiments, greater than about 1% and less than
about 50% of the cancer or tumor cells detectably express one or
more cell surface immune checkpoint receptor proteins (e.g., PD1 or
PD-L; a so called "warm" cancer or tumor). In some embodiments,
less than about 1% of the cancer cells detectably express one or
more cell surface immune checkpoint receptor proteins (e.g., PD1 or
PD-L; a so called "cold" cancer or tumor). In some embodiments, the
subject has a cancer or tumor selected from the group consisting of
an epithelial tumor (e.g., a carcinoma, a squamous cell carcinoma,
a basal cell carcinoma, a squamous intraepithelial neoplasia), a
glandular tumor (e.g., an adenocarcinoma, an adenoma, an
adenomyoma), a mesenchymal or soft tissue tumor (e.g., a sarcoma, a
rhabdomyosarcoma, a leiomyosarcoma, a liposarcoma, a fibrosarcoma,
a dermatofibrosarcoma, a neurofibrosarcoma, a fibrous histiocytoma,
an angiosarcoma, an angiomyxoma, a leiomyoma, a chondroma, a
chondrosarcoma, an alveolar soft-part sarcoma, an epithelioid
hemangioendothelioma, a Spitz tumor, a synovial sarcoma), and a
lymphoma. In some embodiments, the subject has a solid tumor in or
arising from a tissue or organ selected from the group consisting
of. bone (e.g., adamantinoma, aneurysmal bone cysts, angiosarcoma,
chondroblastoma, chondroma, chondromyxoid fibroma, chondrosarcoma,
chordoma, dedifferentiated chondrosarcoma, enchondroma, epithelioid
hemangioendothelioma, fibrous dysplasia of the bone, giant cell
tumour of bone, haemangiomas and related lesions, osteoblastoma,
osteochondroma, osteosarcoma, osteoid osteoma, osteoma, periosteal
chondroma, Desmoid tumor, Ewing sarcoma); lips and oral cavity
(e.g., odontogenic ameloblastoma, oral leukoplakia, oral squamous
cell carcinoma, primary oral mucosal melanoma); salivary glands
(e.g., pleomorphic salivary gland adenoma, salivary gland adenoid
cystic carcinoma, salivary gland mucoepidermoid carcinoma, salivary
gland Warthin's tumors); esophagus (e.g., Barrett's esophagus,
dysplasia and adenocarcinoma); gastrointestinal tract, including
stomach (e.g., gastric adenocarcinoma, primary gastric lymphoma,
gastrointestinal stromal tumors (GISTs), metastatic deposits,
gastric carcinoids, gastric sarcomas, neuroendocrine carcinoma,
gastric primary squamous cell carcinoma, gastric adenoacanthomas),
intestines and smooth muscle (e.g., intravenous leiomyomatosis),
colon (e.g., colorectal adenocarcinoma), rectum, anus; pancreas
(e.g., serous neoplasms, including microcystic or macrocystic
serous cystadenoma, solid serous cystadenoma, Von Hippel-Landau
(VHL)-associated serous cystic neoplasm, serous cystadenocarcinoma;
mucinous cystic neoplasms (MCN), intraductal papillary mucinous
neoplasms (IPMN), intraductal oncocytic papillary neoplasms (IOPN),
intraductal tubular neoplasms, cystic acinar neoplasms, including
acinar cell cystadenoma, acinar cell cystadenocarcinoma, pancreatic
adenocarcinoma, invasive pancreatic ductal adenocarcinomas,
including tubular adenocarcinoma, adenosquamous carcinoma, colloid
carcinoma, medullary carcinoma, hepatoid carcinoma, signet ring
cell carcinoma, undifferentiated carcinoma, undifferentiated
carcinoma with osteoclast-like giant cells, acinar cell carcinoma,
neuroendocrine neoplasms, neuroendocrine microadenoma,
neuroendocrine tumors (NET), neuroendocrine carcinoma (NEC),
including small cell or large cell NEC, insulinoma, gastrinoma,
glucagonoma, serotonin-producing NET, somatostatinoma, VIPoma,
solid-pseudopapillary neoplasms (SPN), pancreatoblastoma); gall
bladder (e.g., carcinoma of the gallbladder and extrahepatic bile
ducts, intrahepatic cholangiocarcinoma); neuro-endocrine (e.g.,
adrenal cortical carcinoma, carcinoid tumors, phaeochromocytoma,
pituitary adenomas); thyroid (e.g., anaplastic (undifferentiated)
carcinoma, medullary carcinoma, oncocytic tumors, papillary
carcinoma, adenocarcinoma); liver (e.g., adenoma, combined
hepatocellular and cholangiocarcinoma, fibrolamellar carcinoma,
hepatoblastoma, hepatocellular carcinoma, mesenchymal, nested
stromal epithelial tumor, undifferentiated carcinoma;
hepatocellular carcinoma, intrahepatic cholangiocarcinoma, bile
duct cystadenocarcinoma, epithelioid hemangioendothelioma,
angiosarcoma, embryonal sarcoma, rhabdomyosarcoma, solitary fibrous
tumor, teratoma, York sac tumor, carcinosarcoma, rhabdoid tumor);
kidney (e.g., ALK-rearranged renal cell carcinoma, chromophobe
renal cell carcinoma, clear cell renal cell carcinoma, clear cell
sarcoma, metanephric adenoma, metanephric adenofibroma, mucinous
tubular and spindle cell carcinoma, nephroma, nephroblastoma (Wilms
tumor), papillary adenoma, papillary renal cell carcinoma, renal
oncocytoma, renal cell carcinoma, succinate dehydrogenase-deficient
renal cell carcinoma, collecting duct carcinoma); breast (e.g.,
invasive ductal carcinoma, including without limitation, acinic
cell carcinoma, adenoid cystic carcinoma, apocrine carcinoma,
cribriform carcinoma, glycogen-rich/clear cell, inflammatory
carcinoma, lipid-rich carcinoma, medullary carcinoma, metaplastic
carcinoma, micropapillary carcinoma, mucinous carcinoma,
neuroendocrine carcinoma, oncocytic carcinoma, papillary carcinoma,
sebaceous carcinoma, secretory breast carcinoma, tubular carcinoma;
lobular carcinoma, including without limitation, pleomorphic
carcinoma, signet ring cell carcinoma, peritoneum (e.g.,
mesothelioma; primary peritoneal cancer); female sex organ tissues,
including ovary (e.g., choriocarcinoma, epithelial tumors, germ
cell tumors, sex cord-stromal tumors), Fallopian tubes (e.g.,
serous adenocarcinoma, mucinous adenocarcinoma, endometrioid
adenocarcinoma, clear cell adenocarcinoma, transitional cell
carcinoma, squamous cell carcinoma, undifferentiated carcinoma,
M0llerian tumors, adenosarcoma, leiomyosarcoma, teratoma, germ cell
tumors, choriocarcinoma, trophoblastic tumors), uterus (e.g.,
carcinoma of the cervix, endometrial polyps, endometrial
hyperplasia, intraepithelial carcinoma (EIC), endometrial carcinoma
(e.g., endometrioid carcinoma, serous carcinoma, clear cell
carcinoma, mucinous carcinoma, squamous cell carcinoma,
transitional carcinoma, small cell carcinoma, undifferentiated
carcinoma, mesenchymal neoplasia), leiomyoma (e.g., endometrial
stromal nodule, leiomyosarcoma, endometrial stromal sarcoma (ESS),
mesenchymal tumors), mixed epithelial and mesenchymal tumors (e.g.,
adenofibroma, carcinofibroma, adenosarcoma, carcinosarcoma
(malignant mixed mesodermal sarcoma--MMMT)), endometrial stromal
tumors, endometrial malignant mullerian mixed tumours, gestational
trophoblastic tumors (partial hydatiform mole, complete hydatiform
mole, invasive hydatiform mole, placental site tumour)), vulva,
vagina; male sex organ tissues, including prostate, testis (e.g.,
germ cell tumors, spermatocytic seminoma), penis; bladder (e.g.,
squamous cell carcinoma, urothelial carcinoma, bladder urothelial
carcinoma); brain, (e.g., gliomas (e.g., astrocytomas, including
non-infiltrating, low-grade, anaplastic, glioblastomas;
oligodendrogliomas, ependymomas), meningiomas, gangliogliomas,
schwannomas (neurilemmomas), craniopharyngiomas, chordomas,
Non-Hodgkin lymphomas (NHLs), indolent non-Hodgkin's lymphoma
(iNHL), refractory iNHL, pituitary tumors; eye (e.g., retinoma,
retinoblastoma, ocular melanoma, posterior uveal melanoma, iris
hamartoma); head and neck (e.g., nasopharyngeal carcinoma,
Endolymphatic Sac Tumor (ELST), epidermoid carcinoma, laryngeal
cancers including squamous cell carcinoma (SCC) (e.g., glottic
carcinoma, supraglottic carcinoma, subglottic carcinoma,
transglottic carcinoma), carcinoma in situ, verrucous, spindle cell
and basaloid SCC, undifferentiated carcinoma, laryngeal
adenocarcinoma, adenoid cystic carcinoma, neuroendocrine
carcinomas, laryngeal sarcoma), head and neck paragangliomas (e.g.,
carotid body, jugulotympanic, vagal); thymus (e.g., thymoma); heart
(e.g., cardiac myxoma); lung (e.g., small cell carcinoma (SCLC),
non-small cell lung carcinoma (NSCLC), including squamous cell
carcinoma (SCC), adenocarcinoma and large cell carcinoma,
carcinoids (typical or atypical), carcinosarcomas, pulmonary
blastomas, giant cell carcinomas, spindle cell carcinomas,
pleuropulmonary blastoma); lymph (e.g., lymphomas, including
Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), indolent
non-Hodgkin's lymphoma (iNHL), refractory iNHL, Epstein-Barr virus
(EBV)-associated lymphoproliferative diseases, including B cell
lymphomas and T cell lymphomas (e.g., Burkitt lymphoma; large B
cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell
lymphoma, indolent B-cell lymphoma, low grade B cell lymphoma,
fibrin-associated diffuse large cell lymphoma; primary effusion
lymphoma; plasmablastic lymphoma; extranodal NK/T cell lymphoma,
nasal type; peripheral T cell lymphoma, cutaneous T cell lymphoma,
angioimmunoblastic T cell lymphoma; follicular T cell lymphoma;
systemic T cell lymphoma), lymphangioleiomyomatosis); central
nervous system (CNS) (e.g., gliomas including astrocytic tumors
(e.g., pilocytic astrocytoma, pilomyxoid astrocytoma, subependymal
giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse
astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma,
protoplasmic astrocytoma, anaplastic astrocytoma, glioblastoma
(e.g., giant cell glioblastoma, gliosarcoma, glioblastoma
multiforme) and gliomatosis cerebri), oligodendroglial tumors
(e.g., oligodendroglioma, anaplastic oligodendroglioma),
oligoastrocytic tumors (e.g., oligoastrocytoma, anaplastic
oligoastrocytoma), ependymal tumors (e.g., subependymom,
myxopapillary ependymoma, ependymomas (e.g., cellular, papillary,
clear cell, tanycytic), anaplastic ependymoma), optic nerve glioma,
and non-gliomas (e.g., choroid plexus tumors, neuronal and mixed
neuronal-glial tumors, pineal region tumors, embryonal tumors,
medulloblastoma, meningeal tumors, primary CNS lymphomas, germ cell
tumors, Pituitary adenomas, cranial and paraspinal nerve tumors,
stellar region tumors); neurofibroma, meningioma, peripheral nerve
sheath tumors, peripheral neuroblastic tumours (including without
limitation neuroblastoma, ganglioneuroblastoma, ganglioneuroma),
trisomy 19 ependymoma);
neuroendocrine tissues (e.g., paraganglionic system including
adrenal medulla (pheochromocytomas) and extra-adrenal paraganglia
((extra-adrenal) paragangliomas); skin (e.g., clear cell
hidradenoma, cutaneous benign fibrous histiocytomas, cylindroma,
hidradenoma, melanoma (including cutaneous melanoma, mucosal
melanoma), pilomatricoma, Spitz tumors); and soft tissues (e.g.,
aggressive angiomyxoma, alveolar rhabdomyosarcoma, alveolar soft
part sarcoma, angiofibroma, angiomatoid fibrous histiocytoma,
synovial sarcoma, biphasic synovial sarcoma, clear cell sarcoma,
dermatofibrosarcoma protuberans, desmoid-type fibromatosis, small
round cell tumor, desmoplastic small round cell tumor,
elastofibroma, embryonal rhabdomyosarcoma, Ewing's tumors/primitive
neurectodermal tumors (PNET), extraskeletal myxoid chondrosarcoma,
extraskeletal osteosarcoma, paraspinal sarcoma, inflammatory
myofibroblastic tumor, lipoblastoma, lipoma, chondroid lipoma,
liposarcoma/malignant lipomatous tumors, liposarcoma, myxoid
liposarcoma, fibromyxoid sarcoma, lymphangioleiomyoma, malignant
myoepithelioma, malignant melanoma of soft parts, myoepithelial
carcinoma, myoepithelioma, myxoinflammatory fibroblastic sarcoma,
undifferentiated sarcoma, pericytoma, rhabdomyosarcoma,
non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), soft tissue
leiomyosarcoma, undifferentiated sarcoma, well-differentiated
liposarcoma. In some embodiments, the subject has a cancer selected
from the group consisting of a lung cancer, a colorectal cancer, a
breast cancer, a prostate cancer, a cervical cancer and a head and
neck cancer. In some embodiments, the subject has neutropenia or
lymphopenia. In some embodiments, the subject has received a
lymphodepleting chemotherapy regimen. In some embodiments, the
subject is naive to or has not received chemotherapy. In some
embodiments, the subject has bone marrow cells, or is not depleted
of bone marrow cells. In some embodiments, the subject does not
have a mutation in the gene encoding the FLT3 receptor that causes
or results in or is associated with cancer. In some embodiments,
the subject has a viral infection. In some embodiments, the subject
is at risk of contracting a viral infection. In certain
embodiments, the subject may have not previously received antiviral
treatment (treatment naive). In certain embodiments, the subject
may have previously received antiviral treatment (treatment
experienced). In some embodiments, the viral infection is an HBV
infection. In some embodiments, the viral infection is an HIV
infection. In some embodiments, the viral infection is a
coronavirus infection. In some embodiments, the coronavirus is a
MERS-associated virus. In some embodiments, the coronavirus is a
SARS-associated virus. In some embodiments, the coronavirus is a
COVID-19-associated virus (SARS-CoV-2). In certain embodiments, the
subject may have previously received antiviral treatment and
developed resistance to the previously received antiviral
treatment. In some embodiments, the FLT3L-Fc fusion protein, the
homodimer, the heterodimer, the conjugate, the polynucleotide, the
vector, the lipoplex, such as an LNP) and/or the pharmaceutical
composition are administered systemically or locally. In some
embodiments, the FLT3L-Fc fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition are
administered intravenously, intratumorally, subcutaneously,
intradermally, intramuscularly, intraperitoneally, intravesically,
intracranially, intrathecally, intracavitary or intraventricularly.
In some embodiments, the FLT3L-Fc fusion protein, the homodimer,
the heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition and
the one or more additional therapeutic agents are administered by
the same routes or by different routes of administration. In some
embodiments, the FLT3L-Fc fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition and
the one or more additional therapeutic agents are administered
concurrently or sequentially. In some embodiments, the FLT3L-Fc
fusion protein has a serum half-life of at least about 7 days,
e.g., at least about 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30 days, or longer. In some embodiments, the methods entail
multiple administrations of the FLT3L-Fc fusion protein, the
homodimer, the heterodimer, the conjugate, the polynucleotide, the
vector, the lipoplex, such as an LNP, and/or the pharmaceutical
composition, optionally with one or more additional therapeutic
agents, at predetermined intervals. In some embodiments, the fusion
protein, the homodimer, the heterodimer, the conjugate, the
polynucleotide, the vector, the lipoplex, such as an LNP, and/or
the pharmaceutical composition is administered once weekly (i.e.,
QW), once bi-weekly (i.e., once every other week, or once every two
weeks or Q2W), once thrice-weekly (i.e., once every three weeks or
Q3W), once monthly (i.e., QM) or once bi-monthly dosing (i.e., once
every other month, or once every two months or Q2M), or less often.
In some embodiments, the FLT3L-Fc fusion protein, the homodimer,
the heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition and
the one or more additional therapeutic agents are co-administered
according to the same schedule (e.g., co-administered at the same
time intervals). In some embodiments, the FLT3L-Fc fusion protein,
the homodimer, the heterodimer, the conjugate, the polynucleotide,
the vector, the lipoplex, such as an LNP, and/or the pharmaceutical
composition and the one or more additional therapeutic agents are
co-administered according to different schedules (e.g.,
co-administered at different time intervals). In some embodiments,
the FLT3L fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide, the vector, the lipoplex, such as an
LNP, and/or the pharmaceutical composition is administered at a
dose in the range of about 0.5 .mu.g/kg to about 5000 .mu.g/kg,
e.g., at least about 0.5 .mu.g/kg per dose and up to about 1
.mu.g/kg, 2 .mu.g/kg, 3 .mu.g/kg, 4 g/kg, 5 .mu.g/kg, 6 .mu.g/kg, 7
.mu.g/kg, 8 .mu.g/kg, 9 .mu.g/kg, 10 .mu.g/kg, 15 .mu.g/kg, 20
.mu.g/kg, 30 .mu.g/kg, 50 g/kg, 100 .mu.g/kg, 150 .mu.g/kg, 300
.mu.g/kg, 400 .mu.g/kg, 500 .mu.g/kg, 600 .mu.g/kg, 700 .mu.g/kg,
800 g/kg, 900 .mu.g/kg, 1000 .mu.g/kg, 1500 .mu.g/kg, 2000
.mu.g/kg, 2500 .mu.g/kg, 3000 .mu.g/kg, 3500 .mu.g/kg, 4000
.mu.g/kg, or 5000 .mu.g/kg, per dose. In some embodiments, the
FLT3L fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide, the vector, the lipoplex, such as an
LNP and/or the pharmaceutical composition is administered at a dose
in the range of about 1 g/kg to about 100 .mu.g/kg, e.g., at least
about 1 .mu.g/kg per dose and up to about 100 .mu.g/kg per dose. In
some embodiments, the FLT3L fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition is
administered at a dose of 1 .mu.g/kg per dose, 3 .mu.g/kg per dose,
10 jg/kg per dose, 30 jg/kg per dose, 60 jg/kg per dose, or 100
jg/kg per dose. In some embodiments, the FLT3L fusion protein, the
homodimer, the heterodimer, the conjugate, the polynucleotide, the
vector, the lipoplex, such as an LNP, and/or the pharmaceutical
composition is administered at a dose of 1 .mu.g/kg per dose. In
some embodiments, the FLT3L fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition is
administered at a dose of 3 .mu.g/kg per dose. In some embodiments,
the FLT3L fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide, the vector, the lipoplex, such as an
LNP, and/or the pharmaceutical composition is administered at a
dose of 10 .mu.g/kg per dose. In some embodiments, the FLT3L fusion
protein, the homodimer, the heterodimer, the conjugate, the
polynucleotide, the vector, the lipoplex, such as an LNP, and/or
the pharmaceutical composition is administered at a dose of 30
.mu.g/kg per dose. In some embodiments, the FLT3L fusion protein,
the homodimer, the heterodimer, the conjugate, the polynucleotide,
the vector, the lipoplex, such as an LNP, and/or the pharmaceutical
composition is administered at a dose of 60 g/kg per dose. In some
embodiments, the FLT3L fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition is
administered at a dose of 100 .mu.g/kg per dose. In some
embodiments, the FLT3L-Fc fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition is
administered at a dose in the range of about 0.5 mg to about 50 mg,
e.g., at least about 0.5 mg per dose and up to about 1 mg, 2 mg, 3,
mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg,
14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35
mg, 40 mg, 45 mg or 50 mg per dose. In some embodiments, the
FLT3L-Fc fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide, the vector, the lipoplex, such as an
LNP, and/or the pharmaceutical composition is administered at a
dose of 10 mg per dose. In some embodiments, the FLT3-expressing
cells (e.g., dendritic cells) are expanded by at least about
10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold,
500-fold, or more, within 3 weeks of a single administration of the
fusion protein, the homodimer, the heterodimer, the conjugate, the
polynucleotide, the vector, the lipoplex, such as an LNP, and/or
the pharmaceutical composition. In some embodiments, the
FLT3-expressing cells (e.g., dendritic cells) are expanded in the
bone marrow and/or in a solid tumor in the subject.
In another aspect, provided are kits. In various embodiments, the
kits comprise one or more unitary doses of the FLT3L-Fc fusion
protein, the homodimer, the heterodimer, the conjugate, the
polynucleotide, the expression cassette, the vector, the lipoplex,
such as an LNP, and/or the pharmaceutical composition, as described
above and herein. In some embodiments, the one or more unitary
doses are in a single container. In some embodiments, the one or
more unitary doses are in two or more separate containers. In some
embodiments, the one or more containers can comprise vials,
ampules, pre-loaded syringes and combinations thereof. In some
embodiments, the kits comprise one or more containers comprising
the FLT3L-Fc fusion protein, the homodimer, the heterodimer, or the
conjugate in an aqueous solution. In some embodiments, the aqueous
solution comprises the FLT3L-Fc fusion protein, the homodimer, the
heterodimer, or the conjugate at a concentration in the range of
about 1-20 mg/ml, e.g., from about 1 mg/ml to about 2 mg/ml, 3
mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10
mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml,
17 mg/ml, 18 mg/ml, 19 mg/ml or 20 mg/ml. In some embodiments, the
aqueous solution comprises the FLT3L-Fc fusion protein, the
homodimer, the heterodimer, or the conjugate at a concentration of
about 2 mg/ml. In various embodiments, the one or more unitary
doses can be the same or different. In some embodiments, each
unitary dose is in the range of about 0.5 mg to about 50 mg, e.g.,
at least about 0.5 mg per dose and up to about 1 mg, 2 mg, 3, mg, 4
mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14
mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg,
40 mg, 45 mg or 50 mg per dose. In some embodiments, each unitary
dose is about 10 mg per dose. In some embodiments, the kits further
comprise one or more unitary doses of one or more additional
therapeutic agents. In some embodiments, the kits further comprise
one or more unitary doses of one or more therapeutic agents
selected from the group consisting of AGEN1884 (zalifrelimab),
AGEN1181, AGEN2034 (balstilimab), AGEN1307, AGEN2373, AGEN1223 and
GS-1423 (AGEN1423; see WO2019/173692). In some embodiments, the
kits further comprise one or more oncolytic viral vectors. In some
embodiments, the viral vector is from a viral family selected from
the group consisting of: Adenoviridae (e.g., Adenovirus),
Arenaviridae (e.g., lymphocytic choriomeningitis mammarenavirus,
Cali mammarenavirus (a.k.a., Pichinde mammarenavirus), Poxviridae
(e.g., Vaccinia virus), Herpesviridae (e.g., Herpesvirus, e.g.,
HSV-1), Parvoviridae (e.g., Parvovirus H1), Reoviridae (e.g.,
Reovirus), Picornaviridae (e.g., Coxsackievirus, Seneca Valley
Virus, Poliovirus), Paramyxoviridae (e.g., Measles virus, Newcastle
disease virus (NDV)), Rhabdoviridae (e.g., Vesicular stomatitis
virus (VSV)), Togaviridae (e.g., Alphavirus, Sindbis virus),
Enteroviridae (e.g., Echovirus). In some embodiments, the kits
comprise one or more antibodies or antigen-binding antibody
fragments thereof, or antibody-drug conjugates thereof,
CD3-targeting multi-specific molecules, CD16-targeting
multi-specific molecules, or non-immunoglobulin antigen-binding
domains or antibody mimetic proteins, or population of immune cells
comprising a chimeric antigen receptor directed against one or more
targets selected from the group consisting of: CD19, MS4A1 (CD20),
CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40,
CD44, CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b,
CD80, CD86, IL3RA (CD123), PROM1 (CD133), CD137, SDC1 (CD138),
alpha fetoprotein (AFP), c-Met; c-Kit; C-type lectin domain family
12 member A (CLEC12A, CLL1, CD371); C-type lectin domain containing
9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic
anhydrase 6 (CA6); carbonic anhydrase 9 (CA9, CAIX);
carcinoembryonic antigen related cell adhesion molecule 3
(CEACAM3); carcinoembryonic antigen related cell adhesion molecule
5 (CEACAM5); carcinoembryonic antigen related cell adhesion
molecule 6 (CEACAM6, CD66c); chorionic somatomammotropin hormone 1
(CSH1, CS1); coagulation factor III, tissue factor (F3, TF);
collectin subfamily member 10 (COLEC10); delta like canonical Notch
ligand 3 (DLL3); ectonucleotide pyrophosphatase/phosphodiesterase 3
(ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor
(EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2);
epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine
kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP);
fibroblast growth factor receptor 2 (FGFR2); fibroblast growth
factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate
receptor 1 (FOLR1, FRa); GD2 ganglioside; glycoprotein NMB (GPNMB,
osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human
papillomavirus (HPV) E6; HPV E7; major histocompatibility complex
(MHC) class I-presented neoantigens, major histocompatibility
complex (MHC) class II-presented neoantigens, major
histocompatibility complex, class I, E (HLA-E); major
histocompatibility complex, class I, F (HLA-F); major
histocompatibility complex, class I, G (HLA-G, MHC-G); integrin
subunit beta 7 (ITGB7); leukocyte immunoglobulin like receptor B1
(LILRB1, ILT2); leukocyte immunoglobulin like receptor B2 (LILRB2,
ILT4); LY6/PLAUR domain containing 3 (LYPD3, C4.4A); glypican 3
(GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family member A1
(MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4
(MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1
(MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1
(MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin
1 (MUC1) and splice variants thereof (e.g., MUC1/C, D, and Z);
mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4
(NECTIN4); SLIT and NTRK like family member 6 (SLITRK6);
promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7
(inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM
family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid
binding Ig like lectin 7 (SIGLEC7); sialic acid binding Ig like
lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate),
member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6;
LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily
member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9
(TNFSF9; 4-1BB-L, CD137L); TNF receptor superfamily member 10a
(TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily member
10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily
member 13B (TNFRSFi3B; CD267, TACI, IGAD2); TNF receptor
superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor
superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF);
transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein
(TPBG, 5T4); trophinin (TRO, MAGED3); tumor associated calcium
signal transducer 2 (TACSTD2, TROP2, EGP1); Fucosyl GM; sialyl
Lewis adhesion molecule (sLe); and Lewis Y antigen. In some
embodiments, the kits further comprise one or more antagonists or
inhibitors of an inhibitory immune checkpoint protein or receptor
and/or one or more activators or agonists of a stimulatory immune
checkpoint protein or receptor. In some embodiments, the one or
more immune checkpoint proteins or receptors are selected from the
group consisting of. CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2),
transmembrane and immunoglobulin domain containing 2 (TMIGD2,
CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244
(SLAMF4); CD276 (B7H3); V-set domain containing T cell activation
inhibitor 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR,
B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3);
natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1,
B7H6); HERV-H LTR-associating 2 (HHLA2, B7H7); inducible T cell
co-stimulator (ICOS, CD278); inducible T cell costimulator ligand
(ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40);
TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8
(CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9
(CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL);
TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte
associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF);
TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related
sequence A (MICA); MHC class I polypeptide-related sequence B
(MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1,
PD1, PD-1); cytotoxic T-lymphocyte associated protein 4 (CTLA4,
CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2
(NECTIN2, CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell
adhesion molecule (PVR, CD155); PVR related immunoglobulin domain
containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM
domains (TIGIT); T cell immunoglobulin and mucin domain containing
4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2,
TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activating 3 (LAG3,
CD223); signaling lymphocytic activation molecule family member 1
(SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3);
SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7,
CD319); UL16 binding protein 1 (ULBP1); UL16 binding protein 2
(ULBP2); UL16 binding protein 3 (ULBP3); retinoic acid early
transcript 1E (RAETIE; ULBP4); retinoic acid early transcript 1G
(RAETIG; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6);
killer cell immunoglobulin like receptor, three Ig domains and long
cytoplasmic tail 1 (KIR, CD158E1); killer cell lectin like receptor
C1 (KLRC1, NKG2A, CD159A); killer cell lectin like receptor K1
(KLRK1, NKG2D, CD314); killer cell lectin like receptor C2 (KLRC2,
CD159c, NKG2C); killer cell lectin like receptor C3 (KLRC3, NKG2E);
killer cell lectin like receptor C4 (KLRC4, NKG2F); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig
domains and long cytoplasmic tail 2 (KIR2DL2); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 3 (KIR2DL3); killer cell immunoglobulin like receptor, three
Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell
lectin like receptor D1 (KLRD1); killer cell lectin like receptor
G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid binding Ig like lectin
7 (SIGLEC7); and sialic acid binding Ig like lectin 9 (SIGLEC9). In
some embodiments, the kits further comprise one or more blockers or
inhibitors of one or more T-cell inhibitory immune checkpoint
proteins or receptors. In some embodiments, the T-cell inhibitory
immune checkpoint proteins or receptors are selected from the group
consisting of CD274 (CD274, PDL1, PD-L1); programmed cell death 1
ligand 2 (PDCDILG2, PD-L2, CD273); programmed cell death 1 (PDCD1,
PD1, PD-1); cytotoxic T-lymphocyte associated protein 4 (CTLA4,
CD152); CD276 (B7H3); V-set domain containing T cell activation
inhibitor 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR,
B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3);
TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte
associated (BTLA)); PVR related immunoglobulin domain containing
(PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains
(TIGIT); lymphocyte activating 3 (LAG3, CD223); hepatitis A virus
cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9);
killer cell immunoglobulin like receptor, three Ig domains and long
cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin like
receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1);
killer cell immunoglobulin like receptor, two Ig domains and long
cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin like
receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and
killer cell immunoglobulin like receptor, three Ig domains and long
cytoplasmic tail 1 (KIR3DL1). In some embodiments, the kits further
comprise one or more agonists or activators of one or more T-cell
stimulatory immune checkpoint proteins or receptors. In some
embodiments, the T-cell stimulatory immune checkpoint proteins or
receptors are selected from the group consisting of CD27, CD70;
CD40, CD40LG; inducible T cell costimulator (ICOS, CD278);
inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor
superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4
(TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR),
TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2
(NECTIN2, CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell
adhesion molecule (PVR, CD155). In some embodiments, the kits
further comprise one or more blockers or inhibitors of one or more
NK-cell inhibitory immune checkpoint proteins or receptors. In some
embodiments, the NK-cell inhibitory immune checkpoint proteins or
receptors are selected from the group consisting of killer cell
immunoglobulin like receptor, three Ig domains and long cytoplasmic
tail 1 (KIR, CD158E1); killer cell immunoglobulin like receptor,
two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 2 (KIR2DL2); killer cell immunoglobulin like receptor, two Ig
domains and long cytoplasmic tail 3 (KIR2DL3); killer cell
immunoglobulin like receptor, three Ig domains and long cytoplasmic
tail 1 (KIR3DL1); killer cell lectin like receptor C1 (KLRC1,
NKG2A, CD159A); killer cell lectin like receptor D1 (KLRD1, CD94);
killer cell lectin like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1);
sialic acid binding Ig like lectin 7 (SIGLEC7); and sialic acid
binding Ig like lectin 9 (SIGLEC9). In some embodiments, the kits
further comprise one or more agonists or activators of one or more
NK-cell stimulatory immune checkpoint proteins or receptors. In
some embodiments, the NK-cell stimulatory immune checkpoint
proteins or receptors include without limitation CD16, CD226
(DNAM-1); killer cell lectin like receptor K1 (KLRK1, NKG2D,
CD314); and SLAM family member 7 (SLAMF7). In some embodiments, the
kits further comprise one or more inhibitors of CD274, PDCD1 or
CTLA4. In some embodiments, the kits further comprise one or more
inhibitors of CD274, PDCD1 or CTLA4 selected from the group
consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181,
AGEN1884 (zalifrelimab), BMS-986249, MK-1308, REGN-4659, ADU-1604,
CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041,
JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, FPT-155
(CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4),
KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717
(PD-1/CTLA4), AK-104 (CTLA4/PD-1), pembrolizumab, nivolumab,
cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514),
spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559,
CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055),
AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091,
AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genolimzumab
(CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210
(camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306,
(MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311,
JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308
(sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001),
BCD-135, FAZ-053, TQB-2450, MDX1105-01, MGD-013 (PD-1/LAG-3),
FS-118 (LAG-3/PD-L1), RO-7121661 (PD-1/TIM4-3), M7824
(PD-L1/TGFP-EC domain) and CA-170 (PD-L1/VISTA), CDX-527
(CD27/PD-L1), LY-3415244 (TIM3/PDL1), INBRX-105 (4-1BB/PDL1),
GS-4224, GS-4416, INCB086550, MAX10181 and BPI-002. In some
embodiments, the kits further comprise one or more containers
comprising one or more populations of immune cells selected from
the group consisting of: natural killer (NK) cells, NK-T cells, T
cells, cytokine-induced killer (CIK) cells, macrophage (MAC) cells,
tumor infiltrating lymphocytes (TILs) and dendritic cell (DCs). In
some embodiments, the population of T cells is selected from the
group consisting of: alpha/beta TCR T cells, gamma/delta TCR T
cells, regulatory T (Treg) cells and TRuC[0020].TM. T cells. In
some embodiments, the kits further comprise a population of NK-92
cells. In some embodiments, the one or more populations of immune
cells comprise one or more chimeric antigen receptors (CARs). In
some embodiments, the cells are allogeneic to an intended
recipient. In some embodiments, the kits further comprises one or
more cytokines or chemokines selected from the group consisting of:
IL 2, IL-12, IL-15, IL-18, IL-21, interferon (IFN)-a, IFN--P,
IFN-.gamma., CXCL9/Mig (monokine induced by interferon-y),
CXCL10/IP10 (interferon-y-inducible 10 kDa protein) and
CXCL11/I-TAC (interferon-inducible T cell a-chemoattractant),
CXCL4/PF4 (platelet factor 4), monocyte chemoattractant protein 2
(MCP-2), macrophage inflammatory protein 1 alpha (MIP-la),
macrophage inflammatory protein 1 beta (MIP-10) and regulated on
activation normal T expressed and secreted protein (RANTES), IL-10,
IL-4, IL-6, IL-8, IL-10, IL-13, IL-23, transforming growth factor
(TGF)-P, colony stimulating factor 3 (CSF3, GCSF), colony
stimulating factor 1 (CSF1), C-C motif chemokine ligand 2 (CCL2,
MCP-1), chemokine (C--X-C motif) ligand 1 (CXCL1/MGSA), stromal
cell-derived factor-1, TNFa and oncostatin M (OSM). In some
embodiments, the kits further comprise one or more activators or
agonists
of: a toll-like receptor (TLR); a stimulator of interferon genes
(STING) receptor; inducible T cell costimulator (ICOS, CD278);
and/or a TNF receptor superfamily (TNFRSF) member. In some
embodiments, the TNF receptor superfamily (TNFRSF) member is
selected from the group consisting of: TNFRSFlA, TNFRSFlB, TNFRSF4
(OX40), TNFRSF5 (CD40), TNFRSF6 (FAS), TNFRSF7 (CD27), TNFRSF8
(CD30), TNFRSF9 (4-1n, CD137), TNFRSF10A (CD261, DR4, TRAILR1),
TNFRSF10B (CD262, DR5, TRALR2), TNFRSF10C (CD263, TRALR3),
TNFRSF10D (CD264, TRATLR4), TNFRSF11A (CD265, RANK), TNFRSF11B,
TNFRSF12A (CD266), TNFRSF13B (CD267), TNFRSF13C (CD268), TNFRSF16
(NGFR, CD271), TNFRSF17 (BCMA, CD269), TNFRSF18 (GITR, CD357),
TNFRSF19, TNFRSF21 (CD358, DR6), and TNFRSF25 (DR3). In some
embodiments, the TNFRSF4 activator or agonist comprises INCAGN1949,
tavolimab (MEDI0562), pogalizumab (MOXR0916/RG7888), MEDI6469, BMS
986178, PF-04518600, GSK3174998, IBI101, ATOR-1015, ABBV-368 or
SL-279252. In some embodiments, the TNFRSF9 (4-1BB or CD137)
activator or agonist comprises urelumab, BMS-663513, utomilumab
(PF-05082566), CTX-471, MP-0310, ADG-106, ATOR-1017 or AGEN2373. In
some embodiments, the TNFRSF18 (GITR or CD357) agonist comprises
GWN323, MEDI1873, MK-1248, MK-4166, TRX518, INCAGN1876, BMS-986156,
BMS-986256, AMG-228, ASP1951 (PTZ 522), FPA-154 or OMP-336B11. In
some embodiments, the kits comprise a molecule that concurrently
binds to TNF receptor superfamily member 4 (TNFRSF4, OX40 or CD134)
and TNF receptor superfamily member 18 (TNFRSF18, GITR or CD357).
In some embodiments, the TLR agonist or activator is selected from
the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4
agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist and a TLR9
agonist. In some embodiments, the TLR7 activator or agonist is
selected from the group consisting of GS 9620, DS-0509, LHC-165,
and TMX-101 (imiquimod), and/or wherein the TLR8 agonist is
selected from the group consisting of GS-9688 and NKTR-262 (dual
TLR7/TLR8 agonist). In some embodiments, the STING receptor
activator or agonist or activator is selected from the group
consisting of ADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291,
AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291,
5,6-dimethylxanthenone-4-acetic acid (DMXAA), cyclic-GAMP (cGAMP)
and cyclic-di-AMP. In some emodiments, the kit comprises an
anti-CD47 antibody. In some embodiments, the anti-CD47 antibody is
magrolimab. In some embodiments, the kit comprises a SIRPalpha
inhibitor. In some embodiments, the SIRPalpha inhibitor is selected
from the group consisting of AL-008, RRx-001, CTX-5861, FSI-189
(GS-0189), ES-004, BI765063, ADU1805, and CC-95251. In some
embodiments, the kits comprise one or more inhibitors or
antagonists of: protein tyrosine phosphatase, non-receptor type 11
(PTPN11 or SHP2), myeloid cell leukemia sequence 1 (MCL1) apoptosis
regulator, mitogen-activated protein kinase kinase kinase kinase 1
(MAP4K1) (also called Hematopoietic Progenitor Kinase 1 (HPK1)),
phosphatidylinositol-4,5-bisphosphate 3-kinase, including catalytic
subunit alpha (PIK3CA), catalytic subunit beta (PIK3CB), catalytic
subunit gamma (PIK3CG) and catalytic subunit delta (PIK3CD),
diacylglycerol kinase alpha (DGKA, DAGK, DAGK1 or DGK-alpha),
5'-nucleotidase ecto (NT5E or CD73), ectonucleoside triphosphate
diphosphohydrolase 1 (ENTPD1 or CD39), transforming growth factor
beta 1 (TGFB1 or TGFO), heme oxygenase 1 (HMOX1, HO-1 or HO1), heme
oxygenase 2 (HMOX2, HO-2 or H02), vascular endothelial growth
factor A (VEGFA or VEGF), erb-b2 receptor tyrosine kinase 2 (ERBB2,
HER2, HER2/neu or CD340), epidermal growth factor receptor (EGFR,
ERBB, ERBB1 or HER1), ALK receptor tyrosine kinase (ALK, CD246),
poly(ADP-ribose) polymerase 1 (PARP1), poly(ADP-ribose) polymerase
2 (PARP2), TCDD inducible poly(ADP-ribose) polymerase (TIPARP,
PARP7), cyclin dependent kinase 4 (CDK4), cyclin dependent kinase 6
(CDK6), TNF receptor superfamily member 14 (TNFRSF14, HVEM, CD270),
T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked
inhibitor of apoptosis (XIAP, BIRC4, IAP-3), baculoviral IAP repeat
containing 2 (BIRC2, cIAP1), baculoviral IAP repeat containing 3
(BIRC3, cIAP2), baculoviral IAP repeat containing 5 (BIRC5,
survivin), C-C motif chemokine receptor 2 (CCR2, CD192), C-C motif
chemokine receptor 5 (CCR5, CD195), C-C motif chemokine receptor 8
(CCR8, CDw198), C-X-C motif chemokine receptor 2 (CXCR2, CD182),
C-X-C motif chemokine receptor 3 (CXCR3, CD182, CD183), C-X-C motif
chemokine receptor 4 (CXCR4, CD184), cytokine inducible SH2
containing protein (CISH), arginase (ARG1, ARG2), carbonic
anhydrase (CA1, CA2, CA3, CA4, CA5A, CA5B, CA6, CA7, CA8, CA9,
CA10, CA11, CA12, CA13, CA14), prostaglandin-endoperoxide synthase
1 (PTGS1, COX-1), prostaglandin-endoperoxide synthase 2 (PTGS2,
COX-2), secreted phospholipase A2, prostaglandin E synthase (PTGES,
PGES), arachidonate 5-lipoxygenase (ALOX5, 5-LOX), soluble epoxide
hydrolase 2 (EPHX2), indoleamine 2,3-dioxygenase 1 (IDO1),
indoleamine 2,3-dioxygenase 2 (IDO2), hypoxia inducible factor 1
subunit alpha (HIF1A), angiopoietin 1 (ANGPT1), Endothelial TEK
tyrosine kinase (TIE-2, TEK), Janus kinase 1 (JAK1), catenin beta 1
(CTNNB1), histone deacetylase 9 (HDAC9), 5'-3' exoribonuclease 1
(XRN1) and/or WRN RecQ like helicase (WRN). In some embodiments,
the activator/agonist or the blocker/inhibitor comprises an
antibody or an antigen-binding fragment thereof, or antibody-drug
conjugate thereof, CD3-targeting multi-specific molecule,
CD16-targeting multi-specific molecule, non-immunoglobulin antigen
binding molecule or antibody mimetic protein. In some embodiments,
the activator/agonist or the blocker/inhibitor comprises a small
organic molecule. In some embodiments, the inhibitor of
5'-nucleotidase ecto (NT5E or CD73) is selected from the group
consisting of MEDI9447 (oleclumab), CPI-006, BMS-986179, IPH5301,
TJ4309 (TJD5), NZV-930, AB-680, PSB-12379, PSB-12441, PSB-12425,
CB-708, GS-1423 (AGEN1423) and PBF-1662. In some embodiments, the
inhibitor of CCR2 and/or CCR5 is selected from the group consisting
of BMS-813160, PF-04136309 and CCX-872. In some embodiments, the
inhibitor of MCL1 is selected from the group consisting of AMG-176,
AMG-397, S-64315, AZD-5991, 483-LM, A 1210477, UMI-77 and
JKY-5-037. In some embodiments, the inhibitor of PTPN11 or SHP2 is
selected from the group consisting of TNO155 (SHP-099), RMC-4550,
JAB-3068 and RMC-4630. In some embodiments, the kits further
comprise an inhibitor or antagonist of a regulatory T cell (Treg).
In some embodiments, the kits further comprise one or more
anti-neoplastic or chemotherapeutic agents. In some embodiments,
the one or more anti-neoplastic or chemotherapeutic agents are
selected from the group consisting of a nucleoside analog (e.g.,
5-fluorouracil, gemcitabine, cytarabine, cladribine, pentostatin,
fludarabine), a taxane (e.g., paclitaxel, nab-paclitaxel,
docetaxel, cabazitaxel), a platinum coordination complex
(cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin
tetranitrate, phenanthriplatin, picoplatin, satraplatin,
dicycloplatin, eptaplatin, lobaplatin, miriplatin), a dihydrofolate
reductase (DHFR) inhibitor (e.g., methotrexate, trimetrexate,
pemetrexed), a topoisomerase inhibitor (e.g., doxorubicin,
daunorubicin, dactinomycin, eniposide, epirubicin, etoposide,
idarubicin, irinotecan, mitoxantrone, pixantrone, sobuzoxane,
topotecan, irinotecan, MM-398 (liposomal irinotecan), vosaroxin and
GPX-150, aldoxorubicin, AR-67, mavelertinib, AST-2818, avitinib
(ACEA-0010), irofulven (MGI-114)), an alkylating agent (e.g., a
nitrogen mustard (e.g., cyclophosphamide, chlormethine, uramustine
or uracil mustard, melphalan, chlorambucil, ifosfamide,
bendamustine), a nitrosourea (e.g., carmustine, lomustine,
streptozocin), an alkyl sulfonate (e.g., busulfan)), and mixtures
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 illustrates induction of mouse IL-6 in a mouse
FLT3-expressing M1 cell line by a titration (50-0.00005 nM) of
recombinant human FLT3-ligand (Recombinant huFLT3L, closed circle),
recombinant human FLT3-ligand human IgG1 fusion protein
(Recombinant huFLT3L-Fc, open triangle), anti-mouse FLT3 agonist
antibody (Comparator 1, closed triangle) or human IgG1 isotype
antibody (Isotype negative control, open square). The x-axis shows
the protein concentration (nM) and the y-axis shows mouse IL-6
concentration (pg/mL). The cross symbol indicates the IL-6 baseline
level in untreated cells. Graph is a combination of two independent
experiments. Experiments were performed in duplicate. Error bars
represent standard deviation of the mean values. EC50 values are
shown in Table 1.
[0022] FIG. 2 illustrates proliferation of a human FLT3-expressing
AML5 cell line in response to a titration (100-0.0025 nM) of
recombinant human FLT3-ligand (Recombinant huFLT3L, open square),
human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1,
open triangle), human FLT3-ligand human IgG1 fusion protein (SEQ ID
NO:21, open circle) or human IgG1 isotype antibody (hIgG1 Isotype,
cross). The x-axis shows the protein concentration (nM) and the
y-axis shows the relative luminescence units (RLU). Graph is a
result of one experiment. Experiments were performed in triplicate.
Error bars represent standard deviation of the mean values. EC50
values are shown in Table 2.
[0023] FIG. 3 illustrates proliferation of a human FLT3-expressing
AML5 cell line induced by a titration (5-0.00008 nM) of human
wildtype FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID
NO:1, open triangle), human FLT3-ligand (H8Y) human hingeless IgG1
fusion protein (SEQ ID NO:22, open circle), human FLT3-ligand
(K84E) human hingeless IgG1 fusion protein (SEQ ID NO:23, open
square), human FLT3-ligand (H8Y+K84E) human hingeless IgG1 fusion
protein (SEQ ID NO:24, closed circle), or human IgG1 isotype
antibody (hIgG1 Isotype, cross). The x-axis shows the protein
concentration (nM) and the y-axis shows the relative luminescence
units (RLU). Graph is a combination of two independent experiments.
Experiments were performed in triplicate. Error bars represent
standard deviation of the mean values. EC50 values are shown in
Table 3.
[0024] FIG. 4 illustrates proliferation of a human FLT3-expressing
AML5 cell line induced by a titration (10-0.0004 nM) of human
FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1, open
triangle), mouse FLT3-ligand mouse IgG2a (LALA-PG) fusion protein
(SEQ ID NO:19, open circle), mouse FLT3-ligand mouse IgG2a (C136S
LALA-PG) fusion protein (SEQ ID NO:20, open square), or human IgG1
isotype antibody (hIgG1 Isotype, cross). The x-axis shows the
protein concentration (nM) and the y-axis shows the relative
luminescence units (RLU). Graph is a result of one experiment of
Experiment was performed in triplicate. Error bars represent
standard deviation of the mean values. EC50 values are shown in
Table 4.
[0025] FIG. 5 illustrates proliferation of a human FLT3-expressing
AML5 cell line induced by a titration (10-0.0004 nM) of human
FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1, open
triangle), human FLT3-ligand (A5 amino acid) human hingeless IgG1
fusion protein (SEQ ID NO:2, closed triangle), human FLT3-ligand
human IgG4 (S228P/L235E) fusion protein (SEQ ID NO:3, open circle),
human FLT3-ligand human IgG4 (S228P/F234A/L235A) fusion protein
(SEQ ID NO:4, closed circle), human FLT3-ligand (S128A/S151A) human
hingeless IgG1 fusion protein (SEQ ID NO:5, open square), human
FLT3-ligand (A5 amino acid) human IgG4 (S228P/F234A/L235A) fusion
protein (SEQ ID NO:6, closed square), human FLT3-ligand (A10 amino
acid) human hingeless IgG1 fusion protein (SEQ ID NO:7, cross), or
human FLT3-ligand (A10 amino acid) human IgG4 (S228P/F234A/L235A)
fusion protein (SEQ ID NO:8, closed diamond). The x-axis shows the
protein concentration (nM) and the y-axis shows the relative
luminescence units (RLU). Graph is a combination of two independent
experiments. Experiment was performed in triplicate. Error bars
represent standard deviation of the mean values. EC50 values are
shown in Table 5.
[0026] FIG. 6 illustrates binding to recombinant human FLT3 of a
titration (35-0.0001 nM) of human FLT3-ligand human hingeless IgG1
fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand (A5
amino acid) human hingeless IgG1 fusion protein (SEQ ID NO:2,
closed triangle), human FLT3-ligand human IgG4 (S228P/L235E) fusion
protein (SEQ ID NO:3, open circle), human FLT3-ligand human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:4, closed circle),
human FLT3-ligand (S128A/S151A) human hingeless IgG1 fusion protein
(SEQ ID NO:5, open square), human FLT3-ligand (A5 amino acid) human
IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:6, closed
square), human FLT3-ligand (A10 amino acid) human hingeless IgG1
fusion protein (SEQ ID NO:7, cross), or human FLT3-ligand (A10
amino acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID
NO:8, closed diamond). The x-axis shows the protein concentration
(nM) and the y-axis shows the optical density (OD) at 450 nm. Graph
is a result of one experiment. EC50 values are shown in Table
6.
[0027] FIG. 7 illustrates binding of recombinant human FcRn with a
dose titration (235-0.035 nM) of human FLT3-ligand human hingeless
IgG1 fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand
(A5 amino acid) human hingeless IgG1 fusion protein (SEQ ID NO:2,
closed triangle), human FLT3-ligand human IgG4 (S228P/L235E) fusion
protein (SEQ ID NO:3, open circle), human FLT3-ligand human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:4, closed circle),
human FLT3-ligand (S128A/S151A) human hingeless IgG1 fusion protein
(SEQ ID NO:5, open square), human FLT3-ligand (A5 amino acid) human
IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:6, closed
square), human FLT3-ligand (A10 amino acid) human hingeless IgG1
fusion protein (SEQ ID NO:7, cross), human FLT3-ligand (A10 amino
acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:8,
closed diamond), human IgG1 isotype antibody (open diamond), or
human IgG4 isotype antibody (open star). The x-axis shows the
protein concentration (nM) and the y-axis shows the optical density
(OD) at 450-650 nm. Graph is a result of one experiment. Experiment
was performed in duplicate. Error bars represent standard deviation
of the mean values. Estimated EC50 values are shown in Table 7.
[0028] FIG. 8 illustrates binding of human IgG to recombinant human
Fc.gamma.RI competed with a titration (294-0.48 nM) of human
FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1, open
triangle), human FLT3-ligand (A5 amino acid) human hingeless IgG1
fusion protein (SEQ ID NO:2, closed triangle), human FLT3-ligand
human IgG4 (S228P/L235E) fusion protein (SEQ ID NO:3, open circle),
human FLT3-ligand human IgG4 (S228P/F234A/L235A) fusion protein
(SEQ ID NO:4, closed circle), human FLT3-ligand (S128A/S151A) human
hingeless IgG1 fusion protein (SEQ ID NO:5, open square), human
FLT3-ligand (A5 amino acid) human IgG4 (S228P/F234A/L235A) fusion
protein (SEQ ID NO:6, closed square), human FLT3-ligand (A10 amino
acid) human hingeless IgG1 fusion protein (SEQ ID NO:7, cross),
human FLT3-ligand (A10 amino acid) human IgG4 (S228P/F234A/L235A)
fusion protein (SEQ ID NO:8, closed diamond), human IgG1 isotype
antibody (open diamond), or human IgG4 isotype antibody (open
star). The x-axis shows the protein concentration (nM) and the
y-axis shows the relative fluorescence units (RFU). Graph is a
result of one experiment. Experiment was performed in duplicate.
Error bars represent standard deviation of the mean values. EC50
values are shown in Table 8.
[0029] FIG. 9 illustrates binding of human IgG to recombinant human
Fc.gamma.RIIIa (V-variant) competed with a dose titration
(1176-1.92 nM) of human FLT3-ligand human hingeless IgG1 fusion
protein (SEQ ID NO:1, open triangle), human FLT3-ligand (A5 amino
acid) human hingeless IgG1 fusion protein (SEQ ID NO:2, closed
triangle), human FLT3-ligand human IgG4 (S228P/L235E) fusion
protein (SEQ ID NO:3, open circle), human FLT3-ligand human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:4, closed circle),
human FLT3-ligand (S128A/S151A) human hingeless IgG1 fusion protein
(SEQ ID NO:5, open square), human FLT3-ligand (A5 amino acid) human
IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:6, closed
square),), human FLT3-ligand (A10 amino acid) human hingeless IgG1
fusion protein (SEQ ID NO:7, cross), human FLT3-ligand (A10 amino
acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:8,
closed diamond), human IgG1 isotype antibody (open diamond), or
human IgG4 isotype antibody (open star). The x-axis shows the
protein concentration (nM) and the y-axis shows the relative
fluorescence units (RFU). Graph is a result of one experiment.
Experiment was performed in duplicate. Error bars represent
standard deviation of the mean values. EC50 values are shown in
Table 9.
[0030] FIG. 10 illustrates binding of recombinant human C1q to a
titration (94-0.74 nM) of human FLT3-ligand human hingeless IgG1
fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand (A5
amino acid) human hingeless IgG1 fusion protein (SEQ ID NO:2,
closed triangle), human FLT3-ligand human IgG4 (S228P/L235E) fusion
protein (SEQ ID NO:3, open circle), human FLT3-ligand human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:4, closed circle),
human FLT3-ligand (S128A/S151A) human hingeless IgG1 fusion protein
(SEQ ID NO:5, open square), human FLT3-ligand (A5 amino acid) human
IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:6, closed
square),), human FLT3-ligand (A10 amino acid) human hingeless IgG1
fusion protein (SEQ ID NO:7, cross), human FLT3-ligand (A10 amino
acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:8,
closed diamond), human IgG1 isotype antibody (open diamond), or
human IgG4 isotype antibody (open star). The x-axis shows the
protein concentration (nM) and the y-axis shows the optical density
(OD) at 450-650 nm. Graph is a result of one experiment. Experiment
was performed in duplicate. Error bars represent standard deviation
of the mean values. EC50 values are shown in Table 10.
[0031] FIGS. 11A-11B illustrate a mouse serum concentration-time
profile following 5 mg/kg intraperitoneal dosing of 8 FLT3-ligand
fusion proteins relative to recombinant FLT3-ligand. Panel A:
linear scale; Panel B: Log 10 scale after a single dose intravenous
administration (5 mg/kg) of human FLT3-ligand human hingeless IgG1
fusion protein produced in Expi293 expression system (SEQ ID NO:1
Expi293, open triangle), human FLT3-ligand human hingeless IgG1
fusion protein produced in ExpiCHO expression system (SEQ ID NO:1
ExpiCHO, closed triangle), human FLT3-ligand (A5 amino acid) human
hingeless IgG1 fusion protein (SEQ ID NO:2, open circle), human
FLT3-ligand human IgG4 (S228P/L235E) fusion protein (SEQ ID NO:3,
closed circle), human FLT3-ligand human IgG4 (S228P/F234A/L235A)
fusion protein (SEQ ID NO:4, open square), human FLT3-ligand
(S128A/S151A) human hingeless IgG1 fusion protein (SEQ ID NO:5,
closed square), human FLT3-ligand (A5 amino acid) human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:6, cross), human
FLT3-ligand (A10 amino acid) human hingeless IgG1 fusion protein
(SEQ ID NO:7, open diamond), human FLT3-ligand (A10 amino acid)
human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:8, closed
diamond), or recombinant human FLT3-ligand (Recombinant huFLT3L,
closed star). Graph is a result of one experiment. The x-axis shows
days post injection and the y-axis shows protein concentration in
serum (pg/mL). Each data point represents the mean value of 4
animals. Error bars represent standard deviation (SD) of the mean
values. Mean pharmacokinetic values SD are shown in Table 11.
[0032] FIG. 12 illustrates day 11 frequency of conventional
dendritic cell subtype 1 (cDC1) in spleens of C57BL/6 mice
administrated intravenously with 5 mg/kg of human FLT3-ligand human
hingeless IgG1 fusion protein produced in Expi293 expression system
(SEQ ID NO:1 Expi293, open triangle), human FLT3-ligand human
hingeless IgG1 fusion protein produced in ExpiCHO expression system
(SEQ ID NO:1 ExpiCHO, closed triangle), human FLT3-ligand (A5 amino
acid) human hingeless IgG1 fusion protein (SEQ ID NO:2, open
circle), human FLT3-ligand human IgG4 (S228P/L235E) fusion protein
(SEQ ID NO:3, closed circle), human FLT3-ligand human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:4, open square),
human FLT3-ligand (S128A/S151A) human hingeless IgG1 fusion protein
(SEQ ID NO:5, closed square), human FLT3-ligand (A5 amino acid)
human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:6, cross),
human FLT3-ligand (A10 amino acid) human hingeless IgG1 fusion
protein (SEQ ID NO:7, open diamond), human FLT3-ligand (A10 amino
acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID NO:8,
closed diamond), or recombinant human FLT3-ligand (Recombinant
huFLT3L, open star) at day 0. Baseline cDC1 frequency is indicated
(closed star). The x-axis shows the percentage of splenic cDC1 in
total mononuclear cells (MNCs). Graph is a result of one
experiment. Each individual symbol represents the data point of a
single mouse. Horizontal bars represent the mean values and the
error bars represent standard deviation of the mean values. Mean
frequency of each group is shown in Table 12.
[0033] FIG. 13 illustrates proliferation of a human FLT3-expressing
AML5 cell line stimulated by a dose titration (10-0.0004 nM) of
human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1,
open triangle), human FLT3-ligand (A5 amino acid) human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:6, open circle),
human FLT3-ligand human hingeless IgG1 (M252Y/S254T/T256E) fusion
protein (SEQ ID NO:9, open square), or human FLT3-ligand (A5 amino
acid) human IgG4 (S228P/F234A/L235A/M252Y/S254T/T256E) fusion
protein (SEQ ID NO:14, cross). The x-axis shows the protein
concentration (nM) and the y-axis shows the relative luminescence
units (RLU). Graph is a result of one experiment. Experiment was
performed in duplicate. Error bars represent standard deviation of
the mean values. EC50 values are shown in Table 13.
[0034] FIG. 14 illustrates differentiation of conventional
dendritic cell subtype 1 (cDC1) cells from human bone marrow CD34+
stem cells by a dose titration (10-0.002 nM) of human FLT3-ligand
human hingeless IgG1 fusion protein (SEQ ID NO:1, open triangle),
human FLT3-ligand (A5 amino acid) human IgG4 (S228P/F234A/L235A)
fusion protein (SEQ ID NO:6, open circle), human FLT3-ligand human
hingeless IgG1 (M252Y/S254T/T256E) fusion protein (SEQ ID NO:9,
open square), or human FLT3-ligand (A5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross). The x-axis shows the protein concentration (nM) and the
y-axis shows percentage of cDC1 in total mononuclear cells (MNCs).
Graph is a summary of 13 bone marrow donors. Error bars represent
standard error mean of the mean values. EC50 values are shown in
Table 14.
[0035] FIG. 15 illustrates a dose titration (10-0.002 nM) potency
of enhancing survival of PBMC-derived conventional dendritic cell
subtype 1 (cDC1) cells by human FLT3-ligand human hingeless IgG1
fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand (A5
amino acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID
NO:6, open circle), human FLT3-ligand human hingeless IgG1
(M252Y/S254T/T256E) fusion protein (SEQ ID NO:9, open square), or
human FLT3-ligand (A5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross). The x-axis shows the protein concentration (nM) and the
y-axis shows percentage of cDC1 in total mononuclear cells (MNCs).
Graph is a summary of 16 PBMC donors. Error bars represent standard
error mean of the mean values. EC50 values are shown in Table
15.
[0036] FIG. 16 illustrates binding of recombinant human FLT3 to a
titration (15-0.007 nM) of human FLT3-ligand human hingeless IgG1
fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand
(.DELTA.5 amino acid) human IgG4 (S228P/F234A/L235A) fusion protein
(SEQ ID NO:6, open circle), human FLT3-ligand human hingeless IgG1
(M252Y/S254T/T256E) fusion protein (SEQ ID NO:9, open square), or
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross). The x-axis shows the protein concentration (nM) and the
y-axis shows optical density (OD) at 450 nm. Graph is a result of
one experiment. Experiment was performed in duplicate. Error bars
represent standard deviation of the mean values. EC50 values are
shown in Table 16.
[0037] FIG. 17 illustrates binding of recombinant human FcRn to a
dose titration (3529-0.55 nM) of human FLT3-ligand human hingeless
IgG1 fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand
(.DELTA.5 amino acid) human IgG4 (S228P/F234A/L235A) fusion protein
(SEQ ID NO:6, open circle), human FLT3-ligand human hingeless IgG1
(M252Y/S254T/T256E) fusion protein (SEQ ID NO:9, open square), or
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross). The x-axis shows the protein concentration (nM) and the
y-axis shows optical density (OD) at 450-650 nm. Graph is a result
of one experiment. Experiment was performed in duplicate. Error
bars represent standard deviation of the mean values. EC50 values
are shown in Table 17.
[0038] FIG. 18 illustrates binding of human IgG to recombinant
human Fc.gamma.RI competed with a titration (294-0.48 nM) of human
FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1, open
triangle), human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:6, open circle),
human FLT3-ligand human hingeless IgG1 (M252Y/S254T/T256E) fusion
protein (SEQ ID NO:9, open square), human FLT3-ligand (.DELTA.5
amino acid) human IgG4 (S228P/F234A/L235A/M252Y/S254T/T256E) fusion
protein (SEQ ID NO:14, cross), human IgG1 isotype antibody (open
diamond), or human IgG4 isotype antibody (open star). The x-axis
shows the protein concentration (nM) and the y-axis shows the
relative fluorescence units (RFU). Graph is a result of one
experiment. Experiment was performed in duplicate. Error bars
represent standard deviation of the mean values. EC50 values are
shown in Table 18.
[0039] FIG. 19 illustrates binding of human IgG to recombinant
human Fc.gamma.RIIIa (V-variant) competed by a dose titration
(1176-1.92 nM) of human FLT3-ligand human hingeless IgG1 fusion
protein (SEQ ID NO:1, open triangle), human FLT3-ligand (.DELTA.5
amino acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID
NO:6, open circle), human FLT3-ligand human hingeless IgG1
(M252Y/S254T/T256E) fusion protein (SEQ ID NO:9, open square),
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross), human IgG1 isotype antibody (open diamond), or human IgG4
isotype antibody (open star). The x-axis shows the protein
concentration (nM) and the y-axis shows the relative fluorescence
units (RFU). Graph is a result of one experiment. Experiment was
performed in duplicate. Error bars represent standard deviation of
the mean values. EC50 values are shown in Table 19.
[0040] FIG. 20 illustrates binding of recombinant human C1q to a
titration (94-0.74 nM) of human FLT3-ligand human hingeless IgG1
fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand
(.DELTA.5 amino acid) human IgG4 (S228P/F234A/L235A) fusion protein
(SEQ ID NO:6, open circle), human FLT3-ligand human hingeless IgG1
(M252Y/S254T/T256E) fusion protein (SEQ ID NO:9, open square),
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross), human IgG1 isotype antibody (open diamond), or human IgG4
isotype antibody (open star). The x-axis shows the protein
concentration (nM) and the y-axis shows optical density (OD) at
450-650 nm. Graph is a result of one experiment. Experiment was
performed in duplicate. Error bars represent standard deviation of
the mean values. EC50 values are shown in Table 20.
[0041] FIGS. 21A-21B illustrate Cynomolgus macaque serum
concentration-time profile following 500 .mu.g/kg intravenous and
subcutaneous dosing of 4 FLT3-ligand fusion proteins relative to
recombinant FLT3-ligand. Average serum concentration-time profiles
after intravenous (Panel A) or subcutaneous (Panel B)
administration of human FLT3-ligand human hingeless IgG1 fusion
protein (SEQ ID NO:1, open triangle), human FLT3-ligand (.DELTA.5
amino acid) human IgG4 (S228P/F234A/L235A) fusion protein (SEQ ID
NO:6, open circle), human FLT3-ligand human hingeless IgG1
(M252Y/S254T/T256E) fusion protein (SEQ ID NO:9, open square),
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross). The x-axis shows days post injection and the y-axis shows
protein concentration in serum (pg/mL). Each data point represents
the mean value of 3 animals. Error bars represent standard
deviation of the mean values. Mean pharmacokinetic values are shown
in Table 21.
[0042] FIGS. 22A-22B illustrate kinetics of conventional dendritic
cell subtype 1 (cDC1) fold-change in peripheral blood of Cynomolgus
macaque administrated intravenously (Panel A) or subcutaneously
(Panel B) with 500 pg/kg of human FLT3-ligand human hingeless IgG1
fusion protein (SEQ ID NO:1, open triangle), human FLT3-ligand
(.DELTA.5 amino acid) human IgG4 (S228P/F234A/L235A) fusion protein
(SEQ ID NO:6, open circle), human FLT3-ligand human hingeless IgG1
(M252Y/S254T/T256E) fusion protein (SEQ ID NO:9, open square),
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID NO:14,
cross) at day 0. Graph is a result of one experiment. Each data
point represents the mean value of 3 animals. Error bars represent
standard deviation of the mean values.
[0043] FIG. 23 illustrates tumor growth kinetics of MC38
tumor-bearing C57BL/6 mice administrated intravenously with 3750
pg/kg (open triangle), 750 pg/kg (open circle), 150 pg/kg (open
square), 30 pg/kg (cross) mouse FLT3-Ligand mouse IgG2a (C136S,
LALA-PG), or 3753 pg/kg mouse IgG2a isotype (open star) at day 0
when tumor volume reached 50 mm.sup.3. The x-axis is days post
dosing. The y-axis is tumor volume in mm.sup.3. Graph is
representative of two independent experiments. Each data point
represents the mean value of 7 animals. Error bars represent
standard deviation of the mean values. Statistical differences in
tumor growth rate of each dose group compared to the isotype group
were determined by repeated mixed ANOVA mode. Data was fitted with
a linear mixed effect model.
[0044] FIGS. 24A-24B illustrate day 7 quantification of
conventional dendritic cell subtype 1 (cDC1) numbers in tumors
(Panel A) or spleens (Panel B) of MC38 tumor-bearing C57BL/6 mice
administrated intravenously with 3750 pg/kg (open triangle), 750
pg/kg (open circle), 150 pg/kg (open square), 30 pg/kg (cross)
mouse FLT3-Ligand mouse IgG2a (C136S, LALA-PG), or 3753 pg/kg mouse
IgG2a isotype (open star) at day 0 when tumor volume reached 50
mm.sup.3. The x-axis indicates dose groups. The y-axis shows the
cDC1 number per gram of tumor (Panel A) or cDC1 number per spleen
(Panel B). Graph is a result of one experiment. Each individual
symbol represents the data point of a single mouse. Horizontal bars
represent the mean values and the error bars represent standard
deviation of the mean values. Statistical differences were
determined one-way ANOVA with Dunnett's post-test. **** p-value
<0.0001; *** p-value <0.001; ** p-value <0.01.
[0045] FIG. 25 illustrates tumor growth kinetics of MC38
tumor-bearing C57BL/6 mice administrated intravenously with QWx2
dose of 3.75 pg/kg (open circle) mouse FLT3-Ligand mouse IgG2a
(C136S, LALA-PG), Q3W dose of 3 mg/kg (open triangle) anti-mouse
PD-1 (clone RMP1-14), combination of the two reagents (closed
circle) or QWx2 dose of 10 mg/kg mouse IgG2a isotype control (open
star) at day 0 when tumor volume reached 50 mm.sup.3. Each data
point represents the mean value of 10 animals. Error bars represent
standard deviation of the mean values.
[0046] FIG. 26 illustrates tumor growth kinetics of CT26
tumor-bearing BALB/c mice administrated intravenously with QWx2
dose of 3.75 pg/kg (open circle) mouse FLT3-Ligand mouse IgG2a
(C136S, LALA-PG), Q3W dose of 3 mg/kg (open triangle) anti-mouse
CTLA4 (clone 9D9), combination of the two reagents (closed circle)
or Q3W dose of 10 mg/kg mouse IgG2a isotype control (open star) at
day 0 when tumor volume reached 65 mm.sup.3. Each data point
represents the mean value of 10 animals. Error bars represent
standard deviation of the mean values.
[0047] FIG. 27 diagrams an immunogenicity study in C57BL/6 mice
that were transduced with an Adeno-Associated Virus (AAV) vector
encoding a 1.2.times. length hepatitis B virus (HBV) genome
(AAV-HBV mice). At indicated times (asterisks) AAV-HBV mice were
administered 3 doses of an HBV vaccine and treated with saline,
mouse FLT3L, anti-mouse inhibitory PD-1, anti-mouse inhibitory
CTLA-4 or anti-mouse stimulatory CD137 antibodies. A control group
of mice received the HBV vaccine alone but no AAV-HBV. HBV-specific
IFN-.gamma. ELISPOT was performed using spleens of all animals at
day 105 post first vaccination.
[0048] FIGS. 28A-C illustrates IFN-.gamma. ELISPOT responses of
AAV-HBV mice specific for HBsAg (FIG. 28A), HBV core (FIG. 28B) and
HBV polymerase (FIG. 28C) observed at the end of the immunogenicity
study diagrammed in FIG. 27 for indicated treatment and control
groups.
DETAILED DESCRIPTION
1. Introduction
[0049] Provided are fms related tyrosine kinase 3 ligand (FLT3L)
extracellular domain-immunoglobulin fragment crystallizable region
(Fc region) fusion proteins that have a serum half-life allowing
for administration intervals that coordinates with other approved
immuno-oncology therapeutic agents, e.g., about once every 1, 2, 3
or 4 weeks, or longer intervals. The FLT3L-Fc fusion proteins
demonstrate favorable expression yields in in vitro culture, have
structural properties that allow for efficient large-scale
purification and long-term storage. The FLT3L-Fc fusion proteins
described herein have an increased serum half-life in comparison to
soluble FLT3L. Whereas soluble FLT3L must be administered on a
daily basis, the herein described FLT3L-Fc fusion proteins can be
administered about once every 1 to 8 weeks, or longer
intervals.
[0050] The FLT3L-Fc fusion proteins described herein are
differentiated from commercially available FLT3L-Fc fusion proteins
(e.g., available from BioXCell or described in Kreiter, et al.,
Cancer Research (2011) 71(19):6132-42) by several structural
modifications that result in improved functionality and the
feasibility for administration to and functional efficacy in
mammalian subjects. For example, the FLT3L-Fc fusion proteins
described herein have been engineered and formulated for an
improved glycosylation profile, allowing for a predictable and
consistent serum half-life or pharmacokinetics (PK). The linker
region between the ligand and the Fc fusion partner has reduced or
eliminated glycosylation sites. In certain FLT3L-Fc fusion variants
described herein, the IgG hinge region is truncated or eliminated.
For example, in FLT3L-Fc fusion variants comprising a human IgG4
Fe, the N-terminal five amino acid residues of the IgG4 hinge
(i.e., ESKYG (SEQ ID NO:97) are truncated or removed. Also,
mutations have been incorporated to stabilize retained amino acids
in a whole or truncated hinge region (e.g., S228P in a human IgG4
hinge), thereby eliminating Fab arm exchange or IgG half chain
exchange, and allowing favorable chemical manufacturing controls.
In addition, mutations have been introduced to reduce or eliminate
Fc gamma receptor (FcR.gamma.) binding (e.g., 234A/235A
substitutions in a human IgG4), effectively reducing or eliminating
antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC). Further, alterations were
made to enhance FcRn binding (e.g., 252Y/254T/256E substitutions in
a human IgG4), resulting in a prolonged serum half-life of the
FLT3L-Fc fusion protein (e.g., in comparison to a wild-type
Fc).
[0051] In certain embodiments, the FLT3L-Fc fusion proteins
described herein are not in an antibody format, and therefore only
bind to the Flt3 and FcRn receptors. In such embodiments, the
FLT3L-Fc fusion proteins described herein are structurally
differentiated from WO 2005/001048 and WO 2006/060021, describing
FLT3L fused to HER2, CD20, TRAL, CD3 or SM5-1. In such embodiments,
the lack of antigen directed binding allows systemic exposure of
FLT3L-Fc which results in a systemic increase in cDC1 cells
allowing the pharmacodynamics of the FLT3L-Fc to be monitored by
liquid biopsy.
2. FLT3L-Fc Fusion Protein Compositions
[0052] Provided are fusion proteins comprising a fms related
tyrosine kinase 3 ligand (FLT3L) extracellular domain operably
linked to an immunoglobulin fragment crystallizable region (Fc
region), wherein at least 5 amino acids are truncated from the
C-terminus of the FLT3L extracellular domain; and/or wherein the Fc
region does not comprise a hinge region.
[0053] In some embodiments, the FLT3L fusion proteins provided
herein are capable of binding to human fms related tyrosine kinase
3 ligand (FLT3). Human fms related tyrosine kinase 3 is identified
as NCBI Gene ID 2322 and is also known as human CD135, FLK-2, FLK2,
or STK1. Binding of FLT3L fusion proteins to FLT3L can be analyzed,
for example, by FACS, SPR, ELISA, immunoprecipitation-western blot,
and other assay formats known in the art.
Fusion Proteins and Homodimers Thereof
[0054] FLT3L Extracellular Domain
[0055] In certain embodiments, the FLT3L extracellular domain
comprises or is derived from a human FLT3L sequence. Human fms
related tyrosine kinase 3 ligand is identified as NCBI Gene ID 2323
and the alternative symbols of FLT3LG, FLT3L, FL and FLG3L. NCBI
identifies two isoforms and five transcriptional variants.
Exemplary polynucleotide and polypeptide sequences of FLT3L include
Ref Seq Nos. NM_001204502.1.fwdarw.NP_001191431.1 (isoform 1,
transcript variant 1); NM_001204503.1.fwdarw.NP_001191432.1
(isoform 1, transcript variant 2); NM_001459.4.fwdarw.NP_001450.2
(isoform 1, transcript variant 3);
NM_001278637.1.fwdarw.NP_001265566.1 (isoform 2, transcript variant
4); and NM_001278638.1.fwdarw.NP_001265567.1 (isoform 2, transcript
variant 5). In some embodiments, the FLT3L extracellular domain
comprises an amino acid sequence having at least 80%, at least 85%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or at least 100% identical to an amino acid sequence of
NP_001191431.1, NP_001191432.1, NP_001450.2, NP_001265566.1 or
NP_001265567.1, wherein the FLT3L extracellular domain binds to and
activates signaling through fms related tyrosine kinase 3 (FLT3,
CD135, FLK2, STK1). In some embodiments, the FLT3L extracellular
domain comprises or is derived from human FLT3L isoform 1. In some
embodiments, the FLT3L extracellular domain comprises or is derived
from human FLT3L isoform 2.
[0056] In some embodiments, the FLT3L portion of the fusion protein
comprises an amino acid sequence having at least 80%, at least 85%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or at least 100% identical to an amino acid sequence
of
TABLE-US-00001 (SEQ ID NO: 71)
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRP; (SEQ ID NO: 72)
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPL; (SEQ ID NO: 73)
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLE; (SEQ ID NO: 74)
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLE A; (SEQ ID NO:
75) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA T; (SEQ ID NO:
76) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA TA; (SEQ ID NO:
77) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA TAP; (SEQ ID NO:
78) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA TAPT; (SEQ ID
NO: 79) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA TAPTA; (SEQ ID
NO: 80) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA TAPTAP; or (SEQ
ID NO: 81) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRL
VLAQRWMERLKTVAGSKMQGLLERVNTEIKFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA TAPTAPQ;
wherein the FLT3L extracellular domain binds to and activates
signaling through fms related tyrosine kinase 3 (Gene ID: 2322;
FLT3, CD135, FLK2, STK1), and promotes or increases the
proliferation of cells expressing FLT3 on their cell surface. In
some embodiments, one or more FLT3L domain amino acid residues
N100, 5102, N123 and S125 are substituted, e.g., to remove the
N--X-S/T motifs, and potential N-linked and/or O-linked
glycosylation sites, e.g., to an amino acid residue selected from
the group consisting of glycine (G), alanine (A), or valine (V),
wherein the amino acid residue positions are with reference to SEQ
ID NOs: 1-18, 21-27 or 71-81. In some embodiments, one or both of
the serine residues at positions 102 and 125 are substituted to
alanine (A), wherein the amino acid residue positions are with
reference to SEQ ID NOs: 1-18, 21-27 or 71-81. In some embodiments,
the FLT3L extracellular domain comprises one or more amino acid
substitutions at the following positions: H8, K84, S102 and/or
S125, wherein the amino acid residue positions are with reference
to SEQ ID NOs: 1-18, 21-27 or 71-81. In some embodiments, the FLT3L
extracellular domain comprises one or more of the following amino
acid substitutions: H8Y, K84E; S102A; and/or S125A; wherein the
amino acid residue positions are with reference to SEQ ID NOs:
1-18, 21-27 or 71-81.
[0057] Modifications may be made in the structure of the FLT3L-Fc
polynucleotides and polypeptides described herein and still obtain
a functional molecule that encodes a variant or derivative
polypeptide with desirable characteristics. When it is desired to
alter the amino acid sequence of a polypeptide to create an
equivalent, or even an improved, variant or portion of a
polypeptide described herein, one skilled in the art will typically
change one or more of the codons of the encoding DNA sequence.
[0058] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of its ability to bind other polypeptides (e.g., antigens) or
cells. Since it is the binding capacity and nature of a protein
that defines that protein's biological functional activity, certain
amino acid sequence substitutions can be made in a protein
sequence, and, of course, its underlying DNA coding sequence, and
nevertheless obtain a protein with like properties. It is thus
contemplated that various changes may be made in the polypeptide
sequences of the disclosed antibodies and antigen-binding fragments
thereof, or corresponding DNA sequences that encode said
polypeptides without appreciable loss of their biological utility
or activity.
[0059] In many instances, a polypeptide variant will contain one or
more conservative substitutions. A "conservative substitution" is
one in which an amino acid is substituted for another amino acid
that has similar properties, such that one skilled in the art of
peptide chemistry would expect the secondary structure and
hydropathic nature of the polypeptide to be substantially
unchanged.
[0060] When comparing polynucleotide and polypeptide sequences, two
sequences are said to be "identical" if the sequence of nucleotides
or amino acids in the two sequences is the same when aligned for
maximum correspondence, as described below. Comparisons between two
sequences are typically performed by comparing the sequences over a
comparison window to identify and compare local regions of sequence
similarity. A "comparison window" as used herein, refers to a
segment of at least about 20 contiguous positions, usually 30 to
about 75, 40 to about 50, or over the full length of a sequence, in
which a sequence may be compared to a reference sequence of the
same number of contiguous positions after the two sequences are
optimally aligned.
[0061] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5: 151-153; Myers, E.
W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971)
Comb. Theor 77: 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
[0062] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0063] One example of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used,
for example with the parameters described herein, to determine
percent sequence identity for the polynucleotides and polypeptides
described herein. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (blast.ncbi.nlm.nih.gov/Blast.cgi).
[0064] In one illustrative example, cumulative scores can be
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a word length (W) of 11, and expectation (E) of
10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff
(1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments, (B) of 50,
expectation (E) of 10, M=5, N=-4 and a comparison of both
strands.
[0065] For amino acid sequences, a scoring matrix can be used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment.
[0066] In one approach, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residues
occur in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e., the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[0067] In some embodiments, the FLT3L extracellular domain does not
comprise a signal peptide. In some embodiments, the FLT3L
extracellular domain comprises an N-terminal signal peptide. The
signal peptide can be an endogenous signal peptide (e.g., from a
native or wild-type FLT3L protein), or from a heterologous
polypeptide. In some embodiments, the heterologous signal peptide
is from a secreted protein, e.g., a serum protein, an
immunoglobulin or a cytokine. In some embodiments, the signal
peptide is from a serum albumin signal peptide (e.g., having the
amino acid sequence KWVTFISLLFLFSSAYS (SEQ ID NO:82). In some
embodiments, the signal peptide is from a FLT3L protein (e.g.,
having the amino acid sequence MTVLAPAWSPTTYLLLLLLLSSGLSG (SEQ ID
NO:83) or MTVLAPAWSPNSSLLLLLLLLSPCLRG (SEQ ID NO:84). The signal
peptide can be designed to be cleaved off, e.g., after secretion
from the cell, to form a mature fusion protein. A modified human
serum albumin signal peptide to secrete proteins in cells that can
find use in expressing the present fusion proteins is described,
e.g., in Attallah, et al., Protein Expr Purf (2017) 132:27-33.
Additional signal peptide sequences for use in expressing the
herein described fusion proteins are described, e.g., in Kober, et
al., Biotechnol Bioeng. (2013) 110(4):1164-73.
[0068] In some embodiments, at least five amino acids are truncated
from the C-terminus of the FLT3L extracellular domain. For example,
in various embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14
or 15 amino acid residues are truncated or removed from the
C-terminus of the FLT3L extracellular domain. In some embodiments,
the FLT3L extracellular domain in the fusion protein is no longer
than 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 or 157 amino
acid residues in length. In some embodiments, the FLT3L
extracellular domain does not comprise the amino acid sequence
PTAPQ (SEQ ID NO:85), APTAPQ (SEQ ID NO:86), TAPTAPQ (SEQ ID
NO:87), ATAPTAPQ (SEQ ID NO:88), EATAPTAPQ (SEQ ID NO:89), or
LEATAPTAPQ (SEQ ID NO:90). In some embodiments, the FLT3L
extracellular domain does not comprise the amino acid sequence
PTAPQPP (SEQ ID NO:91), APTAPQPP (SEQ ID NO:92), TAPTAPQPP (SEQ ID
NO:93), ATAPTAPQPP (SEQ ID NO:94), EATAPTAPQPP (SEQ ID NO:95), or
LEATAPTAPQPP (SEQ ID NO:96).
[0069] In certain embodiments, the FLT3L extracellular domain
comprises or is derived from a mouse or murine FLT3L sequence. Mus
musculus fms related tyrosine kinase 3 ligand is identified as NCBI
Gene ID 14256 and the alternative symbols of Flt31, Ly72L and
Flt3lg. NCBI identifies one validated isoform and three unvalidated
isoforms (X1, X2 and X3). Exemplary polynucleotide and polypeptide
sequences of FLT3L include Ref Seq Nos.
NM_013520.3.fwdarw.NP_038548.3 (validated isoform 1);
XM_006540607.3.fwdarw.XP_006540670.1 (isoform X1);
XM_006540608.3.fwdarw.XP_006540671.1 (isoform X1);
XM_006540606.2.fwdarw.XP_006540669.1 (isoform X1);
XM_011250793.1.fwdarw.XP_011249095.1 (isoform X1);
XM_006540609.3.fwdarw.XP_006540672.1 (isoform X2);
XM_006540610.3.fwdarw.XP_006540673.1 (isoform X2);
XM_006540612.3.fwdarw.XP_006540675.1 (isoform X3); and
XM_011250794.2.fwdarw.XP_011249096.1 (isoform X3). In some
embodiments, the FLT3L extracellular domain comprises an amino acid
sequence having at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or at least
100% identical to an amino acid sequence of NP_038548.3, XP
006540670.1, XP 006540671.1, XP 006540669.1, XP_011249095.1,
XP_006540672.1, XP_006540673.1, XP 006540675.1, XP_011249096.1,
wherein the FLT3L extracellular domain binds to and activates
signaling through fms related tyrosine kinase 3 (FLT3, CD135, FLK2,
STK1), and promotes or increases the proliferation of cells
expressing FLT3 on their cell surface. In some embodiments, the
FLT3L extracellular domain comprises or is derived from murine
FLT3L isoforms 1, X1, X2 or X3. In some embodiments, at least five
amino acids are truncated from the C-terminus of the mouse FLT3L
extracellular domain. For example, in various embodiments, at least
5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues are
truncated or removed from the C-terminus of the mouse FLT3L
extracellular domain. In some embodiments, the FLT3L extracellular
domain in the fusion protein is no longer than 149, 150, 151, 152,
153, 154, 155, 156, 157, 158 or 159 amino acid residues in
length.
[0070] In some embodiments, the mouse FLT3L portion of the fusion
protein comprises an amino acid sequence having at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or at least 100% identical to an amino acid
sequence of:
TABLE-US-00002 (SEQ ID NO: 98)
TPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSL
FLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTN
ISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIAL EATELPEPR,
wherein the mouse FLT3L extracellular domain binds to an activates
signaling through mouse fms related tyrosine kinase 3 (NCBI Human
Gene ID: 14255; Flt3, Flk2; Ly72; wmfl; CD135; Flk-2; Flt-3;
B230315G04). In some embodiments, cysteine at position 109 is
substituted to an amino acid residue selected from the group
consisting of glycine (G), alanine (A), serine (S), threonine (T)
or valine (V), wherein the amino acid residue positions are with
reference to SEQ ID NOs: 19, 20 and 42.
[0071] In certain embodiments, the FLT3L extracellular domain
comprises or is derived from a macaque or macaca FLT3L sequence.
Macaca mulatta (Rhesus monkey) fms related tyrosine kinase 3 ligand
is identified as NCBI Gene ID 719239 and the alternative symbols of
FLT3L and FLT3LG. NCBI identifies five unvalidated isoforms (X1,
X2, X3, X4, X5). Exemplary polynucleotide and polypeptide sequences
of FLT3L include Ref Seq Nos. XM_015124576.1.fwdarw.XP_014980062.1
(isoform X1), XM_015124578.1.fwdarw.XP_014980064.1 (isoform X2),
XM_015124579.1.fwdarw.XP_014980065.1 (isoform X3),
XM_015124580.1.fwdarw.XP_014980066.1 (isoform X4) and
XM_015124581.1.fwdarw.XP_014980067.1 (isoform X5). In some
embodiments, the FLT3L extracellular domain comprises an amino acid
sequence having at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or at least
100% identical to an amino acid sequence of XP_014980062.1,
XP_014980064.1, XP_014980065.1, XP_014980066.1 or XP_014980067.1,
wherein the FLT3L extracellular domain binds to and activates
signaling through fms related tyrosine kinase 3 (FLT3, CD135, FLK2,
STK1), and promotes or increases the proliferation of cells
expressing FLT3 on their cell surface. In some embodiments, the
FLT3L extracellular domain comprises or is derived from macaque
FLT3L isoforms X1, X2, X3, X4 or X5. In some embodiments, at least
five amino acids are truncated from the C-terminus of the macaque
FLT3L extracellular domain. For example, in various embodiments, at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues
are truncated or removed from the C-terminus of the macaque FLT3L
extracellular domain. In some embodiments, the FLT3L extracellular
domain in the fusion protein is no longer than 145, 146, 147, 148,
149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164 or 165 amino acid residues in length.
[0072] As appropriate, in certain embodiments, the FLT3L
extracellular domain is comprised of or derived from a canine or a
feline FLT3L extracellular domain. In some embodiments, the dog or
Canis lupus FLT3L portion of the fusion protein comprises an amino
acid sequence having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or at least
100% identical to an amino acid sequence of. NP_001003350.1,
XP_005615795.1 or XP_022273164.1. In some embodiments, the cat or
Felis catus FLT3L portion of the fusion protein comprises an amino
acid sequence having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or at least
100% identical to an amino acid sequence of: NP_001009842.1 or
XP_011287950.1.
[0073] A "polypeptide variant," as the term is used herein, is a
polypeptide that typically differs from a polypeptide specifically
disclosed herein in one or more substitutions, deletions, additions
and/or insertions. Such variants may be naturally occurring or may
be synthetically generated, for example, by modifying one or more
of the above polypeptide sequences described herein and evaluating
one or more biological activities of the polypeptide as described
herein and/or using any of a number of techniques well known in the
art.
[0074] The term "variant" may also refer to any naturally occurring
or engineered molecule comprising one or more nucleotide or amino
acid mutations. In one embodiment, the multi-specific antigen
binding molecule is a bispecific antigen binding molecule. In one
embodiment, the multi-specific antigen binding molecule is a
bispecific antibody. For example, somatic variants may encompass
all related naturally occurring antibodies that are part of or
derived from the same B-cell lineage. Engineered variants may
encompass all single mutations or combinatorial mutations made to
an antibody.
[0075] Fc Region
[0076] The FLT3L extracellular domain, or truncated fragment
thereof, is operably linked to an Fe domain. Generally, the Fe
domain is comprised of or derived from the same species as the
FLT3L extracellular domain (e.g., human, dog, cat, mouse or
monkey). In some embodiments, the FLT3L extracellular domain, or
truncated fragment thereof, is directly linked or contiguously
linked or abutted to the Fc domain. In some embodiments, the FLT3L
extracellular domain, or truncated fragment thereof, is operably
linked to the Fc domain via a linker. As appropriate, the linker
can be a flexible linker, e.g., a sequence comprising 3 or 4
repeats of a GGGGS motif or "G-S linker" (SEQ ID NO:99) (Desplancq
et al. 1994, Protein Engineering 7:1027-1033).
[0077] In some embodiments, the Fc region is from a human IgG1,
IgG2, IgG3 or IgG4. In some embodiments, the Fc region is from a
human IgG1 or IgG4.
[0078] In certain embodiments the FLT3L extracellular domain, or
truncated fragment thereof, is directly linked to, or linked via an
intervening amino acid sequence (e.g., a G-S linker), to a human
IgG1 (e.g., mutant IgG1m3 sequence), IgG2, IgG3 or IgG4 with 1 to
10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions.
In some embodiments, the Fc modifications can promote one or more
of increased serum half-life or decreased antibody effector
function of the molecule. In other embodiments, certain of these
modifications, decrease antibody effector function and increase
half-life of the antibody. In some embodiments, the FLT3L-Fc fusion
proteins described herein comprise two or more, three or more, four
or more, five or more, six or more, six or fewer, five or fewer,
four or fewer, three or fewer, two or fewer, or one modified Fc
amino acid residue(s). Exemplary amino acid substitutions are
described below.
[0079] In some embodiments, the Fc domain of the fusion protein
does not comprise a hinge region; it is truncated or deleted, in
whole or in part. The structural hinge region of human IgG1, IgG2
and IgG4 antibodies is a peptide linker of about 19 to 23 amino
acids containing two to four cysteine residues, is genetically
encoded on the hinge exon together with the 5'-end of the CH2 exon,
and allows for disulfide bridges between first and second Fc
domains (Roux, et al., J. Immunol. (1998) 161:4083). The structural
hinge region is comprised of amino acid residue positions 216-238
(EU numbering) or 226-251 (Kabat numbering) (identified on
imgt.org). In some embodiments, the Fc region comprises or is
derived from a human IgG4 isotype and does not comprise the amino
acid sequence ESKYGPPCPPCP (SEQ ID NO:100). In some embodiments,
the Fc region comprises or is derived from a human IgG1 isotype and
does not comprise the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID
NO:101) or EPKSCDKTHTCPPCPAPELL (SEQ ID NO:110).
Fc Mutations that Increase Serum Half-Life
[0080] In some embodiments, the Fc region comprises amino acid
modifications that promote an increased serum half-life of the
fusion protein. Mutations that increase the half-life of an
antibody have been described. In one embodiment, the constant
region of a FLT3L-Fc fusion proteins described herein comprise a
methionine to tyrosine substitution at position 252 (EU numbering),
a serine to threonine substitution at position 254 (EU numbering),
and a threonine to glutamic acid substitution at position 256 (EU
numbering). See, e.g., U.S. Pat. No. 7,658,921. This type of
mutant, designated as a "YTE mutant" exhibits a four-fold increased
half-life relative to wild-type versions of the same antibody
(Dall'Acqua, et al., J Biol Chem, 281: 23514-24 (2006); Robbie, et
al., Antimicrob Agents Chemotherap., 57(12):6147-6153 (2013)). In
certain embodiments, the FLT3L-Fc fusion proteins described herein
comprise an IgG constant domain comprising one, two, three or more
amino acid substitutions of amino acid residues at positions
251-257, 285-290, 308-314, 385-389, and 428-436 (EU numbering).
Alternatively, M428L and N434S ("LS") substitutions can increase
the pharmacokinetic half-life of the fusion protein. In other
embodiments, the FLT3L-Fc fusion proteins described herein comprise
a M428L and N434S substitution (EU numbering). In other
embodiments, the FLT3L-Fc fusion proteins described herein comprise
T250Q and M428L (EU numbering) mutations. In other embodiments, the
FLT3L-Fc fusion proteins described herein comprise H433K and N434F
(EU numbering) mutations.
Fc Mutations that Reduce or Eliminate Effector Activity
[0081] In some embodiments, the FLT3L-Fc fusion proteins described
herein can have an Fc domain with amino acid substitutions that
reduce or eliminate Fc effector function (including, e.g.,
antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent
cellular phagocytosis (ADCP), and complement-dependent cytotoxicity
(CDC)).
[0082] In some embodiments, the Fc region is altered by replacing
at least one amino acid residue with a different amino acid residue
to reduce or eliminate effector function(s) of the antibody. For
example, one or more amino acids selected from amino acid residues
234, 235, 236, 237, 297, 318, 320 and 322 (EU numbering) can be
replaced with a different amino acid residue such that the fusion
protein has decreased affinity for an effector ligand. The effector
ligand to which affinity is altered can be, for example, an Fc
receptor (e.g., at residue positions 234, 235, 236, 237, 297 (EU
numbering)) or the C1 component of complement (e.g., at residue
positions 297, 318, 320, 322 (EU numbering)). U.S. Pat. Nos.
5,624,821 and 5,648,260, both by Winter et al.
[0083] Fc modifications reducing or eliminating effector function
include substitutions, insertions, and deletions, e.g., at one or
more positions including 234, 235, 236, 237, 267, 269, 325, and
328, e.g., 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R (EU
numbering). Further, an Fc variant may comprise 236R/328R. Other
modifications for reducing Fc.gamma.R and complement interactions
include substitutions at positions 297A, 234A, 235A, 318A, 228P,
236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and
234V (EU numbering). These and other modifications are reviewed in
Strohl (2009) Current Opinion in Biotechnology 20:685-691. Effector
functions (both ADCC and complement activation) can be reduced,
while maintaining neonatal FcR binding (maintaining half-life), by
mutating IgG residues at one or more of positions 233-236 and
327-331, such as E233P, L234V, L235A, optionally G236A, A327G,
A330S and P331S in IgG1; E233P, F234V, L235A, optionally G236A, in
IgG4; and A330S and P331S in IgG2 (EU numbering). See Armour et al.
(1999) Eur. J. Immunol. 29:2613; WO 99/58572. Other mutations that
reduce effector function include L234A and L235A in IgG1 (Alegre et
al. (1994) Transplantation 57:1537); V234A and G237A in IgG2 (Cole
et al. (1997) J. Immunol. 159:3613; see also U.S. Pat. No.
5,834,597); and S228P and L235E for IgG4 (Reddy et al. (2000) J.
Immunol. 164:1925). Another combination of mutations for reducing
effector function in a human IgG1 include L234F, L235E and P331S.
Oganesyan et al. (2008) Acta Crystallogr. D. Biol. Crystallogr.
64:700. See generally Labrijn et gal. (2008) Curr. Op. Immunol.
20:479. Additional mutations found to decrease effector function in
the context of an Fc (IgG1) fusion protein (abatacept) include
C226S, C229S and P238S (EU numbering). Davis et al. (2007) J.
Immunol. 34:2204.
[0084] ADCC activity may be reduced by modifying the Fc region. In
certain embodiments, sites that affect binding to Fc receptors may
be removed, e.g., sites other than salvage receptor binding sites.
In other embodiments, an Fc region may be modified to remove an
ADCC site. Exemplary ADCC sites have been described with respect to
ADCC sites in IgG1 (Sarmay, et al, (1992) Molec. Immunol. 29 (5):
633-9). In one embodiment, the G236R and L328R variant of human
IgG1 effectively eliminates Fc.gamma.R binding (Horton, et al.
(2011) J. Immunol. 186:4223 and Chu, et al. (2008) Mol. Immunol.
45:3926). In other embodiments, the Fc having reduced binding to
Fc.gamma.Rs comprises the amino acid substitutions L234A, L235E and
G237A. Gross, et al. (2001) Immunity 15:289. Modifications in the
IgG Fc region to decrease binding to Fc.gamma.RI to decrease ADCC
(e.g., 234A; 235E; 236A; G237A) identified in WO 88/007089 can be
used in the present fusion proteins. See also Duncan & Winter
(1988) Nature 332:563; Chappel et al. (1991) Proc. Nat'l Acad. Sci.
(USA) 88:9036; and Sondermann et al. (2000) Nature 406:267
(discussing the effects of these mutations on Fc.gamma.RIII
binding).
[0085] CDC activity may also be reduced by modifying the Fc region.
Mutations at IgG1 positions D270, K322, P329 and P331, specifically
alanine mutations D270A, K322A, P329A and P331A, significantly
reduce the ability of the corresponding antibody to bind C1q and
activate complement (Idusogie et al. (2000) J. Immunol. 164:4178;
WO 99/51642. Modification of position 331 of IgG1 (e.g., P331S) has
been shown to reduce complement binding (Tao et al. (1993) J. Exp.
Med. 178:661; Xu Y, et al. J Biol Chem. 1994. 269:3469-74; and
Canfield & Morrison (1991) J. Exp. Med. 173:1483). In another
example, one or more amino acid residues within amino acid
positions 231 to 239 are altered to thereby reduce the ability of
the antibody to fix complement (WO 94/29351). Modifications in the
IgG Fc region identified in WO 88/007089 that reduce or eliminate
binding to complement component C1q, and therefore reduce or
eliminate CDC (e.g., E318A or V/K320A and K322A/Q) can be used in
the present fusion proteins.
[0086] In some embodiments, the Fc with reduced complement fixation
has the amino acid substitutions A330S and P331S. Gross et al.
(2001) Immunity 15:289.
[0087] Other Fc variants having reduced ADCC and/or CDC are
disclosed at Glaesner et al. (2010) Diabetes Metab. Res. Rev.
26:287 (F234A and L235A to decrease ADCC and ADCP in an IgG4);
Hutchins et al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:11980
(F234A, G237A and E318A in an IgG4); An et al. (2009) MAbs 1:572
and U.S. Pat. App. Pub. 2007/0148167 (H268Q, V309L, A330S and P331S
in an IgG2); McEarchern et al. (2007) Blood 109:1185 (C226S, C229S,
E233P, L234V, L235A in an IgG1); Vafa et al. (2014) Methods 65:114
(V234A, G237A, P238S, H268A, V309L, A330S, P331S in an IgG2) (EU
numbering).
[0088] In certain embodiments, the fusion protein has an Fc having
essentially no effector function, e.g., the Fc has reduced or
eliminated binding to Fc.gamma.Rs and reduced or eliminated
complement fixation, e.g., is effectorless. An exemplary IgG1 Fc
that is effectorless comprises the following five mutations: L234A,
L235E, G237A, A330S and P331S (EU numbering) (Gross et al. (2001)
Immunity 15:289). These five substitutions may be combined with
N297A to eliminate glycosylation as well.
IgG1 Isotype Fc
[0089] In one embodiment, the Fc region comprises or is derived
from a human IgG1. In some embodiments, the antibody has a chimeric
heavy chain constant region (e.g., having the CH1, hinge, CH2
regions of IgG4 and CH3 region of IgG1).
[0090] IgG1 antibodies exist in various allotypes and isoallotypes.
In particular embodiments, the FLT3L-Fc fusion proteins described
herein include an IgG1 heavy chain having an allotype of G1m1;
nG1m2; G1m3; G1m17,1; G1m17,1,2; G1m3,1; or G1m17. Each of these
allotypes or isoallotypes is characterized by the following amino
acid residues at the indicated positions within the IgG1 heavy
chain constant region (Fc) (EU numbering):
[0091] G1m1: D356, L358;
[0092] nG1m1: E356, M358;
[0093] G1m3: R214, E356, M358, A431;
[0094] G1m17,1: K214, D356, L358, A431;
[0095] G1m17,1,2: K214, D356, L358, G431;
[0096] G1m3,1: R214, D356, L358, A431; and
[0097] G1m17: K214, E356, M358, A431.
[0098] In a specific embodiment, the FLT3L extracellular domain, or
truncated fragment thereof, is directly linked to, or linked via an
intervening amino acid sequence (e.g., a G-S linker), to a wild
type IgG1m3 sequence, or fragment thereof, provided below.
TABLE-US-00003 (SEQ ID NO: 102)
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
[0099] In certain embodiments, the FLT3L-Fc fusion protein has an
IgG1 isotype. In some embodiments, the FLT3L-Fc fusion protein
contains a human IgG1 constant region. In some embodiments, the
human IgG1 Fc region contains one or more modifications. For
example, in some embodiments, the Fc region contains one or more
amino acid substitutions (e.g., relative to a wild-type Fc region
of the same isotype). In some embodiments, the one or more amino
acid substitutions are selected from N297A, N297Q (Bolt S et al.
(1993) Eur J Immunol 23:403-411), D265A, L234A, L235A (McEarchem et
al., (2007) Blood, 109:1185-1192), C226S, C229S (McEarchem et al.,
(2007) Blood. 109:1185-1192), P238S (Davis et al., (2007) J
Rheumatol, 34:2204-2210), E233P, L234V (McEarchern et al., (2007)
Blood, 109:1185-1192), P238A, A327Q, A327G, P329A (Shields R L. et
al., (2001) J Biol Chem. 276(9):6591-604), K322A, L234F, L235E
(Hezareh, et al., (2001) J Virol 75, 12161-12168; Oganesyan et al.,
(2008). Acta Crystallographica 64, 700-704), P331S (Oganesyan et
al., (2008) Acta Crystallographica 64, 700-704), T394D (Wilkinson
et al. (2013) MAbs 5(3): 406-417), A330L, M252Y, S254T, and/or
T256E, where the amino acid position is according to the EU
numbering convention. In certain embodiments, the Fc region further
includes an amino acid deletion at a position corresponding to
glycine 236 according to the EU numbering convention. As used
herein, numbering of a given amino acid polymer or nucleic acid
polymer "corresponds to", is "corresponding to" or is "relative to"
the numbering of a selected or reference amino acid polymer or
nucleic acid polymer when the position of any given polymer
component (e.g., amino acid, nucleotide, also referred to
generically as a "residue") is designated by reference to the same
or to an equivalent position (e.g., based on an optimal alignment
or a consensus sequence) in the selected amino acid or nucleic acid
polymer, rather than by the actual numerical position of the
component in the given polymer.
[0100] In some embodiments, the FLT3L-Fc fusion protein has an IgG1
isotype with a heavy chain constant region that contains a C220S
amino acid substitution according to the EU numbering
convention.
[0101] In some embodiments, the Fc region comprises a human IgG1
isotype and comprises one or more amino acid substitutions in the
Fc region at a residue position selected from the group consisting
of. N297A, N297G, N297Q, N297G, D265A, L234A, L235A, C226S, C229S,
P238S, E233P, L234V, P238A, A327Q, A327G, P329A, P329G, K322A,
L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, M428L,
N434S, T366W, T366S, L368A, Y407V and any combination thereof,
wherein the numbering of the residues is according to EU numbering.
In some embodiments, the Fc region comprises a human IgG1 isotype
and comprises one or more amino acid substitutions in the Fc region
at a residue position selected from the group consisting of: L234A,
L234V, L234F, L235A, L235E, A330L, P331S, and any combination
thereof, wherein the numbering of the residues is according to EU
numbering.
IgG4 Isotype Fc
[0102] For uses where effector function is to be avoided
altogether, e.g., when antigen binding alone is sufficient to
generate the desired therapeutic benefit, and effector function
only leads to (or increases the risk of) undesired side effects,
IgG4 antibodies may be used, or antibodies or fragments lacking the
Fc region or a substantial portion thereof can be devised, or the
Fc may be mutated to eliminate glycosylation altogether (e.g.,
N297A). Alternatively, a hybrid construct of human IgG2 (CHI domain
and hinge region) and human IgG4 (CH2 and CH3 domains) has been
generated that is devoid of effector function, lacking the ability
to bind the Fc.gamma.Rs (like IgG2) and unable to activate
complement (like IgG4). (see, Rother et al. (2007) Nat. Biotechnol.
25:1256; Mueller et al. (1997) Mol. Immunol. 34:441; and Labrijn et
al. (2008) Curr. Op. Immunol. 20:479, discussing Fc modifications
to reduce effector function generally).
[0103] In one embodiment, the Fc region comprises or is derived
from a human IgG4. In certain embodiments, the FLT3L-Fc fusion
protein has an IgG4 isotype. In some embodiments, the FLT3L-Fc
fusion protein contains a human IgG4 constant region. In some
embodiments, the human IgG4 constant region includes an Fc region.
In some embodiments, the Fc region contains one or more
modifications. For example, in some embodiments, the Fc region
contains one or more amino acid substitutions (e.g., relative to a
wild-type Fc region of the same isotype). In some embodiments, the
one or more amino acid substitutions are selected from E233P,
F234V, F234A, L235A, G237A, E318A, S228P, L235E, T394D, M252Y,
S254T, T256E, N297A, N297G, N297Q, T366W, T366S, L368A, Y407V,
M428L, N434S, and any combination thereof, where the amino acid
position is according to the EU numbering convention. See, e.g.,
Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984;
Reddy et al., (2000) J Immunol, 164:1925-1933; Angal et al., (1993)
Mol Immunol. 30(1):105-8; U.S. Pat. No. 8,614,299 B2; Vafa O. et
al., (2014) Methods 65:114-126; and Jacobsen et. al., J. Biol.
Chem.(2017) 292(5):1865-1875. In some embodiments, the Fc region
comprises a human IgG4 isotype and comprises one or more amino acid
substitutions in the Fc region at a residue position selected from
the group consisting of: F234V, F234A, L235A, L235E, S228P, and any
combination thereof, wherein the numbering of the residues is
according to EU numbering.
[0104] In some embodiments, an IgG4 variant of the present
disclosure may be combined with an S228P mutation according to the
EU numbering convention (Angal et al., (1993) Mol Immunol,
30:105-108) and/or with one or more mutations described in Peters
et al., (2012) J Biol Chem. 13; 287(29):24525-33) to enhance
antibody stabilization.
IgG2 Isotype Fc
[0105] In certain embodiments, the FLT3L-Fc fusion protein has an
IgG2 isotype. In some embodiments, the FLT3L-Fc fusion protein
contains a human IgG2 constant region. In some embodiments, the
human IgG2 constant region includes an Fc region. In some
embodiments, the Fc region contains one or more modifications. For
example, in some embodiments, the Fc region contains one or more
amino acid substitutions (e.g., relative to a wild-type Fc region
of the same isotype). In some embodiments, the one or more amino
acid substitutions are selected from P238S, V234A, G237A, H268A,
H268Q, H268E, V309L, N297A, N297G, N297Q, V309L, A330S, P331 S,
C232S, C233S, M252Y, S254T, and/or T256E, where the amino acid
position is according to the EU numbering convention (Vafa, et al.,
(2014) Methods 65:114-126).
[0106] In certain embodiments, the FLT3L-Fc fusion proteins
described herein comprise the L234F, L235E, D265A mutations, which
are collectively referred to as "FEA." The FEA mutations decrease
or abrogate effector function. In certain embodiments, the FLT3L-Fc
fusion proteins described herein comprise the L234F, L235E, D265A,
and F405L mutations, which are collectively referred to as "FEAL."
In certain embodiments, the FLT3L-Fc fusion proteins described
herein comprise the L234F, L235E, D265A, and a mutation selected
from the group consisting of F405L, F405A, F405D, F405E, F405H,
F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and
F405Y. In certain embodiments, the FLT3L-Fc fusion proteins
described herein comprise the L234F, L235E, D265A, and K409R
mutations, which are collectively referred to as "FEAR." In certain
embodiments, FEAL and FEAR are comprised in a fusion protein
described herein. In certain embodiments, the FLT3L-Fc fusion
proteins described herein additionally comprise the M428L and N434S
mutations, which are collectively referred to as LS. In certain
embodiments, the FLT3L-Fc fusion proteins described herein comprise
the L234F, L235E, D265A, F405L, M428L, and N434S mutations, which
are collectively referred to as "FEALLS." In certain embodiments,
the FLT3L-Fc fusion proteins described herein comprise the L234F,
L235E, D265A, M428L, and N434S mutations along with one further
mutation selected from the group consisting of F405L, F405A, F405D,
F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T,
F405V, F405W, and F405Y. In certain embodiments, the FLT3L-Fc
fusion proteins described herein comprise the L234F, L235E, D265A,
K409R, M428L, and N434S mutations which are collectively referred
to as "FEARLS." In certain embodiments, FEALLS and FEARLS are
comprised in a fusion protein described herein. By reducing or
abrogating effector function on the Fc domains of the FLT3L-Fc
fusion protein, cells bound by the molecule are not killed by
innate effector cells e.g., NK cells, macrophages.
[0107] In certain embodiments, the one or more modifications are
selected from the following Fc amino acid substitutions (EU
numbering) or combinations thereof. L234F; L235E; G236A; S239D;
F243L; D265E; D265A; S267E; H268F; R292P; N297Q; N297G, N297A;
S298A; S324T; 1332E; S239D; A330L; L234F; L235E; P331S; F243L;
Y300L; V305I; P396L; S298A; E333A; K334A; E345R; L235V; F243L;
R292P; Y300L; P396L; M428L; E430G; N434S; G236A, S267E, H268F,
S324T, and 1332E; G236A, S239D, and 1332E; S239D, A330L, 1332E;
L234F, L235E, and P331S; F243L, R292P, Y300L, V305I, and P396L;
G236A, H268F, S324T, and 1332E; S239D, H268F, S324T, and 1332E;
S298A, E333A, and K334A; L235V, F243L, R292P, Y300L, and P396L;
S239D, 1332E; S239D, S298A, and 1332E; G236A, S239D, 1332E, M428L,
and N434S; G236A, S239D, A330L, 1332E, M428L, and N434S; S239D,
1332E, G236A and A330L; M428L and N4343S; M428L, N434S; G236A,
S239D, A330L, and 1332E; and G236A and 1332E. In certain
embodiments, the one or more modifications is selected from the
group consisting of: D265A, L234F, L235E, N297A, N297G, N297Q, and
P331S. In certain embodiments, the one or more modifications are
selected from N297A and D265A. In certain embodiments, the one or
more modifications are selected from L234F and L235E. In certain
embodiments, the one or more modifications are selected from L234F,
L234E, and D265A. In certain embodiments, the one or more
modifications are selected from L234F, L234E, and N297Q. In certain
embodiments, the one or more modifications are selected from L234F,
L235E, and P331S. In certain embodiments, the one or more
modifications are selected from D265A and N297Q. In certain
embodiments, the one or more modifications are selected from L234F,
L235E, D265A, N297A, N297G, N297Q, and P331S.
[0108] Mutations that reduce Fc-receptor binding and find use in
the herein described fusion proteins include, for example, N297A;
N297G; N297Q; D265A; L234F/L235E; L234F/L235E/N297Q;
L234F/L235E/P331S; D265A/N297Q; and L234F/L235E/D265A/N297Q/P331S
(all EU numbering). In certain embodiments the FLT3L-Fc fusion
proteins described herein described herein comprise L234F and L235E
mutations. In certain embodiments the FLT3L-Fc fusion proteins
described herein described herein comprise L234F, L235E, and D265A
mutations. In certain embodiments the FLT3L-Fc fusion proteins
described herein described herein comprise L234F, L235E, and N297Q
mutations. In certain embodiments the FLT3L-Fc fusion proteins
described herein described herein comprise an N297A or N297Q
mutation. In certain embodiments the FLT3L-Fc fusion proteins
described herein described herein comprise an N297A, N297G or N297Q
mutation as well as L234F, L235E, and D265A mutations. In certain
embodiments, one, two, three, four, or more amino acid
substitutions are introduced into a Fc region to alter the effector
function of the antigen binding molecule. For example, these
substitutions are located at positions selected from the group
consisting of amino acid residues 234, 235, 236, 237, 265, 297,
318, 320, and 322, (according to EU numbering). These positions can
be replaced with a different amino acid residue such that the
antigen binding molecule has an altered (e.g., reduced) affinity
for an effector ligand (e.g., an Fc receptor or the C1 component of
complement), but retains the antigen binding ability of the parent
antibody. In certain embodiments, the FLT3L-Fc fusion proteins
described herein described herein comprise E233P, L234V, L235A,
and/or G236A mutations (EU numbering). In some embodiments, the
FLT3L-Fc fusion proteins described herein comprise A327G, A330S,
and/or P331S mutations (EU numbering). In some embodiments, the
FLT3L-Fc fusion proteins described herein comprise K322A mutations
(EU numbering). In some embodiments the FLT3L-Fc fusion proteins
described herein comprise E318A, K320A, and K322A (EU numbering)
mutations. In certain embodiments, the FLT3L-Fc fusion proteins
described herein comprise a L235E (EU numbering) mutation.
[0109] In some embodiments, the Fc portion of the fusion protein
comprises an amino acid sequence having at least 80%, at least 85%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or at least 100% identical to an amino acid sequence
of
TABLE-US-00004 (SEQ ID NO: 103)
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK;
(SEQ ID NO: 104) GGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK;
(SEQ ID NO: 105) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK; (SEQ ID NO: 106)
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK; or (SEQ ID NO: 107)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0110] Illustrative polypeptide sequences of the FLT3L-Fc fusion
proteins described herein are provided in Table A. In some
embodiments, FLT3-Fc fusion protein comprises an amino acid
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 1-18 and 21-27. In some embodiments, FLT3-Fc fusion protein
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 1-18 and 21-27. In some embodiments, FLT3-Fc fusion
protein comprises an amino acid sequence that is at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% identical to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 19-20. In some
embodiments, FLT3-Fc fusion protein comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs:
19-20.
[0111] In various embodiments, the FLT3L-Fc fusion proteins may be
glycosylated or aglycosylated. In certain embodiments where the
FLT3L-Fc fusion protein is glycosylated, at least 50%, at least
60%, at least 70%, least 80%, at least 85%, at least 90%, or more,
N-linked and/or O-linked glycosylation sites in the fusion protein
are sialylated. In certain embodiments where the FLT3L-Fc fusion
protein is sialylated, the sialylated N-linked and/or O-linked
glycosylation sites in the fusion protein comprise from 2 to 7
sialic acid residues, e.g., from 3 to 6 sialic acid residues, e.g.,
from 4 to 5 sialic acid residues.
[0112] In some embodiments, the FLT3L-Fc fusion proteins have a
serum half-life of at least about 7 days, e.g., in a mammal, e.g.,
in a human, monkey, mouse, cat or dog. In some embodiments, the
FLT3L-Fc fusion proteins have a serum half-life of at least about 7
days, e.g., at least about 8, 9, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30 days, or longer, e.g., in a mammal, e.g., in a human,
monkey, mouse, cat or dog. Generally, a shorter serum half-life is
observed with relatively lower doses. A longer serum half-life is
observed with relatively higher doses.
[0113] Functionally, the FLT3L-Fc fusion proteins described herein
induce, promote and/or increase the growth, proliferation and/or
expansion of cells or populations of cells that express or
overexpress FLT3 on their cell surface. Illustrative cells or
populations of cells that express or overexpress FLT3 include
dendritic cells (e.g., cDC1 cells and/or cDC2 cells),
monocyte-derived dendritic cells (moDCs), and/or progenitor cells
thereof. In some embodiments, the cell or population of cells that
express FLT3 comprise hematopoietic progenitor cells, e.g., Common
Lymphoid Progenitors (CLPs), Early Progenitors with Lymphoid and
Myeloid potential (EPLMs), granulocyte-monocyte (GM) progenitors
(GMP), monocyte-derived dendritic cells (moDCs) progenitors, and
early multi-potent progenitors (MPP) within the Lineage-kit+Sca1
(LSK) compartment.
TABLE-US-00005 TABLE A FLT3L-Fc fusion proteins PROTEIN NO: SEQ ID
NO: Features Polypeptide Sequence (Fc domain is underlined) 1 FLT3L
ECD-hingeless
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
hG1
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 FLT3L ECD (.DELTA.
C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG 5
aa (PTAPQ; SEQ ID
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
NO: 85))-hingeless
QCQPDSSTLPPPWSPRPLEATAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
hG1
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3 FLT3L ECD-hG4
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
S228P/L235E
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
QCQPDSSTLPPPWSPRPLEATAPTAPQESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 4
FLT3L ECD-hG4
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
S228P/F234A/L235A
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
QCQPDSSTLPPPWSPRPLEATAPTAPQESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 5
Aglyco-FLT3L ECD
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
(S128A/S151A)
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNIARLLQETSEQLVALKPWITRQNFARCLEL
hingeless hG1
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 6 FLT3L (.DELTA.
C-term 5 aa
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
(PTAPQ; SEQ ID
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
NO: 85))-hG4
QCQPDSSTLPPPWSPRPLEATAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
S228P/F234A/L235A
VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 7 FLT3L
ECD (.DELTA. C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
10 aa (LEATAPTAPQ;
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
SEQ ID NO: 90))-
QCQPDSSTLPPPWSPRPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
hingeless hG1
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 8 FLT3L ECD (.DELTA. C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
10 aa (LEATAPTAPQ;
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
SEQ ID NO: 90))-hG4
QCQPDSSTLPPPWSPRPESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
S228P/F234A/L235A
SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 9 FLT3L
ECD-hingeless
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
hG1
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
(M252Y/S254T/T256E)
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10 FLT3L ECD (.DELTA.
C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG 5
aa (PTAPQ; SEQ ID
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
NO: 85))-hingeless
QCQPDSSTLPPPWSPRPLEATAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWY
hG1
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
(M252Y/S254T/T256E)
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11 FLT3L ECD-hG4
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
S228P/L235E/
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
M252Y/S254T/T256E)
QCQPDSSTLPPPWSPRPLEATAPTAPQESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLYITRE
PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 12
FLT3L ECD-hG4
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
(S228P/F234A/L235A/
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
M252Y/S254T/T256E)
QCQPDSSTLPPPWSPRPLEATAPTAPQESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITRE
PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 13
Aglyco-FLT3L ECD
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
(S128A/S151A)
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNIARLLQETSEQLVALKPWITRQNFARCLEL
hingeless hG1
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEV
(M252Y/S254T/T256E)
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14 FLT3L ECD (.DELTA.
C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG 5
aa (PTAPQ; SEQ ID
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
NO: 85))-hG4
QCQPDSSTLPPPWSPRPLEATAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTC
(S228P/F234A/L235A/
VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
M252Y/S254T/T256E)
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 15 FLT3L
ECD (.DELTA. C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
10 aa (LEATAPTAPQ;
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
SEQ ID NO: 90))-hG1
QCQPDSSTLPPPWSPRPGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVE
(M252Y/S254T/T256E)
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16 FLT3L ECD (.DELTA. C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
10 aa (LEATAPTAPQ;
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
SEQ ID NO: 90))-hG4
QCQPDSSTLPPPWSPRPESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDV
(S228P/F234A/L235A/
SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
M252Y/S254T/T256E)
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 17 Aglyco-FLT3L
ECD (.DELTA.
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
C-term 10 aa
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNIARLLQETSEQLVALKPWITRQNFARCLEL
(LEATAPTAPQ; SEQ ID
QCQPDSSTLPPPWSPRPESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
NO: 90))
SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS- S
(S128A/S151A)-hG4
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
S228P/F234A/L235A
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 18 Aglyco-FLT3L
ECD (.DELTA.
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
C-term 10 aa
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNIARLLQETSEQLVALKPWITRQNFARCLEL
(LEATAPTAPQ; SEQ ID
QCQPDSSTLPPPWSPRPESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDV
NO: 90))
SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS- S
(S128A/S151A)-hG4
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
(S228P/F234A/L235A/
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
M252Y/S254T/T256E) 19 Murine surrogate
TPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAG
mFLT3L ECD-mG2a
SKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDTCTQLLALKPCIGKACQNFSRCL
Fc
EVQCQPDSSTLLPPRSPIALEATELPEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMI
(L234A/L235A/P329G)
SLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKE
FKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVILTCMVTDFMPEDIYVEWTN
NGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 20
Murine surrogate
TPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAG
mFLT3L ECD (C136S)
SKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDTSTQLLALKPCIGKACQNFSRCL
mG2a Fc
EVQCQPDSSTLLPPRSPIALEATELPEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMI
(L234A/L235A/P329G)
SLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKE
FKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVILTCMVTDFMPEDIYVEWTN
NGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 21
FLT3L ECD-hingeless
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
monoFc
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTKPPSREEMTKNQVSLSCLVKGFYPSDIAVEWESNGQPENNYKTTVPVLDSDGS
FRLASYLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 22 FLT3L ECD (H8Y)-
TQDCSFQYSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
hingeless hG1 Fc
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 23 FLT3L ECD (K84E)-
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
hingeless hG1 Fc
SKMQGLLERVNTEIHFVTECAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 24 FLT3L ECD
TQDCSFQYSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
(H8Y/K84E)
SKMQGLLERVNTEIHFVTECAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
hingeless hG1 Fc
QCQPDSSTLPPPWSPRPLEATAPTAPQGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 25 Aglyco-FLT3L ECD
(.DELTA.
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG
C-term 5 aa (PTAPQ;
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNIARLLQETSEQLVALKPWITRQNFARCLEL
SEQ ID NO: 85))
QCQPDSSTLPPPWSPRPLEATAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTC
(S128A/S151A)-hG4
VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
(S228P/F234A/L235A/
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
M252Y/S254T/T256E)
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26 FLT3L
ECD (.DELTA. C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG 5
aa (PTAPQ; SEQ ID
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
NO: 85))-linker
QCQPDAAALPPPWSPRPLEATAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTC
SST/AAA-hG4
VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
(S228P/F234A/L235A/
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
M252Y/S254T/T256E)
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 27 FLT3L
ECD (.DELTA. C-term
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAG 5
aa (PTAPQ; SEQ ID
SKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLEL
NO: 85))-linker
QCQPDAAALPPPWAPRPLEATAEAKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTC
SST/AAA;
VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN- K
S170A/S180A-hG4
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
(S228P/F234A/L235A/
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
M252Y/S254T/T256E)
Heterodimers and Fusion Proteins Comprising a FLT3L-Fc Fusion
Protein and a Second Polypeptide
[0114] Further provided are fusion proteins comprising (i) a
FLT3L-Fc fusion protein described herein, e.g., having an amino
acid sequence that is at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%,
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 19-20, and (ii) a second polypeptide. In
some embodiments, the second polypeptide comprises a targeting
moiety or domain, a growth factor, a cytokine, a chemokine or a TNF
superfamily (TNFSF) member. In some embodiments, the second
polypeptide is N-terminal to the FLT3L extracellular domain. In
some embodiments, the second polypeptide is C-terminal to the Fc
region. In some embodiments, the second polypeptide is between the
FLT3L extracellular domain and the Fc region. In various
embodiments, the targeting moiety binds to a protein target in
Table B.
[0115] Further provided are heterodimeric molecules comprising (i)
a FLT3L-Fc fusion protein described herein, e.g., having an amino
acid sequence that is at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%,
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 19-20, and (ii) a second polypeptide
fused to a second Fc region. In certain embodiments, the first and
second Fc regions of the heterodimeric molecules are different,
e.g., having complementary "knob (W)-and-hole (S)" amino acid
substitutions at position 366 (EU numbering). In some embodiments,
the second polypeptide comprises a targeting moiety or domain, a
growth factor, a cytokine, a chemokine or a TNF superfamily (TNFSF)
member. In various embodiments, the targeting moiety binds to a
protein target in Table B.
[0116] In some embodiments, the targeting moiety or domain
comprises an antibody fragment (e.g., scFv, sc(Fv).sub.2, Fab,
F(ab).sub.2, Fab', F(ab').sub.2, Facb, and Fv). In some
embodiments, the antibody fragment comprises a Fab or a
single-chain variable fragment (scFv). In some embodiments, both
the first Fc region and the second Fc region do not comprise a
hinge region. In some embodiments, the heterodimer is stabilized by
an interaction between the first Fc region and the second Fc
region. Illustrative interactions that can stabilize the
heterodimer through the Fc region include without limitation
disulfide bonds and complementary amino acid substitutions in the
first and second Fc regions (e.g., knob-in-hole mutations).
[0117] In some embodiments, the targeting moiety or domain
comprises a non-immunoglobulin or antibody mimetic protein.
Examples of non-immunoglobulin or antibody mimetic protein
targeting moieties or domains include without limitation adnectins,
affibody molecules, affilins, affimers, affitins, alphabodies,
anticalins, peptide aptamers, armadillo repeat proteins (ARMs),
atrimers, avimers, designed ankyrin repeat proteins (DARPins.RTM.),
fynomers, knottins, Kunitz domain peptides, monobodies, and
nanoCLAMPs. Non-immunoglobulin or antibody mimetic protein
targeting moieties or domains of use in the herein described
FLT3L-Fc fusion protein heterodimers are described, e.g., in Zhang,
et al., Methods Mol Biol. 2017; 1575:3-13; Ta, et al., Future Med
Chem. 2017 August; 9(12):1301-1304; Yu, et al., Annu Rev Anal Chem
(Palo Alto Calif). 2017 Jun. 12; 10(1):293-320; Baloch, et al.,
Crit Rev Biotechnol. 2016; 36(2):268-75; and Bruce, et al.,
Chembiochem. 2016 Oct. 17; 17(20):1892-1899.
[0118] In some embodiments, the targeting moiety or domain has
T-cell receptor (TCR)-like binding properties, and binds to the
epitope of a target or tumor-associated antigen (TAA) presented in
a major histocompatibility complex (MHC) molecule.
[0119] In some embodiments, the targeting moiety or domain
comprises a binding partner domain, e.g., a soluble or
extracellular domain of the binding partner or ligand of the
protein target or antigen. For example, in some embodiments, the
targeting moiety or domain comprises a binding partner or ligand of
any of the protein or antigen targets listed in Table B. In one
embodiment, the targeting moiety or domain comprises the
extracellular domain of a TGFB1 receptor (e.g., a "TGF beta
trap").
[0120] In homodimers or heterodimer formats of the FLT3L-Fc fusion
proteins, the dimeric molecule comprises first and second Fc
domains. In certain embodiments, amino acid substitutions may be in
one or both of the first and second Fc domains. In certain
embodiments, the one or both of the first and second Fc domains
have one or more (1, 2, 3, 4, or 5) of the following mutations (EU
numbering). In some embodiments, Fc region heterodimerization of
the two different immunoadhesins (Fc fusion proteins) can be
facilitated by so-called `knobs-into-holes` mutations (Atwell et
al. 1997. JMB 270:26-35). The `hole` mutations (T366S, L368A and
Y407V) are incorporated into one Fc-containing chain, the T366W
`knob` mutation is incorporated into the other chain. Knob-and-hole
amino acid substitutions can be incorporated into human IgG1 or
human IgG4 Fc domains. In addition, a C220S mutation can be
incorporated into an IgG1 hinge region of a scFv-containing arm to
eliminate a free cysteine that otherwise forms a disulfide bond
with a corresponding cysteine in the light chain in a wild-type
IgG1. Co-transfection of such constructs leads to preferential
formation of a heterodimeric Fc, with low levels of homodimer
contaminants. Additionally, incorporating a S354C mutation can be
incorporated into the Fc containing the `knob` mutations and a
Y349C mutation into the Fc containing the `hole` mutations can
optionally be used to generate a covalent bond between the two
halves of the heterodimeric Fc if additional thermodynamic
stability is desired (Merchant et al. 1998. Nat. Biotechnol. 16:
677-81). In certain embodiments, R409D, K370E mutations are
introduced in the "knobs chain" and D399K, E357K mutations in the
"hole chain." In other embodiments, Y349C, T366W mutations are
introduced in one of the chains and E356C, T366S, L368A, Y407V
mutations in the counterpart chain. In some embodiments. Y349C,
T366W mutations are introduced in one chain and S354C, T366S,
L368A, Y407V mutations in the counterpart chain. In some
embodiments, Y349C, T366W mutations are introduced in one chain and
S354C, T366S, L368A, Y407V mutations in the counterpart chain. In
yet other embodiments, Y349C, T366W mutations are introduced in one
chain and S354C, T366S, L368A, Y407V mutations in the counterpart
chain (all EU numbering).
[0121] To facilitate purification of the heterodimeric molecule
away from contaminating homodimeric products, the H435R or
H435R+Y436F mutations to reduce or eliminate protein A binding can
be introduced into one but not both of the Fc-containing chains
(Jendeberg, L. et al. 1997 J. Immunol. Methods 201:25-34). This
reduces or eliminates protein A binding of the homodimer
contaminant containing these mutations, and greatly simplifies
purification of the desired heterodimer away from remaining
homodimer contaminant via additional chromatography steps (e.g.,
ion exchange). In embodiments incorporating H435R (or H435R+Y436F)
mutations in the first or second Fc region of a heavy chain, if the
VH region in the same heavy chain is from a VH3 family variable
region, this VH region can also include amino acid substitutions,
as described herein, to reduce or eliminate Protein A binding of
the entire heavy chain.
[0122] Yet another exemplary method of making bispecific antibodies
is by using the Trifunctional Hybrid Antibodies
platform--Triomab.RTM.. This platform employs a chimeric
construction made up of half of two full-length antibodies of
different isotypes, mouse IgG2a and rat IgG2b. This technology
relies on species-preferential heavy/light chain pairing
associations. See, Lindhofer et al., J Immunol., 155:219-225
(1995).
[0123] Yet another method for making bispecific antibodies is the
CrossMab technology. CrossMab are chimeric antibodies constituted
by the halves of two full-length antibodies. For correct chain
pairing, it combines two technologies: (i) the knob-into-hole which
favors a correct pairing between the two heavy chains; and (ii) an
exchange between the heavy and light chains of one of the two Fabs
to introduce an asymmetry which avoids light-chain mispairing. See,
Ridgway et al., Protein Eng., 9:617-621 (1996); Schaefer et al.,
PNAS, 108:11187-11192 (2011). CrossMabs can combine two or more
antigen binding domains for targeting two or more targets or for
introducing bivalency towards one target such as the 2:1
format.
[0124] In some embodiments, the targeting moiety or domain targets
or binds to an effector cell, e.g., engaging or activating a T-cell
or an NK cell. In certain embodiments, the targeting moiety or
domains binds to CD3. In some embodiments, the targeting moiety
binds to CD16. Illustrative proteins and antigens, including
tumor-associated antigens, immune checkpoint proteins and dendritic
cell surface proteins, that can be targeted or bound by the
targeting moiety or domain, include without limitation those listed
in Table B. Target names, symbols (official and alternative) and
Gene IDs identified in Table B are from ncbi.nlm.nih.gov/gene.
TABLE-US-00006 TABLE B Illustrative Antigen/Protein Targets NCBI
Official NCBI Human Alternative Symbols Target Name Symbol Gene ID
No. (also known as) 5'-nucleotidase ecto NT5E 4907 NT; eN; NT5;
NTE; eNT; CD73; E5NT; CALJA ALK receptor tyrosine kinase ALK 238
CD246; NBLST3 Alpha fetoprotein AFP 174 AFPD, FETA, HPAFP B and T
lymphocyte associated BTLA 151888 BTLA1, CD272 cadherin 3 CDH3 1001
CDHP; HJMD; p-cadherin; PCAD carbonic anhydrase 6 CA6 765 CA-VI;
GUSTIN carbonic anhydrase 9 CA9 768 MN; CAIX carcinoembryonic
antigen related cell CEACAM3 1084 CEA; CGM1; W264; W282; CD66D
adhesion molecule 3 carcinoembryonic antigen related cell CEACAM5
1048 CEA; CD66e adhesion molecule 5 carcinoembryonic antigen
related cell CEACAM6 4680 NCA; CEAL; CD66c adhesion molecule 6 C-C
motif chemokine receptor 2 CCR2 729230 CC-CKR-2, CCR-2, CCR2A,
CCR2B, CD192, CKR2, CKR2A, CKR2B, CMKBR2, MCP-1-R C-C motif
chemokine receptor 4 CCR4 1233 CC-CKR-4, CD194, CKR4, CMKBR4,
ChemR13, HGCN: 14099, K5-5 C-C motif chemokine receptor 5 CCR5 1234
CC-CKR-5, CCCKR5, CCR-5, CD195, CKR-5, CKR5, CMKBR5, IDDM22 C-C
motif chemokine receptor 8 CCR8 1237 CC-CKR-8, CCR-8, CDw198,
CKRL1, CMKBR8, CMKBRL2, CY6, GPRCY6, TER1 CD160 molecule CD160
11126 BY55, NK1, NK28 CD19 molecule CD19 930 B4; CVID3 CD1a
molecule CD1A 909 CD1, FCB6, HTA1, R4, T6 CD1c molecule CD1C 911
R7; CD1; CD1A; BDCA1 CD1d molecule CD1D 912 CD1A, R3, R3G1 CD1e
molecule CD1E 913 CD1A, R2 CD22 molecule CD22 933 SIGLEC2; SIGLEC-2
CD226 molecule CD226 10666 DNAM-1, DNAM1, PTA1, TLiSA1 CD24
molecule CD24 100133941 CD24A CD244 molecule CD244 51744 2B4, NAIL,
NKR2B4, Nmrk, SLAMF4 CD27 molecule CD27 939 T14; S152; Tp55;
TNFRSF7; S152. LPFS2 CD207 molecule CD207 50489 CLEC4K CD274
molecule CD274 29126 B7-H; B7H1; PDL1; PD-L1; hPD-L1; PDCD1L1;
PDCD1LG1 CD276 molecule CD276 80381 4Ig-B7-H3, B7-H3, B7H3, B7RP-2
CD28 molecule CD28 940 Tp44 CD33 molecule CD33 945 p67; SIGLEC3;
SIGLEC-3 CD37 molecule CD37 951 GP52-40; TSPAN26 CD38 molecule CD38
952 ADPRC1; ADPRC 1 CD40 ligand CD40LG 959 IGM; IMD3; TRAP; gp39;
CD154; CD40L; HIGM1; T-BAM; TNFSF5; hCD40L CD40 molecule CD40 958
p50; Bp50; CDW40; TNFRSF5 CD44 molecule CD44 960 IN; LHR; MC56;
MDU2; MDU3; MIC4; Pgp1; CDW44; CSPG8; HCELL; HUTCH-I; ECMR-III CD47
molecule CD47 961 IAP, MER6, OA3 CD48 molecule CD48 962 BCM1;
BLAST; hCD48; mCD48; BLAST1; SLAMF2; MEM-102 CD52 molecule CD52
1043 HE5; CDW52; EDDM5 CD70 molecule CD70 970 CD27L; LPFS3; CD27-L;
CD27LG; TNFSF7; TNLG8A CD74 molecule CD74 972 II; p33; DHLAG;
HLADG; Ia-GAMMA CD79a molecule CD79A 973 IGA; MB-1 CD79b molecule
CD79B 974 B29; IGB; AGM6 CD80 molecule CD80 941 B7; BB1; B7-1;
B7.1; LAB7; CD28LG; CD28LG1 CD84 molecule CD84 8832 LY9B; hCD84;
mCD84; SLAMF5 CD86 molecule CD86 942 B70; B7-2; B7.2; LAB72;
CD28LG2 CD96 molecule CD96 10225 TACTILE cell adhesion molecule 1
CADM1 23705 BL2, IGSF4, IGSF4A, NECL2, Necl-2, RA175, ST17, SYNCAM,
TSLC1, sTSLC-1, sgIGSF, synCAM1 chorionic somatomammotropin hormone
1 CSH1 1442 PL; CSA; CS-1; CSMT; GHB3; hCS-1; hCS-A coagulation
factor III, tissue factor F3 2152 TF; TFA; CD142 collagen type IV
alpha 1 chain COL4A1 1282 BSVD, BSVD1, RATOR collagen type IV alpha
2 chain COL4A2 1284 BSVD2, ICH, POREN2 collagen type IV alpha 3
chain COL4A3 1285 ATS2, ATS3 collagen type IV alpha 4 chain COL4A4
1286 ATS2, BFH, CA44 collagen type IV alpha 5 chain COL4A5 1287
ASLN, ATS, ATS1, CA54 collectin subfamily member 10 COLEC10 10584
3MC3; CLL1; CL-34 C-type lectin domain containing 9A CLEC9A 283420
CD370; DNGR1; DNGR-1; UNQ9341 C-type lectin domain family 12 member
A CLEC12A 160364 CLL1; MICL; CD371; CLL-1; DCAL-2 C-type lectin
domain family 4 member C CLEC4C 170482 DLEC; HECL; BDCA2; CD303;
CLECSF7; CLECSF11; PRO34150 C-X-C motif chemokine receptor 1 CXCR1
3577 C-C, C-C-CKR-1, CD128, CD181, CDw128a, CKR-1, CMKAR1, IL8R1,
IL8RA, IL8RBA C-X-C motif chemokine receptor 2 CXCR2 3579 CD182,
CDw128b, CMKAR2, IL8R2, IL8RA, IL8RB C-X-C motif chemokine receptor
3 CXCR3 2833 CD182, CD183, CKR-L2, CMKAR3, GPR9, IP10-R, Mig-R,
MigR C-X-C motif chemokine receptor 4 CXCR4 7852 CD184, D2S201E,
FB22, HM89, HSY3RR, LAP-3, LAP3, LCR1, LESTR, NPY3R, NPYR, NPYRL,
NPYY3R, WHIM, WHIMS cytokine inducible SH2 containing protein CISH
1154 CIS; G18; SOCS; CIS-1; BACTS2 cytotoxic T-lymphocyte
associated protein CTLA4 1493 ALPS5, CD, CD152, CELIAC3, CTLA-4,
GRD4, 4 GSE, IDDM12 delta like canonical Notch ligand 3 DLL3 10683
SCDO1 ectonucleotide pyrophosphatase/ ENPP3 5169 B10; NPP3; PDNP3;
CD203c; PD-IBETA phosphodiesterase 3 ectonucleoside triphosphate
ENTPD1 953 CD39; SPG64; ATPDase; NTPDase-1 diphosphohydrolase 1 EPH
receptor A1 EPHA1 2041 EPH; EPHT; EPHT1 EPH receptor A2 EPHA2 1969
ECK; CTPA; ARCC2; CTPP1; CTRCT6 EPH receptor A4 EPHA4 2043 EK8;
SEK; HEK8; TYRO1 EPH receptor A5 EPHA5 2044 EK7; CEK7; EHK1; HEK7;
EHK-1; TYRO4 EPH receptor A7 EPHA7 2045 EHK3; EK11; EHK-3; HEK11
ephrin A1 EFNA1 1942 B61; EFL1; ECKLG; EPLG1; LERK1; LERK-1;
TNFAIP4 epidermal growth factor receptor, including EGFR (e.g.,
1956 ERBB; HER1; mENA; ERBB1; PIG61; NISBD2 variant III EGFRvIII)
epithelial cell adhesion molecule EPCAM 4072 ESA; KSA; M4S1; MK-1;
DIAR5; EGP-2; EGP40; KS1/4; MIC18; TROP1; EGP314; HNPCC8; TACSTD1
erb-b2 receptor tyrosine kinase 2 ERBB2 2064 NEU; NGL; HER2; TKR1;
CD340; HER-2; MLN 19; HER-2/neu erb-b2 receptor tyrosine kinase 3
ERBB3 2065 ErbB-3, FERLK, HER3, LCCS2, MDA-BF-1, c-erbB- 3,
c-erbB3, erbB3-S, p180-ErbB3, p45-sErbB3, p85- sErbB3 erb-b2
receptor tyrosine kinase 4 ERBB4 2066 ALS19, HER4, p180erbB4 Fc
fragment of IgE receptor Ia FCER1A 2205 FCE1A, FcERI Fc fragment of
IgG receptor IIIa FCGR3A 2214 CD16; FCG3; CD16A; FCGR3; IGFR3;
IMD20; FCR- 10; FCRIII; FCGRIII; FCRIIIA fibroblast activation
protein alpha FAP 2191 DPPIV, FAPA, FAPalpha, SIMP fibroblast
growth factor receptor 1 FGFR1 2260 BFGFR, CD331, CEK, ECCL, FGFBR,
FGFR-1, FLG, FLT-2, FLT2, HBGFR, HH2, HRTFDS, KAL2, N- SAM, OGD,
bFGF-R-1 fibroblast growth factor receptor 2 FGFR2 2263 BEK; JWS;
BBDS; CEK3; CFD1; ECT1; KGFR; TK14; TK25; BFR-1; CD332; K-SAM
fibroblast growth factor receptor 3 FGFR3 2261 ACH, CD333, CEK2,
HSFGFR3EX, JTK4 fms related tyrosine kinase 1 FLT1 2321 FLT, FLT-1,
VEGFR-1, VEGFR1 fms related tyrosine kinase 4 FLT4 2324 FLT-4,
FLT41, LMPH1A, LMPHM1, PCL, VEGFR-3, VEGFR3 folate hydrolase 1
FOLH1 2346 PSM; FGCP; FOLH; GCP2; PSMA; mGCP; GCPII; NAALAD1;
NAALAdase, carboxypeptidase II folate receptor 1 FOLR1 2348 FBP;
FOLR, FR.alpha. galectin 9 LGALS9 3965 HUAT, LGALS9A glypican 3
GPC3 2719 SGB; DGSX; MXR7; SDYS; SGBS; OCI-5; SGBS1; GTR2-2 GPNMB
glycoprotein nmb GPNMB 10457 NMB; HGFIN; PLCA3; osteoactivin
guanylate cyclase 2C GUCY2C 2984 GC-C; STAR; DIAR6; GUC2C; MECIL;
MUCIL hepatitis A virus cellular receptor 2 HAVCR2 84868 TIM3;
CD366; KIM-3; SPTCL; TIMD3; Tim-3; TIMD-3; HAVcr-2 HERV-H
LTR-associating 2 HHLA2 11148 B7-H5, B7-H7, B7H7, B7y
immunoglobulin superfamily member 11 IGSF11 152404 CT119; VSIG3;
Igsf13; BT-IgSF; CXADRL1 inducible T cell costimulator ICOS 29851
AILIM, CD278, CVID1 inducible T cell costimulator ligand ICOSLG
23308 B7-H2, B7H2, B7RP-1, B7RP1, B7h, CD275, GL50, ICOS-L, ICOSL,
LICOS integrin subunit alpha 5 ITGA5 3678 CD49e, FNRA, VLA-5, VLA5A
integrin subunit alpha V ITGAV 3685 CD51, MSK8, VNRA, VTNR integrin
subunit beta 7 ITGB7 3695 interleukin 2 receptor subunit alpha
IL2RA 3559 p55; CD25; IL2R; IMD41; TCGFR; IDDM10 interleukin 3
receptor subunit alpha IL3RA 3563 IL3R; CD123; IL3RX; IL3RY;
IL3RAY; hIL-3Ra killer cell immunoglobulin like receptor, KIR3DL1
3811 CD158E1, KIR, KIR3DL1/S1, NKAT-3, NKAT3, three Ig domains and
long cytoplasmic tail NKB1, NKB1B 1 killer cell immunoglobulin like
receptor, KIR2DL1 3802 CD158A, KIR-K64, KIR221, KIR2DL3, NKAT, two
Ig domains and long cytoplasmic tail 1 NKAT-1, NKAT1, p58.1 killer
cell immunoglobulin like receptor, KIR2DL2 3803 CD158B1, CD158b,
NKAT-6, NKAT6, p58.2 two Ig domains and long cytoplasmic tail 2
killer cell immunoglobulin like receptor, KIR2DL3 3804 p58; NKAT;
GL183; NKAT2; CD158b; KIR2DL; two Ig domains and long cytoplasmic
tail 3 NKAT2A; NKAT2B; CD158B2; KIR-K7b; KIR-K7c; KIR2DS5; KIRCL23;
KIR-023GB killer cell lectin like receptor C1 KLRC1 3821 CD159A,
NKG2, NKG2A killer cell lectin like receptor C2 KLRC2 3822 CD159c,
NKG2-C, NKG2C killer cell lectin like receptor C3 KLRC3 3823 NKG2E;
NKG2-E killer cell lectin like receptor C4 KLRC4 8302 NKG2-F, NKG2F
killer cell lectin like receptor D1 KLRD1 3824 CD94 killer cell
lectin like receptor G1 KLRG1 10219 2F1, CLEC15A, MAFA, MAFA-2F1,
MAFA-L, MAFA-LIKE killer cell lectin like receptor K1 KLRK1 22914
CD314, D12S2489E, KLR, NKG2-D, NKG2D kinase insert domain receptor
KDR 3791 CD309, FLK1, VEGFR, VEGFR2 KIT proto-oncogene, receptor
tyrosine KIT 3815 PBT; SCFR; C-Kit; CD117; MASTC kinase KRAS
proto-oncogene, GTPase KRAS 3845 NS; NS3; CFC2; RALD; K-Ras; KRAS1;
KRAS2; RASK2; KI-RAS; C-K-RAS; K-RAS2A; K-RAS2B; K-RAS4A; K-RAS4B;
c-Ki-ras2 leukocyte immunoglobulin like receptor B1 LILRB1 10859
ILT2; LIR1; MIR7; PIRB; CD85J; ILT-2; LIR-1; MIR- 7; PIR-B
leukocyte immunoglobulin like receptor B2 LILRB2 10288 ILT4; LIR2;
CD85D; ILT-4; LIR-2; MIR10; MIR-10 LY6/PLAUR domain containing 3
LYPD3 27076 C4.4A lymphocyte activating 3 LAG3 3902 CD223
lymphocyte antigen 9 LY9 4063 CD229, SLAMF3, hly9, mLY9 MAGE family
member A1 MAGEA1 4100 CT1.1; MAGE1 MAGE family member A11 MAGEA11
4110 CT1.11; MAGE11; MAGE-11; MAGEA-11 MAGE family member A3 MAGEA3
4102 HIP8; HYPD; CT1.3; MAGE3; MAGEA6 MAGE family member A4 MAGEA4
4103 CT1.4; MAGE4; MAGE4A; MAGE4B; MAGE-41; MAGE-X2 MAGE family
member C1 MAGEC1 9947 CT7; CT7.1 MAGE family member D1 MAGED1 9500
NRAGE; DLXIN-1 MAGE family member D2 MAGED2 10916 11B6; BCG1;
BCG-1; HCA10; BARTS5; MAGE-D2 major histocompatibility complex,
class I, HLA-E 3133 QA1; HLA-6.2 E major histocompatibility
complex, class I, HLA-F 3134 HLAF; CDA12; HLA-5.4; HLA-CDA12 F
major histocompatibility complex, class I, HLA-G 3135 MHC-G G
membrane spanning 4-domains A1 MS4A1 931 B1; S7; Bp35; CD20; CVID5;
MS4A2; LEU-16 Mesothelin MSLN 10232 MPF, SMRP MET proto-oncogene,
receptor tyrosine MET 4233 HGFR; AUTS9; RCCP2; c-Met; DFNB97 kinase
MHC class I polypeptide-related sequence MICA 100507436 MIC-A,
PERB11.1 A MHC class I polypeptide-related sequence MICB 4277
PERB11.2 B mucin 1, cell surface associated, and splice MUC1 4582
ADMCKD, ADMCKD1, CA 15-3, CD227, EMA, variants thereof (e.g,
including MUC1/A, H23AG, KL-6, MAM6, MCD, MCKD, MCKD1, C, D, X, Y,
Z and REP) MUC-1, MUC-1/SEC, MUC-1/X, MUC1/ZD, PEM, PEMT, PUM mucin
16, cell surface associated MUC16 94025 CA125
natural killer cell cytotoxicity receptor 3 NCR3LG1 374383 B7-H6,
B7H6, DKFZp686O24166 ligand 1 necdin, MAGE family member NDN 4692
PWCR; HsT16328 nectin cell adhesion molecule 2 NECTIN2 5819 CD112,
HVEB, PRR2, PVRL2, PVRR2 nectin cell adhesion molecule 4 NECTIN4
81607 EDSS1, LNIR, PRR4, PVRL4, nectin-4 neural cell adhesion
molecule 1 NCAM1 4684 CD56, MSK39, NCAM neurophilin 1 NRP1 8829
NP1; NRP; BDCA4; CD304; VEGF165R Periostin POSTN 10631 OSF-2, OSF2,
PDLPOSTN, PN Poliovirus receptor (PVR) cell adhesion PVR 5817
CD155, HVED, NECL5, Necl-5, PVS, TAGE4 molecule programmed cell
death 1 PDCD1 5133 PD1; PD-1; CD279; SLEB2; hPD-1; hPD-l; hSLE1
programmed cell death 1 ligand 2 PDCD1LG2 80380 B7DC, Btdc, CD273,
PD-L2, PDCD1L2, PDL2, bA574F11.2 prominin 1 PROM1 8842 RP41; AC133;
CD133; MCDR2; STGD4; CORD12; PROML1; MSTP061 promyelocytic leukemia
PML 5371 MYL, PP8675, RNF71, TRIM19 protein tyrosine kinase 7
(inactive) PTK7 5754 CCK-4, CCK4 PVR related immunoglobulin domain
PVRIG 79037 C7orf15, CD112R containing retinoic acid early
transcript 1E RAET1E 135250 LETAL, N2DL-4, NKG2DL4, RAET1E2, RL-4,
ULBP4, bA350J20.7 retinoic acid early transcript 1G RAET1G 353091
ULPB5 retinoic acid early transcript 1L RAET1L 154064 ULPB6
roundabout guidance receptor 4 ROBO4 54538 ECSM4, MRB sialic acid
binding Ig like lectin 9 SIGLEC9 27180 CD329; CDw329; FOAP-9;
siglec-9; OBBP-LIKE sialic acid binding Ig like lectin 10 SIGLEC10
89790 SLG2; PRO940; SIGLEC-10 sialic acid binding Ig like lectin 10
SIGLEC10 89790 SLG2; PRO940; SIGLEC-10 signal regulatory protein
alpha SIRPA 140885 BIT; MFR; P84; SIRP; MYD-1; SHPS1; CD172A;
PTPNS1 signaling lymphocytic activation molecule SLAMF1 6504 SLAM;
CD150; CDw150 family member 1 SLAM family member 6 SLAMF6 114836
CD352, KALI, KALIb, Ly108, NTB-A, NTBA, SF2000 SLAM family member 7
SLAMF7 57823 19A, CD319, CRACC, CS1 SLIT and NTRK like family
member 6 SLITRK6 84189 DFNMYP solute carrier family 34 (sodium
SLC34A2 10568 NPTIIb; NAPI-3B; NAPI-IIb phosphate), member 2 solute
carrier family 39 member 6 SLC39A6 25800 LIV-1, ZIP6 solute carrier
family 44 member 4 SLC44A4 80736 C6orf29, CTL4, DFNA72, NG22, TPPT,
hTPPT1 STEAP family member 1 STEAP1 26872 PRSS24, STEAP syndecan 1
SDC1 6382 SDC; CD138; SYND1; syndecan T cell immunoglobulin and
mucin domain TIMD4 91937 SMUCKLER, TIM4 containing 4 T cell
immunoreceptor with Ig and ITIM TIGIT 201633 VSIG9, VSTM3, WUCAM
domains tenascin C TNC 3371 150-225, DFNA56, GMEM, GP, HXB, JI, TN,
TN-C thrombomodulin THBD 7056 AHUS6, BDCA3, CD141, THPH12, THRM, TM
TNF receptor superfamily member 10a TNFRSF10A 8797 APO2, CD261,
DR4, TRAILR-1, TRAILR1 TNF receptor superfamily member 10b
TNFRSF10B 8795 CD262, DR5, KILLER, KILLER/DR5, TRAIL-R2, TRAILR2,
TRICK2, TRICK2A, TRICK2B, TRICKB, ZTNFR9 TNF receptor superfamily
member 14 TNFRSF14 8764 ATAR, CD270, HVEA, HVEM, LIGHTR, TR2 TNF
receptor superfamily member 17 TNFRSF17 608 BCM, BCMA, CD269,
TNFRSF13A TNF receptor superfamily member 18 TNFRSF18 8784 AITR,
CD357, GITR, GITR-D TNF receptor superfamily member 4 TNFRSF4 7293
OX40; ACT35; CD134; IMD16; TXGP1L TNF receptor superfamily member 8
TNFRSF8 943 CD30; Ki-1; D1S166E TNF receptor superfamily member 9
TNFRSF9 3604 4-1BB, CD137, CDw137, ILA TNF superfamily member 10
TNFSF10 8743 APO2L, Apo-2L, CD253, TL2, TNLG6A, TRAIL TNF
superfamily member 13b TNFSF13B 10673 BAFF, BLYS, CD257, DTL,
TALL-1, TALL1, THANK, TNFSF20, TNLG7A, ZTNF4 TNF superfamily member
14 TNFSF14 8740 CD258, HVEML, LIGHT, LTg TNF superfamily member 18
TNFSF18 8995 AITRL, GITRL, TL6, TNLG2A, hGITRL TNF superfamily
member 4 TNFSF4 7292 CD134L, CD252, GP34, OX-40L, OX4OL, TNLG2B,
TXGP1 TNF superfamily member 8 TNFSF8 944 CD153, CD30L, CD30LG,
TNLG3A TNF superfamily member 9 TNFSF9 8744 4-1BB-L, CD137L, TNLG5A
transferrin TF 7018 HEL-S-71p, PRO1557, PRO2086, TFQTL1
transforming growth factor beta 1 and TGFB1 7040 CED, DPD1,
IBDIMDE, LAP, TGFB, TGFbeta isoforms thereof transmembrane and
immunoglobulin TMIGD2 126259 CD28H, IGPR-1, IGPR1 domain containing
2 trophinin TRO 7216 MAGE-d3, MAGED3 trophoblast glycoprotein TPBG
7162 5T4, 5T4AG, M6P1, WAIF1 tumor associated calcium signal
transducer TACSTD2 4070 EGP-1, EGP1, GA733-1, GA7331, GP50, M1S1, 2
TROP2 UL16 binding protein 1 ULBP1 80329 N2DL-1, NKG2DL1, RAET1I
UL16 binding protein 2 ULBP2 80328 ALCAN-alpha, N2DL2, NKG2DL2,
RAET1H, RAET1L UL16 binding protein 3 ULBP3 79465 N2DL-3, NKG2DL3,
RAET1N V-set domain containing T cell activation VTCN1 79679 B7-H4,
B7H4, B7S1, B7X, B7h.5, PRO1291, VCTN1 inhibitor 1 V-set
immunoregulatory receptor VSIR 64115 B7-H5, B7H5, C10orf54,
DD1alpha, Dies1, GI24, PD- 1H, PP2135, SISP1, VISTA X-C motif
chemokine receptor 1 XCR1 2829 GPR5; CCXCR1
[0125] In some embodiments, the target antigen comprises a
tumor-associated carbohydrate antigen (TACA). Illustrative
carbohydrate antigen targets include, e.g., mucin TACAs, including
truncated glycans Thomsen-nouveau (Tn) (GaNAc.alpha.1-Ser/Thr) and
STn (Neu5Ac.alpha.2,6GalNAc.alpha.1-Ser/Thr), RM2 antigen
hexasaccharide, carbasugars, C-glycosides, gangliosides GM2, GD2
and GD3; globo-H, sialyl Lewis(a), sialyl Lewis(x) and sialyl
Lewis(y) antigens. TACAs are described, e.g., in Sadraei, et al.,
Adv Carbohydr Chem Biochem. (2017) 74:137-237; Sletmoen, et al.,
Glycobiology. (2018) 28(7):437-442; Chuang, et al., J Am Chem Soc.
(2013) 135(30):11140-50; Ragupathi, Cancer Immunol Immunother.
(1996) 43(3):152-7; Ugorski, et al., Acta Biochim Pol. 2002;
49(2):303-11; Takada, et al., Cancer Res. 1993 Jan. 15;
53(2):354-61.
[0126] In some embodiments, the target antigen comprises a
neoantigen presented within a major histocompatibility complex
(MHC) class I or class II molecule. See, e.g., Ott, et al., Nature.
(2017) 547(7662):217-221; Capietto, et al., Curr Opin Immunol.
(2017) 46:58-65; Sun, et al., Cancer Lett. (2017) 392:17-25;
Khodadoust, et al., Nature. (2017) 543(7647):723-727; Kreiter, et
al., Nature. (2015) 520(7549):692-6; Marty, et al., Cell. (2017)
171(6):1272-1283; and Kochin, et al., Oncoimmunology. (2017)
6(4):e1293214 (describing SUV39H2 peptide presented in
HLA-A24).
Conjugated FLT3L-Fc Fusion Proteins
[0127] Any of the FLT3L-Fc fusion proteins, or homodimers or
heterodimers thereof, disclosed herein may be conjugated. FLT3L-Fc
fusion proteins which are bound to various molecules (e.g., labels)
including without limitation macromolecular substances such as
polymers (e.g., polyethylene glycol (PEG), polyethylenimine (PEI)
modified with PEG (PEI-PEG), polyglutamic acid (PGA)
(N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic
acid, radioactive materials (e.g., .sup.90Y, .sup.131I, .sup.125I,
.sup.35S, .sup.3H, .sup.121In, .sup.99Tc), fluorescent substances
(e.g., fluorescein and rhodamine), fluorescent proteins,
luminescent substances (e.g., luminol), Qdots, haptens, enzymes
(e.g., glucose oxidase), metal chelates, biotin, avidin, and
drugs.
[0128] The above-described conjugated FLT3L-Fc fusion proteins can
be prepared according to known methods, e.g., performing chemical
modifications on the FLT3L-Fc fusion proteins described herein. In
certain embodiments, the labelling moiety or therapeutic moiety is
conjugated to the Fc portion of the fusion protein. Methods for
modifying antibody Fc regions are well known in the art (e.g., U.S.
Pat. Nos. 5,057,313 and 5,156,840).
[0129] In some embodiments, the FLT3L-Fc fusion protein, or
homodimer or heterodimer thereof, is conjugated to a drug or
therapeutic agent. In various embodiments, the drug is a small
organic compound or an inhibitory nucleic acid, e.g., a
short-inhibitory RNA (siRNA), a microRNA (miRNA). In some
embodiments, the drug or therapeutic agent is an anti-neoplastic
agent or a chemotherapeutic agent, as known in the art and
described herein. In a particular embodiment, the drug or
therapeutic agent is selected from the group consisting of
monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), a
calicheamicin, ansamitocin, maytansine or an analog thereof (e.g.,
mertansine/emtansine (DM1), ravtansine/soravtansine (DM4)), an
anthracyline (e.g., doxorubicin, daunorubicin, epirubicin,
idarubicin), pyrrolobenzodiazepine (PBD) DNA cross-linking agent
SC-DR002 (D6.5), duocarmycin, a microtubule inhibitors (MTI) (e.g.,
a taxane, a vinca alkaloid, an epothilone), a pyrrolobenzodiazepine
(PBD) or dimer thereof, and a duocarmycin (A, B, B2, C1, C2, D, SA,
CC-1065).
3. Polynucleotides Encoding FLT3L-Fc Fusion Proteins
[0130] Provided are polynucleotides encoding the FLT3L-Fc fusion
proteins, described herein, vectors comprising such
polynucleotides, and host cells (e.g., human cells, mammalian
cells, yeast cells, plant cells, insect cells, bacterial cells,
e.g., E. coli) comprising such polynucleotides or expression
vectors. Provided herein are polynucleotides comprising nucleotide
sequence(s) encoding any of the FLT3L-Fc fusion proteins provided
herein, as well as expression cassettes and vector(s) comprising
such polynucleotide sequences, e.g., expression vectors for their
efficient expression in host cells, e.g., mammalian cells. In
various embodiments, the polynucleotide is a DNA, a cDNA, or an
mRNA.
[0131] The terms "polynucleotide" and "nucleic acid molecule"
interchangeably refer to a polymeric form of nucleotides and
includes both sense and anti-sense strands of RNA, cDNA, genomic
DNA, and synthetic forms and mixed polymers of the above. As used
herein, the term nucleic acid molecule may be interchangeable with
the term polynucleotide. In some embodiments, a nucleotide refers
to a ribonucleotide, deoxynucleotide or a modified form of either
type of nucleotide, and combinations thereof. The terms also
include without limitation, single- and double-stranded forms of
DNA. In addition, a polynucleotide, e.g., a cDNA or mRNA, may
include either or both naturally occurring and modified nucleotides
linked together by naturally occurring and/or non-naturally
occurring nucleotide linkages. The nucleic acid molecules may be
modified chemically or biochemically or may contain non-natural or
derivatized nucleotide bases, as will be readily appreciated by
those of skill in the art. Such modifications include, for example,
labels, methylation, substitution of one or more of the naturally
occurring nucleotides with an analogue, internucleotide
modifications such as uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoramidates, carbamates,
etc.), charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),
intercalators (e.g., acridine, psoralen, etc.), chelators,
alkylators, and modified linkages (e.g., alpha anomeric nucleic
acids, etc.). The above term is also intended to include any
topological conformation, including single-stranded,
double-stranded, partially duplexed, triplex, hairpinned, circular
and padlocked conformations. A reference to a nucleic acid sequence
encompasses its complement unless otherwise specified. Thus, a
reference to a nucleic acid molecule having a particular sequence
should be understood to encompass its complementary strand, with
its complementary sequence. The term also includes codon-biased
polynucleotides for improved expression in a desired host cell.
[0132] A "substitution," as used herein, denotes the replacement of
one or more amino acids or nucleotides by different amino acids or
nucleotides, respectively.
[0133] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location. "Isolated nucleic acid
encoding an FLT3L-Fc fusion protein" refers to one or more nucleic
acid molecules encoding first antigen binding domain, and
optionally second antigen binding domain, antibody heavy and light
chains (or fragments thereof), including such nucleic acid
molecule(s) in a single vector or separate vectors, and such
nucleic acid molecule(s) present at one or more locations in a host
cell.
[0134] An "isolated" polypeptide, such as an isolated FLT3L-Fc
fusion protein provided herein, is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In some embodiments, the
isolated polypeptide will be purified (1) to greater than 95% by
weight of polypeptide as determined by the Lowry method, for
example, more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or silver stain. Isolated polypeptide includes the
polypeptide in situ within recombinant cells since at least one
component of the antibody's natural environment will not be
present. Ordinarily, however, isolated polypeptide will be prepared
by at least one purification step.
[0135] A "polynucleotide variant," as the term is used herein, is a
polynucleotide that typically differs from a polynucleotide
specifically disclosed herein in one or more substitutions,
deletions, additions and/or insertions. Such variants may be
naturally occurring or may be synthetically generated, for example,
by modifying one or more of the polynucleotide sequences described
herein and evaluating one or more biological activities of the
encoded polypeptide as described herein and/or using any of a
number of techniques well known in the art.
[0136] In some embodiments, the nucleic acid molecule is
codon-biased to enhance expression in a desired host cell, e.g., in
human cells, mammalian cells, yeast cells, plant cells, insect
cells, or bacterial cells, e.g., E. coli cells. Accordingly,
provided are polynucleotides encoding a FLT3L-Fc fusion protein
wherein the polynucleotides are codon-biased, comprise replacement
heterologous signal sequences, and/or have mRNA instability
elements eliminated. Methods to generate codon-biased nucleic acids
can be carried out by adapting the methods described in, e.g., U.S.
Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and
6,794,498. Preferred codon usage for expression of the FLT3L-Fc
fusion proteins in desired host cells is provided, e.g., at
kazusa.or.jp/codon/; and
genscript.com/tools/codon-frequency-table.
[0137] In some embodiments, the polynucleotide encoding a FLT3L-Fc
fusion protein, as described herein, has at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% identical, or 100% identical to an nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 28-70,
as provided in Table C.
[0138] As appropriate, in certain embodiments, the 3'-end of the
polynucleotide encoding the FLT3L-Fc fusion protein comprises
multiple tandem stop codons, e.g., two or more tandem TAG
("amber"), TAA ("ochre") or TGA ("opal" or "umber") stop codons.
The multiple tandem stop codons can be the same or different.
TABLE-US-00007 TABLE C Polynucleotides encoding FLT3L-Fc fusion
proteins POLYNUCL. NO. SEQ ID NO: Features Polynucleotide Sequence
28 FLT3L ECD-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC hingeless hG1
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGT-
GAAC
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGGCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC-
CGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG-
ACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT-
CCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC-
CCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG-
AGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG-
GGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC-
TACA
GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC-
TGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 29 FLT3L ECD-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC hingeless hG1
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGACT-
GGT
GTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAAAGAGT-
GAAC
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCT
CCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCCGGCAGAACTTCTCTC-
GGTG
TCTGGAACTGCAGTGTCAGCCCGACTCTTCTACCCTGCCTCCACCTTGGAGCCCCAGACCTTTGGAAGCTAC-
CGCT
CCAACAGCTCCTCAAGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCT-
CGGA
CCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGG-
ACGG
CGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGT-
GCTG
ACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCT-
CCTA
TCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCACCTAGCCGGG-
AAGA
GATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCCTCTGATATCGCCGTGGAATG-
GGAG
AGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTG-
TACT
CCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCTC-
TGCA CAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAA 30 FLT3L ECD
(.DELTA. C-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC term 5 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGAC-
TGTGGCGACTGGT (PTAPQ; SEQ ID
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC NO: 85)-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGC-
AGACCAACATCA hingeless hG1
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCCG-
GTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC-
ACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC-
ATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA-
CCAG
GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC-
ATCT
CCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA-
ACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA-
GCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC-
GTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA-
CGCA GAAGAGCCTCTCCCTGTCTCCGGGTAAA 31 FLT3L ECD (.DELTA. C-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC term 5 aa
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGAC-
TGTGGCGACTGGT (PTAPQ; SEQ ID
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC NO: 85)-
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGC-
AGACCAACATCT hingeless hG1
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTCG-
GTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAAGCTAC-
AGCT
GGCGGCCCAAGCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTG-
ACCT
GCGTGGTGGTGGATGTGTCTCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGC-
ACAA
CGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCA-
CCAG
GATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACC-
ATCT
CCAAGGCCAAGGGCCAGCCTAGGGAACCTCAGGTTTACACCCTGCCACCTAGCCGGGAAGAGATGACCAAAA-
ACCA
GGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCA-
GCCT
GAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACA-
GTGG
ACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCCTGCACAACCACTACA-
CCCA GAAGTCCCTGTCTCTGTCCCCTGGCAAA 32 FLT3L ECD-hG4
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC S228P/L235E
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGT-
GAAC
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGAATCTAAGTACGGCCCTCCCTGCCCTCCTTGCCCAGCCCCTGAATTTGAGGGCGGA-
CCCT
CCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGG-
TGGT
GGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGG-
CCAA
GGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTC-
CCTG
ACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAAC-
AACT
ACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGA-
GCCG
GTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 33 FLT3L ECD-hG4
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC S228P/L235E
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGT-
GAAC
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCT
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTC-
GGTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAGGCTAC-
AGCT
CCTACCGCTCCTCAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAATTTGAAGGCGGC-
CCAA
GCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGG-
TGGT
GGATGTGTCTCAAGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGG-
CCAA
GGGCCAGCCAAGAGAACCTCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTC-
CCTG
ACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAAC-
AACT
ACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGT-
CTAG
ATGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 34 FLT3L ECD-hG4
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC S228P/F234A/
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT L235A
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAA-
AGAGTGAAC
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGC-
CCTT
CCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGG-
TGGT
GGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGG-
CCAA
GGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTC-
CCTG
ACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAAC-
AACT
ACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGT-
CCAG
ATGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 35 FLT3L ECD-hG4
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC S228P/F234A/
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT L235A
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAA-
AGAGTGAAC
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCT
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTC-
GGTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAGGCTAC-
AGCT
CCTACCGCTCCTCAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGA-
CCAA
GCGTTTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGG-
TGGT
GGATGTGTCTCAAGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGG-
CCAA
GGGCCAGCCAAGAGAACCTCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTC-
CCTG
ACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAAC-
AACT
ACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGT-
CTAG
ATGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 36 Aglyco-FLT3L
ATGACAGTTTTGGCTCCAGCTTGGTCCCCTACAACCTACCTGCTGCTGCTGTTGCTGCTCTCCTCTGGCCTGT-
CTG (S128A/S151A)
GCACCCAGGACTGTTCCTTCCAGCACTCCCCTATCTCCAGCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGA-
CTA hingeless hG1
TCTGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGA-
CTG
GTGTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAAAGA-
GTGA
ACACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCA-
ATAT
CGCCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCAGACAGAACTTCGC-
CCGG
TGTCTGGAACTGCAGTGTCAGCCTGACAGCTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTCTGGAAGCT-
ACCG
CTCCAACCGCTCCTCAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT-
CCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT-
GGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC-
GTCC
TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG-
CCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG-
GGAG
GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG-
TGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC-
TCTA
CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC-
TCTG CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 37 Aglyco-FLT3L
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC (S128A/S151A)
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT hingeless hG1
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAT-
ATCG
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCGCCC-
GGTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAGGCTAC-
AGCT
CCTACTGCTCCTCAAGGCGGCCCAAGCGTTTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCT-
CGGA
CCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGG-
ACGG
CGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGT-
GCTG
ACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCT-
CCTA
TCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCTCAGGTTTACACCCTGCCACCTAGCCGGG-
AAGA
GATGACCAAAAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATG-
GGAG
TCTAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTG-
TACT
CCAAGCTGACAGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCC-
TGCA CAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAA 38 FLT3L (.DELTA.
C-term
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC 5 aa (PTAPQ; SEQ
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT ID NO: 85))-hG4
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC S228P/F234A/
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA L235A
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTT-
CAGCCGGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
GAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTG-
TTCC
CTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCC-
AAGA
GGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGA-
GGAA
CAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAG-
TACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTA-
GGGA
ACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGT-
CAAG
GGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACA-
CCTC
CTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGATGGCAAGAGG-
GCAA
CGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCT-
GGGC AAA 39 FLT3L (.DELTA. C-term
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC 5 aa (PTAPQ; SEQ
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT ID NO: 85))-hG4
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC S228P/F234A/
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT L235A
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTT-
CTCTCGGTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTCTGGAAGCTAC-
CGCC
GAGTCTAAGTACGGACCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGACCAAGCGTTTTCCTG-
TTTC
CTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTC-
AAGA
GGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGA-
GGAA
CAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAG-
TACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCAA-
GAGA
ACCTCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGT-
CAAG
GGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACA-
CCTC
CTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGTCTAGATGGCAAGAGG-
GCAA
CGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCT-
GGGC AAA 40 FLT3L ECD (.DELTA. C-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC term 10 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA hingeless hG1
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCCG-
GTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTGGGGGACCGTC-
AGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG-
GACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA-
AGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA-
TGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA-
GGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGA-
CCTG
CCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA-
CAAG
ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGG-
TGGC
AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT-
CCCT GTCTCCGGGTAAA 41 FLT3L ECD (.DELTA. C-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC term 10 aa
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT hingeless hG1
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTCG-
GTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGTCCTAGACCTGGCGGACCAAG-
CGTG
TTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTG-
GATG
TGTCTCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCA-
AGCC
TAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAA-
CGGC
AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAG-
GGCC
AGCCTAGGGAACCTCAGGTTTACACCCTGCCACCTAGCCGGGAAGAGATGACCAAAAACCAGGTGTCCCTGA-
CCTG
CCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAACAACTA-
CAAG
ACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCTCGG-
TGGC
AGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGT-
CTCT GTCCCCTGGCAAA 42 FLT3L ECD (A C-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC term 10 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA hG4 S228P/
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCCG-
GTG F234A/L235A
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTGAGTCTAAGTAC-
GGC
CCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCT-
AAGG
ACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGG-
TGCA
GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTC-
CACC
TACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG-
TCCA
ACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTT-
ACAC
CCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCC-
TTCC
GATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGAC-
TCCG
ACGGCTCCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGATGGCAAGAGGGCAACGTGTTCTCCT-
GCTC
CGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAA 43
FLT3L ECD (A C-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC term 10 aa
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT hG4 S228P/
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTCG-
GTG F234A/L235A
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTCGGCCTGAATCTAAGTAT-
GGC
CCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGACCAAGCGTTTTCCTGTTTCCTCCAAAGCCT-
AAGG
ACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCAAGAGGACCCCGAGG-
TGCA
GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTC-
CACC
TACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG-
TCCA
ACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCAAGAGAACCTCAGGTGT-
ACAC
ACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCC-
ATCC
GATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGAC-
TCCG
ACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGTCTAGATGGCAAGAGGGCAACGTGTTCTCCT-
GCTC
TGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAA 44
FLT3L ECD-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC hingeless hG1
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (M252Y/S254T/
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC T256E)
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAG-
ACCAACATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGGCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCTACATCACC-
CGGG
AACCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG-
ACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT-
CCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC-
CCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG-
AGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG-
GGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC-
TACA
GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC-
TGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 45 FLT3L ECD-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC hingeless hG1
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (M252Y/S254T/
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC T256E)
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAG-
ACCAACATCT
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTC-
GGTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAGGCTAC-
AGCT
CCTACTGCTCCTCAAGGCGGCCCAAGCGTTTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACC-
CGCG
AGCCTGAAGTGACATGCGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGG-
ACGG
CGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGT-
GCTG
ACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCT-
CCTA
TCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCTCAGGTTTACACCCTGCCACCTAGCCGGG-
AAGA
GATGACCAAAAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATG-
GGAG
TCTAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTG-
TACT
CCAAGCTGACAGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCC-
TGCA CAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAA 46 FLT3L ECD
(.DELTA. C-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC term 5 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGAC-
TGTGGCGACTGGT (PTAPQ; SEQ ID
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC NO: 85))-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTG-
CAGACCAACATCA hingeless hG1
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCCG-
GTG (M252Y/S254T/
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCACA-
GCT T256E)
GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCTACATCACCCGGGAACCT-
GAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC-
ATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA-
CCAG
GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC-
ATCT
CCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA-
ACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA-
GCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC-
GTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA-
CGCA GAAGAGCCTCTCCCTGTCTCCGGGTAAA 47 FLT3L ECD (A C-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC term 5 aa
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGAC-
TGTGGCGACTGGT (PTAPQ; SEQ ID
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC NO: 85))-
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTG-
CAGACCAACATCT hingeless hG1
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTCG-
GTG (M252Y/S254T/
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAAGCTACA-
GCT T256E)
GGCGGCCCAAGCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCT-
GAAGTGACAT
GCGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGC-
ACAA
CGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCA-
CCAG
GATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACC-
ATCT
CCAAGGCCAAGGGCCAGCCTAGGGAACCTCAGGTTTACACCCTGCCACCTAGCCGGGAAGAGATGACCAAAA-
ACCA
GGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCA-
GCCT
GAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACA-
GTGG
ACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCCTGCACAACCACTACA-
CCCA GAAGTCCCTGTCTCTGTCCCCTGGCAAA 48 FLT3L ECD-hG4
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC S228P/L235E/
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT M252Y/
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGA-
AAGAGTGAAC S254T/T256E)
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGAATCTAAGTACGGCCCTCCCTGCCCTCCTTGCCCAGCCCCTGAATTTGAGGGCGGA-
CCCT
CCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGTACATCACCCGGGAACCCGAAGTGACCTGCGTGG-
TGGT
GGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGG-
CCAA
GGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTC-
CCTG
ACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAAC-
AACT
ACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGA-
GCCG
GTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 49 FLT3L ECD-hG4
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC S228P/L235E/
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT M252Y/
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGA-
AAGAGTGAAC S254T/T256E)
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTC-
GGTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAGGCTAC-
AGCT
CCTACCGCTCCTCAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAATTTGAAGGCGGC-
CCAA
GCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACATGCGTGG-
TGGT
GGATGTGTCCCAAGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGG-
CCAA
GGGCCAGCCAAGAGAACCTCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTC-
CCTG
ACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAAC-
AACT
ACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGT-
CTAG
ATGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 50 FLT3L ECD-hG4
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC (S228P/F234A/
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT L235A/M252Y/
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC S254T/T256E)
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGC-
CCTT
CCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGGGAACCCGAAGTGACCTGCGTGG-
TGGT
GGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGG-
CCAA
GGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTC-
CCTG
ACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAAC-
AACT
ACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGA-
GCCG
GTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 51 FLT3L ECD-hG4
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC (S228P/F234A/
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT L235A/M252Y/
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC S254T/T256E)
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTC-
GGTG
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGAGGCTAC-
AGCT
CCTACCGCTCCTCAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGA-
CCAA
GCGTTTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACATGCGTGG-
TGGT
GGATGTGTCCCAAGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA-
GACC
AAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG-
CTGA
ACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGG-
CCAA
GGGCCAGCCAAGAGAACCTCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTC-
CCTG
ACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAAC-
AACT
ACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGT-
CTAG
ATGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC-
CCTG TCTCTGTCCCTGGGCAAA 52 Aglyco-FLT3L
ATGACAGTTTTGGCTCCAGCTTGGTCCCCTACAACCTACCTGCTGCTGCTGTTGCTGCTCTCCTCTGGCCTGT-
CTG ECD
GCACCCAGGACTGTTCCTTCCAGCACTCCCCTATCTCCAGCGACTTCGCCGTGAAGATCAGAGAGCTGT-
CCGACTA (S128A/S151A)
TCTGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGA-
CTG hingeless hG1
GTGTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAAAGAG-
TGA (M252Y/S254T/
ACACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAA-
TAT T256E)
CGCCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCAGACAGAA-
CTTCGCCCGG
TGTCTGGAACTGCAGTGTCAGCCTGACAGCTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTCTGGAAGCT-
ACCG
CTCCAACCGCTCCTCAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCTACATCA-
CCCG
GGAACCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT-
GGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC-
GTCC
TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG-
CCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG-
GGAG
GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG-
TGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC-
TCTA
CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC-
TCTG CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 53 Aglyco-FLT3L
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC ECD
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGC-
GACTGGT (S128A/S151A)
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC hingeless hG1
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAATA-
TCG (M252Y/S254T/
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCGCCCG-
GTG T256E)
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTTTGGA-
GGCTACAGCT
CCTACTGCTCCTCAAGGCGGCCCAAGCGTTTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACC-
CGCG
AGCCTGAAGTGACATGCGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGG-
ACGG
CGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGT-
GCTG
ACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCT-
CCTA
TCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCTCAGGTTTACACCCTGCCACCTAGCCGGG-
AAGA
GATGACCAAAAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATG-
GGAG
TCTAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTG-
TACT
CCAAGCTGACAGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCC-
TGCA CAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAA 54 FLT3L ECD (A C-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC term 5 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGAC-
TGTGGCGACTGGT (PTAPQ; SEQ ID
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC NO: 85))-hG4
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA (S228P/F234A/
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCCG-
GTG L235A/M252Y/
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCACA-
GCT S254T/T256E)
GAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGT-
TCC
CTCCAAAGCCCAAGGACACCCTGTACATCACCCGGGAACCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCC-
AGGA
AGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGA-
GGAA
CAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAG-
TACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCC-
GCGA
ACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGT-
GAAA
GGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC-
CCCC
CTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAG-
GCAA
CGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCT-
GGGC AAA 55 FLT3L ECD (.DELTA. C-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC term 5 aa
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGAC-
TGTGGCGACTGGT (PTAPQ; SEQ ID
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC NO: 85))-hG4
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT (S228P/F234A/
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCTCTCG-
GTG L235A/M252Y/
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTCTGGAAGCTACC-
GCC S254T/T256E)
GAGTCTAAGTACGGACCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGACCAAGCGTTTTCCTGT-
TTC
CTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACATGCGTGGTGGTGGATGTGTCCC-
AAGA
GGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGA-
GGAA
CAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAG-
TACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCAA-
GAGA
ACCTCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGT-
CAAG
GGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACA-
CCTC
CTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGTCTAGATGGCAAGAGG-
GCAA
CGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCT-
GGGC AAA 56 FLT3L ECD (.DELTA. C-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC term 10 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA hG1
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCA-
GCCGGTG (M252Y/S254T/
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTGGGGGACCGTCA-
GTC T256E)
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCTACATCACCCGGGAACCTGAGGTCACATGCGTG-
GTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA-
AGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA-
TGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA-
GGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGA-
CCTG
CCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA-
CAAG
ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGG-
TGGC
AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT-
CCCT GTCTCCGGGTAAA 57 FLT3L ECD (A C-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC term 10 aa
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT hG1
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCT-
CTCGGTG (M252Y/S254T/
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGTCCTAGACCTGGCGGACCAAGC-
GTG T256E)
TTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACATGCGTG-
GTGGTGGATG
TGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCA-
AGCC
TAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAA-
CGGC
AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAG-
GGCC
AGCCTAGGGAACCTCAGGTTTACACCCTGCCACCTAGCCGGGAAGAGATGACCAAAAACCAGGTGTCCCTGA-
CCTG
CCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAACAACTA-
CAAG
ACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCTCGG-
TGGC
AGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGT-
CTCT GTCCCCTGGCAAA 58 FLT3L ECD (.DELTA. C-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC term 10 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCA hG4
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCA-
GCCGGTG (S228P/F234A/
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTGAGTCTAAGTAC-
GGC L235A/M252Y/
CCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTA-
AGG S254T/T256E)
ACACCCTGTACATCACCCGGGAACCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGT-
GCA
GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAG-
CACC
TACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG-
TCCA
ACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGT-
ACAC
ACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCC-
CTCC
GATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC-
AGCG
ACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTGTTCAGCT-
GCAG
CGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAA 59
FLT3L ECD (.DELTA. C-
ACCCAGGACTGCTCCTTCCAGCACTCCCCTATCTCTTCCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ACC term 10 aa
TGCTGCAGGACTATCCTGTGACCGTGGCCAGCAACCTGCAGGATGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT (LEATAPTAPQ;
GCTGGCTCAGAGATGGATGGAAAGACTGAAAACCGTGGCCGGCTCCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC SEQ ID NO: 90))-
ACAGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT hG4
CCCGGCTGCTGCAAGAGACATCTGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCCGGCAGAACTTCT-
CTCGGTG (S228P/F234A/
CCTGGAACTGCAGTGTCAGCCTGATTCTTCTACCCTGCCTCCACCTTGGAGCCCTCGGCCTGAATCTAAGTAT-
GGC L235A/M252Y/
CCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGACCAAGCGTTTTCCTGTTTCCTCCAAAGCCTA-
AGG S254T/T256E)
ACACCCTGTACATCACCCGCGAGCCTGAAGTGACATGCGTGGTGGTGGATGTGTCCCAAGAGGACCCCGAGGT-
GCA
GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTC-
CACC
TACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG-
TCCA
ACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCAAGAGAACCTCAGGTGT-
ACAC
ACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCC-
ATCC
GATATCGCCGTGGAATGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGAC-
TCCG
ACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGTCTAGATGGCAAGAGGGCAACGTGTTCTCCT-
GCTC
TGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAA 60
Aglyco-FLT3L
ACCCAGGACTGTTCCTTCCAGCACTCCCCTATCTCCAGCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ATC ECD (.DELTA. C-term
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGACT-
GGT 10 aa
GTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAA-
AGAGTGAAC (LEATAPTAPQ;
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAATA-
TCG SEQ ID NO: 90))
CCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCAGACAGAACTTCGCCCG-
GTG (S128A/S151A)-
TCTGGAACTGCAGTGTCAGCCTGACAGCTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTGAGTCTAAGTAC-
GGC hG4
CCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAG-
CCTAAGG S228P/F234A/
ACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGT-
GCA L235A
GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTC-
AACTCCACC
TACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG-
TCCA
ACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTT-
ACAC
CCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCC-
TTCC
GATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGAC-
TCCG
ACGGCTCCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGATGGCAAGAGGGCAACGTGTTCTCCT-
GCTC
CGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAA 61
Aglyco-FLT3L
ACCCAGGACTGTTCCTTCCAGCACTCCCCTATCTCCAGCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ATC ECD (.DELTA. C-term
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGACT-
GGT 10 aa
GTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAA-
AGAGTGAAC (LEATAPTAPQ;
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAATA-
TCG SEQ ID NO: 90))
CCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCAGACAGAACTTCGCCCG-
GTG (S128A/S151A)-
TCTGGAACTGCAGTGTCAGCCTGACAGCTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTGAGTCTAAGTAC-
GGC hG4
CCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAG-
CCCAAGG (S228P/F234A/
ACACCCTGTACATCACCCGGGAACCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGT-
GCA L235A/M252Y/
GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGC-
ACC S254T/T256E)
TACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGT-
CCA
ACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGT-
ACAC
ACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCC-
CTCC
GATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC-
AGCG
ACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTGTTCAGCT-
GCAG
CGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAA 62
Murine
ACCCCTGACTGCTACTTCAGCCACTCTCCTATCTCCAGCAACTTCAAAGTGAAGTTCCGCGAGC-
TGACCGACCATC surrogate
TGCTGAAGGACTATCCTGTGACCGTGGCCGTGAACCTGCAGGACGAAAAGCACTGCAAGGCCC-
TGTGGTCCCTGTT mFLT3L ECD-
TCTGGCCCAGAGATGGATCGAGCAGCTGAAAACCGTGGCTGGCTCCAAGATGCAGACCCTGCTGGAAGATGTG-
AAC mG2a Fc
ACCGAGATCCACTTCGTGACCAGCTGCACCTTCCAGCCTCTGCCTGAGTGCCTGAGATTCGTGCA-
GACCAACATCT (L234A/L235A/
CCCACCTGTTGAAGGACACATGCACCCAGCTGCTGGCCCTGAAGCCTTGTATCGGCAAGGCCTGCCAGAACTT-
CTC P329G)
CCGGTGTCTGGAAGTGCAGTGCCAGCCTGACTCCTCCACACTGCTGCCACCTAGAAGCCCTATCGC-
TCTGGAAGCT
ACCGAGCTGCCTGAGCCTAGAGGCCCTACCATCAAGCCTTGTCCTCCATGCAAGTGCCCCGCTCCTAATGCT-
GCTG
GTGGCCCTTCCGTGTTCATCTTCCCACCTAAGATCAAGGACGTGCTGATGATCTCCCTGTCTCCTATCGTGA-
CCTG
CGTGGTGGTGGACGTGTCCGAGGATGATCCTGACGTGCAGATCAGTTGGTTCGTGAACAACGTGGAAGTGCA-
CACC
GCTCAGACCCAGACACACAGAGAGGACTACAACAGCACCCTGAGAGTGGTGTCTGCCCTGCCTATCCAGCAC-
CAGG
ATTGGATGTCCGGCAAAGAATTCAAGTGCAAAGTGAACAACAAGGACCTGGGCGCTCCCATCGAGCGGACCA-
TCTC
TAAGCCTAAGGGATCCGTCAGAGCCCCTCAGGTGTACGTTCTGCCTCCACCTGAGGAAGAGATGACCAAGAA-
ACAA
GTGACCCTGACCTGCATGGTCACCGACTTCATGCCCGAGGACATCTACGTGGAATGGACCAACAACGGCAAG-
ACCG
AGCTGAACTACAAGAACACCGAGCCTGTGCTGGACTCCGACGGCTCCTACTTCATGTACTCCAAGCTGCGCG-
TCGA
GAAGAAGAACTGGGTCGAGAGAAACTCCTACTCCTGCTCCGTGGTGCACGAGGGCCTGCACAATCACCACAC-
CACC AAGTCCTTCTCTCGGACCCCTGGCAAA 63 Murine
ACCCCTGACTGCTACTTCAGCCACTCTCCTATCTCCAGCAACTTCAAAGTGAAGTTCCGCGAGC-
TGACCGACCATC surrogate
TGCTGAAGGACTATCCTGTGACCGTGGCCGTGAACCTGCAGGACGAAAAGCACTGCAAGGCCC-
TGTGGTCCCTGTT mFLT3L ECD
TCTGGCCCAGAGATGGATCGAGCAGCTGAAAACCGTGGCTGGCTCCAAGATGCAGACCCTGCTGGAAGATGTG-
AAC (C136S) mG2a Fc
ACCGAGATCCACTTCGTGACCAGCTGCACCTTCCAGCCTCTGCCTGAGTGCCTGAGATTCGTGCAGACCAACA-
TCT (L234A/L235A/
CCCACCTGTTGAAGGACACATCCACCCAGCTGCTGGCCCTGAAGCCTTGTATCGGCAAGGCCTGCCAGAACTT-
CTC P329G)
CCGGTGTCTGGAAGTGCAGTGCCAGCCTGACTCCTCCACACTGCTGCCACCTAGAAGCCCTATCGC-
TCTGGAAGCT
ACCGAGCTGCCTGAGCCTAGAGGCCCTACCATCAAGCCTTGTCCTCCATGCAAGTGCCCCGCTCCTAATGCT-
GCTG
GTGGCCCTTCCGTGTTCATCTTCCCACCTAAGATCAAGGACGTGCTGATGATCTCCCTGTCTCCTATCGTGA-
CCTG
CGTGGTGGTGGACGTGTCCGAGGATGATCCTGACGTGCAGATCAGTTGGTTCGTGAACAACGTGGAAGTGCA-
CACC
GCTCAGACCCAGACACACAGAGAGGACTACAACAGCACCCTGAGAGTGGTGTCTGCCCTGCCTATCCAGCAC-
CAGG
ATTGGATGTCCGGCAAAGAATTCAAGTGCAAAGTGAACAACAAGGACCTGGGCGCTCCCATCGAGCGGACCA-
TCTC
TAAGCCTAAGGGATCCGTCAGAGCCCCTCAGGTGTACGTTCTGCCTCCACCTGAGGAAGAGATGACCAAGAA-
ACAA
GTGACCCTGACCTGCATGGTCACCGACTTCATGCCCGAGGACATCTACGTGGAATGGACCAACAACGGCAAG-
ACCG
AGCTGAACTACAAGAACACCGAGCCTGTGCTGGACTCCGACGGCTCCTACTTCATGTACTCCAAGCTGCGCG-
TCGA
GAAGAAGAACTGGGTCGAGAGAAACTCCTACTCCTGCTCCGTGGTGCACGAGGGCCTGCACAATCACCACAC-
CACC AAGTCCTTCTCTCGGACCCCTGGCAAA 64 FLT3L ECD-
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC hingeless
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGAC-
TGTGGCGACTGGT mono Fc
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGG-
AAAGAGTGAAC
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGGCGGACCGTCAGTCTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGC-
AGAA
CCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGG-
ACGG
CGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGT-
GCTG
ACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCT-
CCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACAAAGCCTCCAAGCCGGG-
AAGA
GATGACCAAGAACCAGGTGTCCCTGAGCTGCCTGGTCAAGGGCTTTTACCCCAGCGACATTGCCGTGGAATG-
GGAG
AGCAATGGCCAGCCTGAGAACAACTACAAGACCACCGTGCCTGTGCTGGACAGCGACGGCTCTTTTAGACTG-
GCCA
GCTACCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC-
TGCA CAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 65 FLT3L ECD
(H8Y)-
ACACAGGATTGCAGCTTCCAGTACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC hingeless hG1
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT Fc
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGA-
GTGAAC
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAAC-
ATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGGCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC-
CGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG-
ACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT-
CCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC-
CCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG-
AGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG-
GGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC-
TACA
GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC-
TGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 66 FLT3L ECD
ACACAGGATTGCAGCTTCCAGCACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC (K84E)-
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTG-
TGGCGACTGGT hingeless hG1
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC Fc
ACCGAGATCCACTTCGTGACCGAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCA-
ACATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGGCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC-
CGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG-
ACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT-
CCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC-
CCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG-
AGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG-
GGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC-
TACA
GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC-
TGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 67 FLT3L ECD
ACACAGGATTGCAGCTTCCAGTACAGCCCCATCAGCAGCGATTTCGCCGTGAAGATCAGAGAGCTGAGCGACT-
ACC (H8Y/K84E)
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGACTGTGGCGACT-
GGT hingeless hG1
GCTGGCTCAGAGATGGATGGAACGGCTGAAAACAGTGGCCGGCAGCAAGATGCAGGGACTGCTGGAAAGAGTG-
AAC Fc
ACCGAGATCCACTTCGTGACCGAGTGCGCCTTCCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCA-
ACATCA
GCAGACTGCTGCAAGAGACAAGCGAGCAGCTGGTGGCCCTGAAGCCTTGGATCACCAGACAGAACTTCAGCC-
GGTG
CCTGGAACTGCAGTGTCAGCCCGATAGCAGCACACTGCCTCCGCCTTGGAGTCCTAGACCTCTGGAAGCCAC-
AGCT
CCCACCGCTCCTCAAGGCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC-
CGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG-
ACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT-
CCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC-
CCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG-
AGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG-
GGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC-
TACA
GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC-
TGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 68 Aglyco-FLT3L
ACCCAGGACTGTTCCTTCCAGCACTCCCCTATCTCCAGCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ATC ECD (.DELTA. C-term
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGACT-
GGT 5 aa (PTAPQ;
GTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAAAGAGTG-
AAC SEQ ID
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAG-
ACCAATATCG NO: 85))
CCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCAGACAGAA-
CTTCGCCCGGTG (S128A/S151A)-
TCTGGAACTGCAGTGTCAGCCTGACAGCTCTACCCTGCCTCCACCTTGGAGCCCTAGACCTCTGGAAGCTACC-
GCT hG4
GAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTT-
CTGTTCC (S228P/F234A/
CTCCAAAGCCcAAGGACACCCTGtacATCaccCGGgaaCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCA-
GGA L235A/M252Y/
AGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAG-
GAA S254T/T256E)
CAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGT-
ACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCC-
GCGA
ACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGT-
GAAA
GGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC-
CCCC
CTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAG-
GCAA
CGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCT-
GGGC AAA 69 FLT3L ECD (.DELTA.
ACCCAGGACTGTTCCTTCCAGCACTCCCCTATCTCCAGCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ATC C-term 5 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGACT-
GGT (PTAPQ; SEQ
GTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAAAGAGTG-
AAC ID NO: 85))-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT linker
CCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCCGGCAGAACT-
TCTCTCGGTG SST/AAA-hG4
TCTGGAACTGCAGTGTCAGCCTGATGCTGCCGCTTTGCCTCCACCTTGGAGCCCTAGACCTCTGGAAGCTACC-
GCC (S228P/F234A/
GAGTCTAAGTACGGACCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGT-
TCC L235A/M252Y/
CTCCAAAGCCCAAGGACACCCTGTACATCACCCGGGAACCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCA-
GGA S254T/T256E)
AGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAG-
GAA
CAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAG-
TACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCC-
GCGA
ACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGT-
GAAA
GGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC-
CCCC
CTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAG-
GCAA
CGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCOT-
GGGC AAA 70 FLT3L ECD (.DELTA.
ACCCAGGACTGTTCCTTCCAGCACTCCCCTATCTCCAGCGACTTCGCCGTGAAGATCAGAGAGCTGTCCGACT-
ATC C-term 5 aa
TGCTGCAGGACTACCCTGTGACCGTGGCCAGCAATCTGCAGGACGAAGAACTGTGTGGTGGCCTGTGGCGACT-
GGT (PTAPQ; SEQ
GTTGGCTCAGAGATGGATGGAACGGCTGAAAACCGTGGCCGGCTCTAAGATGCAGGGCCTGCTGGAAAGAGTG-
AAC ID NO: 85))-
ACCGAGATCCACTTCGTGACCAAGTGCGCCTTTCAGCCTCCTCCATCCTGCCTGAGATTCGTGCAGACCAACA-
TCT linker
CCCGGCTGCTGCAAGAGACATCCGAGCAGCTGGTGGCTCTGAAGCCCTGGATCACCCGGCAGAACT-
TCTCTCGGTG SST/AAA;
TCTGGAACTGCAGTGTCAGCCTGATGCTGCCGCTTTGCCTCCTCCTTGGGCTCCTCGACCTCTG-
GAAGCTACAGCC S170A/S180A-
GAGGCTAAGTATGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCOTTCCGTGTTTCTGT-
TCC hG4
CTCCAAAGCCCAAGGACACCOTGTACATCACCCGGGAACCCGAAGTGACCTGCGTGGTGGTGGATGTGT-
CCCAGGA (S228P/F234A/
AGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAG-
GAA L235A/M252Y/
CAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGT-
ACA S254T/T256E)
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCG-
CGA
ACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGT-
GAAA
GGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC-
CCCC
CTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAG-
GCAA
CGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCOT-
GGGC AAA
4. Vectors and Host Cells
[0139] Further provided are vectors comprising one or more
polynucleotides encoding one or more of the FLT3L-Fc fusion
proteins, described herein. A vector can be of any type, for
example, a recombinant vector such as an expression vector. Vectors
include without limitation, plasmids, cosmids, bacterial artificial
chromosomes (BAC) and yeast artificial chromosomes (YAC) and
vectors derived from bacteriophages or plant or animal (including
human) viruses. Vectors can comprise an origin of replication
recognized by the proposed host cell and in the case of expression
vectors, promoter and other regulatory regions recognized by the
host cell. In additional embodiments, a vector comprises a
polynucleotide encoding an antibody of the disclosure operably
linked to a promoter and optionally additional regulatory elements.
Certain vectors are capable of autonomous replication in a host
into which they are introduced (e.g., vectors having a bacterial
origin of replication can replicate in bacteria). Other vectors can
be integrated into the genome of a host upon introduction into the
host, and thereby are replicated along with the host genome.
Vectors include without limitation, those suitable for recombinant
production of the antibodies disclosed herein.
[0140] The choice of the vector is dependent on the recombinant
procedures followed and the host used. Introduction of vectors into
host cells can be affected by inter alia calcium phosphate
transfection, virus infection, DEAE-dextran-mediated transfection,
lipofectamine transfection or electroporation. Vectors may be
autonomously replicating or may replicate together with the
chromosome into which they have been integrated. In certain
embodiments, the vectors contain one or more selection markers. The
choice of the markers may depend on the host cells of choice. These
include without limitation, kanamycin, neomycin, puromycin,
hygromycin, zeocin, thymidine kinase gene from Herpes simplex virus
(HSV-TK), and dihydrofolate reductase gene from mouse (dhfr).
Vectors comprising one or more nucleic acid molecules encoding the
FLT3L-Fc fusion proteins described herein, operably linked to one
or more nucleic acid molecules encoding proteins or peptides that
can be used to isolate the FLT3L-Fc fusion proteins, are also
covered by the disclosure. These proteins or peptides include
without limitation, glutathione-S-transferase, maltose binding
protein, metal-binding polyhistidine, green fluorescent protein,
luciferase and beta-galactosidase.
[0141] In other embodiments, the vector that is used is
pcDNA.TM.3.1+(ThermoFisher, MA).
[0142] In some embodiments, the viral vector comprises an oncolytic
viral vector. As appropriate, the oncolytic viral vector can be a
DNA virus or a RNA virus. In some embodiments, the viral vector is
from a viral family selected from the group consisting of:
Adenoviridae (e.g., Adenovirus), Arenaviridae (e.g., lymphocytic
choriomeningitis mammarenavirus, Cali mammarenavirus (a.k.a.,
Pichinde mammarenavirus), Poxviridae (e.g., Vaccinia virus),
Herpesviridae (e.g., Herpesvirus, e.g., HSV-1), Parvoviridae (e.g.,
Parvovirus H1), Reoviridae (e.g., Reovirus), Picornaviridae (e.g.,
Coxsackievirus, Seneca Valley Virus, Poliovirus), Paramyxoviridae
(e.g., Measles virus, Newcastle disease virus (NDV)), Rhabdoviridae
(e.g., Vesicular stomatitis virus (VSV)), Togaviridae (e.g.,
Alphavirus, Sindbis virus), Enteroviridae (e.g., Echovirus). The
use of oncolytic viruses in cancer therapy is described e.g.,
Fukuhara, et al., Cancer Sci. (2016) 107(10):1373-1379; Kaufman, et
al., Nat Rev Drug Discov. (2015) 14(9):642-62; Hamid, et al.,
Cancer Immunol Immunother. (2017) 66(10):1249-1264; Taguchi, et
al., Int J Urol. (2017) 24(5):342-351; and Buijs, et al., Hum
Vaccin Immunother. (2015) 11(7):1573-84.
[0143] The disclosure also provides host cells comprising a nucleic
acid or a vector described herein. Any of a variety of host cells
can be used. In one embodiment, a host cell is a prokaryotic cell,
for example, E. coli. In another embodiment, a host cell is a
eukaryotic cell, for example, a yeast cell, a plant cell, an insect
cell, a mammalian cell, such as a Chinese Hamster Ovary (CHO)-based
or CHO-origin cell line (e.g., CHO-S, CHO DG44, ExpiCHO.TM.,
CHOZN.RTM. ZFN-modified GS-/- CHO cell line, CHO-K1, CHO-K1a), COS
cells, BHK cells, NSO cells or Bowes melanoma cells. Examples of
human host cells are, inter alia, HeLa, 911, AT1080, A549 and
HEK293 (e.g., HEK293E, HEK293T, Expi293.TM.). In addition, the
FLT3L-Fc fusion proteins can be expressed in a yeast cell such as
Pichia (see, e.g., Powers et al., J Immunol Methods. 251:123-35
(2001)), Hanseula, or Saccharomyces.
[0144] In some embodiments, the host cell predominantly sialylates
N-linked glycosylation sites of the fusion protein. In some
embodiments, the polynucleotides encoding a FLT3L-Fc fusion
protein, as described herein, are expressed in a host cell that
sialylates at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, or more, N-linked glycosylation sites in the expressed
FLT3L-Fc fusion proteins.
[0145] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Some vectors are
suitable for delivering the nucleic acid molecule or polynucleotide
of the present application. Certain vectors are capable of
directing the expression of nucleic acids to which they are
operatively linked. Such vectors are referred to herein as
expression vectors.
[0146] The term "operably linked" refers to two or more nucleic
acid sequence or polypeptide elements that are usually physically
linked and are in a functional relationship with each other. For
instance, a promoter is operably linked to a coding sequence if the
promoter is able to initiate or regulate the transcription or
expression of a coding sequence, in which case, the coding sequence
should be understood as being "under the control of" the
promoter.
[0147] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0148] As appropriate, the host cells can be stably or transiently
transfected with a polynucleotide encoding a FLT3L-Fc fusion
protein, as described herein.
5. Methods of Producing FLT3L-Fc Fusion Proteins
[0149] The FLT3L-Fc fusion proteins described herein can be
produced by any method known in the art for the synthesis of fusion
proteins, for example, by chemical synthesis or by recombinant
expression techniques.
[0150] Methods of recombinant expression of fusion proteins are
known and can be applied to the recombinant production and
isolation/purification of the FLT3L-Fc fusion proteins. Methods of
recombinantly expressing proteins, including fusion proteins, are
described, for example, in Green and Sambrook, "Molecular Cloning:
A Laboratory Manual," 4.sup.th Edition, 2012, Cold Spring Harbor
Laboratory Press; Current Protocols in Protein Science, Wiley,
1995-2109
(currentprotocols.onlinelibrary.wiley.com/journal/19343663/); and
Current Protocols in Molecular Biology, Wiley, 1987-2019
(currentprotocols.onlinelibrary.wiley.com/journal/19343647/). In
addition, other publications relating to producing recombinantly
expressed fusion proteins include, e.g., Argelia Lorence (Editor),
"Recombinant Gene Expression" (Methods in Molecular Biology) 2012,
Humana Press; James L Hartley (Editor), "Protein Expression in
Mammalian Cells: Methods and Protocols" (Methods in Molecular
Biology) 2012, Humana Press; Roslyn M. Bill (Editor), "Recombinant
Protein Production in Yeast: Methods and Protocols" (Methods in
Molecular Biology) 2012, Humana Press; and MacDonald, Kolotilin and
Menassa (Editors) "Recombinant Proteins from Plants: Methods and
Protocols" (Methods in Molecular Biology), 2d Edition, 2016, Humana
Press.
[0151] In various embodiments, the FLT3L-Fc fusion proteins
described herein may be produced in bacterial or eukaryotic cells.
The FLT3L-Fc fusion proteins can also be produced in eukaryotic
cells such as transformed cell lines (e.g., CHO, CHO-S, CHO DG44,
ExpiCHO.TM., CHOZN.RTM., CHO-K1, CHO-K1a, 293E, 293T, COS, NIH3T3).
In addition, the FLT3L-Fc fusion proteins described herein can be
expressed in a yeast cell such as Pichia (see, e.g., Powers et al.,
J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces.
In one embodiment, the FLT3L-Fc fusion proteins described herein
are produced in a CHO cell line, e.g., a CHO-S, CHO DG44,
ExpiCHO.TM., CHOZN.RTM., CHO-K1, CHO-K1a cell line, or a HEK293
(e.g., HEK293E, HEK293T, Expi293.TM.) cell line. To produce the
FLT3L-Fc fusion proteins of interest (including a heterodimer
comprising the FLT3-Fc fusion protein), one or more polynucleotides
encoding the FLT3L-Fc fusion proteins is constructed, introduced
into an expression vector, and then expressed in one or more
suitable host cells. In some embodiments, three polynucleotides
encoding a FLT3L-Fc fusion, a Fab heavy chain and a Fab light chain
comprising a second antigen binding domain are co-expressed in a
single host cell. Standard molecular biology techniques are used to
prepare the recombinant expression vector, transfect the host
cells, select for transformants, culture the host cells, and
recover the FLT3L-Fc fusion proteins.
[0152] In some embodiments, the host cell predominantly sialylates
N-linked glycosylation sites of the fusion protein. In some
embodiments, the polynucleotides encoding a FLT3L-Fc fusion
protein, as described herein, are expressed in a host cell that
sialylates at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, or more, N-linked glycosylation sites in the expressed
FLT3L-Fc fusion proteins.
[0153] If the FLT3L-Fc fusion proteins are to be expressed in
bacterial cells (e.g., E. coli), the expression vector should have
characteristics that permit amplification of the vector in the
bacterial cells. Additionally, when E. coli such as JM109, DH5a,
113101, or XL1-Blue is used as a host, the vector must have a
promoter, for example, a lacZ promoter (Ward et al., 341:544-546
(1989), araB promoter (Better et al., Science, 240:1041-1043
(1988)), or T7 promoter that can allow efficient expression in E.
coli. Examples of such vectors include, for example, M13-series
vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script,
pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET
(when this expression vector is used, the host is preferably BL21
expressing T7 RNA polymerase). The expression vector may contain a
signal sequence for secretion of the FLT3L-Fc fusion proteins. For
production into the periplasm of E. coli, the pelB signal sequence
(Lei et al., J. Bacteriol., 169: 4379 (1987)) may be used as the
signal sequence for secretion of the FLT3L-Fc fusion proteins. For
bacterial expression, calcium chloride methods or electroporation
methods may be used to introduce the expression vector into the
bacterial cell.
[0154] If the FLT3L-Fc fusion proteins are to be expressed in
mammalian cells (e.g., such as CHO-S, CHO DG44, ExpiCHO.TM., CHOZN,
CHO-K1, CHO-K1a, 293E, 293T, Expi293.TM., COS, NIH3T3 cells), the
expression vector includes a promoter to promote expression in
these cells, for example, an SV40 promoter (Mulligan et al.,
Nature, 277:108 (1979)), MMLV-LTR promoter, EF1.alpha. promoter
(Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or CMV
promoter. In addition to the nucleic acid sequence encoding the
immunoglobulin or domain thereof, the recombinant expression
vectors may carry additional sequences, such as sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin, or
methotrexate, on a host cell into which the vector has been
introduced. Examples of vectors with selectable markers include
pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
[0155] In one embodiment, the FLT3L-Fc fusion proteins are produced
in mammalian cells. Exemplary mammalian host cells for expressing
FLT3L-Fc fusion proteins include Chinese Hamster Ovary (e.g., CHO,
CHO-S, CHO DG44, ExpiCHO.TM., CHOZN.RTM., CHO-K1, CHO-K1a)
(including dhfr-CHO cells, described in Urlaub and Chasin (1980)
Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR
selectable marker, e.g., as described in Kaufman and Sharp (1982)
Mol. Biol. 159:601 621), human embryonic kidney 293 cells (e.g.,
293, 293E, 293T, Expi293.TM.), COS cells, NIH3T3 cells, lymphocytic
cell lines, e.g., NSO myeloma cells and SP2 cells, and a cell from
a transgenic animal, e.g., a transgenic mammal. For example, in
some embodiments, the cell is a mammary epithelial cell.
[0156] In an exemplary system for expression of the FLT3L-Fc fusion
proteins, recombinant expression vectors encoding the FLT3L-Fc
fusion protein are introduced into dhfr-CHO cells by calcium
phosphate-mediated transfection. In a specific embodiment, the
dhfr-CHO cells are cells of the DG44 cell line, such as DG44i (see,
e.g., Derouaz et al., Biochem Biophys Res Commun., 340(4):1069-77
(2006)). Within the recombinant expression vectors, the
polynucleotide encoding the FLT3L-Fc fusion protein, and optionally
a second polynucleotide encoding a second Fc fusion protein for
forming a heterodimer, are operatively linked to enhancer/promoter
regulatory elements (e.g., derived from SV40, CMV, adenovirus and
the like, such as a CMV enhancer/AdMLP promoter regulatory element
or an SV40 enhancer/AdMLP promoter regulatory element) to drive
high levels of transcription of the genes. The recombinant
expression vectors also carry a DHFR gene, which allows for
selection of CHO cells that have been transfected with the vector
using methotrexate selection/amplification. The selected
transformant host cells are cultured to allow for expression and
secretion of the FLT3L-Fc fusion protein, and the fusion protein is
recovered from the culture medium.
[0157] The FLT3L-Fc fusion proteins can also be produced by a
transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a
method of expressing an antibody in the mammary gland of a
transgenic mammal. A transgene is constructed that includes a
milk-specific promoter and one or more polynucleotides encoding the
FLT3L-Fc fusion protein of interest and a signal sequence for
secretion. The milk produced by females of such transgenic mammals
includes, secreted-therein, the FLT3L-Fc fusion protein of
interest. The FLT3L-Fc fusion protein can be purified from the
milk, or for some applications, used directly. Animals are also
provided comprising one or more of the FLT3L-Fc-encoding nucleic
acids described herein.
[0158] The FLT3L-Fc fusion proteins can be isolated from inside or
outside (such as medium) of the host cell and purified as
substantially pure and homogenous, non-aggregated FLT3L-Fc fusion
proteins (e.g, including monomeric, homodimeric and/or
heterodimeric bispecific FLT3L-Fc fusion proteins). Methods for
isolation and purification commonly used for protein purification,
including antibody purification, may be used for the isolation and
purification of herein described FLT3L-Fc fusion proteins, and are
not limited to any particular method. Applicable protein
purification techniques are described, e.g., in Labrou,
Chronopoulou and Ataya (Editors), "Handbook on Protein
Purification: Industry Challenges and Technological Developments,
2018, Nova Science Pub Inc; Gottschalk (Editor), "Process Scale
Purification of Antibodies," 2nd Edition, 2017, Wiley; Staby,
Rathore and Ahuja (Editors), "Preparative Chromatography for
Separation of Proteins, 2017, Wiley; and Labrou (Editor), "Protein
Downstream Processing: Design, Development and Application of High
and Low-Resolution Methods, 2014, Human Press. The FLT3L-Fc fusion
proteins may be isolated and purified by appropriately selecting
and combining, for example, column chromatography, filtration,
ultrafiltration, salting out, solvent precipitation, solvent
extraction, distillation, immunoprecipitation, SDS-polyacrylamide
gel electrophoresis, isoelectric focusing, dialysis, and
recrystallization. Chromatography includes, for example, affinity
chromatography, ion exchange chromatography, hydrophobic
chromatography, gel filtration, reverse-phase chromatography, and
adsorption chromatography (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography
can be carried out using liquid phase chromatography such as HPLC
and FPLC. Columns used for affinity chromatography include protein
A column and protein G column. Examples of columns using protein A
column include Hyper D, POROS, and Sepharose FF (GE Healthcare
Biosciences). The present disclosure also includes FLT3L-Fc fusion
proteins that are highly purified using these purification
methods.
6. Pharmaceutical Compositions
[0159] Provided are pharmaceutical compositions comprising a
FLT3L-Fc fusion protein, as described herein, or a polynucleotide
encoding a FLT3L-Fc fusion protein, as described herein, and a
pharmaceutically acceptable diluent, carrier or excipient. In
certain embodiments, the pharmaceutical composition comprises a
therapeutically effective amount of the FLT3L-Fc fusion protein, or
polynucleotide encoding such FLT3L-Fc fusion protein.
[0160] Various pharmaceutically acceptable diluents, carriers, and
excipients, and techniques for the preparation and use of
pharmaceutical compositions will be known to those of skill in the
art in light of the present disclosure. Illustrative pharmaceutical
compositions and pharmaceutically acceptable diluents, carriers,
and excipients are also described in, e.g., Loyd V. Allen Jr
(Editor), "Remington: The Science and Practice of Pharmacy,"
22.sup.nd Edition, 2012, Pharmaceutical Press; Brunton, Knollman
and Hilal-Dandan, "Goodman and Gilman's The Pharmacological Basis
of Therapeutics," 13th Edition, 2017, McGraw-Hill
Education/Medical; McNally and Hastedt (Editors), "Protein
Formulation and Delivery," 2nd Edition, 2007, CRC Press; Banga,
"Therapeutic Peptides and Proteins: Formulation, Processing, and
Delivery Systems," 3rd Edition, 2015, CRC Press; Lars Hovgaard,
Frokjaer and van de Weert (Editors), "Pharmaceutical Formulation
Development of Peptides and Proteins," 2nd Edition, 2012, CRC
Press; Carpenter and Manning (Editors), "Rational Design of Stable
Protein Formulations: Theory and Practice," 2002, Springer
(Pharmaceutical Biotechnology (Book 13)); Meyer (Editor),
"Therapeutic Protein Drug Products: Practical Approaches to
Formulation in the Laboratory, Manufacturing, and the Clinic,"
2012, Woodhead Publishing; and Shire, "Monoclonal Antibodies:
Meeting the Challenges in Manufacturing, Formulation, Delivery and
Stability of Final Drug Product," 2015, Woodhead Publishing.
[0161] In some embodiments, each carrier, diluent or excipient is
"acceptable" in the sense of being compatible with the other
ingredients of the pharmaceutical composition and not injurious to
the subject. Often, the pharmaceutically acceptable carrier is an
aqueous pH-buffered solution. Some examples of materials which can
serve as pharmaceutically-acceptable carriers, diluents or
excipients include: water; buffers, e.g., phosphate-buffered
saline; sugars, such as lactose, trehalose, glucose and sucrose;
starches, such as corn starch and potato starch; cellulose, and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; glycols, such as
propylene glycol; polyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl
laurate; agar; buffering agents, such as magnesium hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; amino acids (e.g., charged amino acids, including
without limitation, aspartate, asparagine, glutamate, glutamine,
histidine, lysine); and other non-toxic compatible substances
employed in pharmaceutical formulations. Wetting agents,
emulsifiers and lubricants, such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, release agents,
coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the
compositions.
[0162] The formulation of and delivery methods of pharmaceutical
compositions will generally be adapted according to the site and
the disease to be treated. Exemplary formulations include without
limitation, those suitable for parenteral administration, e.g.,
intratumoral, intravenous, intra-arterial, intramuscular, or
subcutaneous administration, including formulations encapsulated in
micelles, liposomes or drug-release capsules (active agents
incorporated within a biocompatible coating designed for
slow-release); ingestible formulations; formulations for topical
use, such as creams, ointments and gels; and other formulations
such as inhalants, aerosols and sprays. In some embodiments, the
pharmaceutical compositions are formulated for parenteral, e.g.,
intravenous, subcutaneous, or oral administration. In some
embodiments, the pharmaceutical compositions are formulated for
intratumoral administration.
[0163] In certain embodiments, pharmaceutical compositions are
sterile. In certain embodiments, the pharmaceutical composition has
a pH in the range of 4.5 to 8.5, 4.5 to 6.5, 6.5 to 8.5, or a pH of
about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5,
about 8.0 or about 8.5. In some embodiments, the pharmaceutical
composition has a pH of 5.9. In one embodiment, the pharmaceutical
composition has an osmolarity in the range of 240-260 or 250-330
mOsmol/L. In certain embodiments, the pharmaceutical composition is
isotonic or near isotonic.
[0164] In some embodiments, the pharmaceutical compositions are
liquids or solids. In some embodiments, the pharmaceutical
composition comprises an aqueous solution, e.g., at a concentration
in the range of about 1 mg/ml to about 2 mg/ml, 3 mg/ml, 4 mg/ml, 5
mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12
mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml,
19 mg/ml or 20 mg/ml. In some embodiments, the pharmaceutical
composition comprises an aqueous solution of FLT3L-Fc fusion
protein at a concentration of about 2 mg/ml. In some embodiments,
the pharmaceutical composition comprises an aqueous solution of
FLT3L-Fc fusion protein at a concentration of 2 mg/ml. In some
embodiments, the pharmaceutical composition is lyophilized. In
certain embodiments, the pharmaceutical composition is formulated
for intravenous administration and has a concentration of FLT3L-Fc
fusion protein of about 1-100 mg/ml, 1-10 mg/ml, 2-20 mg/ml or
about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/ml. In certain
embodiments, the pharmaceutical composition is formulated for
intravenous administration and has a concentration of FLT3L-Fc
fusion protein of about 2 mg/ml. In certain embodiments, the
pharmaceutical composition is formulated for intravenous
administration and has a concentration of FLT3L-Fc fusion protein
of 2 mg/ml. In some embodiments, the pharmaceutical composition is
formulated for subcutaneous injection and has a concentration of
FLT3L-Fc fusion protein of 1-100 mg/ml, 1-10 mg/ml, 2-20 mg/ml or
about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/ml, and a viscosity less
than 50 cP, less than 30 cP, less than 20 cP, or about 10 cP.
[0165] In some embodiments, the pharmaceutical composition is an
aqueous solution containing 2 mg/mL FLT3L-Fc fusion protein in 20
mM histidine, 90 g/L sucrose, 0.2 g/L polysorbate 80, pH 5.9.
[0166] In some embodiments, the pharmaceutical composition further
comprises one or more additional therapeutic agents, e.g., a second
therapeutic agent, or second and third therapeutic agents.
7. Methods of Treatment
[0167] The FLT3L-Fc fusion proteins described herein find use for
treating cancer or another proliferative disorder. In some
embodiments, the methods entail administering a FLT3L-Fc fusion
protein, homodimer or heterodimer comprising such fusion protein,
polynucleotide encoding such fusion protein, or pharmaceutical
composition comprising such fusion protein or polynucleotide, to a
patient with cancer or another proliferative disorder. In certain
embodiments, the method of treating cancer or another proliferative
disorder comprises administering a provided compound or composition
thereof to a mammal.
[0168] In another aspect, provided herein are methods for inducing
the immune system in a subject in need thereof comprising
administering an FLT3L-Fc fusion protein or lipoplex, such as an
LNP, provided herein or a polynucleotide or vector encoding an
FLT3L-Fc fusion protein provided herein to the subject.
[0169] In another aspect, provided herein are compounds for use in
treating cancer comprising an FLT3L-Fc fusion protein provided
herein, or a polynucleotide or vector encoding an FLT3L-Fc fusion
protein provided herein.
[0170] In another aspect, the FLT3L-Fc fusion proteins described
herein find use for treating or preventing a viral infection. In
some embodiments, the viral infection is an infection caused by
HIV. In some embodiments the viral infection is an infection caused
by HBV. In some embodiments, the viral infection is caused by a
coronavirus. In some embodiments, the coronavirus infection is
caused by the SARS virus, the MERS virus, or the 2019-nCoV
(COVID-19) virus. In some embodiments, the methods entail
administering a FLT3L-Fc fusion protein, homodimer or heterodimer
comprising such fusion protein, polynucleotide encoding such fusion
protein, or pharmaceutical composition comprising such fusion
protein or polynucleotide, to a patient having or at risk of having
an infection caused by a virus. In some embodiments, the patient
has or is at risk of having an infection caused by HIV, HBV, or
coronavirus (e.g., SARS virus, MERS, virus, or COVID-19 virus). In
certain embodiments, the method of treating or preventing a viral
infection, such as an infection caused by a virus such as HIV, HBV,
or coronavirus (e.g., SARS virus, MERS, virus, or COVID-19 virus),
comprises administering a provided compound or composition thereof
to a mammal.
[0171] As used herein, the terms "inhibition of cancer" and
"inhibition of cancer cell proliferation" refer to the inhibition
of the growth, division, maturation or viability of cancer cells,
and/or causing the death of cancer cells, individually or in
aggregate with other cancer cells, by cytotoxicity, nutrient
depletion, or the induction of apoptosis.
[0172] As used herein, the terms "treatment," "treat," and
"treating" refer to reversing, alleviating, delaying the onset of,
or inhibiting the progress of a disease or disorder, or one or more
symptoms thereof, as described herein. In some embodiments,
treatment may be administered after one or more symptoms have
developed. In other embodiments, treatment may be administered in
the absence of symptoms. For example, treatment may be administered
to a susceptible individual prior to the onset of symptoms (e.g.,
in light of a history of symptoms and/or in light of genetic or
other susceptibility factors). Treatment may also be continued
after symptoms have resolved, for example to prevent or delay their
recurrence.
[0173] As used herein, "delaying" development of a disease or
disorder, or one or more symptoms thereof, means to defer, hinder,
slow, retard, stabilize and/or postpone development of the disease,
disorder, or symptom thereof. This delay can be of varying lengths
of time, depending on the history of the disease and/or subject
being treated. As is evident to one skilled in the art, a
sufficient or significant delay can, in effect, encompass
prevention, in that the subject does not develop the disease,
disorder, or symptom thereof. For example, a method that "delays"
development of AIDS is a method that reduces the probability of
disease development in a given time frame and/or reduces extent of
the disease in a given time frame, when compared to not using the
method. Such comparisons may be based on clinical studies, using a
statistically significant number of subjects. For example, the
development of AIDS can be detected using known methods, such as
confirming a subject's HIV.sup.+ status and assessing the subject's
T-cell count or other indication of AIDS development, such as
extreme fatigue, weight loss, persistent diarrhea, high fever,
swollen lymph nodes in the neck, armpits or groin, or presence of
an opportunistic condition that is known to be associated with AIDS
(e.g., a condition that is generally not present in subjects with
functioning immune systems but does occur in AIDS patients).
Development may also refer to disease progression that may be
initially undetectable and includes occurrence, recurrence and
onset.
[0174] As used herein, "prevention" or "preventing" refers to a
regimen that protects against the onset of the disease or disorder
such that the clinical symptoms of the disease do not develop.
Thus, "prevention" relates to administration of a therapy (e.g.,
administration of a therapeutic substance) to a subject before
signs of the disease are detectable in the subject (e.g.,
administration of a therapeutic substance to a subject in the
absence of detectable infectious agent (e.g., virus) in the
subject). The subject may be an individual at risk of developing
the disease or disorder, such as an individual who has one or more
risk factors known to be associated with development or onset of
the disease or disorder. For example, the term "preventing HIV
infection" refers to administering to a subject who does not have a
detectable HIV infection an anti-HIV therapeutic substance. It is
understood that the subject for anti-HIV preventative therapy may
be an individual at risk of contracting the HIV virus. Further, it
is understood that prevention may not result in complete protection
against onset of the disease or disorder. In some instances,
prevention includes reducing the risk of developing the disease or
disorder. The reduction of the risk may not result in complete
elimination of the risk of developing the disease or disorder.
[0175] With respect to subjects, in some embodiments, the methods
of treatment provided herein may be used to treat a subject (e.g.,
human, monkey, dog, cat, mouse) who has been diagnosed with or is
suspected of having cancer. In some embodiments, the methods of
treatment provided herein can be used to treat a subject (e.g.,
human, monkey, dog, cat, mouse) who has been diagnosed with or is
suspected of having a viral infection. As used herein, a subject
refers to a mammal, including, for example, a human.
[0176] In some embodiments, the subject may be a human who exhibits
one or more symptoms associated with cancer or hyperproliferative
disease (e.g., a tumor). In some embodiments, the subject may be a
human who exhibits one or more symptoms associated with cancer. Any
of the methods of cancer treatment provided herein may be used to
treat cancer at various stages. By way of example, the cancer stage
includes but is not limited to early, advanced, locally advanced,
remission, refractory, reoccurred after remission and progressive.
In some embodiments, the subject is at an early stage of a cancer.
In other embodiments, the subject is at an advanced stage of
cancer. In various embodiments, the subject has a stage I, stage
II, stage III or stage IV cancer. One or more administrations of
the FLT3L-Fc fusion protein, optionally with one or more additional
therapeutic agents, can promote reduction or retraction of a tumor,
decrease or inhibit tumor growth or cancer cell proliferation,
and/or induce, increase or promote tumor cell killing. In some
embodiments, the subject is in cancer remission. One or more
administrations of the FLT3L-Fc fusion protein, optionally with one
or more additional therapeutic agents, can prevent or delay
metastasis or recurrence of cancer.
[0177] In some embodiments, the subject may be a human who exhibits
one or more symptoms associated with a viral infection (e.g., a
detectable viral titer). In some embodiments, the subject may be a
human who exhibits one or more symptoms associated with a viral
infection. Any of the methods of antiviral treatment provided
herein may be used to treat a viral infection at various stages. In
some embodiments, the subject is at an early stage of a viral
infection. In other embodiments, the subject is at an advanced
stage of a viral infection. In some embodiments, one or more
administrations of the FLT3L-Fc fusion protein, optionally with one
or more additional therapeutic agents, can promote the reduction of
a viral titer in a subject.
[0178] In some embodiments, the subject may be a human who is at
risk, or genetically or otherwise predisposed (e.g., risk factor)
to developing cancer or hyperproliferative disease who has or has
not been diagnosed. In some embodiments, the subject may be a human
who is at risk, or genetically or otherwise predisposed (e.g., risk
factor) to a disease, disorder, or symptoms thereof, caused by a
viral infection who has or has not been diagnosed.
[0179] As used herein, an "at risk" individual is an individual who
is at risk of developing a condition to be treated. In some
embodiments, an "at risk" subject is a subject who is at risk of
developing cancer. Generally, an"at risk" subject may or may not
have detectable disease, and may or may not have displayed
detectable disease prior to the treatment methods described herein.
"At risk" denotes that an individual has one or more so-called risk
factors, which are measurable parameters that correlate with
development of a disease or condition and are known in the art. For
example, an at risk subject may have one or more risk factors,
which are measurable parameters that correlate with development of
cancer, which are described herein. A subject having one or more of
these risk factors has a higher probability of developing cancer
than an individual without these risk factor(s). In general, risk
factors may include, for example, age, sex, race, diet, history of
previous disease, presence of precursor disease, genetic (e.g.,
hereditary) considerations, and environmental exposure. In some
embodiments, the subjects at risk for cancer include, for example,
those having relatives who have experienced the disease, and those
whose risk is determined by analysis of genetic or biochemical
markers. In some embodiments, the at risk subject is at risk of
developing symptoms of a viral infection. For example, individuals
at risk for AIDS are those infected with HIV.
[0180] In addition, the subject may be a human who is undergoing
one or more standard therapies, such as chemotherapy, radiotherapy,
immunotherapy, surgery, or combination thereof. Accordingly, one or
more kinase inhibitors may be administered before, during, or after
administration of chemotherapy, radiotherapy, immunotherapy,
surgery or combination thereof.
[0181] In certain embodiments, the subject may be a human who is
(i) substantially refractory to at least one chemotherapy
treatment, or (ii) is in relapse after treatment with chemotherapy,
or both (i) and (ii). In some of embodiments, the subject is
refractory to at least two, at least three, or at least four
chemotherapy treatments (including standard or experimental
chemotherapies).
[0182] The FLT3L-Fc fusion proteins described herein find use as a
vaccine adjuvant, promoting, increasing, supplementing and/or
boosting the immune response induced by the vaccine. In various
embodiments, the vaccine can be an anticancer vaccine, antiviral
vaccine, or antibacterial vaccine. In some embodiments, the
anticancer vaccine is a neoantigen vaccine, wherein a neoantigen
refers to a class of HLA-bound peptides that arise from
tumor-specific mutations. Illustrative neoantigen anticancer
vaccines are described, e.g., in Ott, et al., Nature. 2017 Jul. 13;
547(7662):217-221; Li, et al., Ann Oncol. 2017 Dec. 1;
28(suppl_12):xii11-xii17; Aldous, et al., BioorgMed Chem. 2018 Jun.
1; 26(10):2842-2849; and Linette, et al, Trends Mol Med. 2017
October; 23(10):869-871. In various embodiments, the vaccine
comprises an antiviral vaccine against a virus selected from the
group consisting of hepatitis A virus (HAV), hepatitis B virus
(HBV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), a
herpes simplex virus (HSV), Epstein-Barr virus (EBV), human
orthopneumovirus or human respiratory syncytial virus (RSV), human
papillomavirus (HPV), varicella-zoster virus, measles virus, mumps
virus, poliovirus vaccine, influenza virus, paramyxovirus,
rotavirus, Zika virus, Dengue virus and Ebola virus. In some
embodiments, the vaccine comprises an antibacterial vaccine against
a bacterium selected from the group consisting of Mycobacterium
tuberculosis, pertussis, tetanus, diphtheria, meningococcus,
pneumococcus, Haemophilus influenza, cholera, typhoid, and anthrax.
Illustrative anticancer vaccines include without limitation
Bacillus Calmettle-Guerin (TheraCys.RTM.)--a live attenuated strain
of Mycobacterium bovis for non-muscle invasive bladder carcinoma;
Sipuleucel-T (Provenge.RTM.)--a dendritic cell (DC) vaccine for
metastatic castration resistant prostate cancer (mCRPC); talimogene
laherparepvec (T-VEC or Imlygic.RTM.)--an oncolytic viral-based
vaccine for advanced melanoma; and recombinant viral prostate
cancer vaccine PROSTVAC.RTM.-VF/TRICOM.TM.. In some embodiments,
the anticancer vaccine is an antiviral vaccine. In some embodiments
the anticancer vaccine is an HPV vaccine. In some embodiments, the
HPV vaccine is PRGN-2009 (Precigen; PGEN Therapeutics). In some
embodiments the HPV vaccine is Gardasil or Gardasil-9
(Merck&Co). In some embodiments, the HPV vaccine is Cervarix
(GaxoSmithKline Biologicals). In some embodiments the HSV vaccine
is HSV529 (Sanofi Pasteur).
[0183] Accordingly, in one embodiment, provided are methods for
promoting, inducing and/or increasing the expansion and/or
proliferation of a cell or a population of cells that express fms
related tyrosine kinase 3 (FLT3, CD135). In some embodiments, the
methods comprise contacting the cell or population of cells with an
effective amount of a FLT3L-Fc fusion protein, as described herein,
a homodimer or heterodimer comprising such fusion protein, a
polynucleotide encoding such fusion protein, a vector or lipoplex,
such as a lipid nanoparticle (LNP), comprising such polynucleotide,
or pharmaceutical composition comprising such fusion protein or
polynucleotide. As used herein, a "lipoplex" refers to cationic
liposomes that are nonviral (synthetic) lipid carriers of DNA. In
some embodiments the lipoplex is a lipid nanoparticle (LNP). As
used herein, the term "lipid nanoparticle" refers to one or more
spherical nanoparticles with an average diameter of between about
10 to about 1000 nanometers, and which comprise a solid lipid core
matrix that can solubilize lipophilic molecules. In certain
embodiments, the lipid core is stabilized by surfactants (e.g.,
emulsifiers), and can comprise one or more of triglycerides (e.g.,
tristearin), diglycerides (e.g., glycerol bahenate), monoglycerides
(e.g., glycerol monostearate), fatty acids (e.g., stearic acid),
steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate),
including combinations thereof. Lipid nanoparticles are described,
for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55,
2014; and U.S. Pat. Nos. 6,217,912; 6,881,421; 7,402,573;
7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8,871,509;
9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130, each of
which is incorporated by reference in its entirety.
[0184] In some embodiments, the cell or population of cells that
express FLT3 comprise dendritic cells (e.g., cDC1 cells and/or cDC2
cells), monocyte-derived dendritic cells (moDCs), and/or progenitor
cells thereof. In some embodiments, the cell or population of cells
that express FLT3 comprise hematopoietic progenitor cells. In some
embodiments, the hematopoietic progenitor cells comprise Common
Lymphoid Progenitors (CLPs), Early Progenitors with Lymphoid and
Myeloid potential (EPLMs), granulocyte-monocyte (GM) progenitors
(GMP), monocyte-derived dendritic cells (moDCs) progenitors, and/or
early multi-potent progenitors (MPP) within the Lineage-kit+Sca1
(LSK) compartment. As appropriate, the cell can be contacted in
vitro or in vivo. In some embodiments, conventional dendritic cells
(e.g., cDC1 and/or cDC2) are expanded. In some embodiments, cDC1
dendritic cells (e.g., positive for surface expression of X-C motif
chemokine receptor 1 (XCR1), thrombomodulin (THBD, CD141), and
C-type lectin domain containing 9A (CLEC9A)) are expanded or
induced to proliferate. In some embodiments, cDC2 dendritic cells
(e.g., positive for surface expression of CDlc molecule (BDCA1))
are expanded or induced to proliferate. In some embodiments,
dendritic cells positive for surface expression of BDCA1 (cDC1),
BDCA2 (CLEC4c), BDCA3 (THBD) and/or BDCA4 (NRP1) are expanded or
induced to proliferate. In some embodiments, the FLT3-expressing
cells (e.g., dendritic cells) are expanded by at least about
10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold,
500-fold, or more, e.g., in the tumor, in the lymph nodes, within 3
weeks of a single administration of the fusion protein, the
homodimer, the heterodimer, the conjugate, the polynucleotide, the
vector, the lipoplex (e.g., LNP) and/or the pharmaceutical
composition.
[0185] In a further embodiment, provided are methods of expanding
hematopoietic stem cells (HSCs) ex vivo, comprising culturing HSCs
in vitro, in the presence of mesenchymal lineage precursor or stem
cells (MLPSCs) and an effective amount of a FLT3L-Fc fusion
protein, as described herein, a homodimer or heterodimer comprising
such fusion protein, a polynucleotide encoding such fusion protein,
or a vector or lipoplex, such as an lipid nanoparticle (LNP),
comprising such polynucleotide such that HSCs having the phenotype
CD34+ are expanded. In some embodiments, the HSC are derived from
bone marrow, umbilical cord, peripheral blood, liver, thymus,
lymph, or spleen. In some embodiments, the HSCs are further
cultured in the presence of one or more histone deacetylase
inhibitors (HDACi), such as valproic acid (VPA), trichostatin A
(TSA), DLS3, MS275, or SAHA. In some embodiments, the HSCs have the
phenotype CD34+, CD90+ or CD34+, CD45RA-, CD49f+. In some
embodiments, the HSC are expanded at least 5-fold, at least
10-fold, at least 20-fold, or at least 40-fold. In some
embodiments, the HSCs are further cultured in the presence of one
or more factors selected from the group consisting of stem cell
factor (SCF), thrombopoietin (TPO), interleukin 3 (IL3), and
interleukin 6 (IL6). In some embodiments, the methods further
comprise isolating cells having the phenotype the phenotype CD34+,
CD90+ or CD34+, CD45RA-, CD49f+ to provide an enriched population
of cells having the phenotype CD34+, CD90+ or CD34+, CD45RA-,
CD49f+. In a further embodiment, provided are compositions
comprising HSCs having of the phenotype CD34+, CD90+ or CD34+,
CD45RA-, CD49f+ obtained using a method described herein.
Illustrative examples of methods of HSC ex vivo expansion in which
a FLT3L-Fc fusion protein described herein, a homodimer or
heterodimer comprising such fusion protein, a polynucleotide
encoding such fusion protein, or a vector or lipoplex, such as a
lipid nanoparticle (LNP), comprising such polynucleotide could be
applied are described for example, without limitation, in WO
2020/089411.
[0186] In a further embodiment, provided are methods of preventing,
reducing and/or inhibiting the recurrence, growth, proliferation,
migration and/or metastasis of a cancer cell or population of
cancer cells in a subject in need thereof. Further provided are
methods of enhancing, improving, and/or increasing the response to
an anticancer therapy in a subject in need thereof. In some
embodiments, the methods entail administering to the subject an
effective amount of a FLT3L-Fc fusion protein, as described herein,
a homodimer or heterodimer comprising such fusion protein, a
polynucleotide encoding such fusion protein, a vector or lipoplex,
such as a lipid nanoparticle (LNP), comprising such polynucleotide,
or pharmaceutical composition comprising such fusion protein or
polynucleotide.
[0187] In a further embodiment, provided are methods of treating or
preventing a virus infection, comprising administering to a subject
in need thereof an effective amount of a FLT3L-Fc fusion protein,
as described herein, a homodimer or heterodimer comprising such
fusion protein, a polynucleotide encoding such fusion protein, a
vector or lipoplex, such as an lipid nanoparticle (LNP), comprising
such polynucleotide. Further provided are methods of enhancing,
improving, and/or increasing the response to an antiviral therapy
in a subject in need thereof comprising administering to the
subject an effective amount of a FLT3L-Fc fusion protein, as
described herein, a homodimer or heterodimer comprising such fusion
protein, a polynucleotide encoding such fusion protein, a vector or
lipoplex, such as a lipid nanoparticle (LNP), comprising such
polynucleotide. In some embodiments, the virus infection is a human
hepatitis B virus (HBV) infection. In some embodiments, the virus
infection is a human immunodeficiency virus (HIV) infection. In
some embodiments, the virus infection is a coronavirus infection.
In some embodiments the coronavirus is a Severe Acute Respiratory
Syndrome (SARS)-associated virus. In some embodiments the
coronavirus is a MERS-associated virus. In some embodiments, the
coronavirus is a COVID-19-associated virus (e.g., SARS-CoV-2).
[0188] As used herein, "HBV" refers to a virus described by NCBI
Taxonomy ID: NCBI:txid10407.
[0189] As used herein, "HIV" refers to a virus described by NCBI
Taxonomy ID: NCBI:txid11676.
[0190] As used herein, "SARS"-associated virus refers to a virus
described by NCBI Taxonomy ID: NCBI:txid694009.
[0191] As used herein, "MERS"-associated virus refers to a virus
described by NCBI Taxonomy ID: NCBI:txid1335626.
[0192] As used herein, "COVID-19-associated virus" or "SARS-CoV-2"
refers to a virus described by NCBI Taxonomy ID:
NCB:txid2697049.
[0193] In a further embodiment, provided are methods of treating or
preventing a human hepatitis B virus (HBV) infection comprising
administering to a subject in need thereof an effective amount of a
FLT3L-Fc fusion protein, as described herein, a homodimer or
heterodimer comprising such fusion protein, a polynucleotide
encoding such fusion protein, a vector or lipoplex, such as an
lipid nanoparticle (LNP), comprising such polynucleotide. In
certain embodiments, provided are methods of treating an HBV
infection comprising administering to a subject in need thereof an
effective amount of a FLT3L-Fc fusion protein, as described herein,
a homodimer or heterodimer comprising such fusion protein, a
polynucleotide encoding such fusion protein, a vector or lipoplex,
such as a lipid nanoparticle (LNP), comprising such polynucleotide.
In certain embodiments, the method comprises administering a
FLT3L-Fc fusion protein, as described herein, a homodimer or
heterodimer comprising such fusion protein, a polynucleotide
encoding such fusion protein, a vector or lipoplex, such as a lipid
nanoparticle (LNP), comprising such polynucleotide, in combination
with one or more (e.g., one, two, three, one or two, or one to
three) additional therapeutic agents. In certain embodiments, the
subject is at risk of contracting the HBV virus, such as a subject
who has one or more risk factors known to be associated with
contracting the HBV virus. In certain embodiments, the subject may
have not previously received antiviral treatment (treatment naive).
In certain embodiments, the subject may have previously received
antiviral treatment (treatment experienced). In certain
embodiments, the subject may have previously received antiviral
treatment and developed resistance to the previously received
antiviral treatment. In some embodiments, a method for inhibiting
the replication of the HBV virus, treating an HBV infection or
delaying the onset of symptoms of an HBV infection in a subject
(e.g., a human), comprising administering an effective amount of a
FLT3L-Fc fusion protein, as described herein to the subject is
disclosed.
[0194] In a further embodiment, provided are methods of treating or
preventing a human immunodeficiency virus (HIV) infection
comprising administering to a subject in need thereof an effective
amount of a FLT3L-Fc fusion protein, as described herein, a
homodimer or heterodimer comprising such fusion protein, a
polynucleotide encoding such fusion protein, a vector or lipoplex,
such as an lipid nanoparticle (LNP), comprising such
polynucleotide. In certain embodiments, provided are methods of
treating an HIV infection comprising administering to a subject in
need thereof an effective amount of a FLT3L-Fc fusion protein, as
described herein, a homodimer or heterodimer comprising such fusion
protein, a polynucleotide encoding such fusion protein, a vector or
lipoplex, such as a lipid nanoparticle (LNP), comprising such
polynucleotide. In certain embodiments, the method comprises
administering a FLT3L-Fc fusion protein, as described herein, a
homodimer or heterodimer comprising such fusion protein, a
polynucleotide encoding such fusion protein, a vector or lipoplex,
such as a lipid nanoparticle (LNP), comprising such polynucleotide,
in combination with one or more (e.g., one, two, three, one or two,
or one to three) additional therapeutic agents. In certain
embodiments, the subject is at risk of contracting the HIV virus,
such as a subject who has one or more risk factors known to be
associated with contracting the HIV virus. In certain embodiments,
the subject may have not previously received antiviral treatment
(treatment naive). In certain embodiments, the subject may have
previously received antiviral treatment (treatment experienced). In
certain embodiments, the subject may have previously received
antiviral treatment and developed resistance to the previously
received antiviral treatment. In some embodiments, a method for
inhibiting the replication of the HIV virus, treating AIDS or
delaying the onset of AIDS in a subject (e.g., a human), comprising
administering an effective amount of a FLT3L-Fc fusion protein, as
described herein, to the subject is disclosed.
[0195] In a further embodiment, provided are methods of treating or
preventing a coronavirus infection comprising administering to a
subject in need thereof an effective amount of a FLT3L-Fc fusion
protein, as described herein, a homodimer or heterodimer comprising
such fusion protein, a polynucleotide encoding such fusion protein,
a vector or lipoplex, such as an lipid nanoparticle (LNP),
comprising such polynucleotide. In certain embodiments, provided
herein are methods of treating a coronavirus infection comprising
administering to a subject in need thereof an effective amount of a
FLT3L-Fc fusion protein, as described herein, a homodimer or
heterodimer comprising such fusion protein, a polynucleotide
encoding such fusion protein, a vector or lipoplex, such as a lipid
nanoparticle (LNP) comprising such polynucleotide. In certain
embodiments, the method comprises administering a compound
disclosed herein, or a pharmaceutically acceptable salt thereof, in
combination with one or more (e.g., one, two, three, one or two, or
one to three) additional therapeutic agents. In certain
embodiments, the subject is at risk of contracting the coronavirus.
In certain embodiments, the subject may have not previously
received antiviral treatment (treatment naive). In certain
embodiments, the subject may have previously received antiviral
treatment (treatment experienced). In certain embodiments, the
subject may have previously received antiviral treatment and
developed resistance to the previously received antiviral
treatment. In some embodiments, a method for inhibiting the
replication of a coronavirus, treating a coronavirus or delaying
the onset of symptoms of a coronavirus infection in a subject
(e.g., a human), comprising administering an effective amount of a
FLT3L-Fc fusion protein, as described herein, to the subject is
disclosed. In certain embodiments, the coronavirus infection is an
infection caused by the SARS virus, the MERS virus, or the
2019-nCoV (COVID-19) virus. In certain embodiments, the coronavirus
infection is an infection caused by the SARS virus. In certain
embodiments, the coronavirus infection is an infection caused by
the MERS virus. In certain embodiments, the coronavirus infection
is an infection caused by the 2019-nCoV (COVID-19) virus.
[0196] With respect to route of administration, in various
embodiments, the FLT3L-Fc fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition are
administered systemically or locally. In some embodiments, the
FLT3L-Fc fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide, the vector, the lipoplex, such as an
LNP, and/or the pharmaceutical composition can be administered
intravenously, intratumorally, subcutaneously, intradermally,
intramuscularly, intraperitoneally, intravesically, intracranially,
intrathecally, intracavitary or intraventricularly. In embodiments
involving combination therapies, as appropriate, the FLT3L-Fc
fusion protein, the homodimer, the heterodimer, the conjugate, the
polynucleotide, the vector, the lipoplex, such as an LNP, and/or
the pharmaceutical composition and the one or more additional
therapeutic agents can be administered by the same or different
routes of administration. As appropriate, in certain embodiments,
administration is via injection or infusion.
[0197] With respect to dosing, a therapeutically effective amount
of FLT3L-Fc fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide, the vector, the lipoplex, such as an
LNP, and/or the pharmaceutical composition is administered to the
subject. As used herein, a "therapeutically effective amount" means
an amount sufficient to induce, promote and/or increase expansion
and/or proliferation of FLT3+ cells, and thereby treat a subject
(such as a human) suffering an indication, or to alleviate the
existing symptoms of the indication (e.g., cancer, viral infection,
bacterial infection). Determination of a therapeutically effective
amount is within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein.
[0198] In some embodiments, a therapeutically effective amount of a
FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate,
polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, optionally, with
one or more additional therapeutic agents, as described herein, can
(i) reduce the number of diseased cells; (ii) reduce tumor size;
(iii) inhibit, retard, slow to some extent, and preferably stop the
diseased cell infiltration into peripheral organs; (iv) inhibit
(e.g., slow to some extent and preferably stop) tumor metastasis;
(v) inhibit tumor growth; (vi) prevent or delay occurrence and/or
recurrence of a tumor; and/or (vii) relieve to some extent one or
more of the symptoms associated with cancer or myeloproliferative
disease. In some embodiments, a therapeutically effective amount of
a FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate,
polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, optionally, with
one or more additional therapeutic agents, as described herein, can
(i) reduce the number of cancer cells; (ii) reduce tumor size;
(iii) inhibit, retard, slow to some extent, and preferably stop
cancer cell infiltration into peripheral organs; (iv) inhibit
(e.g., slow to some extent and preferably stop) tumor metastasis;
(v) inhibit tumor growth; (vi) prevent or delay occurrence and/or
recurrence of a tumor; and/or (vii) relieve to some extent one or
more of the symptoms associated with the cancer. In various
embodiments, the amount is sufficient to ameliorate, palliate,
lessen, and/or delay one or more of symptoms of cancer.
[0199] In some embodiments, a therapeutically effective amount of a
FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate,
polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, optionally, with
one or more additional therapeutic agents, as described herein, can
inhibit the proliferation of a virus in a subject and/or delay to
some extent one or more of the symptoms associated with viral
infection (e.g., AIDS, SARS, MERS, liver disease caused by HBV). In
various embodiments, the amount is sufficient to ameliorate,
palliate, lessen, and/or delay one or more of symptoms of a viral
infection.
[0200] An "increased" or "enhanced" amount (e.g., with respect to
FLT3L+ cell expansion, antitumor response, cancer cell metastasis)
refers to an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30,
40, or 50 or more times (e.g., 100, 500, 1000 times) (including all
integers and decimal points in between and above 1, e.g., 2.1, 2.2,
2.3, 2.4, etc.) an amount or level described herein. It may also
include an increase of at least 10%, at least 20%, at least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 100%, at least 150%, at least 200%, at least
500%, or at least 1000% of an amount or level described herein.
[0201] A "decreased" or "reduced" or "lesser" amount (e.g., with
respect to tumor size, cancer cell proliferation or growth) refers
to a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8,
1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or
50 or more times (e.g., 100, 500, 1000 times) (including all
integers and decimal points in between and above 1, e.g., 1.5, 1.6,
1.7. 1.8, etc.) an amount or level described herein. It may also
include a decrease of at least 10%, at least 20%, at least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
or at least 90%, at least 100%, at least 150%, at least 200%, at
least 500%, or at least 1000% of an amount or level described
herein.
[0202] In some embodiments, the FLT3L-Fc fusion protein, homodimer,
heterodimer, conjugate, polynucleotide, vector, lipoplex, such as
an LNP, and/or pharmaceutical composition, as described herein, is
administered at a dose in the range of about 0.5 .mu.g/kg to about
5000 .mu.g/kg, e.g., at least about 0.5 .mu.g/kg per dose and up to
about 1 .mu.g/kg, 2 .mu.g/kg, 3 .mu.g/kg, 4 .mu.g/kg, 5 .mu.g/kg, 6
.mu.g/kg, 7 .mu.g/kg, 8 .mu.g/kg, 9 .mu.g/kg, 10 .mu.g/kg, 15
.mu.g/kg, 20 .mu.g/kg, 30 .mu.g/kg, 50 .mu.g/kg, 100 .mu.g/kg, 150
.mu.g/kg, 300 .mu.g/kg, 400 .mu.g/kg, 500 .mu.g/kg, 600 .mu.g/kg,
700 .mu.g/kg, 800 .mu.g/kg, 900 .mu.g/kg, 1000 .mu.g/kg, 1500
.mu.g/kg, 2000 .mu.g/kg, 2500 .mu.g/kg, 3000 .mu.g/kg, 3500
.mu.g/kg, 4000 .mu.g/kg, or 5000 .mu.g/kg, per dose. In some
embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer,
conjugate, polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, is administered at
a dose in the range of about 1 .mu.g/kg to about 100 .mu.g/kg, per
dose. In some embodiments, the FLT3L-Fc fusion protein, homodimer,
heterodimer, conjugate, polynucleotide, vector, lipoplex, such as
an LNP, and/or pharmaceutical composition, as described herein, is
administered at a dose of 1 .mu.g/kg, per dose. In some
embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer,
conjugate, polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, is administered at
a dose of 3 .mu.g/kg, per dose. In some embodiments, the FLT3L-Fc
fusion protein, homodimer, heterodimer, conjugate, polynucleotide,
vector, lipoplex, such as an LNP, and/or pharmaceutical
composition, as described herein, is administered at a dose of 10
.mu.g/kg, per dose. In some embodiments, the FLT3L-Fc fusion
protein, homodimer, heterodimer, conjugate, polynucleotide, vector,
lipoplex, such as an LNP, and/or pharmaceutical composition, as
described herein, is administered at a dose of 30 .mu.g/kg, per
dose. In some embodiments, the FLT3L-Fc fusion protein, homodimer,
heterodimer, conjugate, polynucleotide, vector, lipoplex, such as
an LNP, and/or pharmaceutical composition, as described herein, is
administered at a dose of 60 .mu.g/kg, per dose. In some
embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer,
conjugate, polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, is administered at
a dose of 100 .mu.g/kg, per dose. In some embodiments, the FLT3L-Fc
fusion protein, homodimer, heterodimer, conjugate, polynucleotide,
vector, lipoplex, such as an LNP, and/or pharmaceutical
composition, as described herein, is administered at a dose in the
range of about 0.5 mg to about 50 mg, e.g., at least about 0.5 mg
per dose and up to about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg,
8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg,
18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg per
dose. In some embodiments, the FLT3L-Fc fusion protein, homodimer,
heterodimer, conjugate, polynucleotide, vector, lipoplex, such as
an LNP, and/or pharmaceutical composition, as described herein, is
administered at a dose of dose about 10 mg, per dose. In some
embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer,
conjugate, polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, is administered at
a dose that saturates FLT3 receptors in the tumor. In some
embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer,
conjugate, polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein, is administered at
a dose that saturates FLT3 receptors in the subject.
[0203] With respect to scheduling of administrations, in various
embodiments, the methods comprise administering multiple
administrations or doses of the fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition,
optionally with one or more additional therapeutic agents, at
predetermined intervals. As appropriate, in various embodiments,
the FLT3L-Fc fusion protein, the homodimer, the heterodimer, the
conjugate, the polynucleotide, the vector, the lipoplex, such as an
LNP, and/or the pharmaceutical composition can be administered once
weekly (i.e., QW), once bi-weekly (i.e., once every other week, or
once every two weeks or Q2W), once thrice-weekly (i.e., once every
three weeks or Q3W), once monthly (i.e., QM) or once bi-monthly
dosing (i.e., once every other month, or once every two months or
Q2M), or less often. As appropriate, the fusion protein, the
homodimer, the heterodimer, the conjugate, the polynucleotide, the
vector, the lipoplex, such as an LNP, and/or the pharmaceutical
composition and the one or more additional therapeutic agents can
be co-administered according to the same schedule (e.g.,
co-administered at the same time intervals) or different schedules
(e.g., co-administered at different time intervals). in various
embodiments, the FLT3L-Fc fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition can
be administered first, followed by administration of the one or
more additional therapeutic agents, e.g., 1, 2 or 3 weeks later,
e.g., after detectable or sufficient expansion of FLT3-expressing
cells, e.g., cDC1 dendritic cells.
[0204] Examples of tissues containing cancerous cells whose
proliferation is inhibited by the FLT3L-Fc fusion proteins
described herein and against which the methods described herein are
useful include but are not limited to breast, prostate, brain,
blood, bone marrow, liver, pancreas, skin, kidney, colon, ovary,
lung, testicle, penis, thyroid, parathyroid, pituitary, thymus,
retina, uvea, conjunctiva, spleen, head, neck, trachea, gall
bladder, rectum, salivary gland, adrenal gland, throat, esophagus,
lymph nodes, sweat glands, sebaceous glands, muscle, heart, and
stomach.
[0205] In some embodiments, the subject has a solid tumor. In
various embodiments, the cancer or tumor is malignant and/or a
metastatic. In various embodiments, the subject has a cancer
selected from the group consisting of an epithelial tumor (e.g., a
carcinoma, a squamous cell carcinoma, a basal cell carcinoma, a
squamous intraepithelial neoplasia), a glandular tumor (e.g., an
adenocarcinoma, an adenoma, an adenomyoma), a mesenchymal or soft
tissue tumor (e.g., a sarcoma, a rhabdomyosarcoma, a
leiomyosarcoma, a liposarcoma, a fibrosarcoma, a
dermatofibrosarcoma, a neurofibrosarcoma, a fibrous histiocytoma,
an angiosarcoma, an angiomyxoma, a leiomyoma, a chondroma, a
chondrosarcoma, an alveolar soft-part sarcoma, an epithelioid
hemangioendothelioma, a Spitz tumor, a synovial sarcoma), and a
lymphoma.
[0206] In various embodiments, the subject has a solid tumor in or
arising from a tissue or organ selected from the group consisting
of: [0207] bone (e.g., adamantinoma, aneurysmal bone cysts,
angiosarcoma, chondroblastoma, chondroma, chondromyxoid fibroma,
chondrosarcoma, chordoma, dedifferentiated chondrosarcoma,
enchondroma, epithelioid hemangioendothelioma, fibrous dysplasia of
the bone, giant cell tumour of bone, haemangiomas and related
lesions, osteoblastoma, osteochondroma, osteosarcoma, osteoid
osteoma, osteoma, periosteal chondroma, Desmoid tumor, Ewing
sarcoma); [0208] lips and oral cavity (e.g., odontogenic
ameloblastoma, oral leukoplakia, oral squamous cell carcinoma,
primary oral mucosal melanoma); salivary glands (e.g., pleomorphic
salivary gland adenoma, salivary gland adenoid cystic carcinoma,
salivary gland mucoepidermoid carcinoma, salivary gland Warthin's
tumors); [0209] esophagus (e.g., Barrett's esophagus, dysplasia and
adenocarcinoma); [0210] gastrointestinal tract, including stomach
(e.g., gastric adenocarcinoma, primary gastric lymphoma,
gastrointestinal stromal tumors (GISTs), metastatic deposits,
gastric carcinoids, gastric sarcomas, neuroendocrine carcinoma,
gastric primary squamous cell carcinoma, gastric adenoacanthomas),
intestines and smooth muscle (e.g., intravenous leiomyomatosis),
colon (e.g., colorectal adenocarcinoma), rectum, anus; [0211]
pancreas (e.g., serous neoplasms, including microcystic or
macrocystic serous cystadenoma, solid serous cystadenoma, Von
Hippel-Landau (VHL)-associated serous cystic neoplasm, serous
cystadenocarcinoma; mucinous cystic neoplasms (MCN), intraductal
papillary mucinous neoplasms (IPMN), intraductal oncocytic
papillary neoplasms (IOPN), intraductal tubular neoplasms, cystic
acinar neoplasms, including acinar cell cystadenoma, acinar cell
cystadenocarcinoma, pancreatic adenocarcinoma, invasive pancreatic
ductal adenocarcinomas, including tubular adenocarcinoma,
adenosquamous carcinoma, colloid carcinoma, medullary carcinoma,
hepatoid carcinoma, signet ring cell carcinoma, undifferentiated
carcinoma, undifferentiated carcinoma with osteoclast-like giant
cells, acinar cell carcinoma, neuroendocrine neoplasms,
neuroendocrine microadenoma, neuroendocrine tumors (NET),
neuroendocrine carcinoma (NEC), including small cell or large cell
NEC, insulinoma, gastrinoma, glucagonoma, serotonin-producing NET,
somatostatinoma, VIPoma, solid-pseudopapillary neoplasms (SPN),
pancreatoblastoma); [0212] gall bladder (e.g., carcinoma of the
gallbladder and extrahepatic bile ducts, intrahepatic
cholangiocarcinoma); [0213] neuro-endocrine (e.g., adrenal cortical
carcinoma, carcinoid tumors, phaeochromocytoma, pituitary
adenomas); [0214] thyroid (e.g., anaplastic (undifferentiated)
carcinoma, medullary carcinoma, oncocytic tumors, papillary
carcinoma, adenocarcinoma); [0215] liver (e.g., adenoma, combined
hepatocellular and cholangiocarcinoma, fibrolamellar carcinoma,
hepatoblastoma, hepatocellular carcinoma, mesenchymal, nested
stromal epithelial tumor, undifferentiated carcinoma;
hepatocellular carcinoma, intrahepatic cholangiocarcinoma, bile
duct cystadenocarcinoma, epithelioid hemangioendothelioma,
angiosarcoma, embryonal sarcoma, rhabdomyosarcoma, solitary fibrous
tumor, teratoma, York sac tumor, carcinosarcoma, rhabdoid tumor);
[0216] kidney (e.g., ALK-rearranged renal cell carcinoma,
chromophobe renal cell carcinoma, clear cell renal cell carcinoma,
clear cell sarcoma, metanephric adenoma, metanephric adenofibroma,
mucinous tubular and spindle cell carcinoma, nephroma,
nephroblastoma (Wilms tumor), papillary adenoma, papillary renal
cell carcinoma, renal oncocytoma, renal cell carcinoma, succinate
dehydrogenase-deficient renal cell carcinoma, collecting duct
carcinoma); [0217] breast (e.g., invasive ductal carcinoma,
including without limitation, acinic cell carcinoma, adenoid cystic
carcinoma, apocrine carcinoma, cribriform carcinoma,
glycogen-rich/clear cell, inflammatory carcinoma, lipid-rich
carcinoma, medullary carcinoma, metaplastic carcinoma,
micropapillary carcinoma, mucinous carcinoma, neuroendocrine
carcinoma, oncocytic carcinoma, papillary carcinoma, sebaceous
carcinoma, secretory breast carcinoma, tubular carcinoma; lobular
carcinoma, including without limitation, pleomorphic carcinoma,
signet ring cell carcinoma); [0218] peritoneum (e.g., mesothelioma;
primary peritoneal cancer); [0219] female sex organ tissues,
including ovary (e.g., choriocarcinoma, epithelial tumors, germ
cell tumors, sex cord-stromal tumors), Fallopian tubes (e.g.,
serous adenocarcinoma, mucinous adenocarcinoma, endometrioid
adenocarcinoma, clear cell adenocarcinoma, transitional cell
carcinoma, squamous cell carcinoma, undifferentiated carcinoma,
mullerian tumors, adenosarcoma, leiomyosarcoma, teratoma, germ cell
tumors, choriocarcinoma, trophoblastic tumors), uterus (e.g.,
carcinoma of the cervix, endometrial polyps, endometrial
hyperplasia, intraepithelial carcinoma (EIC), endometrial carcinoma
(e.g., endometrioid carcinoma, serous carcinoma, clear cell
carcinoma, mucinous carcinoma, squamous cell carcinoma,
transitional carcinoma, small cell carcinoma, undifferentiated
carcinoma, mesenchymal neoplasia), leiomyoma (e.g., endometrial
stromal nodule, leiomyosarcoma, endometrial stromal sarcoma (ESS),
mesenchymal tumors), mixed epithelial and mesenchymal tumors (e.g.,
adenofibroma, carcinofibroma, adenosarcoma, carcinosarcoma
(malignant mixed mesodermal sarcoma-MMMT)), endometrial stromal
tumors, endometrial malignant mullerian mixed tumours, gestational
trophoblastic tumors (partial hydatiform mole, complete hydatiform
mole, invasive hydatiform mole, placental site tumour)), vulva,
vagina; [0220] male sex organ tissues, including prostate, testis
(e.g., germ cell tumors, spermatocytic seminoma), penis; [0221]
bladder (e.g., squamous cell carcinoma, urothelial carcinoma,
bladder urothelial carcinoma); [0222] brain, (e.g., gliomas (e.g.,
astrocytomas, including non-infiltrating, low-grade, anaplastic,
glioblastomas; oligodendrogliomas, ependymomas), meningiomas,
gangliogliomas, schwannomas (neurilemmomas), craniopharyngiomas,
chordomas, Non-Hodgkin lymphomas (NHLs), indolent non-Hodgkin's
lymphoma (iNHL), refractory iNHL, pituitary tumors; [0223] eye
(e.g., retinoma, retinoblastoma, ocular melanoma, posterior uveal
melanoma, iris hamartoma); [0224] head and neck (e.g.,
nasopharyngeal carcinoma, Endolymphatic Sac Tumor (ELST),
epidermoid carcinoma, laryngeal cancers including squamous cell
carcinoma (SCC) (e.g., glottic carcinoma, supraglottic carcinoma,
subglottic carcinoma, transglottic carcinoma), carcinoma in situ,
verrucous, spindle cell and basaloid SCC, undifferentiated
carcinoma, laryngeal adenocarcinoma, adenoid cystic carcinoma,
neuroendocrine carcinomas, laryngeal sarcoma), head and neck
paragangliomas (e.g., carotid body, jugulotympanic, vagal); [0225]
thymus (e.g., thymoma); [0226] heart (e.g., cardiac myxoma); [0227]
lung (e.g., small cell carcinoma (SCLC), non-small cell lung
carcinoma (NSCLC), including squamous cell carcinoma (SCC),
adenocarcinoma and large cell carcinoma, carcinoids (typical or
atypical), carcinosarcomas, pulmonary blastomas, giant cell
carcinomas, spindle cell carcinomas, pleuropulmonary blastoma);
[0228] lymph (e.g., lymphomas, including Hodgkin's lymphoma,
non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma
(iNHL), refractory iNHL, Epstein-Barr virus (EBV)-associated
lymphoproliferative diseases, including B cell lymphomas and T cell
lymphomas (e.g., Burkitt lymphoma; large B cell lymphoma, diffuse
large B-cell lymphoma (DLBCL), mantle cell lymphoma, indolent
B-cell lymphoma, low grade B cell lymphoma, fibrin-associated
diffuse large cell lymphoma; primary effusion lymphoma;
plasmablastic lymphoma; extranodal NK/T cell lymphoma, nasal type;
peripheral T cell lymphoma, cutaneous T cell lymphoma,
angioimmunoblastic T cell lymphoma; follicular T cell lymphoma;
systemic T cell lymphoma), lymphangioleiomyomatosis); [0229]
central nervous system (CNS) (e.g., gliomas including astrocytic
tumors (e.g., pilocytic astrocytoma, pilomyxoid astrocytoma,
subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma,
diffuse astrocytoma, fibrillary astrocytoma, gemistocytic
astrocytoma, protoplasmic astrocytoma, anaplastic astrocytoma,
glioblastoma (e.g., giant cell glioblastoma, gliosarcoma,
glioblastoma multiforme) and gliomatosis cerebri), oligodendroglial
tumors (e.g., oligodendroglioma, anaplastic oligodendroglioma),
oligoastrocytic tumors (e.g., oligoastrocytoma, anaplastic
oligoastrocytoma), ependymal tumors (e.g., subependymom,
myxopapillary ependymoma, ependymomas (e.g., cellular, papillary,
clear cell, tanycytic), anaplastic ependymoma), optic nerve glioma,
and non-gliomas (e.g., choroid plexus tumors, neuronal and mixed
neuronal-glial tumors, pineal region tumors, embryonal tumors,
medulloblastoma, meningeal tumors, primary CNS lymphomas, germ cell
tumors, Pituitary adenomas, cranial and paraspinal nerve tumors,
stellar region tumors); neurofibroma, meningioma, peripheral nerve
sheath tumors, peripheral neuroblastic tumours (including without
limitation neuroblastoma, ganglioneuroblastoma, ganglioneuroma),
trisomy 19 ependymoma); [0230] neuroendocrine tissues (e.g.,
paraganglionic system including adrenal medulla (pheochromocytomas)
and extra-adrenal paraganglia ((extra-adrenal) paragangliomas);
[0231] skin (e.g., clear cell hidradenoma, cutaneous benign fibrous
histiocytomas, cylindroma, hidradenoma, melanoma (including
cutaneous melanoma, mucosal melanoma), basal cell carcinoma,
pilomatricoma, Spitz tumors); and [0232] soft tissues (e.g.,
aggressive angiomyxoma, alveolar rhabdomyosarcoma, alveolar soft
part sarcoma, angiofibroma, angiomatoid fibrous histiocytoma,
synovial sarcoma, biphasic synovial sarcoma, clear cell sarcoma,
dermatofibrosarcoma protuberans, desmoid-type fibromatosis, small
round cell tumor, desmoplastic small round cell tumor,
elastofibroma, embryonal rhabdomyosarcoma, Ewing's tumors/primitive
neurectodermal tumors (PNET), extraskeletal myxoid chondrosarcoma,
extraskeletal osteosarcoma, paraspinal sarcoma, inflammatory
myofibroblastic tumor, lipoblastoma, lipoma, chondroid lipoma,
liposarcoma/malignant lipomatous tumors, liposarcoma, myxoid
liposarcoma, fibromyxoid sarcoma, lymphangioleiomyoma, malignant
myoepithelioma, malignant melanoma of soft parts, myoepithelial
carcinoma, myoepithelioma, myxoinflammatory fibroblastic sarcoma,
undifferentiated sarcoma, pericytoma, rhabdomyosarcoma,
non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), soft tissue
leiomyosarcoma, undifferentiated sarcoma, well-differentiated
liposarcoma.
[0233] In some embodiments, the subject has a hematological cancer,
e.g., a leukemia (e.g., Acute Myelogenous Leukemia (AML), Acute
Lymphoblastic Leukemia (ALL), B-cell ALL, Myelodysplastic Syndrome
(MDS), myeloproliferative disease (MPD), Chronic Myelogenous
Leukemia (CML), Chronic Lymphocytic Leukemia (CLL),
undifferentiated leukemia), a lymphoma (e.g., small lymphocytic
lymphoma (SLL), mantle cell lymphoma (MCL), follicular lymphoma
(FL), T-cell lymphoma, B-cell lymphoma, diffuse large B-cell
lymphoma (DLBCL), marginal zone lymphoma (MZL), Waldestrom's
macroglobulinemia (WM)) and/or a myeloma (e.g., multiple myeloma
(MM)).
[0234] In some embodiments, the subject has a tumor that is
infiltrated with conventional dendritic cells (cDCs). In some
embodiments, the tumor infiltrating dendritic cells express C-C
motif chemokine receptor 5 (CCR5, CD195) and/or X-C motif chemokine
receptor 1 (XCR1) on their cell surface and/or produce CXCL9/10.
Expression of XCR1 and CCR5 by cDC1 enables local recruitment by
cytotoxic lymphocytes producing the ligands for these chemokine
receptors, XCL1 and CCL4/5. cDC1 ability to produce CXCL9/10
promotes local recruitment of effector and memory CTLs expressing
CXCR3. Cancel, et al., Front Immunol. (2019) 10:9. In some
embodiments, the tumor infiltrating dendritic cells express one or
more cell surface proteins selected from the group consisting of
XCR1, interferon regulatory factor 8 (IRF8), cell adhesion molecule
1 (CADM1), C-type lectin domain containing 9A (CLEC9A, CD370), and
thrombomodulin (THBD), which identify a cDC1 subtype. In some
embodiments, the tumor infiltrating dendritic cells express one or
more proteins selected from the group consisting of XCR1, IRF8,
CADM1, CLEC9A, THBD, copine 3 (CPNE3), carboxypeptidase
vitellogenic like (CPVL), N-acylethanolamine acid amidase (NAAA),
cystatin C (CST3), WDFY family member 4 (WDFY4) and galectin 2
(LGALS2), which identify a cDC.sub.1 subtype. cDC.sub.1 cells are
efficient antigen cross-presenters to CD8+ T cells. In some
embodiments, the tumor infiltrating dendritic cells express one or
more cell surface proteins selected from the group consisting of
CD1A, CD1C, CD1E, signal regulatory protein alpha (SIRPA; CD172A),
CD207 and Fc fragment of IgE receptor Ia (FCER1A), which identify a
cDC.sub.2 subtype. In some embodiments, the tumor infiltrating
dendritic cells express one or more cell surface proteins selected
from the group consisting of CD1A, CD1C, CD1E, SIRPA, FCER1A,
CD207, HLA-DQA2, HLA-DQB2, Fc fragment of IgG binding protein
(FCGBP), S100 calcium binding protein B (S100B), NDRG family member
2 (NDRG2), interleukin 22 receptor subunit alpha 2 (IL22RA2), and
chondroadherin (CHAD), which identify a cDC2 subtype. cDC.sub.2
cells preferentially interact with CD4+ T cells. In some
embodiments, the tumor infiltrating dendritic cells expresses one
or more proteins selected from the group consisting of basic
leucine zipper ATF-like transcription factor 3 (BATF3) and
interferon regulatory factor 8 (RF8), identifying an "activated" DC
phenotype or hDC.sub.3 subtype. In some embodiments, the tumor
infiltrating dendritic cells expresses one or more proteins
selected from the group consisting of BATF3, IRF8, C-C motif
chemokine ligand 22 (CCL22), lymphocyte antigen 75 (LY75), C-C
motif chemokine receptor 7 (CCR7), protein O-glucosyltransferase 1
(POGLUTI), lysine demethylase 2B (KDM2B), INSM transcriptional
repressor 1 (INSM1), and UV radiation resistance associated
(UVRAG), identifying an "activated" DC phenotype or hDC.sub.3
subtype. Expression signatures of various dendritic cell subtypes
are described in Zilionis et al., Immunity (2019) 50, 1317-1334. In
some embodiments, the tumor infiltrating dendritic cells express
one or more cell surface proteins selected from the group
consisting of XCR1, BATF3, RF8, CLEC9A and THBD.
[0235] Administration of the FLT3L-Fc proteins described herein can
promote or increase expansion and/or infiltration of myeloid cells
(e.g., T-cells, NK cells and dendritic cells) into a tumor.
Further, administration of the FLT3L-Fc proteins described herein
can improve, increase, enhance and/or promote the antitumor effects
or efficacy of an immune checkpoint inhibitor. In some embodiments,
the subject has a cancer that detectably expresses or overexpresses
one or more cell surface immune checkpoint receptors. In certain
embodiments, greater than about 50% of the cells within the tumor
(e.g., tumor cells, T cells and/or NK cells within the tumor)
detectably express one or more cell surface immune checkpoint
proteins (e.g., the subject has a so-called "hot" cancer or tumor).
In some embodiments, greater than about 1% and less than about 50%
of the cells within the tumor (e.g., tumor cells, T cells and/or NK
cells within the tumor) detectably express one or more cell surface
immune checkpoint proteins (e.g., the subject has a so called
"warm" cancer or tumor). In some embodiments, the one or more cell
surface immune checkpoint receptors are selected from the group
consisting of. CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2),
transmembrane and immunoglobulin domain containing 2 (TMIGD2,
CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244
(SLAMF4); CD276 (B7H3); V-set domain containing T cell activation
inhibitor 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR,
B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3);
natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1,
B7H6); HERV-H LTR-associating 2 (HHLA2, B7H7); inducible T cell
costimulator (ICOS, CD278); inducible T cell costimulator ligand
(ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40);
TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8
(CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9
(CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL);
TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte
associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF);
TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related
sequence A (MICA); MHC class I polypeptide-related sequence B
(MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand
2 (PDCDILG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PDi,
PD-1); cytotoxic T-lymphocyte associated protein 4 (CTLA4, CD152);
CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2,
CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell adhesion
molecule (PVR, CD155); PVR related immunoglobulin domain containing
(PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains
(TIGIT); T cell immunoglobulin and mucin domain containing 4
(TIMD4; TTM4); hepatitis A virus cellular receptor 2 (HAVCR2,
TIMD3, TIM3); galectin 9 (LGALS9); signaling lymphocytic activation
molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen
9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM
family member 7 (SLAMF7, CD319); UL16 binding protein 1 (ULBP1);
UL16 binding protein 2 (ULBP2); UL16 binding protein 3 (ULBP3);
retinoic acid early transcript 1E (RAETIE; ULBP4); retinoic acid
early transcript 1G (RAETIG; ULBP5); retinoic acid early transcript
1L (RAET1L; ULBP6); lymphocyte activating 3 (CD223); killer cell
immunoglobulin like receptor, three Ig domains and long cytoplasmic
tail 1 (KIR, CD158E1); killer cell lectin like receptor C1 (KLRC1,
NKG2A, CD159A); killer cell lectin like receptor K1 (KLRK1, NKG2D,
CD314); killer cell lectin like receptor C2 (KLRC2, CD159c, NKG2C);
killer cell lectin like receptor C3 (KLRC3, NKG2E); killer cell
lectin like receptor C4 (KLRC4, NKG2F); killer cell immunoglobulin
like receptor, two Ig domains and long cytoplasmic tail 1
(KIR2DL1); killer cell immunoglobulin like receptor, two Ig domains
and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin
like receptor, two Ig domains and long cytoplasmic tail 3
(KIR2DL3); killer cell immunoglobulin like receptor, three Ig
domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin
like receptor D1 (KLRD1); killer cell lectin like receptor G1
(KLRG1; CLEC15A, MAFA, 2F1); sialic acid binding Ig like lectin 7
(SIGLEC7); and sialic acid binding Ig like lectin 9 (SIGLEC9).
[0236] The FLT3L-Fc variants described herein can be used to
promote or accelerate the recovery from or reverse the effects of
lymphopenia or neutropenia. Accordingly, in some embodiments, the
subject has neutropenia or lymphopenia, e.g., as a result of having
received or undergone a lymphodepleting chemotherapy regimen, e.g.,
an alkylating agent such as chlorambucil or cyclophosphamide, or a
nucleoside analog, including pyrimidine nucleosides such as
cytarabine and purine nucleosides such as cladribine, pentostatin
and fludarabine. See, e.g., Lowe, et al., Gene Therapy (2018)
25:176-191. In certain embodiments, the methods comprise (a)
subjecting a patient to a lymphodepleting chemotherapy regimen; (b)
administering a FLT3L-Fc fusion protein, homodimer, heterodimer,
conjugate, polynucleotide, vector, lipoplex, such as an LNP, and/or
pharmaceutical composition, as described herein; and (c)
administering to the patient a cellular therapy, as described
herein. Illustrative lymphodepleting chemotherapy regimens, along
with correlative beneficial biomarkers, are described in WO
2016/191756 and WO 2019/079564, incorporated herein by reference in
their entireties for all purposes. In certain embodiments, the
lymphodepleting chemotherapy regimen comprises administering to the
patient doses of cyclophosphamide (between 200 mg/m.sup.2/day and
2000 mg/m.sup.2/day) and doses of fludarabine (between 20
mg/m.sup.2/day and 900 mg/m.sup.2/day). One such dose regimen
involves treating a patient comprising administering daily to the
patient about 500 mg/m.sup.2/day of cyclophosphamide and about 60
mg/m.sup.2/day of fludarabine for three days, e.g., prior to
administration of a therapeutically effective amount of a cellular
therapy (e.g., an effector cell having a chimeric antigen receptor)
to the patient. In another example, in some embodiments, a
lymphodepleting chemotherapy regimen of cyclophosphamide 500
mg/m.sup.2 IV and fludarabine 30 mg/m2 IV on the fifth, fourth, and
third day e.g., prior to administration of a therapeutically
effective amount of a cellular therapy (e.g., an effector cell
having a chimeric antigen receptor) to the patient. In some
embodiments, the subject is naive to or has not received
chemotherapy. In some embodiments, the subject has bone marrow
cells (e.g., is not depleted of bone marrow cells).
[0237] In some embodiments, the subject does not have a mutation in
the gene encoding the FLT3 receptor that causes or results in or is
associated with cancer, e.g., FLT3 mutations associated with
constitutive signaling of the FLT3 receptor, e.g., FLT3 mutations
associated with Acute Myeloid Leukemia (AML). For example, in
certain embodiments, the subject does not have internal tandem
duplication (ITD) of the FMS-related tyrosine kinase 3 (FLT3) gene,
which occurs in exons 14 and 15, and is one of the most prevalent
somatic mutations in adult acute myeloid leukemia (AML). In some
embodiments, the subject does not have a mutation in the FLT3 gene
in exon 20 that affects codon 835, encoding the tyrosine kinase
domain (TKD) mutation, which occurs relatively frequently in adult
AML. In some embodiments, the subject does not have point mutations
affecting amino acid positions D835 (e.g., resulting in D835Y,
D835V, and D835H amino acid substitutions) and/or 1836 in the TKD.
See, e.g., Azari-Yam, et al., Clin Lab. (2016) 62(10):2011-2017;
Han, et al., Zhongguo Shi Yan Xue Ye Xue Za Zhi. (2009)
17(5):1135-9; Shoji, et al., Rinsho Byori. (2017) 65(1):44-5; and
Liang, et al., Leukemia. (2003) 17(5):883-6.
8. Combination Therapies
[0238] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplex, such
as an LNPs, and/or pharmaceutical compositions, as described
herein, are combined with one or more additional therapeutic
agents, e.g., an inhibitory immune checkpoint blocker or inhibitor,
a stimulatory immune checkpoint stimulator, agonist or activator, a
chemotherapeutic agent, an anticancer agent, an antiviral agent, a
radiotherapeutic agent, an anti-neoplastic agent, an
anti-proliferation agent, an anti-angiogenic agent, an
anti-inflammatory agent, an immunotherapeutic agent, a therapeutic
antigen-binding molecule (mono- and multi-specific antibodies and
fragments thereof in any format (e.g., including without limitation
DARTs.RTM., Duobodies.RTM., BiTEs.RTM., BiKEs, TriKEs, XmAbs.RTM.,
TandAbs.RTM., scFvs, Fabs, Fab derivatives), bi-specific
antibodies, non-immunoglobulin antibody mimetics (e.g., including
without limitation adnectins, affibody molecules, affilins,
affimers, affitins, alphabodies, anticalins, peptide aptamers,
armadillo repeat proteins (ARMs), atrimers, avimers, designed
ankyrin repeat proteins (DARPins.RTM.), fynomers, knottins, Kunitz
domain peptides, monobodies, and nanoCLAMPs), antibody-drug
conjugates (ADC)), an oncolytic virus, a gene modifier or editor, a
cell comprising a chimeric antigen receptor (CAR), e.g., including
a T-cell immunotherapeutic agent, an NK-cell immunotherapeutic
agent, or a macrophage immunotherapeutic agent, a cell comprising
an engineered T-cell receptor (TCR-T), or any combination
thereof.
[0239] Illustrative Targets
[0240] In some embodiments, the one or more additional therapeutic
agents include, without limitation, an inhibitor, agonist,
antagonist, ligand, modulator, stimulator, blocker, activator or
suppressor of a target (e.g., polypeptide or polynucleotide)
including without limitation: Abelson murine leukemia viral
oncogene homolog 1 gene (ABL, such as ABL1), Acetyl-CoA carboxylase
(such as ACC1/2), activated CDC kinase (ACK, such as ACK1),
Adenosine deaminase, adenosine receptor (such as A2B, A2a, A3),
Adenylate cyclase, ADP ribosyl cyclase-1, adrenocorticotropic
hormone receptor (ACTH), Aerolysin, AKT1 gene, Alk-5 protein
kinase, Alkaline phosphatase, Alpha 1 adrenoceptor, Alpha 2
adrenoceptor, Alpha-ketoglutarate dehydrogenase (KGDH),
Aminopeptidase N, AMP activated protein kinase, anaplastic lymphoma
kinase (ALK, such as ALK1), Androgen receptor, Angiopoietin (such
as ligand-1, ligand-2), Angiotensinogen (AGT) gene, murine thymoma
viral oncogene homolog 1 (AKT) protein kinase (such as AKT1, AKT2,
AKT3), apolipoprotein A-I (APOA1) gene, Apoptosis inducing factor,
apoptosis protein (such as 1, 2), apoptosis signal-regulating
kinase (ASK, such as ASK1), Arginase (I), Arginine deiminase,
Aromatase, Asteroid homolog 1 (ASTEl) gene, ataxia telangiectasia
and Rad 3 related (ATR) serine/threonine protein kinase, Aurora
protein kinase (such as 1, 2), Axl tyrosine kinase receptor, 4-1BB
ligand (CD137L), Baculoviral IAP repeat containing 5 (BIRC5) gene,
Basigin, B-cell lymphoma 2 (BCL2) gene, Bcl2 binding component 3,
Bl2 protein, BCL2L11 gene, BCR (breakpoint cluster region) protein
and gene, Beta adrenoceptor, Beta-catenin, B-lymphocyte antigen
CD19, B-lymphocyte antigen CD20, B-lymphocyte cell adhesion
molecule, B-lymphocyte stimulator ligand, Bone morphogenetic
protein-10 ligand, Bone morphogenetic protein-9 ligand modulator,
Brachyury protein, Bradykinin receptor, B-Raf proto-oncogene
(BRAF), Brc-Abl tyrosine kinase, Bromodomain and external domain
(BET) bromodomain containing protein (such as BRD2, BRD3, BRD4),
Bruton's tyrosine kinase (BTK), Calmodulin, calmodulin-dependent
protein kinase (CaMK, such as CAMKII), Cancer testis antigen 2,
Cancer testis antigen NY-ESO-1, cancer/testis antigen 1B (CTAG1)
gene, Cannabinoid receptor (such as CB1, CB2), Carbonic anhydrase,
casein kinase (CK, such as CKI, CKII), Caspase (such as caspase-3,
caspase-7, Caspase-9), caspase 8 apoptosis-related cysteine
peptidase CASP8-FADD-like regulator, Caspase recruitment domain
protein-15, Cathepsin G, CCR5 gene, CDK-activating kinase (CAK),
Checkpoint kinase (such as CHK1, CHK2), chemokine (C-C motif)
receptor (such as CCR2, CCR4, CCR5, CCR8), chemokine (C--X-C motif)
receptor (such as CXCR1, CXCR2, CXCR3 and CXCR4), Chemokine CC21
ligand, Cholecystokinin CCK2 receptor, Chorionic gonadotropin,
c-Kit (tyrosine-protein kinase Kit or CD117), CISH
(Cytokine-inducible SH2-containing protein), Claudin (such as 6,
18), cluster of differentiation (CD) such as CD4, CD27, CD29, CD30,
CD33, CD37, CD40, CD40ligand receptor, CD40ligand, CD40LG gene,
CD44, CD45, CD47, CD49b, CD51, CD52, CD55, CD58, CD66e (CEACAM6),
CD70 gene, CD74, CD79, CD79b, CD79B gene, CD80, CD95, CD99, CD117,
CD122, CDw123, CD134, CDw137, CD158a, CD158b1, CD158b2, CD223,
CD276 antigen; clusterin (CLU) gene, Clusterin, c-Met (hepatocyte
growth factor receptor (HGFR)), Complement C3, Connective tissue
growth factor, COP9 signalosome subunit 5, CSF-1
(colony-stimulating factor 1 receptor), CSF2 gene, CTLA-4
(cytotoxic T-lymphocyte protein 4) receptor, C-type lectin domain
protein 9A (CLEC9A), Cyclin D1, Cyclin G, cyclin-dependent kinases
(CDK, such as CDK1, CDK1B, CDK2-9), cyclooxygenase (such as COX1,
COX2), CYP2B1 gene, Cysteine palmitoyltransferase porcupine,
Cytochrome P450 11B2, Cytochrome P450 17, cytochrome P450 17A1,
Cytochrome P450 2D6, cytochrome P450 3A4, Cytochrome P450
reductase, cytokine signalling-1, cytokine signalling-3,
Cytoplasmic isocitrate dehydrogenase, Cytosine deaminase, cytosine
DNA methyltransferase, cytotoxic T-lymphocyte protein-4, DDR2 gene,
Death receptor 5 (DR5, TRAILR2), Death receptor 4 (DR4, TRAILR1),
Delta-like protein ligand (such as 3, 4), Deoxyribonuclease,
Dickkopf-1 ligand, dihydrofolate reductase (DHFR),
Dihydropyrimidine dehydrogenase, Dipeptidyl peptidase IV, discoidin
domain receptor (DDR, such as DDR1), DNA binding protein (such as
HU-beta), DNA dependent protein kinase, DNA gyrase, DNA
methyltransferase, DNA polymerase (such as alpha), DNA primase,
dUTP pyrophosphatase, L-dopachrome tautomerase, echinoderm
microtubule like protein 4, EGFR tyrosine kinase receptor,
Elastase, Elongation factor 1 alpha 2, Elongation factor 2,
Endoglin, Endonuclease, Endoplasmin, Endosialin, Endostatin,
endothelin (such as ET-A, ET-B), Enhancer of zeste homolog 2
(EZH2), Ephrin (EPH) tyrosine kinase (such as Epha3, Ephb4), Ephrin
B2 ligand, epidermal growth factor, epidermal growth factor
receptors (EGFR), epidermal growth factor receptor (EGFR) gene,
Epigen, Epithelial cell adhesion molecule (EpCAM), Erb-b2 (v-erb-b2
avian erythroblastic leukemia viral oncogene homolog 2) tyrosine
kinase receptor, Erb-b3 tyrosine kinase receptor, Erb-b4 tyrosine
kinase receptor, E-selectin, Estradiol 17 beta dehydrogenase,
Estrogen receptor (such as alpha, beta), Estrogen related receptor,
Eukaryotic translation initiation factor 5A (EIF5A) gene, Exportin
1, Extracellular signal related kinase (such as 1, 2),
Extracellular signal-regulated kinases (ERK), Factor (such as Xa,
VIIa), farnesoid x receptor (FXR), Fas ligand, Fatty acid synthase
(FASN), Ferritin, FGF-2 ligand, FGF-5 ligand, fibroblast growth
factor (FGF, such as FGF1, FGF2, FGF4), Fibronectin, focal adhesion
kinase (FAK, such as FAK2), folate hydrolase prostate-specific
membrane antigen 1 (FOLH1), Folate receptor (such as alpha),
Folate, Folate transporter 1, FYN tyrosine kinase, paired basic
amino acid cleaving enzyme (FURIN), Beta-glucuronidase,
Galactosyltransferase, Galectin-3, Ganglioside GD2, Glucocorticoid,
glucocorticoid-induced TNFR-related protein GITR receptor,
Glutamate carboxypeptidase II, glutaminase, Glutathione
S-transferase P, glycogen synthase kinase (GSK, such as 3-beta),
Glypican 3 (GPC3), gonadotropin-releaseing hormone (GNRH),
Granulocyte macrophage colony stimulating factor (GM-CSF) receptor,
Granulocyte-colony stimulating factor (GCSF) ligand, growth factor
receptor-bound protein 2 (GRB2), Grp78 (78 kDa glucose-regulated
protein) calcium binding protein, molecular chaperone groEL2 gene,
Heme oxygenase 1 (HO1), Heme oxygenase 2 (H02), Heat shock protein
(such as 27, 70, 90 alpha, beta), Heat shock protein gene, Heat
stable enterotoxin receptor, Hedgehog protein, Heparanase,
Hepatocyte growth factor, HERV-H LTR associating protein 2, Hexose
kinase, Histamine H2 receptor, Histone methyltransferase (DOT1L),
histone deacetylase (HDAC, such as 1, 2, 3, 6, 10, 11), Histone H1,
Histone H3, HLA class I antigen (A-2 alpha), HLA class II antigen,
HLA class I antigen alpha G (HLA-G), Non-classical HLA, Homeobox
protein NANOG, HSPB1 gene, Human leukocyte antigen (HLA), Human
papillomavirus (such as E6, E7) protein, Hyaluronic acid,
Hyaluronidase, Hypoxia inducible factor-1 alpha (HIFIa), Imprinted
Maternally Expressed Transcript (H19) gene, mitogen-activated
protein kinase kinase kinase kinase 1 (MAP4K1), tyrosine-protein
kinase HCK, I-Kappa-B kinase (IKK, such as IKKbe), IL-1 alpha, IL-1
beta, IL-12, IL-12 gene, IL-15, IL-17, IL-2 gene, IL-2 receptor
alpha subunit, IL-2, IL-3 receptor, IL-4, IL-6, IL-7, IL-8,
immunoglobulin (such as G, G1, G2, K, M), Immunoglobulin Fc
receptor, Immunoglobulin gamma Fc receptor (such as I, III, IIIA),
indoleamine 2,3-dioxygenase (IDO, such as IDO1 and IDO2),
indoleamine pyrrole 2,3-dioxygenase 1 inhibitor, insulin receptor,
Insulin-like growth factor (such as 1, 2), Integrin alpha-4/beta-1,
integrin alpha-4/beta-7, Integrin alpha-5/beta-1, Integrin
alpha-V/beta-3, Integrin alpha-V/beta-5, Integrin alpha-V/beta-6,
Intercellular adhesion molecule 1 (ICAM-1), interferon (such as
alpha, alpha 2, beta, gamma), Interferon inducible protein absent
in melanoma 2 (AIM2), interferon type I receptor, Interleukin 1
ligand, Interleukin 13 receptor alpha 2, interleukin 2 ligand,
interleukin-1 receptor-associated kinase 4 (IRAK4), Interleukin-2,
Interleukin-29 ligand, isocitrate dehydrogenase (such as IDH1,
IDH2), Janus kinase (JAK, such as JAK1, JAK2), Jun N terminal
kinase, kallikrein-related peptidase 3 (KLK3) gene, Killer cell Ig
like receptor, Kinase insert domain receptor (KDR), Kinesin-like
protein KIF11, Kirsten rat sarcoma viral oncogene homolog (KRAS)
gene, Kisspeptin (KiSS-1) receptor, KIT gene, v-kit Hardy-Zuckerman
4 feline sarcoma viral oncogene homolog (KIT) tyrosine kinase,
lactoferrin, Lanosterol-14 demethylase, LDL receptor related
protein-1, Leukocyte immunoglobulin-like receptor subfamily B
member 1 (ILT2), Leukocyte immunoglobulin-like receptor subfamily B
member 2 (ILT4), Leukotriene A4 hydrolase, Listeriolysin,
L-Selectin, Luteinizing hormone receptor, Lyase, lymphocyte
activation gene 3 protein (LAG-3), Lymphocyte antigen 75,
Lymphocyte function antigen-3 receptor, lymphocyte-specific protein
tyrosine kinase (LCK), Lymphotactin, Lyn (Lck/Yes novel) tyrosine
kinase, lysine demethylases (such as KDM1, KDM2, KDM4, KDM5, KDM6,
A/B/C/D), Lysophosphatidate-1 receptor, lysosomal-associated
membrane protein family (LAMP) gene, Lysyl oxidase homolog 2, lysyl
oxidase protein (LOX), lysyl oxidase-like protein (LOXL, such as
LOXL2), 5-Lipoxygenase (5-LOX), Hematopoietic Progenitor Kinase 1
(HPK1), Hepatocyte growth factor receptor (MET) gene, macrophage
colony-stimulating factor (MCSF) ligand, Macrophage migration
inhibitory fact, MAGEC1 gene, MAGEC2 gene, Major vault protein,
MAPK-activated protein kinase (such as MK2), Mas-related G-protein
coupled receptor, matrix metalloprotease (MMP, such as MMP2, MMP9),
Mcl-1 differentiation protein, Mdm2 p53-binding protein, Mdm4
protein, Melan-A (MART-1) melanoma antigen, Melanocyte protein Pmel
17, melanocyte stimulating hormone ligand, melanoma antigen family
A3 (MAGEA3) gene, Melanoma associated antigen (such as 1, 2, 3, 6),
Membrane copper amine oxidase, Mesothelin, MET tyrosine kinase,
Metabotropic glutamate receptor 1, Metalloreductase STEAPI (six
transmembrane epithelial antigen of the prostate 1), Metastin,
methionine aminopeptidase-2, Methyltransferase, Mitochondrial 3
ketoacyl CoA thiolase, mitogen-activate protein kinase (MAPK),
mitogen-activated protein kinase (MEK, such as MEK1, MEK2), mTOR
(mechanistic target of rapamycin (serine/threonine kinase), mTOR
complex (such as 1,2), mucin (such as 1, 5A, 16), mut T homolog
(MTH, such as MTH1), Myc proto-oncogene protein, myeloid cell
leukemia 1 (MCL1) gene, myristoylated alanine-rich protein kinase C
substrate (MARCKS) protein, NAD ADP ribosyltransferase, natriuretic
peptide receptor C, Neural cell adhesion molecule 1, Neurokinin 1
(NK1) receptor, Neurokinin receptor, Neuropilin 2, NF kappa B
activating protein, NIMA-related kinase 9 (NEK9), Nitric oxide
synthase, NK cell receptor, NK3 receptor, NKG2 A B activating NK
receptor, NLRP3 (NACHT LRR PYD domain protein 3) modulators,
Noradrenaline transporter, Notch (such as Notch-2 receptor, Notch-3
receptor, Notch-4 receptor), Nuclear erythroid 2-related factor 2,
Nuclear Factor (NF) kappa B, Nucleolin, Nucleophosmin,
nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), 2 oxoglutarate
dehydrogenase, 2,5-oligoadenylate synthetase, O-methylguanine DNA
methyltransferase, Opioid receptor (such as delta), Ornithine
decarboxylase, Orotate phosphoribosyltransferase, orphan nuclear
hormone receptor NR4A1, Osteocalcin, Osteoclast differentiation
factor, Osteopontin, OX-40 (tumor necrosis factor receptor
superfamily member 4 TNFRSF4, or CD134) receptor, P3 protein, p38
kinase, p38 MAP kinase, p53 tumor suppressor protein, Parathyroid
hormone ligand, peroxisome proliferator-activated receptors (PPAR,
such as alpha, delta, gamma), P-Glycoprotein (such as 1),
phosphatase and tensin homolog (PTEN), phosphatidylinositol
3-kinase (PI3K), phosphoinositide-3 kinase (PI3K such as alpha,
delta, gamma), phosphorylase kinase (PK), PKN3 gene, placenta
growth factor,platelet-derived growth factor (PDGF, such as alpha,
beta), Platelet-derived growth factor (PDGF, such as alpha, beta),
Pleiotropic drug resistance transporter, Plexin B1, PLK1 gene,
polo-like kinase (PLK), Polo-like kinase 1, Poly (ADP-ribose)
polymerase (PARP, such as PARP1, PARP2 and PARP3, PARP7, and
mono-PARPs), Preferentially expressed antigen in melanoma (PRAME)
gene, Prenyl-binding protein (PrPB), Probable transcription factor
PML, Progesterone receptor, Programmed cell death 1 (PD-1),
Programmed cell death ligand 1 inhibitor (PD-L1), Prosaposin (PSAP)
gene, Prostanoid receptor (EP4), Prostaglandin E2 synthase,
prostate specific antigen, Prostatic acid phosphatase, proteasome,
Protein E7, Protein farnesyltransferase, protein kinase (PK, such
as A, B, C), protein tyrosine kinase, Protein tyrosine phosphatase
beta, Proto-oncogene serine/threonine-protein kinase (PIM, such as
PIM-1, PIM-2, PIM-3), P-Selectin, Purine nucleoside phosphorylase,
purinergic receptor P2X ligand gated ion channel 7 (P2X7), Pyruvate
dehydrogenase (PDH), Pyruvate dehydrogenase kinase, Pyruvate kinase
(PYK), 5-Alpha-reductase, Raf protein kinase (such as 1, B), RAF1
gene, Ras gene, Ras GTPase, RET gene, Ret tyrosine kinase receptor,
retinoblastoma associated protein, retinoic acid receptor (such as
gamma), Retinoid X receptor, Rheb (Ras homolog enriched in brain)
GTPase, Rho (Ras homolog) associated protein kinase 2,
ribonuclease, Ribonucleotide reductase (such as M2 subunit),
Ribosomal protein S6 kinase, RNA polymerase (such as I, II), Ron
(Recepteur d'Origine Nantais) tyrosine kinase, ROS1 (ROS
proto-oncogene 1, receptor tyrosine kinase) gene, Ros1 tyrosine
kinase, Runt-related transcription factor 3, Gamma-secretase, S100
calcium binding protein A9, Sarco endoplasmic calcium ATPase,
Second mitochondria-derived activator of caspases (SMAC) protein,
Secreted frizzled related protein-2, Secreted phospholipase A2,
Semaphorin-4D, Serine protease, serine/threonine kinase (STK),
serine/threonine-protein kinase (TBK, such as TBK1), signal
transduction and transcription (STAT, such as STAT-1, STAT-3,
STAT-5), Signaling lymphocytic activation molecule (SLAM) family
member 7, six-transmembrane epithelial antigen of the prostate
(STEAP) gene, SL cytokine ligand, smoothened (SMO) receptor, Sodium
iodide cotransporter, Sodium phosphate cotransporter 2B,
Somatostatin receptor (such as 1, 2, 3, 4, 5), Sonic hedgehog
protein, Son of sevenless (SOS), Specific protein 1 (SpI)
transcription factor, Sphingomyelin synthase, Sphingosine kinase
(such as 1, 2), Sphingosine-1-phosphate receptor-1, spleen tyrosine
kinase (SYK), SRC gene, Src tyrosine kinase, STAT3 gene, Steroid
sulfatase, Stimulator of interferon genes (STING) receptor,
stimulator of interferon genes protein, Stromal cell-derived factor
1 ligand, SUMO (small ubiquitin-like modifier), Superoxide
dismutase, Suppressor of cytokine signaling modulators (SOCS),
Survivin protein, Synapsin 3, Syndecan-1, Synuclein alpha, T cell
surface glycoprotein CD28, tank-binding kinase (TBK), TATA
box-binding protein-associated factor RNA polymerase I subunit B
(TAF1B) gene, T-cell CD3 glycoprotein zeta chain, T-cell
differentiation antigen CD6, T-cell immunoglobulin and mucin-domain
containing-3 (TIM-3), T-cell surface glycoprotein CD8, Tec protein
tyrosine kinase, Tek tyrosine kinase receptor, telomerase,
Telomerase reverse transcriptase (TERT) gene, Tenascin,
Thrombopoietin receptor, Thymidine kinase, Thymidine phosphorylase,
Thymidylate synthase, Thymosin (such as alpha 1), Thyroid hormone
receptor, Thyroid stimulating hormone receptor, Tissue factor, TNF
related apoptosis inducing ligand, TNFR1 associated death domain
protein, TNF-related apoptosis-inducing ligand (TRAIL) receptor,
TNFSFI Igene, TNFSF9 gene, Toll-like receptor (TLR such as 1-13),
topoisomerase (such as I, II, III), Transcription factor,
Transferase, transferrin (TF); transforming growth factor beta 1
(TGFB1) and isoforms thereof, TGF beta 2 ligand, Transforming
growth factor TGF-P receptor kinase, Transglutaminase,
Translocation associated protein, Transmembrane glycoprotein NMB,
Trop-2 calcium signal transducer, trophoblast glycoprotein (TPBG)
gene, Trophoblast glycoprotein, Tropomyosin receptor kinase (Trk)
receptor (such as TrkA, TrkB, TrkC), Tryptophan 5-hydroxylase,
Tubulin, Tumor necrosis factor (TNF, such as alpha, beta), Tumor
necrosis factor 13C receptor, tumor progression locus 2 (TPL2),
Tumor protein 53 (TP53) gene, Tumor suppressor candidate 2 (TUSC2)
gene, Tumor specific neoantigens, Tyrosinase, Tyrosine hydroxylase,
tyrosine kinase (TK), Tyrosine kinase receptor, Tyrosine kinase
with immunoglobulin-like and EGF-like domains (TIE) receptor,
Tyrosine protein kinase ABL1 inhibitor, Ubiquitin, Ubiquitin
carboxyl hydrolase isozyme L5, Ubiquitin thioesterase-14,
Ubiquitin-conjugating enzyme E21 (UBE2I, UBC9), Urease, Urokinase
plasminogen activator, Uteroglobin, Vanilloid VR1, Vascular cell
adhesion protein 1, vascular endothelial growth factor receptor
(VEGFR), V-domain Ig suppressor of T-cell activation (VISTA),
VEGF-1 receptor, VEGF-2 receptor, VEGF-3 receptor, VEGF-A, VEGF-B,
Vimentin, Vitamin D3 receptor, Proto-oncogene tyrosine-protein
kinase,
Mer (Mer tyrosine kinase receptor modulators), YAP (Yes-associated
protein modulators)es, Wee-1 protein kinase, Wilms' tumor antigen
1, Wilms' tumor protein, WW domain containing transcription
regulator protein 1 (TAZ), X-linked inhibitor of apoptosis protein,
Zinc finger protein transcription factor or any combination
thereof.
[0241] In some embodiments, the one or more additional therapeutic
agents include without limitation an HBV DNA polymerase inhibitor,
immunomodulator, TLR modulator, HBsAg inhibitor, HBsAg secretion or
assembly inhibitor, HBV therapeutic vaccine, HBV antibody, such as
HBV antibodies targeting the surface antigens of the hepatitis B
virus and bispecific antibodies and "antibody-like" therapeutic
proteins (such as DARTs.RTM., DUOBODIES, BITES.RTM., XmAbs.RTM.,
TandAbs.RTM., Fab derivatives, or TCR-like antibodies), cyclophilin
inhibitor, stimulator of retinoic acid-inducible gene 1, stimulator
of RIG-I like receptor, PD-1 inhibitor, PD-L1 inhibitor, arginase
inhibitor, PI3K inhibitor, IDO inhibitor, stimulator of NOD2, HBV
viral entry inhibitors, NTCP inhibitor, HBx inhibitor, cccDNA
inhibitor, HBV antibody targeting the surface antigens of the
hepatitis B virus, siRNA, miRNA gene therapy agents, sshRNAs, KDM5
inhibitors, or nucleoprotein modulator (HBV core or capsid protein
modulator).
[0242] In some embodiments, the one or more additional therapeutic
agents include without limitation an HBV DNA polymerase inhibitor,
immunomodulator, TLR modulator, HBsAg inhibitor, HBV therapeutic
vaccine, HBV antibody, such as an HBV antibody targeting a surface
antigens of the hepatitis B virus, bispecific antibody and
"antibody-like" therapeutic protein (such as DARPins.RTM.,
anti-pMHC TCR-like antibodies, DARTs.RTM., DUOBODIES.RTM.,
BITES.RTM., XmAbs.RTM., TandAbs.RTM., Fab derivatives, or TCR-like
antibodies), cyclophilin inhibitor, stimulator of retinoic
acid-inducible gene 1, stimulator of RIG-I like receptor, PD-1
inhibitor, PD-L1 inhibitor, arginase inhibitor, PI3K inhibitor, IDO
inhibitor, and stimulator of NOD2.
[0243] In some embodiments, the one or more additional therapeutic
agents include without limitation an HBV DNA polymerase inhibitor,
HBV viral entry inhibitor, NTCP inhibitor, HBx inhibitor, cccDNA
inhibitor, an HBV antibody targeting a surface antigen of the
hepatitis B virus, siRNA, miRNA gene therapy agent, sshRNA, KDM5
inhibitor, and nucleoprotein modulator (HBV core or capsid protein
inhibitors).
[0244] In some embodiments, the one or more additional therapeutic
agents include without limitation an inhibitor of certain HCV
nonstructural proteins, such as a NS5A inhibitor, a NS5B inhibitor,
a NS3 inhibitor, or combinations thereof.
[0245] In some embodiments, the one or more additional therapeutic
agents include without limitation a combination drug for HIV,
another drug for treating HIV, HIV protease inhibitor, HIV
non-nucleoside or non-nucleotide inhibitor of reverse
transcriptase, HIV nucleoside or nucleotide inhibitor of reverse
transcriptase, HIV integrase inhibitor, HIV non-catalytic site (or
allosteric) integrase inhibitor, HIV entry inhibitor, HIV
maturation inhibitor, immunomodulator, immunotherapeutic agent,
antibody-drug conjugate, gene modifier, gene editor (such as
CRISPR/Cas9, zinc finger nuclease, homing nuclease, synthetic
nuclease, TALEN), cell therapy (such as chimeric antigen receptor
T-cell, CAR-T, and engineered T cell receptor, TCR-T, autologous T
cell therapy), latency reversing agent, compound that targets the
HIV capsid, immune-based therapy, phosphatidylinositol 3-kinase
(PI3K) inhibitor, HIV antibody, bispecific antibody and
"antibody-like" therapeutic protein, HIV p17 matrix protein
inhibitor, IL-13 antagonist, peptidyl-prolyl cis-trans isomerase A
modulator, protein disulfide isomerase inhibitor, complement C5a
receptor antagonist, DNA methyltransferase inhibitor, HIV vif gene
modulator, Vif dimerization antagonist, HIV-1 viral infectivity
factor inhibitor, TAT protein inhibitor, HIV-1 Nef modulator, Hck
tyrosine kinase modulator, mixed lineage kinase-3 (MLK-3)
inhibitor, HIV-1 splicing inhibitor, Rev protein inhibitor,
integrin antagonist, nucleoprotein inhibitor, splicing factor
modulator, COMM domain containing protein 1 modulator, CD4
modulator, CD4 antagonist, HIV ribonuclease H inhibitor,
retrocyclin modulator, CDK-9 inhibitor, CCR5 chemokine antagonist,
CCR5 gene modulator, dendritic ICAM-3 grabbing nonintegrin 1
inhibitor, HIV GAG protein inhibitor, HIV POL protein inhibitor,
hyaluronidase inhibitor, Nef antagonist, Nef inhibitor,
protease-activated receptor-1 antagonist, TNF alpha ligand
inhibitor, PDE4 inhibitor, Complement Factor H modulator, ubiquitin
ligase inhibitor, deoxycytidine kinase inhibitor, cyclin dependent
kinase inhibitor, proprotein convertase PC9 stimulator, ATP
dependent RNA helicase DDX3X inhibitor, reverse transcriptase
priming complex inhibitor, G6PD and NADH-oxidase inhibitor,
pharmacokinetic enhancer, HIV gene therapy, and HIV vaccines, a
long-acting HIV regimen, and a contraceptive agent, or any
combination thereof. In certain embodiments, the one or more
additional therapeutic agents are selected from the group
consisting of HIV protease inhibiting compounds, HIV non-nucleoside
inhibitors of reverse transcriptase, HIV non-nucleotide inhibitors
of reverse transcriptase, HIV nucleoside inhibitors of reverse
transcriptase, HIV nucleotide inhibitors of reverse transcriptase,
HIV integrase inhibitors, gp41 inhibitors, CXCR4 inhibitors, gpl20
inhibitors, CCR5 inhibitors, capsid polymerization inhibitors,
pharmacokinetic enhancers, and other drugs for treating HIV, or any
combinations thereof. In certain embodiments, the one or more
additional therapeutic agents do not include a pharmacokinetic
enhancer.
[0246] In some embodiments, the one or more additional therapeutic
agents include without limitation an HIV protease inhibitor, HIV
reverse transcriptase inhibitor, HIV integrase inhibitor, HIV
non-catalytic site (or allosteric) integrase inhibitor, HIV entry
(fusion) inhibitor, HIV maturation inhibitor, HIV latency reversing
agent, HIV capsid inhibitor, anti-HIV antibody, or combination
thereof.
[0247] In some embodiments, the one or more additional therapeutic
agents include an HIV nucleoside or nucleotide inhibitor of reverse
transcriptase and an HIV non-nucleoside inhibitor of reverse
transcriptase. In some embodiments, the one or more additional
therapeutic agents include an HIV nucleoside or nucleotide
inhibitor of reverse transcriptase, and an HIV protease inhibiting
compound. In some embodiments, the one or more additional
therapeutic agents include an HIV nucleoside or nucleotide
inhibitor of reverse transcriptase, an HIV non-nucleoside inhibitor
of reverse transcriptase, and a pharmacokinetic enhancer. In some
embodiments, the one or more additional therapeutic agents include
at least one HIV nucleoside inhibitor of reverse transcriptase, an
integrase inhibitor, and a pharmacokinetic enhancer. In some
embodiments, the one or more additional therapeutic agents include
two HIV nucleoside or nucleotide inhibitors of reverse
transcriptase.
[0248] In some embodiments, the one or more additional therapeutic
agents include one or more antiviral agents. Any suitable antiviral
agent can be used in the methods described herein. In some
embodiments, the one or more antiviral agents include without
limitation a 5-substituted 2'-deoxyuridine analogue, a nucleoside
analogue, a pyrophosphate analogue, a nucleoside reverse
transcriptase inhibitor, a non-nucleoside reverse transcriptase
inhibitor, a protease inhibitor, an integrase inhibitor, an entry
inhibitor, an acyclic guanosine analogue, an acyclic nucleoside
phosphonate analogue, a HCV NS5A/NS5B inhibitor, an influenza virus
inhibitors, an interferon, an immunostimulator, an oligonucleotide,
an antimitotic inhibitor, and combinations thereof. In some
embodiments the one or more therapeutic agents include an RNA
polymerase inhibitor.
[0249] Illustrative Mechanisms of Action
[0250] In various embodiments, the one or more additional
therapeutic agents may be categorized by their mechanism of action
into, for example, the following groups: [0251]
anti-metabolites/anticancer agents, such as pyrimidine analogs
floxuridine, capecitabine, cytarabine, CPX-351 (liposomal
cytarabine, daunorubicin), and TAS-118; [0252] purine analogs,
folate antagonists (such as pralatrexate), cladribine, pentostatin,
fludarabine and related inhibitors; [0253]
antiproliferative/antimitotic agents including natural products,
such as vinca alkaloids (vinblastine, vincristine) and microtubule
disruptors such as taxane (paclitaxel, docetaxel), vinblastin,
nocodazole, epothilones, vinorelbine (NAVELBINE.RTM.), and
epipodophyllotoxins (etoposide, teniposide); [0254] DNA damaging
agents, such as actinomycin, amsacrine, busulfan, carboplatin,
chlorambucil, cisplatin, cyclophosphamide (CYTOXAN.RTM.),
dactinomycin, daunorubicin, doxorubicin, epirubicin, iphosphamide,
melphalan, merchlorethamine, mitomycin C, mitoxantrone,
nitrosourea, procarbazine, taxol, Taxotere, teniposide, etoposide,
and triethylenethiophosphoramide; [0255] DNA-hypomethylating
agents, such as guadecitabine (SGI-110), ASTX727; [0256]
antibiotics such as dactinomycin, daunorubicin, doxorubicin,
idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin
(mithramycin); [0257] enzymes such as L-asparaginase which
systemically metabolizes L-asparagine and deprives cells which do
not have the capacity to synthesize their own asparagine; [0258]
DNAi oligonucleotides targeting Bcl-2, such as PNT2258; agents that
activate or reactivate latent human immunodeficiency virus (HIV),
such as panobinostat and romidepsin; [0259] asparaginase
stimulators, such as crisantaspase (Erwinase.RTM.) and GRASPA
(ERY-001, ERY-ASP), calaspargase pegol; [0260] pan-Trk, ROS1 and
ALK inhibitors, such as entrectinib, TPX-0005; anaplastic lymphoma
kinase (ALK) inhibitors, such as alectinib, ceritinib; [0261]
antiproliferative/antimitotic alkylating agents, such as nitrogen
mustard cyclophosphamide and analogs (e.g., melphalan,
chlorambucil, hexamethylmelamine, thiotepa), alkyl nitrosoureas
(e.g., carmustine) and analogs, streptozocin, and triazenes (e.g.,
dacarbazine); [0262] antiproliferative/antimitotic antimetabolites,
such as folic acid analogs (methotrexate); [0263] platinum
coordination complexes (e.g., cisplatin, oxiloplatinim, and
carboplatin), procarbazine, hydroxyurea, mitotane, and
aminoglutethimide; [0264] hormones, hormone analogs (e.g.,
estrogen, tamoxifen, goserelin, bicalutamide, and nilutamide), and
aromatase inhibitors (e.g., letrozole and anastrozole); [0265]
antiplatelet agents; anticoagulants such as heparin, synthetic
heparin salts, and other inhibitors of thrombin; [0266]
fibrinolytic agents such as tissue plasminogen activator,
streptokinase, urokinase, aspirin, dipyridamole, ticlopidine, and
clopidogrel; [0267] antimigratory agents; antisecretory agents
(e.g., breveldin); [0268] immunosuppressives, such as tacrolimus,
sirolimus, azathioprine, and mycophenolate; [0269] growth factor
inhibitors, and vascular endothelial growth factor inhibitors;
[0270] fibroblast growth factor inhibitors, such as FPA14; [0271]
anti-VEGFR antibodies, such as IMC-3C5, GNR-011, tanibirumab,
LYN-00101; [0272] anti-VEGF/DDL4 antibodies, such as ABT-165;
[0273] anti-cadherin antibodies, such as HKT-288; [0274] anti-CD52
antibodies, such as alemtuzumab; [0275] anti-CD70 antibodies, such
as AMG-172; [0276] anti-leucine-rich repeat containing 15 (LRRC15)
antibodies, such as ABBV-085, ARGX-110; [0277] angiotensin receptor
blockers, nitric oxide donors; [0278] antisense oligonucleotides,
such as AEG35156, IONIS-KRAS-2.5Rx, EZN-3042, RX-0201,
IONIS-AR-2.5Rx, BP-100 (prexigebersen), IONIS-STAT3-2.5Rx; [0279]
DNA interference oligonucleotides, such as PNT2258, AZD-9150;
[0280] anti-angiopoietin (ANG)-2 antibodies, such as MEDI3617, and
LY3127804; [0281] anti-ANG-1/ANG-2 antibodies, such as AMG-780;
[0282] anti-CSF1R antibodies, such as emactuzumab, LY3022855,
AMG-820, FPA-008 (cabiralizumab); [0283] anti-CD40 antibodies, such
as RG7876, SEA-CD40, APX-005M, ABBV-428; [0284] anti-endoglin
antibodies, such as TRC105 (carotuximab); [0285] anti-CD45
antibodies, such as 131I-BC8 (lomab-B); anti-HER3 antibodies, such
as LJM716, GSK2849330; [0286] anti-MET/EGFR antibodies, such as
LY3164530; [0287] anti-EGFR antibodies, such as ABT-414, AMG-595,
necitumumab, ABBV-221, depatuxizumab mafodotin (ABT-414),
tomuzotuximab, ABT-806, vectibix, modotuximab, RM-1929; [0288]
anti-HER2 antibodies, such as HERCEPTIN.RTM. (trastuzumab),
margetuximab, MEDI4276, BAT-8001, Pertuzumab (Perjeta), ZW25 (a
bispecific HER2-directed antibody targeting the extracellular
domains 2 and 4; Cancer Discov. 2019 January; 9(1):8; PMID:
30504239); [0289] HER2 inhibitors, such as neratinib, tucatinib
(ONT-380); [0290] EGFR/ErbB2/Ephb4 inhibitors, such as tesevatinib;
[0291] anti-ERBB antibodies, such as CDX-3379, HLX-02,
seribantumab; [0292] EGFR/ErbB-2 inhibitors, such as varlitinib;
[0293] Mutant selective EGFR inhibitors, such as PF-06747775,
EGF816 (nazartinib), ASP8273, ACEA-0010, BI-1482694; [0294]
anti-HLA-DR antibodies, such as IMMU-114; [0295] anti-IL-3
antibodies, such as JNJ-56022473; [0296] anti-TNF receptor
superfamily member 4 (TNFRSF4, OX40; NCBI Gene ID: 7293)
antibodies, such as MEDI6469, MEDI6383, MEDI0562 (tavolixizumab),
MOXR0916, PF-04518600, RG-7888, GSK-3174998, INCAGN1949,
BMS-986178, GBR-8383, ABBV-368; and those described in Intl. Patent
Publ. Nos. WO 2016/179517, WO 2017/096179, WO 2017/096182, WO
2017/096281 and WO 2018/089628; [0297] anti-TNF receptor
superfamily member 18 (TNFRSF18, GITR; NCBI Gene ID: 8784)
antibodies, such as MEDI1873, FPA-154, INCAGN-1876, TRX-518,
BMS-986156, MK-1248, GWN-323; and those described, e.g., in Intl.
Patent Publ. Nos. WO 2017/096179, WO 2017/096276, WO 2017/096189;
and WO 2018/089628; [0298] anti-TNFRSF4 (OX40)/TNFRSF18(GITR)
bi-specific antibodies, such as those described in Intl. Patent
Publ. Nos. WO 2017/096179 and WO 2018/089628; [0299] anti-EphA3
antibodies, such as KB-004; [0300] anti-CD20 antibodies, such as
obinutuzumab, IGN-002; [0301] anti-CD37 antibodies, such as AGS67E,
otlertuzumab (TRU-016); [0302] anti-ENPP3 antibodies, such as
AGS-16C3F; [0303] anti-FGFR-3 antibodies, such as LY3076226, B-701;
[0304] anti-FGFR-2 antibodies, such as GAL-F2; [0305] anti-C5
antibodies, such as ALXN-1210; [0306] anti-CD27 antibodies, such as
varlilumab (CDX-1127); [0307] anti-TROP-2 antibodies, such as
IMMU-132; [0308] anti-NKG2a antibodies, such as monalizumab; [0309]
anti-VISTA antibodies, such as HMBD-002; [0310] anti-PVRIG
antibodies, such as COM-701; [0311] anti-EpCAM antibodies, such as
VB4-845; [0312] antibodies against TNF receptor superfamily member
17 (TNFRSF17, BCMA), such as GSK-2857916; [0313] anti-CEA
antibodies, such as RG-7813; [0314] anti-cluster of differentiation
3 (CD3) antibodies, such as MGD015; anti-folate receptor alpha
antibodies, such as IMGN853; [0315] epha2 inhibitors, such as
MM-310; [0316] anti LAG-3 (Lymphocyte-activation) antibodies, such
as relatlimab (ONO-4482), LAG-525, MK-4280, REGN-3767, INCAGN2385;
[0317] raf kinase/VEGFR inhibitors, such as RAF-265; [0318]
polycomb protein (EED) inhibitors, such as MAK683; [0319]
anti-fibroblast activation protein (FAP)/IL-2R antibodies, such as
RG7461; [0320] anti-fibroblast activation protein (FAP)/TRAIL-R2
antibodies, such as RG7386; [0321] anti-fucosyl-GM1 antibodies,
such as BMS-986012; [0322] p38 MAP kinase inhibitors, such as
ralimetinib; [0323] PRMT1 inhibitors, such as MS203; [0324]
Sphingosine kinase 2 (SK2) inhibitors, such as opaganib; [0325]
Nuclear erythroid 2-related factor 2 stimulators, such as
omaveloxolone (RTA-408); [0326] Tropomyosin receptor kinase (TRK)
inhibitors, such as LOXO-195, ONO-7579; [0327] anti-ICOS
antibodies, such as JTX-2011, GSK3359609; [0328] ICOS agonists,
such as ICOS-L.COMP (Gariepy, J. et al. 106th Annu Meet Am Assoc
Immunologists (AAI) (May 9-13, San Diego) 2019, Abst 71.5); [0329]
anti-TNF receptor superfamily member 10b (TNFRSF10B, DR5, TRAILR2)
antibodies, such as DS-8273, CTB-006, INBRX-109, GEN-1029; [0330]
anti-Carcinoembryonic-antigen-related-cell-adhesion-molecule-6
(CEACAM6, CD66C) antibodies, such as BAY-1834942, NEO-201 (CEACAM
5/6); [0331] anti-GD2 antibodies, such as APN-301; [0332]
anti-interleukin-17 (IL-17) antibodies, such as CJM-112; [0333]
anti-carbonic anhydrase 9 (CA9, CAIX) antibodies, such as TX-250;
[0334] anti-CD38 antibodies, such as isatuximab, MOR-202; [0335]
anti-CD38-attenukine, such as TAK573; [0336] anti-Mucin 1 (MUC1)
antibodies, such as gatipotuzumab, Mab-AR-20.5; [0337] Mucin 1
inhibitors, such as GO-203-2C; [0338] MARCKS protein inhibitors,
such as BIO-11006; [0339] Folate antagonists, such as
arfolitixorin; [0340] Galectin-3 inhibitors, such as GR-MD-02;
[0341] Phosphorylated P68 inhibitors, such as RX-5902; [0342]
CD95/TNF modulators, such as ofranergene obadenovec; [0343]
PI3K/Akt/mTOR inhibitors, such as ABTL-0812; [0344] pan-PIM kinase
inhibitors, such as INCB-053914; [0345] IL-12 gene stimulators,
such as EGEN-001, tavokinogene telseplasmid; [0346] Heat shock
protein HSP90 inhibitors, such as TAS-116, PEN-866; [0347] VEGF/HGF
antagonists, such as MP-0250; [0348] SYK tyrosine kinase/JAK
tyrosine kinase inhibitors, such as ASN-002; [0349] JAK3/JAK1/TBK1
kinase inhibitors, such as CS-12912; [0350] IL-24 antagonist, such
as AD-IL24; [0351] NLRP3 (NACHT LRR PYD domain protein 3)
modulators, such as BMS-986299; [0352] RIG-I agonists, such as
RGT-100; [0353] Aerolysin stimulators, such as topsalysin; [0354]
P-Glycoprotein 1 inhibitors, such as HM-30181A; [0355] CSF-1
antagonists, such as ARRY-382, BLZ-945; [0356] CCR8 inhibitors,
such as I-309, SB-649701, HG-1013, RAP-310; [0357] anti-CCR8
antibodies, such as neutralizing anti-CCR8 antibodies, or anti-CCR8
antibodies having ADCC activity; [0358] anti-Mesothelin antibodies,
such as SEL-403; [0359] Thymidine kinase stimulators, such as
aglatimagene besadenovec; [0360] Polo-like kinase 1 inhibitors,
such as PCM-075; [0361] NEDD8 inhibitors, such as pevonedistat
(MLN-4924), TAS-4464; [0362] Pleiotropic pathway modulators, such
as avadomide (CC-122); [0363] FoxM1 inhibitors, such as
thiostrepton; [0364] UBA1 inhibitors, such as TAK-243; [0365] Src
tyrosine kinase inhibitors, such as VAL-201; [0366] VDAC/HK
inhibitors, such as VDA-1102; [0367] BRAF/PI3K inhibitors, such as
ASN-003; [0368] Elf4a inhibitors, such as rohinitib, eFT226; [0369]
TP53 gene stimulators, such as ad-p53; [0370] Retinoic acid
receptor alpha (RARa) inhibitors, such as SY-1425; [0371] SIRT3
inhibitors, such as YC8-02; [0372] Stromal cell-derived factor 1
ligand inhibitors, such as olaptesed pegol (NOX-A12); [0373] IL-4
receptor modulators, such as MDNA-55; [0374] Arginase-I
stimulators, such as pegzilarginase; [0375] Topoisomerase I
inhibitor/hypoxia inducible factor-1 alpha inhibitors, such as
PEG-SN38 (firtecan pegol); [0376] Hypoxia inducible factor-1 alpha
inhibitors, such as PT-2977, PT-2385; [0377] CD122 agonists, such
as NKTR-214; [0378] TLR7/TLR8 agonist, such as NKTR-262; [0379]
TLR7 agonists, such as DS-0509, GS-9620 (versatolimod), LHC-165,
TMX-101 (imiquimod); [0380] p53 tumor suppressor protein
stimulators such as kevetrin; [0381] Mdm4/Mdm2 p53-binding protein
inhibitors, such as ALRN-6924; [0382] kinesin spindle protein (KSP)
inhibitors, such as filanesib (ARRY-520); [0383] CD80-fc fusion
protein inhibitors, such as FPT-155; [0384] Menin and mixed lineage
leukemia (MLL) inhibitors such as KO-539; [0385] Liver x receptor
agonists, such as RGX-104; [0386] IL-10 agonists, such as AM-0010;
[0387] VEGFR/PDGFR inhibitors, such as vorolanib; [0388] IRAK4
inhibitors, such as CA-4948; [0389] anti-TLR-2 antibodies, such as
OPN-305; [0390] Calmodulin modulators, such as CBP-501; [0391]
Glucocorticoid receptor antagonists, such as relacorilant
(CORT-125134); [0392] Second mitochondria-derived activator of
caspases (SMAC) protein inhibitors, such as BI-891065; [0393]
Lactoferrin modulators, such as LTX-315; [0394] KIT proto-oncogene,
receptor tyrosine kinase (KIT) inhibitors, such as PLX-9486; [0395]
platelet derived growth factor receptor alpha (PDGFRA)/KIT
proto-oncogene, receptor tyrosine kinase (KIT) mutant-specific
antagonists/inhibitors such as BLU-285, DCC-2618; [0396] Exportin 1
inhibitors, such as eltanexor; [0397] anti-CD33 antibodies, such as
IMGN-779; [0398] anti-KMA antibodies, such as MDX-1097; [0399]
anti-TIM-3 antibodies, such as TSR-022, LY-3321367, MBG-453; [0400]
anti-CD55 antibodies, such as PAT-SC1; [0401] anti-PSMA antibodies,
such as ATL-101; [0402] anti-CD100 antibodies, such as VX-15;
[0403] anti-EPHA3 antibodies, such as fibatuzumab; [0404]
anti-APRIL antibodies, such as BION-1301; [0405] anti-TIGIT
antibodies, such as BMS-986207, RG-6058, AGEN-1307, AGEN-1327,
AGEN-1777, AB154; [0406] anti-TIM-3 antibodies, such as
INCAGN-2390; [0407] CHST15 gene inhibitors, such as STNM-01; [0408]
RAS inhibitors, such as NEO-100; [0409] Somatostatin receptor
antagonist, such as OPS-201; [0410] CEBPA gene stimulators, such as
MTL-501; [0411] DKK3 gene modulators, such as MTG-201; [0412]
Chemokine (CXCR1/CXCR2) inhibitors, such as SX-682; [0413] p70s6k
inhibitors, such as MSC2363318A; [0414] methionine aminopeptidase 2
(MetAP2) inhibitors, such as M8891, APL-1202; [0415] arginine
N-methyltransferase 5 inhibitors, such as GSK-3326595; [0416]
anti-programmed cell death protein 1 (anti-PD-1) antibodies, such
as nivolumab (OPDIVO.RTM., BMS-936558, MDX-1106), pembrolizumab
(KEYTRUDA.RTM., MK-3477, SCH-900475, lambrolizumab, CAS Reg. No.
1374853-91-4), pidilizumab, PF-06801591, BGB-A317 (tislelizumab),
GLS-010 (WBP-3055), AK-103 (HX-008), CS-1003, HLX-10, MGA-012,
BI-754091, REGN-2810 (cemiplimab), AGEN-2034 (balstilimab), JS-001
(toripalimab), JNJ-63723283, genolimzumab (CBT-501), LZM-009,
BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021,
ABBV-181, AK-105, PD1-PIK, BAT-1306, zimberelimab, and
anti-programmed death-ligand 1 (anti-PD-L1) antibodies such as
BMS-936559, atezolizumab (MPDL3280A), durvalumab (MEDI-4736),
avelumab, CK-301 (MSB0010718C), MEDI-0680, CX-072, CBT-502, PDR-001
(spartalizumab), TSR-042 (dostarlimab), MSB-2311, JTX-4014,
BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308
(sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001),
BCD-135, FAZ-053, TQB-2450, and MDX1105-01; [0417] PD-L1/VISTA
antagonists such as CA-170; [0418] PD-1/PD-L1 inhibitors, such as
INCB086550, GS-4224, GS-4416; [0419] anti-PD-L1/TGFO antibodies,
such as M-7824; [0420] PD-L1/EGFR inhibitors, such as GNS-1480
(lazertinib); [0421] PD-1/CTLA-4 inhibitors, such as PF-06936308;
[0422] anti-CD73/TGFO inhibitors, such as GS-1423 (AGEN1423;
published in WO2019/173692); [0423] anti-CTLA-4 (cytotoxic
T-lymphocyte protein-4) antibodies, such as tremelimumab,
ipilimumab (BMS-734016), AGEN-1884, BMS-986218, AGEN1181,
BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145,
APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044,
CG-0161, ATOR-1144, PBI-5D3H5, BA-3071;
[0424] CTLA-4 (cytotoxic T-lymphocyte protein-4) inhibitors, such
as BPI-002; TLR-3 agonist/interferon inducers, such as Poly-ICLC
(NSC-301463); [0425] anti-transferrin antibodies, such as CX-2029;
[0426] anti-IL-8 (Interleukin-8) antibodies, such as HuMax-Inflam;
[0427] ATM (ataxia telangiectasia) inhibitors, such as AZD0156;
[0428] CHK1 inhibitors, such as GDC-0575, LY2606368 (prexasertib),
SRA737, RG7741 (CHK1/2); [0429] CXCR4 antagonists, such as BL-8040,
LY2510924, burixafor (TG-0054), X4P-002, X4P-001-IO, Plerixafor;
[0430] EXH2 inhibitors, such as GSK2816126; [0431] KDM1 inhibitors,
such as ORY-1001, IMG-7289, INCB-59872, GSK-2879552; [0432] CXCR2
antagonists, such as AZD-5069; [0433] GM-CSF antibodies, such as
lenzilumab; [0434] DNA dependent protein kinase inhibitors, such as
MSC2490484A (nedisertib), VX-984, AsiDNA (DT-01); protein kinase C
(PKC) inhibitors, such as LXS-196, sotrastaurin; [0435] Selective
estrogen receptor downregulators (SERD), such as fulvestrant
(Faslodex.RTM.), RG6046, RG6047, elacestrant (RAD-1901) and
AZD9496; [0436] Selective estrogen receptor covalent antagonists
(SERCAs), such as H3B-6545; [0437] selective androgen receptor
modulator (SARM), such as GTX-024, darolutamide; [0438]
transforming growth factor-beta (TGF-beta) kinase antagonists, such
as galunisertib; TGF-beta inhibitors described in WO 2019/103203;
[0439] anti-transforming growth factor-beta (TGF-beta) antibodies,
such as LY3022859, NIS793, XOMA 089, SRK-181; [0440] bispecific
antibodies, such as MM-141 (IGF-1/ErbB3), MM-111 (Erb2/Erb3),
JNJ-64052781 (CD19/CD3), PRS-343 (CD-137/HER2), AFM26 (BCMA/CD16A),
JNJ-61186372 (EGFR/cMET), AMG-211 (CEA/CD3), RG7802 (CEA/CD3),
ERY-974 (CD3/GPC3) vancizumab (angiopoietins/VEGF), PF-06671008
(Cadherins/CD3), AFM-13 (CD16/CD30), APV0436 (CD123/CD3),
flotetuzumab (CD123/CD3), REGN-1979 (CD20/CD3), MCLA-117
(CD3/CLEC12A), MCLA-128 (HER2/HER3), JNJ-0819, JNJ-7564 (CD3/heme),
AMG-757 (DLL3-CD3), MGD-013 (PD-1/LAG-3), FS-118 (LAG-3/PD-L1)
MGD-019 (PD-1/CTLA-4), KN-046 (PD-1/CTLA-4), MEDI-5752
(CTLA-4/PD-1), RO-7121661 (PD-1/TIM4-3), XmAb-20717 (PD-1/CTLA-4),
AK-104 (CTLA-4/PD-1), AMG-330 (CD33/CD3), AMG-420 (BCMA/CD3),
BI-836880 (VEFG/ANG2), JNJ-63709178 (CD123/CD3), MGD-007
(CD3/gpA33), MGD-009 (CD3/B7H3), AGEN1223, IMCgp100 (CD3/gp100),
AGEN-1423 (GS-1423; CD73/TGF-beta), ATOR-1015 (CTLA-4/OX40),
LY-3415244 (TIM3/PDL1), INHIBRX-105 (4-1BB/PDL1), faricimab
(VEGF-A/ANG-2), FAP-4-IBBL (4-1BB/FAP), XmAb-13676 (CD3/CD20),
TG-1801 (CD19/CD47), XmAb-18087 (SSTR2/CD3), catumaxomab
(CD3/EpCAM), SAR-156597 (IL4/IL13), EMB-01 (EGFR/cMET), REGN-4018
(MUC16/CD3), RG-7828 (CD20/CD3), CC-93269 (CD3/BCMA), REGN-5458
(CD3/BCMA), navicixizumab (DLL4/VEGF), GRB-1302 (CD3/Erbb2),
vanucizumab (VEGF-A/ANG-2), GRB-1342 (CD38/CD3), GEM-333
(CD3/CD33), IMM-0306 (CD47/CD20); [0441] anti-delta-like protein
ligand 3 (DDL3) antibodies, such as rovalpituzumab tesirine; [0442]
anti-clusterin antibodies, such as AB-16B5; [0443] anti-Ephrin-A4
(EFNA4) antibodies, such as PF-06647263; [0444] anti-RANKL
antibodies, such as denosumab; [0445] anti-mesothelin antibodies,
such as BMS-986148, Anti-MSLN-MMAE; [0446] anti-sodium phosphate
cotransporter 2B (NaP2B) antibodies, such as lifastuzumab; [0447]
anti-c-Met antibodies, such as ABBV-399; [0448] Adenosine A2A
receptor antagonists, such as CPI-444, AZD-4635, preladenant,
PBF-509; [0449] Dual adenosine A2A/A2B receptor antagonists, such
as AB-928; [0450] Alpha-ketoglutarate dehydrogenase (KGDH)
inhibitors, such as CPI-613; [0451] XPO1 inhibitors, such as
selinexor (KPT-330); [0452] Isocitrate dehydrogenase 2 (IDH2)
inhibitors, such as enasidenib (AG-221); [0453] IDH1 inhibitors
such as AG-120, and AG-881 (IDH1 and IDH2), IDH-305, BAY-1436032;
interleukin-3 receptor (IL-3R) modulators, such as SL-401; [0454]
Arginine deiminase stimulators, such as pegargiminase (ADI-PEG-20);
[0455] antibody-drug conjugates, such as MLN0264 (anti-GCC,
guanylyl cyclase C), T-DM1 (trastuzumab emtansine, Kadcycla);
SYD985 (anti-HER2, Duocarmycin), milatuzumab-doxorubicin
(hCD74-DOX), brentuximab vedotin, DCDT2980S, polatuzumab vedotin
(RG-7596), SGN-CD70A, SGN-CD19A, inotuzumab ozogamicin (CMC-544),
lorvotuzumab mertansine, SAR3419, isactuzumab govitecan, enfortumab
vedotin (ASG-22ME), ASG-15ME, DS-8201 ((trastuzumab deruxtecan),
225Ac-lintuzumab, U3-1402, 177Lu-tetraxetan-tetuloma, tisotumab
vedotin, anetumab ravtansine, CX-2009, SAR-566658, W-0101,
polatuzumab vedotin, ABBV-085, gemtuzumab ozogamicin, ABT-414,
glembatumumab vedotin (CDX-011), labetuzumab govitecan (IMMU-130),
sacituzumab govitecan (INMU-132), lifastuzumab vedotin, (RG-7599),
milatuzumab-doxorubicin (IMMU-110), indatuximab ravtansine
(BT-062), pinatuzumab vedotin (RG-7593), SGN-LIV1A, SGN-CD33A,
SAR566658, MLN2704, SAR408701, rovalpituzumab tesirine, ABBV-399,
AGS-16C3F, ASG-22ME, AGS67E, AMG 172, AMG 595, AGS-15E, BAY1129980,
BAY1187982, BAY94-934 (anetumab ravtansine), GSK2857916,
Humax-TF-ADC (tisotumab vedotin), IMGN289, IMGN529, IMGN853
(mirvetuximab soravtansine), LOP628, PCA062, MDX-1203, MEDI-547,
PF-06263507, PF-06647020, PF-06647263, PF-06664178, RG7450, RG7458,
RG7598, SAR566658, SGN-CD33A, DS-1602 and DS-7300, DS-6157,
DS-6000; [0456] claudin-18 inhibitors, such as claudiximab; [0457]
.beta.-catenin inhibitors, such as CWP-291; [0458] anti-CD73
antibodies, such as MEDI-9447 (oleclumab), CPX-006, IPH-53,
BMS-986179, NZV-930, GS-1423 (AGEN-1423); [0459] CD73 inhibitors,
such as AB-680, PSB-12379, PSB-12441, PSB-12425, CB-708, GS-1423
(AGEN-1423); [0460] CD39/CD73 inhibitors, such as PBF-1662; [0461]
anti-CD39 antibodies, such as TTX-030; [0462] chemokine receptor 2
(CCR) inhibitors, such as PF-04136309, CCX-872, BMS-813160
(CCR2/CCR5); [0463] thymidylate synthase inhibitors, such as
ONX-0801; [0464] ALK/ROS1 inhibtors, such as lorlatinib; [0465]
tankyrase inhibitors, such as G007-LK; [0466] Mdm2 p53-binding
protein inhibitors, such as CMG-097, HDM-201; [0467] c-PIM
inhibitors, such as PIM447; [0468] BRAF inhibitors, such as
dabrafenib, vemurafenib, encorafenib (LGX818), PLX8394; [0469]
sphingosine kinase-2 (SK2) inhibitors, such as Yeliva.RTM.
(ABC294640); [0470] cell cycle inhibitors, such as selumetinib
(MEK1/2), and sapacitabine; [0471] AKT inhibitors such as MK-2206,
ipatasertib, afuresertib, AZD5363, and ARQ-092, capivasertib,
triciribine; [0472] c-MET inhibitors, such as AMG-337, savolitinib,
tivantinib (ARQ-197), capmatinib, and tepotinib, ABT-700, AG213,
AMG-208, JNJ-38877618 (OMO-1), merestinib, HQP-8361; [0473]
c-Met/VEGFR inhibitors, such as BMS-817378, TAS-115; [0474]
c-Met/RON inhibitors, such as BMS-777607; [0475] BRAF/EGFR
inhibitors, such as BGB-283; [0476] bcr/abl inhibitors, such as
rebastinib, asciminib; [0477] MNK1/MNK2 inhibitors, such as
eFT-508; [0478] mTOR inhibitor/cytochrome P450 3A4 stimulators,
such as TYME-88; [0479] lysine-specific demethylase-1 (LSD1)
inhibitors, such as CC-90011; [0480] Pan-RAF inhibitors, such as
LY3009120, LXH254, TAK-580; [0481] Raf/MEK inhibitors, such as
RG7304; [0482] CSF1R/KIT and FLT3 inhibitors, such as pexidartinib
(PLX3397); [0483] kinase inhibitors, such as vandetanib; [0484] E
selectin antagonists, such as GMI-1271; [0485] differentiation
inducers, such as tretinoin; [0486] epidermal growth factor
receptor (EGFR) inhibitors, such as osimertinib (AZD-9291); [0487]
topoisomerase inhibitors, such as doxorubicin, daunorubicin,
dactinomycin, eniposide, epirubicin, etoposide, idarubicin,
irinotecan, mitoxantrone, pixantrone, sobuzoxane, topotecan,
irinotecan, MM-398 (liposomal irinotecan), vosaroxin and GPX-150,
aldoxorubicin, AR-67, mavelertinib, AST-2818, avitinib (ACEA-0010),
irofulven (MGI-114); [0488] corticosteroids, such as cortisone,
dexamethasone, hydrocortisone, methylprednisolone, prednisone,
prednisolone; [0489] growth factor signal transduction kinase
inhibitors; [0490] nucleoside analogs, such as DFP-10917; [0491]
Axl inhibitors, such as BGB-324 (bemcentinib), SLC-0211; [0492]
Inhibitors of bromodomain and extraterminal motif (BET) proteins,
including BRD2 (NCBI Gene ID: 6046), BRD3 (NCBI Gene ID: 8019),
BRD4 (NCBI Gene ID: 23476), and bromodomain testis-specific protein
(BRDT; NCBI Gene ID: 676), such as INCB-054329, INCB057643,
TEN-010, AZD-5153, ABT-767, BMS-986158, CC-90010, GSK525762
(molibresib), NHWD-870, ODM-207, GSK-2820151, GSK-1210151A, ZBC246,
ZBC260, ZEN3694, FT-1101, RG-6146, CC-90010, mivebresib, BI-894999,
PLX-2853, PLX-51107, CPI-0610, GS-5829; [0493] PARP inhibitors,
such as olaparib, rucaparib, veliparib, talazoparib, ABT-767,
BGB-290, fluzolepali (SHR-3162), niraparib (JNJ-64091742),
bendamustine hydrochloride; [0494] PARP/Tankyrase inhibitors such
as 2X-121 (e-7499); [0495] IMP-4297, SC-10914, IDX-1197, HWH-340,
CK-102, simmiparib; [0496] Proteasome inhibitors, such as ixazomib,
carfilzomib (Kyprolis.RTM.), marizomib; [0497] Glutaminase
inhibitors, such as CB-839 (telaglenastat),
bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide
(BPTES); [0498] mitochondrial complex I inhibitors, such as
metformin, phenformin; [0499] Vaccines, such as peptide vaccine
TG-01 (RAS), GALE-301, GALE-302, nelipepimut-s, SurVaxM, DSP-7888,
TPIV-200, PVX-410, VXL-100, DPX-E7, ISA-101, 6MHP, OSE-2101,
galinpepimut-S, SVN53-67/M57-KLH, IMU-131; bacterial vector
vaccines such as CRS-207/GVAX, axalimogene filolisbac (ADXS11-001);
adenovirus vector vaccines such as nadofaragene firadenovec;
autologous Gp96 vaccine; dendritic cells vaccines, such as CVactm,
tapuldencel-T, eltrapuldencel-T, SL-701, BSKO1TM, rocapuldencel-T
(AGS-003), DCVAC, CVactm, stapuldencel-T, eltrapuldencel-T, SL-701,
BSKO1TM, ADXS31-142; oncolytic vaccines such as, talimogene
laherparepvec, pexastimogene devacirepvec, GL-ONC1, MG1-MA3,
parvovirus H-1, ProstAtak, enadenotucirev, MG1MA3, ASN-002
(TG-1042); therapeutic vaccines, such as CVAC-301, CMP-001,
CreaVax-BC, PF-06753512, VBI-1901, TG-4010, ProscaVax.TM.; tumor
ell vaccines, such as Vigil.RTM. (IND-14205), Oncoquest-L vaccine;
live attenuated, recombinant, serotype 1 poliovirus vaccine, such
as PVS-RIPO; Adagloxad simolenin; MEDI-0457; DPV-001 a
tumor-derived, autophagosome enriched cancer vaccine; RNA vaccines
such as, CV-9209, LV-305; DNA vaccines, such as MEDI-0457, MVI-816,
INO-5401; modified vaccinia virus Ankara vaccine expressing p53,
such as MVA-p53; DPX-Survivac; BriaVax.TM.; GI-6301; GI-6207;
GI-4000; I0-103; Neoantigen peptide vaccines, such as AGEN-2017,
GEN-010, NeoVax, RG-6180, GEN-009, PGV-001 (TLR-3 agonist),
GRANITE-001, NEO-PV-01; Peptide vaccines that target heat shock
proteins, such as PhosphoSynVax.TM.; Vitespen (HSPPC-96-C); [0500]
anti-DLL4 (delta like ligand 4) antibodies, such as demcizumab;
[0501] STAT-3 inhibitors, such as napabucasin (BBI-608); [0502]
ATPase p97 inhibitors, such as CB-5083; [0503] smoothened (SMO)
receptor inhibitors, such as Odomzo.RTM. (sonidegib, formerly
LDE-225), LEQ506, vismodegib (GDC-0449), BMS-833923, glasdegib
(PF-04449913), LY2940680, and itraconazole; [0504] interferon alpha
ligand modulators, such as interferon alpha-2b, interferon alpha-2a
biosimilar (Biogenomics), ropeginterferon alfa-2b (AOP-2014,
P-1101, PEG IFN alpha-2b), Multiferon (Alfanative, Viragen),
interferon alpha 1b, Roferon-A (Canferon, Ro-25-3036), interferon
alfa-2a follow-on biologic (Biosidus)(Inmutag, Inter 2A),
interferon alfa-2b follow-on biologic (Biosidus--Bioferon,
Citopheron, Ganapar, Beijing Kawin Technology--Kaferon),
Alfaferone, pegylated interferon alpha-1b, peginterferon alfa-2b
follow-on biologic (Amega), recombinant human interferon alpha-1b,
recombinant human interferon alpha-2a, recombinant human interferon
alpha-2b, veltuzumab-IFN alpha 2b conjugate, Dynavax (SD-101), and
interferon alfa-n1 (Humoferon, SM-10500, Sumiferon); [0505]
interferon gamma ligand modulators, such as interferon gamma
(OH-6000, Ogamma 100); [0506] IL-6 receptor modulators, such as
tocilizumab, siltuximab, AS-101 (CB-06-02, IVX-Q-101); [0507]
Telomerase modulators, such as, tertomotide (GV-1001, HR-2802,
Riavax) and imetelstat (GRN-163, JNJ-63935937); [0508] DNA
methyltransferases inhibitors, such as temozolomide (CCRG-81045),
decitabine, guadecitabine (S-110, SGI-110), KRX-0402, RX-3117,
RRx-001, and azacitidine; [0509] DNA gyrase inhibitors, such as
pixantrone and sobuzoxane; [0510] Bcl-2 family protein inhibitors,
such as ABT-263, venetoclax (ABT-199), ABT-737, and AT-101; [0511]
Notch inhibitors, such as LY3039478 (crenigacestat), tarextumab
(anti-Notch2/3), BMS-906024; [0512] anti-myostatin inhibitors, such
as landogrozumab; [0513] hyaluronidase stimulators, such as
PEGPH-20; [0514] Wnt pathway inhibitors, such as SM-04755, PRI-724,
WNT-974; [0515] gamma-secretase inhibitors, such as PF-03084014,
MK-0752, RO-4929097; [0516] Grb-2 (growth factor receptor bound
protein-2) inhibitors, such as BP1001; [0517] TRAIL
pathway-inducing compounds, such as ONC201, ABBV-621; [0518] Focal
adhesion kinase inhibitors, such as VS-4718, defactinib,
GSK2256098; [0519] hedgehog inhibitors, such as saridegib,
sonidegib (LDE225), glasdegib and vismodegib; [0520] Aurora kinase
inhibitors, such as alisertib (MLN-8237), and AZD-2811, AMG-900,
barasertib, ENMD-2076; [0521] HSPB1 modulators (heat shock protein
27, HSP27), such as brivudine, apatorsen; [0522] ATR inhibitors,
such as BAY-937, AZD6738, AZD6783, VX-803, VX-970 (berzosertib) and
VX-970; [0523] mTOR inhibitors, such as sapanisertib and
vistusertib (AZD2014), ME-344; [0524] mTOR/PI3K inhibitors, such as
gedatolisib, GSK2141795, omipalisib, RG6114; [0525] Hsp90
inhibitors, such as AUY922, onalespib (AT13387), SNX-2112, SNX5422;
[0526] Murine double minute (mdm2) oncogene inhibitors, such as
DS-3032b, RG7775, AMG-232, HDM201, and idasanutlin (RG7388); [0527]
CD137 agonists, such as urelumab, utomilumab (PF-05082566),
AGEN2373, ADG-106; STING agonists, such as ADU-S100 (MIW-815),
SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532, SYN-STING, MSA-1,
SR-8291; [0528] FGFR inhibitors, such as FGF-401, INCB-054828,
BAY-1163877, AZD4547, JNJ-42756493, LY2874455, Debio-1347; [0529]
fatty acid synthase (FASN) inhibitors, such as TVB-2640; [0530]
Anti-killer cell immunoglobulin like receptor, three Ig domains and
long cytoplasmic tail 1 (KIR3DL1; KIR; NCBI Gene ID: 3811)
monoclonal antibodies, such as lirilumab (IPH-2102), IPH-4102;
[0531] Antigen CD19 inhibitors, such as MOR208, MEDI-551, AFM-11,
inebilizumab; [0532] CD44 binders, such as A6; [0533] protein
phosphatease 2A (PP2A) inhibitors, such as LB-100; [0534] CYP17
inhibitors, such as seviteronel (VT-464), ASN-001, ODM-204, CFG920,
abiraterone acetate; [0535] RXR agonists, such as IRX4204; [0536]
hedgehog/smoothened (hh/Smo) antagonists, such as taladegib,
patidegib; [0537] complement C3 modulators, such as Imprime PGG;
[0538] IL-15 agonists, such as ALT-803, NKTR-255, and hetIL-15;
[0539] EZH2 (enhancer of zeste homolog 2) inhibitors, such as
tazemetostat, CPI-1205, GSK-2816126; [0540] Oncolytic viruses, such
as pelareorep, CG-0070, MV-NIS therapy, HSV-1716, DS-1647, VCN-01,
ONCOS-102, TBI-1401, tasadenoturev (DNX-2401), vocimagene
amiretrorepvec, RP-1, CVA21, Celyvir, LOAd-703, OBP-301;
[0541] DOT1L (histone methyltransferase) inhibitors, such as
pinometostat (EPZ-5676); [0542] toxins such as Cholera toxin,
ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase
toxin, diphtheria toxin, and caspase activators; [0543] DNA
plasmids, such as BC-819; [0544] PLK inhibitors of PLK 1, 2, and 3,
such as volasertib (PLK1); [0545] WEEl inhibitors, such as AZD-1775
(adavosertib); Rho kinase (ROCK) inhibitors, such as AT13148,
KD025; [0546] ERK inhibitors, such as GDC-0994, LY3214996, MK-8353;
[0547] Inhibition of Apoptosis Protein (IAP) inhibitors, such as
ASTX660, debio-1143, birinapant, APG-1387, LCL-161; [0548] RNA
polymerase inhibitors, such has lurbinectedin (PM-1183), CX-5461;
[0549] Tubulin inhibitors, such as PM-184, BAL-101553
(lisavanbulin), and OXI-4503, fluorapacin (AC-0001), plinabulin;
[0550] Toll-like receptor 4 (TL4) agonists, such as G100,
GSK1795091, and PEPA-10; [0551] Elongation factor 1 alpha 2
inhibitors, such as plitidepsin; [0552] CD95 inhibitors, such as
APG-101, APO-010, asunercept; [0553] WT1 inhibitors, such as
DSP-7888; [0554] splicing factor 3B subunit1 (SF3B1) inhibitors,
such as H3B-8800; [0555] retinoid Z receptor gamma (RORy) agonists,
such as LYC-55716; and [0556] Microbiome modulators, such as
SER-401, EDP-1503, MRx-0518.
[0557] In some embodiments, the fusion protein, the homodimer, the
heterodimer, the conjugate, the polynucleotide, the vector, the
lipoplex, such as an LNP, and/or the pharmaceutical composition is
co-administered with one or more additional therapeutic agents
comprising an inhibitor or antagonist of: protein tyrosine
phosphatase, non-receptor type 11 (PTPN11 or SHP2; NCBI Gene ID:
5781); myeloid cell leukemia sequence 1 (MCL1) apoptosis regulator
(NCBI Gene ID: 4170); mitogen-activated protein kinase kinase
kinase kinase 1 (MAP4K1) (also called Hematopoietic Progenitor
Kinase 1 (HPK1), NCBI Gene ID: 11184);
phosphatidylinositol-4,5-bisphosphate 3-kinase, including catalytic
subunit alpha (PIK3CA; NCBI Gene ID: 5290), catalytic subunit beta
(PIK3CB; NCBI Gene ID: 5291), catalytic subunit gamma (PIK3CG; NCBI
Gene ID: 5294) and catalytic subunit delta (PIK3CD; NCBI Gene ID:
5293), diacylglycerol kinase alpha (DGKA, DAGK, DAGK1 or DGK-alpha;
NCBI Gene ID: 1606); 5'-nucleotidase ecto (NT5E or CD73; NCBI Gene
ID: 4907); ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1
or CD39; NCBI Gene ID: 593); transforming growth factor beta 1
(TGFB1 or TGFO; NCBI Gene ID: 7040); heme oxygenase 1 (HMOX1, HO-1
or HOl; NCBI Gene ID: 3162); heme oxygenase 2 (HMOX2, HO-2 or H02;
NCBI Gene ID: 3163); vascular endothelial growth factor A (VEGFA or
VEGF; NCBI Gene ID: 7422); erb-b2 receptor tyrosine kinase 2
(ERBB2, HER2, HER2/neu or CD340; NCBI Gene ID: 2064), epidermal
growth factor receptor (EGFR, ERBB, ERBB1 or HER1; NCBI Gene ID:
1956); ALK receptor tyrosine kinase (ALK, CD246; NCBI Gene ID:
238); poly(ADP-ribose) polymerase 1 (PARP1; NCBI Gene ID: 142);
poly(ADP-ribose) polymerase 2 (PARP2; NCBI Gene ID: 10038); TCDD
inducible poly(ADP-ribose) polymerase (TIPARP, PARP7; NCBI Gene ID:
25976); cyclin dependent kinase 4 (CDK4; NCBI Gene ID: 1019);
cyclin dependent kinase 6 (CDK6; NCBI Gene ID: 1021); TNF receptor
superfamily member 14 (TNFRSF14, HVEM, CD270; NCBI Gene ID: 8764);
T cell immunoreceptor with Ig and ITIM domains (TIGIT; NCBI Gene
ID: 201633); X-linked inhibitor of apoptosis (XIAP, BIRC4, IAP-3;
NCBI Gene ID: 331); baculoviral IAP repeat containing 2 (BIRC2,
cIAP1; NCBI Gene ID: 329); baculoviral IAP repeat containing 3
(BIRC3, cIAP2; NCBI Gene ID: 330); baculoviral IAP repeat
containing 5 (BIRC5, surviving; NCBI Gene ID: 332); C-C motif
chemokine receptor 2 (CCR2, CD192; NCBI Gene ID: 729230); C-C motif
chemokine receptor 5 (CCR5, CD195; NCBI Gene ID: 1234); C-C motif
chemokine receptor 8 (CCR8, CDw198; NCBI Gene ID: 1237); C--X-C
motif chemokine receptor 2 (CXCR2, CD182; NCBI Gene ID: 3579);
C--X-C motif chemokine receptor 3 (CXCR3, CD182, CD183; NCBI Gene
ID: 2833); C--X-C motif chemokine receptor 4 (CXCR4, CD184; NCBI
Gene ID: 7852); cytokine inducible SH2 containing protein (CISH;
NCBI Gene ID: 1154); arginase (ARG1 (NCBI Gene ID: 383), ARG2 (NCBI
Gene ID: 384)), carbonic anhydrase (CA1 (NCBI Gene ID: 759), CA2
(NCBI Gene ID: 760), CA3 (NCBI Gene ID: 761), CA4 (NCBI Gene ID:
762), CA5A (NCBI Gene ID: 763), CAB (NCBI Gene ID: 11238), CA6
(NCBI Gene ID: 765), CA7 (NCBI Gene ID: 766), CA8 (NCBI Gene ID:
767), CA9 (NCBI Gene ID: 768), CA10 (NCBI Gene ID: 56934), CA11
(NCBI Gene ID: 770), CA12 (NCBI Gene ID: 771), CA13 (NCBI Gene ID:
377677), CA14 (NCBI Gene ID: 23632)), prostaglandin-endoperoxide
synthase 1 (PTGS1, COX-1; NCBI Gene ID: 5742),
prostaglandin-endoperoxide synthase 2 (PTGS2, COX-2; NCBI Gene ID:
5743), secreted phospholipase A2, prostaglandin E synthase (PTGES,
PGES; Gene ID: 9536), arachidonate 5-lipoxygenase (ALOX5, 5-LOX;
NCBI Gene ID: 240) and/or soluble epoxide hydrolase 2 (EPHX2, SEH;
NCBI Gene ID: 2053); a secreted phospholipase A2 (e.g., PLA2G1B
(NCBI Gene ID: 5319); PLA2G7 (NCBI Gene ID: 7941), PLA2G3 (NCBI
Gene ID: 50487), PLA2G2A (NCBI Gene ID: 5320); PLA2G4A (NCBI Gene
ID: 5321); PLA2G12A (NCBI Gene ID: 81579); PLA2G12B (NCBI Gene ID:
84647); PLA2G10 (NCBI Gene ID: 8399); PLA2G5 (NCBI Gene ID: 5322);
PLA2G2D (NCBI Gene ID: 26279); PLA2G15 (NCBI Gene ID: 23659));
indoleamine 2,3-dioxygenase 1 (IDO1; NCBI Gene ID: 3620);
indoleamine 2,3-dioxygenase 2 (IDO2; NCBI Gene ID: 169355); hypoxia
inducible factor 1 subunit alpha (HIF1A; NCBI Gene ID: 3091);
angiopoietin 1 (ANGPT1; NCBI Gene ID: 284); Endothelial TEK
tyrosine kinase (TIE-2, TEK, CD202B; NCBI Gene ID: 7010); Janus
kinase 1 (JAK1; NCBI Gene ID: 3716); catenin beta 1 (CTNNB1; NCBI
Gene ID: 1499); histone deacetylase 9 (HDAC9; NCBI Gene ID: 9734),
5'-3' exoribonuclease 1 (XRN1; NCBI Gene ID: 54464); and/or WRN
RecQ like helicase (WRN; NCBI Gene ID: 7486).
Immune Checkpoint Modulators
[0558] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with one or more blockers or inhibitors of
inhibitory immune checkpoint proteins or receptors and/or with one
or more stimulators, activators or agonists of one or more
stimulatory immune checkpoint proteins or receptors. Blockade or
inhibition of inhibitory immune checkpoints can positively regulate
T-cell or NK cell activation and prevent immune escape of cancer
cells within the tumor microenvironment. Activation or stimulation
of stimulatory immune check points can augment the effect of immune
checkpoint inhibitors in cancer therapeutics. In various
embodiments, the immune checkpoint proteins or receptors regulate T
cell responses (e.g., reviewed in Xu, et al., J Exp Clin Cancer
Res. (2018) 37:110). In various embodiments, the immune checkpoint
proteins or receptors regulate NK cell responses (e.g., reviewed in
Davis, et al., Semin Immunol. (2017) 31:64-75 and Chiossone, et
al., Nat Rev Immunol. (2018) 18(11):671-688).
[0559] Examples of immune checkpoint proteins or receptors include
without limitation CD27 (NCBI Gene ID: 939), CD70 (NCBI Gene ID:
970); CD40 (NCBI Gene ID: 958), CD40LG (NCBI Gene ID: 959); CD47
(NCBI Gene ID: 961), SIRPA (NCBI Gene ID: 140885); CD48 (SLAMF2;
NCBI Gene ID: 962), transmembrane and immunoglobulin domain
containing 2 (TMIGD2, CD28H; NCBI Gene ID: 126259), CD84 (LY9B,
SLAMF5; NCBI Gene ID: 8832), CD96 (NCBI Gene ID: 10225), CD160
(NCBI Gene ID: 11126), MS4A1 (CD20; NCBI Gene ID: 931), CD244
(SLAMF4; NCBI Gene ID: 51744); CD276 (B7H3; NCBI Gene ID: 80381);
V-set domain containing T cell activation inhibitor 1 (VTCN1,
B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA; NCBI
Gene ID: 64115); immunoglobulin superfamily member 11 (IGSF11,
VSIG3; NCBI Gene ID: 152404); natural killer cell cytotoxicity
receptor 3 ligand 1 (NCR3LG1, B7H6; NCBI Gene ID: 374383); HERV-H
LTR-associating 2 (HHLA2, B7H7; NCBI Gene ID: 11148); inducible T
cell co-stimulator (ICOS, CD278; NCBI Gene ID: 29851); inducible T
cell co-stimulator ligand (ICOSLG, B7H2; NCBI Gene ID: 23308); TNF
receptor superfamily member 4 (TNFRSF4, OX40; NCBI Gene ID: 7293);
TNF superfamily member 4 (TNFSF4, OX40L; NCBI Gene ID: 7292);
TNFRSF8 (CD30; NCBI Gene ID: 943), TNFSF8 (CD30L; NCBI Gene ID:
944); TNFRSF10A (CD261, DR4, TRAILR1; NCBI Gene ID: 8797), TNFRSF9
(CD137; NCBI Gene ID: 3604), TNFSF9 (CD137L; NCBI Gene ID: 8744);
TNFRSF10B (CD262, DR5, TRAILR2; NCBI Gene ID: 8795), TNFRSF10
(TRAIL; NCBI Gene ID: 8743); TNFRSF14 (HVEM, CD270; NCBI Gene ID:
8764), TNFSF14 (HVEML; NCBI Gene ID: 8740); CD272 (B and T
lymphocyte associated (BTLA); NCBI Gene ID: 151888); TNFRSF17
(BCMA, CD269; NCBI Gene ID: 608), TNFSF13B (BAFF; NCBI Gene ID:
10673); TNFRSF18 (GITR; NCBI Gene ID: 8784), TNFSF18 (GITRL; NCBI
Gene ID: 8995); MHC class I polypeptide-related sequence A (MICA;
NCBI Gene ID: 100507436); MHC class I polypeptide-related sequence
B (MICB; NCBI Gene ID: 4277); CD274 (CD274, PDL1, PD-L; NCBI Gene
ID: 29126); programmed cell death 1 (PDCD1, PD1, PD-1; NCBI Gene
ID: 5133); cytotoxic T-lymphocyte associated protein 4 (CTLA4,
CD152; NCBI Gene ID: 1493); CD80 (B7-1; NCBI Gene ID: 941), CD28
(NCBI Gene ID: 940); nectin cell adhesion molecule 2 (NECTIN2,
CD112; NCBI Gene ID: 5819); CD226 (DNAM-1; NCBI Gene ID: 10666);
Poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155; NCBI
Gene ID: 5817); PVR related immunoglobulin domain containing
(PVRIG, CD112R; NCBI Gene ID: 79037); T cell immunoreceptor with Ig
and ITIM domains (TIGIT; NCBI Gene ID: 201633); T cell
immunoglobulin and mucin domain containing 4 (TIMD4; TIM4; NCBI
Gene ID: 91937); hepatitis A virus cellular receptor 2 (HAVCR2,
TIMD3, TIM3; NCBI Gene ID: 84868); galectin 9 (LGALS9; NCBI Gene
ID: 3965); lymphocyte activating 3 (LAG3, CD223; NCBI Gene ID:
3902); signaling lymphocytic activation molecule family member 1
(SLAMFI, SLAM, CD150; NCBI Gene ID: 6504); lymphocyte antigen 9
(LY9, CD229, SLAMF3; NCBI Gene ID: 4063); SLAM family member 6
(SLAMF6, CD352; NCBI Gene ID: 114836); SLAM family member 7
(SLAMF7, CD319; NCBI Gene ID: 57823); UL16 binding protein 1
(ULBPl; NCBI Gene ID: 80329); UL16 binding protein 2 (ULBP2; NCBI
Gene ID: 80328); UL16 binding protein 3 (ULBP3; NCBI Gene ID:
79465); retinoic acid early transcript 1E (RAETIE; ULBP4; NCBI Gene
ID: 135250); retinoic acid early transcript 1G (RAETIG; ULBP5; NCBI
Gene ID: 353091); retinoic acid early transcript 1L (RAET1L; ULBP6;
NCBI Gene ID: 154064); killer cell immunoglobulin like receptor,
three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1; NCBI
Gene ID: 3811, e.g., lirilumab (IPH-2102, IPH-4102)); killer cell
lectin like receptor C1 (KLRC1, NKG2A, CD159A; NCBI Gene ID: 3821);
killer cell lectin like receptor K1 (KLRK1, NKG2D, CD314; NCBI Gene
ID: 22914); killer cell lectin like receptor C2 (KLRC2, CD159c,
NKG2C; NCBI Gene ID: 3822); killer cell lectin like receptor C3
(KLRC3, NKG2E; NCBI Gene ID: 3823); killer cell lectin like
receptor C4 (KLRC4, NKG2F; NCBI Gene ID: 8302); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 1 (KIR2DL1; NCBI Gene ID: 3802); killer cell immunoglobulin
like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2;
NCBI Gene ID: 3803); killer cell immunoglobulin like receptor, two
Ig domains and long cytoplasmic tail 3 (KIR2DL3; NCBI Gene ID:
3804); killer cell immunoglobulin like receptor, three Ig domains
and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin like
receptor D1 (KLRD1; NCBI Gene ID: 3824); killer cell lectin like
receptor G1 (KLRG1; CLEC15A, MAFA, 2F1; NCBI Gene ID: 10219);
sialic acid binding Ig like lectin 7 (SIGLEC7; NCBI Gene ID:
27036); and sialic acid binding Ig like lectin 9 (SIGLEC9; NCBI
Gene ID: 27180).
[0560] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with one or more blockers or inhibitors of one
or more T-cell inhibitory immune checkpoint proteins or receptors.
Illustrative T-cell inhibitory immune checkpoint proteins or
receptors include without limitation CD274 (CD274, PDL1, PD-L1);
programmed cell death 1 ligand 2 (PDCDILG2, PD-L2, CD273);
programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T-lymphocyte
associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain
containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set
immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin
superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270),
TNFSF14 (HVEML); CD272 (B and T lymphocyte associated (BTLA)); PVR
related immunoglobulin domain containing (PVRIG, CD112R); T cell
immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte
activating 3 (LAG3, CD223); hepatitis A virus cellular receptor 2
(HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell
immunoglobulin like receptor, three Ig domains and long cytoplasmic
tail 1 (KIR, CD158E1); killer cell immunoglobulin like receptor,
two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell
immunoglobulin like receptor, two Ig domains and long cytoplasmic
tail 2 (KIR2DL2); killer cell immunoglobulin like receptor, two Ig
domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell
immunoglobulin like receptor, three Ig domains and long cytoplasmic
tail 1 (KIR3DL1). In various embodiments, the FLT3L-Fc fusion
proteins, homodimers, heterodimers, polynucleotides, vectors,
lipoplexes, such as LNPs, and/or pharmaceutical compositions, as
described herein, are combined with one or more agonist or
activators of one or more T-cell stimulatory immune checkpoint
proteins or receptors. Illustrative T-cell stimulatory immune
checkpoint proteins or receptors include without limitation CD27,
CD70; CD40, CD40LG; inducible T cell costimulator (ICOS, CD278);
inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor
superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4
(TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR),
TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2
(NECTIN2, CD112); CD226 (DNAM-1); CD244 (2B4, SLAMF4), Poliovirus
receptor (PVR) cell adhesion molecule (PVR, CD155). See, e.g., Xu,
et al., J Exp Clin Cancer Res. (2018) 37:110.
[0561] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with one or more blockers or inhibitors of one
or more NK-cell inhibitory immune checkpoint proteins or receptors.
Illustrative NK-cell inhibitory immune checkpoint proteins or
receptors include without limitation killer cell immunoglobulin
like receptor, three Ig domains and long cytoplasmic tail 1 (KIR,
CD158E1); killer cell immunoglobulin like receptor, two Ig domains
and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin
like receptor, two Ig domains and long cytoplasmic tail 2
(KIR2DL2); killer cell immunoglobulin like receptor, two Ig domains
and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin
like receptor, three Ig domains and long cytoplasmic tail 1
(KIR3DL1); killer cell lectin like receptor C1 (KLRC1, NKG2A,
CD159A); killer cell lectin like receptor D1 (KLRD1, CD94), killer
cell lectin like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic
acid binding Ig like lectin 7 (SIGLEC7); and sialic acid binding Ig
like lectin 9 (SIGLEC9). In various embodiments, the FLT3L-Fc
fusion proteins, homodimers, heterodimers, polynucleotides,
vectors, lipoplexes, such as LNPs, and/or pharmaceutical
compositions, as described herein, are combined with one or more
agonist or activators of one or more NK-cell stimulatory immune
checkpoint proteins or receptors. Illustrative NK-cell stimulatory
immune checkpoint proteins or receptors include without limitation
CD16, CD226 (DNAM-1); CD244 (2B4, SLAMF4); killer cell lectin like
receptor K1 (KLRK1, NKG2D, CD314); SLAM family member 7 (SLAMF7).
See, e.g., Davis, et al., Semin Immunol. (2017) 31:64-75; Fang, et
al., Semin Immunol. (2017) 31:37-54; and Chiossone, et al., Nat Rev
Immunol. (2018) 18(11):671-688.
[0562] In some embodiments, the one or more immune checkpoint
inhibitors comprises a proteinaceous (e.g., antibody or fragment
thereof, or antibody mimetic) inhibitor of PD-L1 (CD274), PD-1
(PDCD1) or CTLA4. In some embodiments, the one or more immune
checkpoint inhibitors comprises a small organic molecule inhibitor
of PD-L1 (CD274), PD-1 (PDCD1) or CTLA4.
[0563] Examples of inhibitors of CTLA4 that can be co-administered
include without limitation ipilimumab, tremelimumab, BMS-986218,
AGEN1181, AGEN1884 (zalifrelimab), BMS-986249, MK-1308, REGN-4659,
ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392,
AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5,
BPI-002, as well as multi-specific inhibitors FPT-155
(CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4),
KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717
(PD-1/CTLA4), and AK-104 (CTLA4/PD-1).
[0564] Examples of inhibitors of PD-L1 (CD274) or PD-1 (PDCD1) that
can be co-administered include without limitation pembrolizumab,
nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514),
spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559,
CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055),
AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091,
AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genolimzumab
(CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210
(camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306,
(MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311,
JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308
(sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001),
BCD-135, FAZ-053, TQB-2450, MDX1105-01, GS-4224, GS-4416,
INCB086550, MAX10181, AGEN2034 (balstilimab), zimberelimab, as well
as multi-specific inhibitors FPT-155 (CTLA4/PD-L1/CD28),
PF-06936308 (PD-1/CTLA4), MGD-013 (PD-1/LAG-3), FS-118
(LAG-3/PD-L1) MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752
(CTLA4/PD-1), RO-7121661 (PD-1/TIM4-3), XmAb-20717 (PD-1/CTLA4),
AK-104 (CTLA4/PD-1), M7824 (PD-L1/TGFO-EC domain), CA-170
(PD-L1/VISTA), CDX-527 (CD27/PD-L1), LY-3415244 (TIM3/PDL1), and
INBRX-105 (4-1BB/PDL1).
TNF Receptor Superfamily (TNFRSF) Member Agonists or Activators
[0565] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an agonist of one or more TNF receptor
superfamily (TNFRSF) members, e.g., an agonist of one or more of
TNFRSFlA (NCBI Gene ID: 7132), TNFRSFlB (NCBI Gene ID: 7133),
TNFRSF4 (OX40, CD134; NCBI Gene ID: 7293), TNFRSF5 (CD40; NCBI Gene
ID: 958), TNFRSF6 (FAS, NCBI Gene ID: 355), TNFRSF7 (CD27, NCBI
Gene ID: 939), TNFRSF8 (CD30, NCBI Gene ID: 943), TNFRSF9 (4-1BB,
CD137, NCBI Gene ID: 3604), TNFRSF10A (CD261, DR4, TRAILR1, NCBI
Gene ID: 8797), TNFRSF10B (CD262, DR5, TRAILR2, NCBI Gene ID:
8795), TNFRSF10C (CD263, TRAILR3, NCBI Gene ID: 8794), TNFRSF10D
(CD264, TRAILR4, NCBI Gene ID: 8793), TNFRSFIIA (CD265, RANK, NCBI
Gene ID: 8792), TNFRSFIIB (NCBI Gene ID: 4982), TNFRSF12A (CD266,
NCBI Gene ID: 51330), TNFRSF13B (CD267, NCBI Gene ID: 23495),
TNFRSF13C (CD268, NCBI Gene ID: 115650), TNFRSF16 (NGFR, CD271,
NCBI Gene ID: 4804), TNFRSF17 (BCMA, CD269, NCBI Gene ID: 608),
TNFRSF18 (GITR, CD357, NCBI Gene ID: 8784), TNFRSF19 (NCBI Gene ID:
55504), TNFRSF21 (CD358, DR6, NCBI Gene ID: 27242), and TNFRSF25
(DR3, NCBI Gene ID: 8718).
[0566] Example anti-TNFRSF4 (OX40) antibodies that can be
co-administered include without limitation, MEDI6469, MEDI6383,
MEDI0562 (tavolixizumab), MOXR0916, PF-04518600, RG-7888,
GSK-3174998, INCAGN1949, BMS-986178, GBR-8383, ABBV-368, and those
described in WO2016179517, WO2017096179, WO2017096182,
WO2017096281, and WO2018089628.
[0567] Example anti-TNFRSF5 (CD40) antibodies that can be
co-administered include without limitation RG7876, SEA-CD40,
APX-005M and ABBV-428.
[0568] In some embodiments, the anti-TNFRSF7 (CD27) antibody
varlilumab (CDX-1127) is co-administered.
[0569] Example anti-TNFRSF9 (4-1iB, CD137) antibodies that can be
co-administered include without limitation urelumab, utomilumab
(PF-05082566), AGEN2373 and ADG-106.
[0570] In some embodiments, the anti-TNFRSF17 (BCMA) antibody
GSK-2857916 is co-administered.
[0571] Example anti-TNFRSF18 (GITR) antibodies that can be
co-administered include without limitation, MEDI1873, FPA-154,
INCAGN-1876, TRX-518, BMS-986156, MK-1248, GWN-323, and those
described in WO2017096179, WO2017096276, WO2017096189, and
WO2018089628. In some embodiments, an antibody, or fragment
thereof, co-targeting TNFRSF4 (OX40) and TNFRSF18 (GITR) is
co-administered. Such antibodies are described, e.g., in
WO2017096179 and WO2018089628.
[0572] Bi-specific antibodies targeting TNFRSF family members that
can be co-administered include without limitation PRS-343
(CD-137/HER2), AFM26 (BCMA/CD16A), AFM-13 (CD16/CD30), REGN-1979
(CD20/CD3), AMG-420 (BCMA/CD3), INHIBRX-105 (4-1BB/PDL1),
FAP-4-IBBL (4-1BB/FAP), XmAb-13676 (CD3/CD20), RG-7828 (CD20/CD3),
CC-93269 (CD3/BCMA), REGN-5458 (CD3/BCMA), and IMM-0306
(CD47/CD20).
Bi-Specific T-Cell Engagers
[0573] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a bi-specific T-cell engager (e.g., not
having an Fc) or an anti-CD3 bi-specific antibody (e.g., having an
Fc). Illustrative anti-CD3 bi-specific antibodies or BiTEs that can
be co-administered include JNJ-64052781 (CD19/CD3), AMG-211
(CEA/CD3), RG7802 (CEA/CD3), ERY-974 (CD3/GPC3), PF-06671008
(Cadherins/CD3), APV0436 (CD123/CD3), flotetuzumab (CD123/CD3),
REGN-1979 (CD20/CD3), MCLA-117 (CD3/CLEC12A), JNJ-0819, JNJ-7564
(CD3/heme), AMG-757 (DLL3-CD3), AMG-330 (CD33/CD3), AMG-420
(BCMA/CD3), JNJ-63709178 (CD123/CD3), MGD-007 (CD3/gpA33), MGD-009
(CD3/B7H3), IMCgp100 (CD3/gp100), XmAb-14045 (CD123/CD3),
XmAb-13676 (CD3/CD20), XmAb-18087 (SSTR2/CD3), catumaxomab
(CD3/EpCAM), REGN-4018 (MUC16/CD3), RG-7828 (CD20/CD3), CC-93269
(CD3/BCMA), REGN-5458 (CD3/BCMA), GRB-1302 (CD3/Erbb2), GRB-1342
(CD38/CD3), GEM-333 (CD3/CD33). As appropriate, the anti-CD3
binding bi-specific molecules may or may not have an Fc.
Illustrative bi-specific T-cell engagers that can be
co-administered target CD3 and a tumor-associated antigen as
described herein, including, e.g., CD19 (e.g., blinatumomab); CD33
(e.g., AMG330); CEA (e.g., MEDI-565); receptor tyrosine kinase-like
orphan receptor 1 (ROR1) (Gohil, et al., Oncoimmunology. (2017) May
17; 6(7):e1326437); PD-L1 (Horn, et al., Oncotarget. 2017 Aug. 3;
8(35):57964-57980); and EGFRvIII (Yang, et al., Cancer Lett. 2017
Sep. 10; 403:224-230).
Bi- and Tri-Specific Natural Killer (NK)-Cell Engagers
[0574] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a bi-specific NK-cell engager (BiKE) or a
tri-specific NK-cell engager (TriKE) (e.g., not having an Fc) or
bi-specific antibody (e.g., having an Fc) against an NK cell
activating receptor, e.g., CD16A, C-type lectin receptors
(CD94/NKG2C, NKG2D, NKG2E/H and NKG2F), natural cytotoxicity
receptors (NKp30, NKp44 and NKp46), killer cell C-type lectin-like
receptor (NKp65, NKp80), Fc receptor Fc.gamma.R (which mediates
antibody-dependent cell cytotoxicity), SLAM family receptors (e.g.,
2B4, SLAM6 and SLAM7), killer cell immunoglobulin-like receptors
(KIR) (KIR-2DS and KIR-3DS), DNAM-1 and CD137 (41BB). Illustrative
anti-CD16 bi-specific antibodies, BiKEs or TriKEs that can be
co-administered include AFM26 (BCMA/CD16A) and AFM-13 (CD16/CD30).
As appropriate, the anti-CD16 binding bi-specific molecules may or
may not have an Fc. Illustrative bi-specific NK-cell engagers that
can be co-administered target CD16 and one or more tumor-associated
antigens as described herein, including, e.g., CD19, CD20, CD22,
CD30, CD33, CD123, EGFR, EpCAM, ganglioside GD2, HER2/neu, HLA
Class II and FOLR1. BiKEs and TriKEs are described, e.g., in
Felices, et al., Methods Mol Biol. (2016) 1441:333-346; Fang, et
al., Semin Immunol. (2017) 31:37-54.
MCL1 Apoptosis Regulator, BCL2 Family Member (MCL1) Inhibitors
[0575] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of MCL1 apoptosis regulator,
BCL2 family member (MCL1, TM; EAT; MCL1L; MCL1S; Mcl-1; BCL2L3;
MCL1-ES; bc2-L-3; mcll/EAT; NCBI Gene ID: 4170). Examples of MCL1
inhibitors include AMG-176, AMG-397, -64315, and AZD-5991, 483-LM,
A-1210477, UMI-77, JKY-5-037, and those described in WO2018183418,
WO2016033486, and WO2017147410.
SHP2 Inhibitors
[0576] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of protein tyrosine
phosphatase non-receptor type 11 (PTPN11; BPTP3, CFC, JMML, METCDS,
NS1, PTP-1D, PTP2C, SH-PTP2, SH-PTP3, SHP2; NCBI Gene ID: 5781).
Examples of SHP2 inhibitors include TNO155 (SHP-099), RMC-4550,
JAB-3068, RMC-4630, and those described in WO2018172984 and
WO2017211303.
Hematopoietic Progenitor Kinase 1 (HPK1) Inhibitors
[0577] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of mitogen-activated protein
kinase kinase kinase kinase 1 (MAP4K1, HPK1; NCBI Gene ID: 11184).
Examples of Hematopoietic Progenitor Kinase 1 (HPK1) inhibitors
include without limitation, those described in WO-2018183956,
WO-2018183964, WO-2018167147, WO-2018183964, WO-2016205942,
WO-2018049214, WO-2018049200, WO-2018049191, WO-2018102366,
WO-2018049152 and WO-2016090300;
Apoptosis Signal-Regulating Kinase (ASK) Inhibitors
[0578] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of an ASK inhibitor, e.g.,
mitogen-activated protein kinase kinase kinase 5 (MAP3K5; ASK1,
MAPKKK5, MEKK5; NCBI Gene ID: 4217). Examples of ASK1 inhibitors
include without limitation, those described in WO 2011/008709
(Gilead Sciences) and WO 2013/112741 (Gilead Sciences).
Bruton Tyrosine Kinase (BTK) Inhibitors
[0579] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplex, such
as LNPs, and/or pharmaceutical compositions, as described herein,
are combined with an inhibitor of Bruton tyrosine kinase (BTK,
AGMX1, AT, ATK, BPK, IGHD3, IMD1, PSCTK1, XLA; NCBI Gene ID: 695).
Examples of BTK inhibitors include without limitation,
(S)-6-amino-9-(1-(but-2-ynoyl)pyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-pur-
in-8(9H)-one, acalabrutinib (ACP-196), BGB-3111, CB988, HM71224,
ibrutinib, M-2951 (evobrutinib), M7583, tirabrutinib (ONO-4059),
PRN-1008, spebrutinib (CC-292), TAK-020, vecabrutinib, ARQ-531,
SHR-1459, DTRMWXHS-12, TAS-5315.
Cluster of Differentiation 47 (CD47) Inhibitors
[0580] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplex, such
as LNPs, and/or pharmaceutical compositions, as described herein,
are combined with an inhibitor of CD47 (IAP, MER6, OA3; NCBI Gene
ID: 961). Examples of CD47 inhibitors include without limitation
anti-CD47 mAbs (Vx-1004), anti-human CD47 mAbs (CNTO-7108),
CC-90002, CC-90002-ST-001, humanized anti-CD47 antibody (Hu5F9-G4),
NI-1701, NI-1801, RCT-1938, and TTI-621. In some embodiments, the
CD47 inhibitor is magrolimab.
SIRP.alpha. Targeting Agents
[0581] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a SIRP.alpha. targeting agent (NCBI Gene
ID: 140885; UniProt P78324). Examples of SIRP.alpha. targeting
agents include without limitation SIRP.alpha. inhibitors, such as
AL-008, RRx-001, and CTX-5861, and anti-SIRP.alpha. antibodies,
such as FSI-189 (GS-0189), ES-004, BI765063, ADU1805, and CC-95251.
Additional SIRPa-targeting agents of use are described, for
example, in W200140307, W2002092784, WO2007133811, WO2009046541,
WO2010083253, WO2011076781, WO2013056352, WO2015138600,
WO2016179399, WO2016205042, WO2017178653, WO2018026600,
WO2018057669, WO2018107058, WO2018190719, WO2018210793,
WO2019023347, WO2019042470, WO2019175218, WO2019183266,
WO2020013170 and WO2020068752.
Cyclin-dependent Kinase (CDK) Inhibitors
[0582] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of cyclin dependent kinase 1
(CDK1, CDC2; CDC28A; P34CDC2; NCBI Gene ID: 983); cyclin dependent
kinase 2 (CDK2, CDKN2; p33(CDK2); NCBI Gene ID: 1017); cyclin
dependent kinase 3 (CDK3, NCBI Gene ID: 1018); cyclin dependent
kinase 4 (CDK4, CMM3; PSK-J3; NCBI Gene ID: 1019); cyclin dependent
kinase 6 (CDK6, MCPH12; PLSTIRE; NCBI Gene ID: 1021); cyclin
dependent kinase 7 (CDK7, CAK; CAK1; HCAK; MO15; STK1; CDKN7;
p39MO15; NCBI Gene ID: 1022); cyclin dependent kinase 9 (CDK9, TAK;
C-2k; CTK1; CDC2L4; PITALRE; NCBI Gene ID: 1025). Inhibitors of CDK
1, 2, 3, 4, 6, 7 and/or 9, include without limitation abemaciclib,
alvocidib (HMR-1275, flavopiridol), AT-7519, dinaciclib, ibrance,
FLX-925, LEE001, palbociclib, ribociclib, rigosertib, selinexor,
UCN-01, SY1365, CT-7001, SY-1365, G1T38, milciclib, trilaciclib,
and TG-02.
Discoidin Domain Receptor (DDR) Inhibitors
[0583] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of discoidin domain receptor
tyrosine kinase 1 (DDR1, CAK, CD167, DDR, EDDR1, HGK2, MCK10, NEP,
NTRK4, PTK3, PTK3A, RTK6, TRKE; NCBI Gene ID: 780); and/or
discoidin domain receptor tyrosine kinase 2 (DDR2, MIG20a, NTRKR3,
TKT, TYRO10, WRCN; NCBI Gene ID: 4921). Examples of DDR inhibitors
include without limitation, dasatinib and those disclosed in
WO2014/047624 (Gilead Sciences), US 2009-0142345 (Takeda
Pharmaceutical), US 2011-0287011 (Oncomed Pharmaceuticals), WO
2013/027802 (Chugai Pharmaceutical), and WO2013/034933 (Imperial
Innovations).
Targeted E3 Ligase Ligand Conjugates
[0584] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a targeted E3 ligase ligand conjugate.
Such conjugates have a target protein binding moiety and an E3
ligase binding moiety (e.g., an inhibitor of apoptosis protein
(lAP) (e.g., XIAP, c-IAP1, c-IAP2, NIL-IAP, Bruce, and surviving)
E3 ubiquitin ligase binding moiety, Von Hippel-Lindau E3 ubiquitin
ligase (VHL) binding moiety, a cereblon E3 ubiquitin ligase binding
moiety, mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase
binding moiety), and can be used to promote or increase the
degradation of targeted proteins, e.g., via the ubiquitin pathway.
In one embodiment, the targeted E3 ligase ligand conjugates
comprise a targeting or binding moiety that targets or binds a
protein identified in Table B, and an E3 ligase ligand or binding
moiety. In one embodiment, the targeted E3 ligase ligand conjugates
comprise a targeting or binding moiety that targets or binds a
protein selected from Cbl proto-oncogene B (CBLB; Cbl-b, Nbla00127,
RNF56; NCBI Gene ID: 868) and hypoxia inducible factor 1 subunit
alpha (HIF1A; NCBI Gene ID: 3091). In one embodiment, the targeted
E3 ligase ligand conjugates comprise a kinase inhibitor (e.g., a
small molecule kinase inhibitor, e.g., of BTK and an E3 ligase
ligand or binding moiety. See, e.g., WO2018098280. In another
embodiment, the targeted E3 ligase ligand conjugates comprise a
binding moiety targeting or binding to Interleukin-1 (IL-1)
Receptor-Associated Kinase-4 (IRAK-4); Rapidly Accelerated
Fibrosarcoma (RAF, such as c-RAF, A-RAF and/or B-RAF), c-Met/p38,
or a BRD protein; and an E3 ligase ligand or binding moiety. See,
e.g., WO2019099926, WO2018226542, WO2018119448, WO2018223909,
WO2019079701. Additional targeted E3 ligase ligand conjugates that
can be co-administered are described, e.g., in WO2018237026,
WO2019084026, WO2019084030, WO2019067733, WO2019043217,
WO2019043208 and WO2018144649.
Histone Deacetylase (HDAC) Inhibitors
[0585] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of a histone deacetylase,
e.g., histone deacetylase 9 (HDAC9, HD7, HD7b, HD9, HDAC, HDAC7,
HDAC7B, HDAC9B, HDAC9FL, HDRP, MITR; Gene ID: 9734). Examples of
HDAC inhibitors include without limitation, abexinostat, ACY-241,
AR-42, BEBT-908, belinostat, CKD-581, CS-055 (HBI-8000), CUDC-907
(fimepinostat), entinostat, givinostat, mocetinostat, panobinostat,
pracinostat, quisinostat (JNJ-26481585), resminostat, ricolinostat,
SHP-141, valproic acid (VAL-001), vorinostat, tinostamustine,
remetinostat, entinostat.
Indoleamine-Pyrrole-2,3-Dioxygenase (IDO1) Inhibitors
[0586] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of indoleamine
2,3-dioxygenase 1 (IDO; NCBI Gene ID: 3620). Examples of IDO1
inhibitors include without limitation, BLV-0801, epacadostat,
F-001287, GBV-1012, GBV-1028, GDC-0919, indoximod, NKTR-218,
NLG-919-based vaccine, PF-06840003, pyranonaphthoquinone
derivatives (SN-35837), resminostat, SBLK-200802, BMS-986205, and
shIDO-ST, EOS-200271, KHK-2455, LY-3381916.
Janus Kinase (JAK) Inhibitors
[0587] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of Janus kinase 1 (JAK1,
JAK1A, JAK1B, JTK3; NCBI Gene ID: 3716); Janus kinase 2 (JAK2,
JTK10, THCYT3; NCBI Gene ID: 3717); and/or Janus kinase 3 (JAK3,
JAK-3, JAK3_HUMAN, JAKL, L-JAK, LJAK; NCBI Gene ID: 3718). Examples
of JAK inhibitors include without limitation, AT9283, AZD1480,
baricitinib, BMS-911543, fedratinib, filgotinib (GLPG0634),
gandotinib (LY2784544), INCB039110 (itacitinib), lestaurtinib,
momelotinib (CYT0387), NS-018, pacritinib (SB1518), peficitinib
(ASP015K), ruxolitinib, tofacitinib (formerly tasocitinib),
INCB052793, and XL019.
Lysyl Oxidase-Like Protein (LOXL) Inhibitors
[0588] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of a LOXL protein, e.g.,
LOXL1 (NCBI Gene ID: 4016), LOXL2 (NCBI Gene ID: 4017), LOXL3 (NCBI
Gene ID: 84695), LOXL4 (NCBI Gene ID: 84171), and/or LOX (NCBI Gene
ID: 4015). Examples of LOXL inhibitors include without limitation,
the antibodies described in WO 2009/017833 (Arresto Biosciences).
Examples of LOXL2 inhibitors include without limitation, the
antibodies described in WO 2009/017833 (Arresto Biosciences), WO
2009/035791 (Arresto Biosciences), and WO 2011/097513 (Gilead
Biologics).
Matrix Metalloprotease (MMP) Inhibitors
[0589] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of a matrix metallopeptidase
(MMP), e.g., an inhibitor of MMP1 (NCBI Gene ID: 4312), MMP2 (NCBI
Gene ID: 4313), MMP3 (NCBI Gene ID: 4314), MMP7 (NCBI Gene ID:
4316), MMP8 (NCBI Gene ID: 4317), MMP9 (NCBI Gene ID: 4318); MMP10
(NCBI Gene ID: 4319); MMP11 (NCBI Gene ID: 4320); MMP12 (NCBI Gene
ID: 4321), MMP13 (NCBI Gene ID: 4322), MMP14 (NCBI Gene ID: 4323),
MMP15 (NCBI Gene ID: 4324), MMP16 (NCBI Gene ID: 4325), MMP17 (NCBI
Gene ID: 4326), MMP19 (NCBI Gene ID: 4327), MMP20 (NCBI Gene ID:
9313), MMP21 (NCBI Gene ID: 118856), MMP24 (NCBI Gene ID: 10893),
MMP25 (NCBI Gene ID: 64386), MMP26 (NCBI Gene ID: 56547), MMP27
(NCBI Gene ID: 64066) and/or MMP28 (NCBI Gene ID: 79148). Examples
of MMP9 inhibitors include without limitation, marimastat
(BB-2516), cipemastat (Ro 32-3555), GS-5745 (andecaliximab) and
those described in WO 2012/027721 (Gilead Biologics).
RAS and RAS Pathway Inhibitors
[0590] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of KRAS proto-oncogene,
GTPase (KRAS; a.k.a., NS; NS3; CFC2; RALD; K-Ras; KRAS1; KRAS2;
RASK2; KI-RAS; C--K-RAS; K-RAS2A; K-RAS2B; K-RAS4A; K-RAS4B;
c-Ki-ras2; NCBI Gene ID: 3845); NRAS proto-oncogene, GTPase (NRAS;
a.k.a., NS6; CMNS; NCMS; ALPS4; N-ras; NRAS1; NCBI Gene ID: 4893);
HRas proto-oncogene, GTPase (HRAS; a.k.a., CTLO; KRAS; HAMSV;
HRAS1; KRAS2; RASH1; RASK2; Ki-Ras; p2lras; C-H-RAS; c-K-ras;
H-RASIDX; c-Ki-ras; C-BAS/HAS; C-HA-RAS1; NCBI Gene ID: 3265). The
Ras inhibitors can inhibit Ras at either the polynucleotide (e.g.,
transcriptional inhibitor) or polypeptide (e.g., GTPase enzyme
inhibitor) level. In some embodiments, the inhibitors target one or
more proteins in the Ras pathway, e.g., inhibit one or more of
EGFR, Ras, Raf (A-Raf, B-Raf, C-Raf), MEK (MEK1, MEK2), ERK, PI3K,
AKT and mTOR. Illustrative K-Ras inhibitors that can be
co-administered include ARS-1620 (G12C), SML-8-73-1 (G12C),
Compound 3144 (G12D), Kobe0065/2602 (Ras GTP), RT11, MRTX-849
(G12C) and K-Ras(G12D)-selective inhibitory peptides, including
KRpep-2 (Ac-RRCPLYISYDPVCRR-NH.sub.2) (SEQ ID NO:108) and KRpep-2d
(Ac-RRRRCPLYISYDPVCRRRR-NH.sub.2) (SEQ ID NO:109). Illustrative
KRAS mRNA inhibitors include anti-KRAS U1 adaptor, AZD-4785,
siG12D-LODER.TM., and siG12D exosomes. Illustrative MEK inhibitors
that can be co-administered include binimetinib, cobimetinib,
PD-0325901, pimasertib, RG-7304, selumetinib, trametinib, and those
described below and herein. Illustrative Raf dimer inhibitors that
can be co-administered BGB-283, HM-95573, LXH-254, LY-3009120,
RG7304 and TAK-580. Illustrative ERK inhibitors that can be
co-administered include LTT-462, LY-3214996, MK-8353, ravoxertinib
and ulixertinib. Illustrative Ras GTPase inhibitors that can be
co-administered include rigosertib. Illustrative PI3K inhibitors
that can be co-administered include idelalisib (Zydelig.RTM.),
alpelisib, buparlisib, pictilisib, and those described below and
herein. Illustrative PI3K/mTOR inhibitors that can be
co-administered include dactolisib, omipalisib and voxtalisib. In
certain embodiments, Ras-driven cancers (e.g., NSCLC) having CDKN2A
mutations can be inhibited by co-administration of the MEK
inhibitor selumetinib and the CDK4/6 inhibitor palbociclib. See,
e.g., Zhou, et al., Cancer Lett. 2017 Nov. 1; 408:130-137. Also,
K-RAS and mutant N-RAS can be reduced by the irreversible ERBB1/2/4
inhibitor neratinib. See, e.g., Booth, et al., Cancer Biol Ther.
2018 Feb. 1; 19(2):132-137.
Mitogen-Activated Protein Kinase (MEK) Inhibitors
[0591] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of mitogen-activated protein
kinase kinase 7 (MAP2K7, JNKK2, MAPKK7, MEK, MEK 7, MKK7, PRKMK7,
SAPKK-4, SAPKK4; NCBI Gene ID: 5609). Examples of MEK inhibitors
include antroquinonol, binimetinib, cobimetinib (GDC-0973, XL-518),
MT-144, selumetinib (AZD6244), sorafenib, trametinib (GSK1120212),
uprosertib+trametinib, PD-0325901, pimasertib, LTT462, AS703988,
CC-90003, refametinib.
Phosphatidylinositol 3-Kinase (PI3K) Inhibitors
[0592] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as an LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of a
phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit,
e.g., phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic
subunit alpha (PIK3CA, CLAPO, CLOVE, CWS5, MCAP, MCM, MCMTC, PI3K,
PI3K-alpha, p110-alpha; NCBI Gene ID: 5290);
phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit
beta (PIK3CB, PIOBETA, PI3K, PI3KBETA, PIK3C1; NCBI Gene ID: 5291);
phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit
gamma (PIK3CG, PI3CG, PI3K, PI3Kgamma, PIK3, p110gamma, p120-PI3K;
Gene ID: 5494); and/or phosphatidylinositol-4,5-bisphosphate
3-kinase catalytic subunit delta (PIK3CD, APDS, IMD14, P110DELTA,
PI3K, p.sup.110D, NCBI Gene ID: 5293). In some embodiments, the
PI3K inhibitor is a pan-PI3K inhibitor. Examples of PI3K inhibitors
include without limitation, ACP-319, AEZA-129, AMG-319, AS252424,
AZD8186, BAY 10824391, BEZ235, buparlisib (BKM120), BYL719
(alpelisib), CH5132799, copanlisib (BAY 80-6946), duvelisib,
GDC-0032, GDC-0077, GDC-0941, GDC-0980, GSK2636771, GSK2269557,
idelalisib (Zydelig.RTM.), INCB50465, IPI-145, IPI-443, IPI-549,
KAR4141, LY294002, LY3023414, MLN1117, OXY111A, PA799, PX-866,
RG7604, rigosertib, RP5090, RP6530, SRX3177, taselisib, TG100115,
TGR-1202 (umbralisib), TGX221, WX-037, X-339, X-414, XL147
(SAR245408), XL499, XL756, wortmannin, ZSTK474, and the compounds
described in WO 2005/113556 (ICOS), WO 2013/052699 (Gilead
Calistoga), WO 2013/116562 (Gilead Calistoga), WO 2014/100765
(Gilead Calistoga), WO 2014/100767 (Gilead Calistoga), and WO
2014/201409 (Gilead Sciences).
Spleen Tyrosine Kinase (SYK) Inhibitors
[0593] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an inhibitor of spleen associated
tyrosine kinase (SYK, p72-Syk, Gene ID: 6850). Examples of SYK
inhibitors include without limitation,
6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine,
BAY-61-3606, cerdulatinib (PRT-062607), entospletinib, fostamatinib
(R788), HIMPL-523, NVP-QAB 205 AA, R112, R343, tamatinib (R406),
and those described in U.S. Pat. No. 8,450,321 (Gilead Connecticut)
and those described in U.S. 2015/0175616.
Toll-Like Receptor (TLR) Agonists
[0594] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an agonist of a toll-like receptor (TLR),
e.g., an agonist of TLR1 (NCBI Gene ID: 7096), TLR2 (NCBI Gene ID:
7097), TLR3 (NCBI Gene ID: 7098), TLR4 (NCBI Gene ID: 7099), TLR5
(NCBI Gene ID: 7100), TLR6 (NCBI Gene ID: 10333), TLR7 (NCBI Gene
ID: 51284), TLR8 (NCBI Gene ID: 51311), TLR9 (NCBI Gene ID: 54106),
and/or TLR10 (NCBI Gene ID: 81793). Example TLR7 agonists that can
be co-administered include without limitation DS-0509, GS-9620
(vesatolimod), vesatolimod analogs, LHC-165, TMX-101 (imiquimod),
GSK-2245035, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465,
MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202,
RG-7863, RG-7795, and the compounds disclosed in US20100143301
(Gilead Sciences), US20110098248 (Gilead Sciences), and
US20090047249 (Gilead Sciences), US20140045849 (Janssen),
US20140073642 (Janssen), WO2014/056953 (Janssen), WO2014/076221
(Janssen), WO2014/128189 (Janssen), US20140350031 (Janssen),
WO2014/023813 (Janssen), US20080234251 (Array Biopharma),
US20080306050 (Array Biopharma), US20100029585 (Ventirx Pharma),
US20110092485 (Ventirx Pharma), US20110118235 (Ventirx Pharma),
US20120082658 (Ventirx Pharma), US20120219615 (Ventirx Pharma),
US20140066432 (Ventirx Pharma), US20140088085 (Ventirx Pharma),
US20140275167 (Novira Therapeutics), and US20130251673 (Novira
Therapeutics). An TLR7/TLR8 agonist that can be co-administered is
NKTR-262. Example TLR8 agonists that can be co-administered include
without limitation E-6887, IMO-4200, IMO-8400, IMO-9200, MCT-465,
MEDI-9197, motolimod, resiquimod, GS-9688, VTX-1463, VTX-763,
3M-051, 3M-052, and the compounds disclosed in US20140045849
(Janssen), US20140073642 (Janssen), WO2014/056953 (Janssen),
WO2014/076221 (Janssen), WO2014/128189 (Janssen), US20140350031
(Janssen), WO2014/023813 (Janssen), US20080234251 (Array
Biopharma), US20080306050 (Array Biopharma), US20100029585 (Ventirx
Pharma), US20110092485 (Ventirx Pharma), US20110118235 (Ventirx
Pharma), US20120082658 (Ventirx Pharma), US20120219615 (Ventirx
Pharma), US20140066432 (Ventirx Pharma), US20140088085 (Ventirx
Pharma), US20140275167 (Novira Therapeutics), and US20130251673
(Novira Therapeutics). Example TLR9 agonists that can be
co-administered include without limitation AST-008, CMP-001,
IMO-2055, IMO-2125, litenimod, MGN-1601, BB-001, BB-006, IMO-3100,
IMO-8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079,
DV-1179, AZD-1419, leftolimod (MGN-1703), CYT-003, CYT-003-QbG10
and PUL-042. Examples of TLR3 agonist include rintatolimod,
poly-ICLC, RIBOXXON.RTM., Apoxxim, RIBOXXIM.RTM., IPH-33, MCT-465,
MCT-475, and ND-1.1.
Tyrosine-Kinase Inhibitors (TKIs)
[0595] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a tyrosine kinase inhibitor (TKI). TKIs
may target epidermal growth factor receptors (EGFRs) and receptors
for fibroblast growth factor (FGF), platelet-derived growth factor
(PDGF), and vascular endothelial growth factor (VEGF). Examples of
TKIs include without limitation, afatinib, ARQ-087 (derazantinib),
asp5878, AZD3759, AZD4547, bosutinib, brigatinib, cabozantinib,
cediranib, crenolanib, dacomitinib, dasatinib, dovitinib, E-6201,
erdafitinib, erlotinib, gefitinib, gilteritinib (ASP-2215),
FP-1039, HM61713, icotinib, imatinib, KX2-391 (Src), lapatinib,
lestaurtinib, lenvatinib, midostaurin, nintedanib, ODM-203,
osimertinib (AZD-9291), ponatinib, poziotinib, quizartinib,
radotinib, rociletinib, sulfatinib (HMPL-012), sunitinib, famitinib
L-malate, (MAC-4), tivoanib, TH-4000, and MEDI-575 (anti-PDGFR
antibody).
Chemotherapeutic Agents
[0596] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a chemotherapeutic agent or
anti-neoplastic agent.
[0597] As used herein, the term "chemotherapeutic agent" or
"chemotherapeutic" (or "chemotherapy" in the case of treatment with
a chemotherapeutic agent) is meant to encompass any
non-proteinaceous (e.g., non-peptidic) chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include but not limited to: alkylating agents such as thiotepa and
cyclophosphamide (CYTOXAN.RTM.); alkyl sulfonates such as busulfan,
improsulfan, and piposulfan; aziridines such as benzodepa,
carboquone, meturedepa, and uredepa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide, and
trimemylolomelamine; acetogenins, e.g., bullatacin and
bullatacinone; a camptothecin, including synthetic analog
topotecan; bryostatin, callystatin; CC-1065, including its
adozelesin, carzelesin, and bizelesin synthetic analogs;
cryptophycins, particularly cryptophycin 1 and cryptophycin 8;
dolastatin; duocarmycin, including the synthetic analogs KW-2189
and CBI-TMI; eleutherobin; 5-azacytidine; pancratistatin; a
sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cyclophosphamide, glufosfamide, evofosfamide,
bendamustine, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, and uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, foremustine,
lomustine, nimustine, and ranimustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammaII and calicheamicin phiIl), dynemicin including dynemicin A,
bisphosphonates such as clodronate, an esperamicin,
neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromomophores, aclacinomycins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
carrninomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogs such as demopterin, methotrexate, pteropterin,
and trimetrexate; purine analogs such as cladribine, pentostatin,
fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;
pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
and floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, and testolactone;
anti-adrenals such as aminoglutethimide, mitotane, and trilostane;
folic acid replinishers such as frolinic acid; radiotherapeutic
agents such as Radium-223; trichothecenes, especially T-2 toxin,
verracurin A, roridin A, and anguidine; taxoids such as paclitaxel
(TAXOL.RTM.), abraxane,docetaxel (TAXOTERE.RTM.), cabazitaxel,
BIND-014, tesetaxel; platinum analogs such as cisplatin and
carboplatin, NC-6004 nanoplatin; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; hestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformthine; elliptinium acetate; an epothilone; etoglucid; gallium
nitrate; hydroxyurea; lentinan; leucovorin; lonidamine;
maytansinoids such as maytansine and ansamitocins; mitoguazone;
mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;
pirarubicin; losoxantrone; fluoropyrimidine; folinic acid;
podophyllinic acid; 2-ethylhydrazide; procarbazine;
polysaccharide-K (PSK); razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; trabectedin, triaziquone;
2,2',2''-trichlorotriemylamine; urethane; vindesine; dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); cyclophosphamide; thiopeta; chlorambucil;
gemcitabine (GEMZAR.RTM.); 6-thioguanine; mercaptopurine;
methotrexate; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitroxantrone; vancristine; vinorelbine (NAVELBINE.RTM.);
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DFMO); retinoids such as retinoic acid;
capecitabine; NUC-1031; FOLFOX (folinic acid, 5-fluorouracil,
oxaliplatin); FOLFIRI (folinic acid, 5-fluorouracil, irinotecan);
FOLFOXIRI (folinic acid, 5-fluorouracil, oxaliplatin, irinotecan),
FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, oxaliplatin),
and pharmaceutically acceptable salts, acids, or derivatives of any
of the above. Such agents can be conjugated onto an antibody or any
targeting agent described herein to create an antibody-drug
conjugate (ADC) or targeted drug conjugate.
Anti-Hormonal Agents
[0598] Also included in the definition of "chemotherapeutic agent"
are anti-hormonal agents such as anti-estrogens and selective
estrogen receptor modulators (SERMs), inhibitors of the enzyme
aromatase, anti-androgens, and pharmaceutically acceptable salts,
acids or derivatives of any of the above that act to regulate or
inhibit hormone action on tumors.
[0599] Examples of anti-estrogens and SERMs include, for example,
tamoxifen (including NOLVADEX.TM.), raloxifene, droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and toremifene (FARESTON.RTM.).
[0600] Inhibitors of the enzyme aromatase regulate estrogen
production in the adrenal glands. Examples include 4(5)-imidazoles,
aminoglutethimide, megestrol acetate (MEGACE.RTM.), exemestane,
formestane, fadrozole, vorozole (RIVISOR.RTM.), letrozole
(FEMARA.RTM.), and anastrozole (ARIMIDEX.RTM.).
[0601] Examples of anti-androgens include apalutamide, abiraterone,
enzalutamide, flutamide, galeterone, nilutamide, bicalutamide,
leuprolide, goserelin, ODM-201, APC-100, ODM-204.
[0602] An example progesterone receptor antagonist includes
onapristone.
Anti-Angiogenic Agents
[0603] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an anti-angiogenic agent. Anti-angiogenic
agents that can be co-administered include, but are not limited to,
retinoid acid and derivatives thereof, 2-methoxyestradiol,
ANGIOSTATIN.RTM., ENDOSTATIN.RTM., regorafenib, necuparanib,
suramin, squalamine, tissue inhibitor of metalloproteinase-1,
tissue inhibitor of metalloproteinase-2, plasminogen activator
inhibitor-1, plasminogen activator inbibitor-2, cartilage-derived
inhibitor, paclitaxel (nab-paclitaxel), platelet factor 4,
protamine sulphate (clupeine), sulphated chitin derivatives
(prepared from queen crab shells), sulphated polysaccharide
peptidoglycan complex (sp-pg), staurosporine, modulators of matrix
metabolism including proline analogs such as
1-azetidine-2-carboxylic acid (LACA), cishydroxyproline,
d,I-3,4-dehydroproline, thiaproline, .alpha.,.alpha.'-dipyridyl,
beta-aminopropionitrile fumarate,
4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone, methotrexate,
mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chicken
inhibitor of metalloproteinase-3 (ChIMP-3), chymostatin,
beta-cyclodextrin tetradecasulfate, eponemycin, fumagillin, gold
sodium thiomalate, d-penicillamine, beta-1-anticollagenase-serum,
alpha-2-antiplasmin, bisantrene, lobenzarit disodium,
n-2-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA",
thalidomide, angiostatic steroid, carboxy aminoimidazole,
metalloproteinase inhibitors such as BB-94, inhibitors of S100A9
such as tasquinimod. Other anti-angiogenesis agents include
antibodies, preferably monoclonal antibodies against these
angiogenic growth factors: beta-FGF, alpha-FGF, FGF-5, VEGF
isoforms, VEGF-C, HGF/SF, and Ang-1/Ang-2.
Anti-Fibrotic Agents
[0604] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an anti-fibrotic agent. Anti-fibrotic
agents that can be co-administered include, but are not limited to,
the compounds such as beta-aminoproprionitrile (BAPN), as well as
the compounds disclosed in U.S. Pat. No. 4,965,288 relating to
inhibitors of lysyl oxidase and their use in the treatment of
diseases and conditions associated with the abnormal deposition of
collagen and U.S. Pat. No. 4,997,854 relating to compounds which
inhibit LOX for the treatment of various pathological fibrotic
states, which are herein incorporated by reference. Further
exemplary inhibitors are described in U.S. Pat. No. 4,943,593
relating to compounds such as 2-isobutyl-3-fluoro-, chloro-, or
bromo-allylamine, U.S. Pat. Nos. 5,021,456, 5,059,714, 5,120,764,
5,182,297, 5,252,608 relating to
2-(1-naphthyloxymemyl)-3-fluoroallylamine, and US 2004-0248871,
which are herein incorporated by reference.
[0605] Exemplary anti-fibrotic agents also include the primary
amines reacting with the carbonyl group of the active site of the
lysyl oxidases, and more particularly those which produce, after
binding with the carbonyl, a product stabilized by resonance, such
as the following primary amines: emylenemamine, hydrazine,
phenylhydrazine, and their derivatives; semicarbazide and urea
derivatives; aminonitriles such as BAPN or 2-nitroethylamine;
unsaturated or saturated haloamines such as 2-bromo-ethylamine,
2-chloroethylamine, 2-trifluoroethylamine, 3-bromopropylamine, and
p-halobenzylamines; and selenohomocysteine lactone.
[0606] Other anti-fibrotic agents are copper chelating agents
penetrating or not penetrating the cells. Exemplary compounds
include indirect inhibitors which block the aldehyde derivatives
originating from the oxidative deamination of the lysyl and
hydroxylysyl residues by the lysyl oxidases. Examples include the
thiolamines, particularly D-penicillamine, and its analogs such as
2-amino-5-mercapto-5-methylhexanoic acid,
D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid,
p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid,
sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane
sulphurate, 2-acetamidoethyl-2-acetamidoethanethiol sulphanate, and
sodium-4-mercaptobutanesulphinate trihydrate.
Anti-Inflammatory Agents
[0607] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an anti-inflammatory agent. Example
anti-inflammatory agents include without limitation inhibitors of
one or more of arginase (ARG1 (NCBI Gene ID: 383), ARG2 (NCBI Gene
ID: 384)), carbonic anhydrase (CA1 (NCBI Gene ID: 759), CA2 (NCBI
Gene ID: 760), CA3 (NCBI Gene ID: 761), CA4 (NCBI Gene ID: 762),
CA5A (NCBI Gene ID: 763), CA5B (NCBI Gene ID: 11238), CA6 (NCBI
Gene ID: 765), CA7 (NCBI Gene ID: 766), CA8 (NCBI Gene ID: 767),
CA9 (NCBI Gene ID: 768), CA10 (NCBI Gene ID: 56934), CA11 (NCBI
Gene ID: 770), CA12 (NCBI Gene ID: 771), CA13 (NCBI Gene ID:
377677), CA14 (NCBI Gene ID: 23632)), prostaglandin-endoperoxide
synthase 1 (PTGS1, COX-1; NCBI Gene ID: 5742),
prostaglandin-endoperoxide synthase 2 (PTGS2, COX-2; NCBI Gene ID:
5743), secreted phospholipase A2, prostaglandin E synthase (PTGES,
PGES; Gene ID: 9536), arachidonate 5-lipoxygenase (ALOX5, 5-LOX;
NCBI Gene ID: 240), soluble epoxide hydrolase 2 (EPHX2, SEH; NCBI
Gene ID: 2053) and/or mitogen-activated protein kinase kinase
kinase 8 (MAP3K8, TPL2; NCBI Gene ID: 1326). In some embodiments,
the inhibitor is a dual inhibitor, e.g., a dual inhibitor of
COX-2/COX-1, COX-2/SEH, COX-2/CA, COX-2/5-LOX.
[0608] Examples of inhibitors of prostaglandin-endoperoxide
synthase 1 (PTGS1, COX-1; NCBI Gene ID: 5742) that can be
co-administered include without limitation mofezolac, GLY-230, and
TRK-700.
[0609] Examples of inhibitors of prostaglandin-endoperoxide
synthase 2 (PTGS2, COX-2; NCBI Gene ID: 5743) that can be
co-administered include without limitation diclofenac, meloxicam,
parecoxib, etoricoxib, AP-101, celecoxib, AXS-06, diclofenac
potassium, DRGT-46, AAT-076, meisuoshuli, lumiracoxib, meloxicam,
valdecoxib, zaltoprofen, nimesulide, Anitrazafen, Apricoxib,
Cimicoxib, Deracoxib, Flumizole, Firocoxib, Mavacoxib, NS-398,
Pamicogrel, Parecoxib, Robenacoxib, Rofecoxib, Rutecarpine,
Tilmacoxib, and Zaltoprofen. Examples of dual COX1/COX2 inhibitors
that can be co-administered include without limitation, HP-5000,
lornoxicam, ketorolac tromethamine, bromfenac sodium, ATB-346,
HP-5000. Examples of dual COX-2/carbonic anhydrase (CA) inhibitors
that can be co-administered include without limitation polmacoxib
and imrecoxib.
[0610] Examples of inhibitors of secreted phospholipase A2,
prostaglandin E synthase (PTGES, PGES; Gene ID: 9536) that can be
co-administered include without limitation LY3023703, GRC 27864,
and compounds described in WO2015158204, WO2013024898,
WO2006063466, WO2007059610, WO2007124589, WO2010100249,
WO2010034796, WO2010034797, WO2012022793, WO2012076673,
WO2012076672, WO2010034798, WO2010034799, WO2012022792,
WO2009103778, WO2011048004, WO2012087771, WO2012161965,
WO2013118071, WO2013072825, WO2014167444, WO2009138376,
WO2011023812, WO2012110860, WO2013153535, WO2009130242,
WO2009146696, WO2013186692, WO2015059618, WO2016069376,
WO2016069374, WO2009117985, WO2009064250, WO2009064251,
WO2009082347, WO2009117987, and WO2008071173. Metformin has further
been found to repress the COX2/PGE2/STAT3 axis, and can be
co-administered. See, e.g., Tong, et al., Cancer Lett. (2017)
389:23-32; and Liu, et al., Oncotarget. (2016) 7(19):28235-46.
[0611] Examples of inhibitors of carbonic anhydrase (e.g., one or
more of CA1 (NCBI Gene ID: 759), CA2 (NCBI Gene ID: 760), CA3 (NCBI
Gene ID: 761), CA4 (NCBI Gene ID: 762), CA5A (NCBI Gene ID: 763),
CA5B (NCBI Gene ID: 11238), CA6 (NCBI Gene ID: 765), CA7 (NCBI Gene
ID: 766), CA8 (NCBI Gene ID: 767), CA9 (NCBI Gene ID: 768), CA10
(NCBI Gene ID: 56934), CA11 (NCBI Gene ID: 770), CA12 (NCBI Gene
ID: 771), CA13 (NCBI Gene ID: 377677), CA14 (NCBI Gene ID: 23632))
that can be co-administered include without limitation
acetazolamide, methazolamide, dorzolamide, zonisamide, brinzolamide
and dichlorphenamide. A dual COX-2/CA1/CA2 inhibitor that can be
co-administered includes CG100649.
[0612] Examples of inhibitors of arachidonate 5-lipoxygenase
(ALOX5, 5-LOX; NCBI Gene ID: 240) that can be co-administered
include without limitation meclofenamate sodium, zileuton.
[0613] Examples of inhibitors of soluble epoxide hydrolase 2
(EPHX2, SEH; NCBI Gene ID: 2053) that can be co-administered
include without limitation compounds described in WO2015148954.
Dual inhibitors of COX-2/SEH that can be co-administered include
compounds described in WO2012082647. Dual inhibitors of SEH and
fatty acid amide hydrolase (FAAH; NCBI Gene ID: 2166) that can be
co-administered include compounds described in WO2017160861.
[0614] Examples of inhibitors of mitogen-activated protein kinase
kinase kinase 8 (MAP3K8, tumor progression loci-2, TPL2; NCBI Gene
ID: 1326) that can be co-administered include without limitation
GS-4875, GS-5290, BHM-078 and those described, e.g., in
WO2006124944, WO2006124692, WO2014064215, WO2018005435, Teli, et
al., JEnzyme Inhib Med Chem. (2012) 27(4):558-70; Gangwall, et al.,
Curr Top Med Chem. (2013) 13(9):1015-35; Wu, et al., Bioorg Med
Chem Lett. (2009) 19(13):3485-8; Kaila, et al., Bioorg Med Chem.
(2007) 15(19):6425-42; and Hu, et al., Bioorg Med Chem Lett. (2011)
21(16):4758-61.
Tumor Oxygenation Agents
[0615] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an agent that promotes or increases tumor
oxygenation or reoxygenation, or prevents or reduces tumor hypoxia.
Illustrative agents that can be co-administered include, e.g.,
Hypoxia inducible factor-1 alpha (HIF-1a) inhibitors, such as
PT-2977, PT-2385; VEGF inhibitors, such as bevasizumab, IMC-3C5,
GNR-011, tanibirumab, LYN-00101, ABT-165; and/or an oxygen carrier
protein (e.g., a heme nitric oxide and/or oxygen binding protein
(HNOX)), such as OMX-302 and HNOX proteins described in WO
2007/137767, WO 2007/139791, WO 2014/107171, and WO
2016/149562.
Immunotherapeutic Agents
[0616] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an immunotherapeutic agent. Example
immunotherapeutic agents that can be co-administered include
without limitation abagovomab, ABP-980, adecatumumab, afutuzumab,
alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab,
bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab,
brentuximab, cantuzumab, catumaxomab, CC49, cetuximab, citatuzumab,
cixutumumab, clivatuzumab, conatumumab, dacetuzumab, dalotuzumab,
daratumumab, detumomab, dinutuximab, drozitumab, duligotumab,
dusigitumab, ecromeximab, elotuzumab, emibetuzumab, ensituximab,
ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab,
flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab,
glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab,
inotuzumab, intetumumab, ipilimumab (YERVOY.RTM., MDX-010,
BMS-734016, and MDX-101), iratumumab, labetuzumab, lexatumumab,
lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab,
milatuzumab, minretumomab, mitumomab, mogamulizumab, moxetumomab,
naptumomab, narnatumab, necitumumab, nimotuzumab, nofetumomab,
OBI-833, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab,
onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab,
pasudotox, patritumab, pemtumomab, pertuzumab, pintumomab,
pritumumab, racotumomab, radretumab, ramucirumab (Cyramza.RTM.),
rilotumumab, rituximab, robatumumab, samalizumab, satumomab,
sibrotuzumab, siltuximab, solitomab, simtuzumab, tacatuzumab,
taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab,
trastuzumab, tucotuzumab, ubilituximab, veltuzumab, vorsetuzumab,
votumumab, zalutumumab, and 3F8. Rituximab can be used for treating
indolent B-cell cancers, including marginal-zone lymphoma, WM, CLL
and small lymphocytic lymphoma. A combination of Rituximab and
chemotherapy agents is especially effective.
[0617] The exemplified therapeutic antibodies may be further
labeled or combined with a radioisotope particle such as
indium-111, yttrium-90 (90Y-clivatuzumab), or iodine-131.
[0618] In some embodiments, the immunotherapeutic agent is an
antibody-drug conjugate (ADC). Illustrative ADCs that can be
co-administered include without limitation drug-conjugated
antibodies, fragments thereof, or antibody mimetics targeting the
proteins or antigens listed above and herein (e.g., in Table B).
Example ADCs that can be co-administered include without limitation
gemtuzumab, brentuximab, trastuzumab, inotuzumab, glembatumumab,
anetumab, mirvetuximab, depatuxizumab, rovalpituzumab,
vadastuximab, labetuzumab, sacituzumab, lifastuzumab, indusatumab,
polatzumab, pinatuzumab, coltuximab, indatuximab, milatuzumab,
rovalpituzumab, ABBV-399, AGS-16C3F, ASG-22ME, AGS67E, AMG172,
AMG575, BAY1129980, BAY1187982, BAY94-9343, GSK2857916,
Humax-TF-ADC, 1MGN289, IMGN529, I1MGN853, LOP628, PCA062, MDX-1203
(BMS936561), MEDI-547, PF-06263507, PF-06647020, PF-06647263,
PF-06664178, RG7450, RG7458, RG7598, SAR566658, SGN-CD19A,
SGN-CD33A, SGN-CD70A, SGN-LIV1A and SYD985. ADCs that can be
co-administered are described, e.g., in Lambert, et al., Adv Ther
(2017) 34:1015-1035 and in de Goeij, Current Opinion in Immunology
(2016) 40:14-23.
[0619] Illustrative therapeutic agents (e.g., anticancer or
antineoplastic agents) that can be conjugated to the
drug-conjugated antibodies, fragments thereof, or antibody mimetics
include without limitation monomethyl auristatin E (MMAE),
monomethyl auristatin F (MMAF), a calicheamicin, ansamitocin,
maytansine or an analog thereof (e.g., mertansine/emtansine (DM1),
ravtansine/soravtansine (DM4)), an anthracyline (e.g., doxorubicin,
daunorubicin, epirubicin, idarubicin), pyrrolobenzodiazepine (PBD)
DNA cross-linking agent SC-DR002 (D6.5), duocarmycin, a microtubule
inhibitors (MTI) (e.g., a taxane, a vinca alkaloid, an epothilone),
a pyrrolobenzodiazepine (PBD) or dimer thereof, a duocarmycin (A,
B1, B2, C1, C2, D, SA, CC-1065), and other anticancer or
anti-neoplastic agents described herein. In some embodiments, the
therapeutic agents (e.g., anticancer or antineoplastic agents) that
can be conjugated to the drug-conjugated antibodies, fragments
thereof, or antibody mimetics include an immune checkpoint
inhibitor. In some embodiments, the conjugated immune checkpoint
inhibitor is a conjugated small molecule inhibitor of CD274 (PDL1,
PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1) or CTLA4. In
some embodiments, the conjugated small molecule inhibitor of CD274
or PDCD1 is selected from the group consisting of GS-4224, GS-4416,
INCB086550 and MAX10181. In some embodiments, the conjugated small
molecule inhibitor of CTLA4 comprises BPI-002.
Cancer Gene Therapy and Cell Therapy
[0620] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a cancer gene therapy and cell therapy.
Cancer gene therapies and cell therapies include the insertion of a
normal gene into cancer cells to replace a mutated or altered gene;
genetic modification to silence a mutated gene; genetic approaches
to directly kill the cancer cells; including the infusion of immune
cells designed to replace most of the patient's own immune system
to enhance the immune response to cancer cells, or activate the
patient's own immune system (T cells or Natural Killer cells) to
kill cancer cells, or find and kill the cancer cells; genetic
approaches to modify cellular activity to further alter endogenous
immune responsiveness against cancer.
Cellular Therapies
[0621] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with one or more cellular therapies.
Illustrative cellular therapies include without limitation
co-administration of one or more of a population of natural killer
(NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK)
cells, macrophage (MAC) cells, tumor infiltrating lymphocytes
(TILs) and/or dendritic cells (DCs). In some embodiments, the
cellular therapy entails a T cell therapy, e.g., co-administering a
population of alpha/beta TCR T cells, gamma/delta TCR T cells,
regulatory T (Treg) cells and/or TRuC.TM. T cells. In some
embodiments, the cellular therapy entails a NK cell therapy, e.g.,
co-administering NK-92 cells. As appropriate, a cellular therapy
can entail the co-administration of cells that are autologous,
syngeneic or allogeneic to the subject.
[0622] In some embodiments, the cellular therapy entails
co-administering cells comprising chimeric antigen receptors
(CARs). In such therapies, a population of immune effector cells
engineered to express a CAR, wherein the CAR comprises a tumor
antigen-binding domain. In T cell therapies, the T cell receptors
(TCRs) are engineered to target tumor derived peptides presented on
the surface of tumor cells.
[0623] With respect to the structure of a CAR, in some embodiments,
the CAR comprises an antigen binding domain, a transmembrane
domain, and an intracellular signaling domain. In some embodiments,
the intracellular domain comprises a primary signaling domain, a
costimulatory domain, or both of a primary signaling domain and a
costimulatory domain. In some embodiments, the primary signaling
domain comprises a functional signaling domain of one or more
proteins selected from the group consisting of CD3 zeta, CD3 gamma,
CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc
Epsilon Rlb), CD79a, CD79b, Fcgamma RIIa, DAP10, and DAP12.
[0624] In some embodiments, the costimulatory domain comprises a
functional domain of one or more proteins selected from the group
consisting of CD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, PD-1,
ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically
binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7,
NKp80 (KLRFI), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R
gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,
VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1A (NCBI Gene ID: 909),
CD1B (NCBI Gene ID: 910), CD1C (NCBI Gene ID: 911), CD1D (NCBI Gene
ID: 912), CD1E (NCBI Gene ID: 913), ITGAM, ITGAX, ITGB1, CD29,
ITGB2 (CD18, LFA-1), ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226),
SLAMF4 (CD244,2B4), CD84, CD96 (Tactile), CEACAMI, CRTAM, Ly9
(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,
Ly108), SLAM (SLAMFI, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and
NKG2D.
[0625] In some embodiments, the transmembrane domain comprises a
transmembrane domain of a protein selected from the group
consisting of the alpha, beta or zeta chain of the T-cell receptor,
CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,
CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2,
CD27, ICOS (CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR),
SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R,
ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,
CD1A, CD1B, CD1C, CD1D, CDE, ITGAE, CD103, ITGAL, ITGAM, ITGAX,
ITGB1, CD29, ITGB2 (LFA-1, CD18), ITGB7, TNFR2, DNAM1 (CD226),
SLAMF4 (CD244,22B4), CD84, CD96 (TACTILE), CEACAMI, CRTAM, Ly9
(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A,
Ly108), SLAM (SLAMFI, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.
[0626] In some embodiments, the TCR or CAR antigen binding domain
or the immunotherapeutic agent described herein (e.g., monospecific
or multi-specific antibody or antigen-binding fragment thereof or
antibody mimetic) binds a tumor-associated antigen (TAA). In some
embodiments, the tumor-associated antigen is selected from the
group consisting of: CD19; CD123; CD22; CD30; CD171; CS-1 (also
referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24);
C-type lectin-like molecule-1 (CLL-1 or CLECLI); CD33; epidermal
growth factor receptor variant III (EGFRvlll); ganglioside G2
(GD2); ganglioside GD3
(aNeuSAc(2-8)aNeuSAc(2-3)PDGaip(1-4)bDGIcp(1-1)Cer); ganglioside
GM3 (aNeuSAc(2-3)PDGalp(1-4)PDGlcp(1-1)Cer); TNF receptor
superfamily member 17 (TNFRSF17, BCMA); Tn antigen ((Tn Ag) or
(GaINAcu-Ser/Thr)); prostate-specific membrane antigen (PSMA);
Receptor tyrosine kinase-like orphan receptor 1 (RORI);
Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;
Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule
(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit
alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor
alpha (IL-1IRa); prostate stem cell antigen (PSCA); Protease Serine
21 (Testisin or PRSS21); vascular endothelial growth factor
receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived
growth factor receptor beta (PDGFR-beta); Stage-specificembryonic
antigen-4 (SSEA-4); CD20; delta like 3 (DLL3); Folate receptor
alpha; Receptor tyrosine-protein kinase, ERBB2 (Her2/neu); Mucin 1,
cell surface associated (MUC1); epidermal growth factor receptor
(EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic
acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin
B2; fibroblast activation protein alpha (FAP); insulin-like growth
factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);
Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);
glycoprotein 100 (gp100); oncogene fusion protein consisting of
breakpoint cluster region (BCR) and Abelson murine leukemia viral
oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A
receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule
(sLe); transglutaminase 5 (TGS5); high molecular
weight-melanomaassociatedantigen (HMWMAA); o-acetyl-GD2 ganglioside
(OAcGD2); Folate receptor beta; tumor endothelial marker 1
(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); six
transmembrane epithelial antigen of the prostate I (STEAPI);
claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G
protein-coupled receptor class C group 5, member D (GPRCSD);
chromosome X open reading frame 61 (CXORF61); CD97; CD179a;
anaplastic lymphoma kinase (ALK); Polysialic acid;
placenta-specific 1 (PLAC1); hexasaccharide portion of globoH
glycoceramide (GloboH); mammary gland differentiation antigen
(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor
1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G
protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex,
locus K 9 (LY6K); Olfactory receptor 51E2 (ORS IE2); TCR Gamma
Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1);
Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2
(LAGE-la); Melanoma associated antigen 1 (MAGE-A1); ETS
translocation-variant gene 6, located on chromosome 12p (ETV6-AML);
sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGEl);
angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma
cancer testis antigen-1 (MADCT-1); melanoma cancer testis antigen-2
(MAD-CT-2); Fos-related antigen 1; tumor protein p53, (p53); p53
mutant; prostein; survivin; telomerase; prostate carcinoma tumor
antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T
cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human
Telomerase reverse transcriptase (hTERT); sarcoma translocation
breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG
(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene);
N-Acetyl glucosaminyl-transferase V (NA17); paired box protein
Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian
myelocytomatosis viral oncogene neuroblastoma derived homolog
(MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related
protein 2 (TRP-2); Cytochrome P450 1B1(CYP IBI); CCCTC-Binding
Factor (Zinc Finger Protein)-Like (BORIS or Brother of the
Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen
Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5);
proacrosin binding protein sp32 (OY-TES I); lymphocyte-specific
protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);
synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced
Glycation Endproducts (RAGE-I); renal ubiquitous 1 (RUI); renal
ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6);
human papilloma virus E7 (HPV E7); intestinal carboxyl esterase;
heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72;
Leukocyte-associated immunoglobulin-like receptor 1 (LAIRI); Fc
fragment of IgA receptor (FCAR or CD89); Leukocyte
immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300
molecule-like family member f (CD300LF); C-type lectin domain
family 12 member A (CLEC12A); bone marrow stromal cell antigen 2
(BST2); EGF-like module containing mucin-like hormone receptor-like
2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc
receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide
1 (IGLL1). In some embodiments, the target is an epitope of the
tumor associated antigen presented in an MHC.
[0627] In some embodiments, the tumor antigen is selected from
CD150, 5T4, ActRIIA, B7, TNF receptor superfamily member 17
(TNFRSF17, BCMA), CA-125, CCNA1, CD123, CD126, CD138, CD14, CD148,
CD15, CD19, CD20, CD200, CD21, CD22, CD23, CD24, CD25, CD26, CD261,
CD262, CD30, CD33, CD362, CD37, CD38, CD4, CD40, CD40L, CD44, CD46,
CD5, CD52, CD53, CD54, CD56, CD66a-d, CD74, CD8, CD80, CD92, CE7,
CS-1, CSPG4, ED-B fibronectin, EGFR, EGFRvIII, EGP-2, EGP-4, EPHa2,
ErbB2, ErbB3, ErbB4, FBP, HER1-HER2 in combination, HER2-HER3 in
combination, HERV--K, HIV-1 envelope glycoprotein gp120, HIV-1
envelope glycoprotein gp41, HLA-DR, HM1.24, HMW-MAA, Her2,
Her2/neu, IGF-1R, IL-11Ralpha, IL-13R-alpha2, IL-2, IL-22R-alpha,
IL-6, IL-6R, Ia, Ii, L1-CAM, L1-cell adhesion molecule, Lewis Y,
L1-CAM, MAGE A3, MAGE-A1, MART-1, MUC1, NKG2C ligands, NKG2D
Ligands, NYESO-1, OEPHa2, PIGF, PSCA, PSMA, ROR1, T101, TAC, TAG72,
TIM-3, TRAIL-R1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), VEGF, VEGFR2,
WT-I, a G-protein coupled receptor, alphafetoprotein (AFP), an
angiogenesis factor, an exogenous cognate binding molecule
(ExoCBM), oncogene product, anti-folate receptor, c-Met,
carcinoembryonic antigen (CEA), cyclin (D 1), ephrinB2, epithelial
tumor antigen, estrogen receptor, fetal acetylcholine e receptor,
folate binding protein, gp100, hepatitis B surface antigen, kappa
chain, kappa light chain, kdr, lambda chain, livin,
melanoma-associated antigen, mesothelin, mouse double minute 2
homolog (MDM2), mucin 16 (MUC16), mutated p53, mutated ras,
necrosis antigens, oncofetal antigen, ROR2, progesterone receptor,
prostate specific antigen, tEGFR, tenascin, P2-Microgiobuiin, Fc
Receptor-like 5 (FcRL5).
[0628] In some embodiments, the antigen binding domain binds to an
epitope of a target or tumor associated antigen (TAA) presented in
a major histocompatibility complex (MHC) molecule. In some
embodiments, the TAA is a cancer testis antigen. In some
embodiments, the cancer testis antigen is selected from the group
consisting of acrosin binding protein (ACRBP; CT23, OY-TES-1, SP32;
NCBI Gene ID: 84519), alpha fetoprotein (AFP; AFPD, FETA, HPAFP;
NCBI Gene ID: 174); A-kinase anchoring protein 4 (AKAP4; AKAP 82,
AKAP-4, AKAP82, CT99, FSC1, HI, PRKA4, hAKAP82, p82; NCBI Gene ID:
8852), ATPase family AAA domain containing 2 (ATAD2; ANCCA, CT137,
PR02000; NCBI Gene ID: 29028), kinetochore scaffold 1 (KNL1;
AF15Q14, CASC5, CT29, D40, MCPH4, PPP1R55, Spc7, hKNL-1, hSpc105;
NCBI Gene ID: 57082), centrosomal protein 55 (CEP55; C10orf3,
CT111, MARCH, URCC6; NCBI Gene ID: 55165), cancer/testis antigen 1A
(CTAG1A; ESO1; CT6.1; LAGE-2; LAGE2A; NY-ESO-1; NCBI Gene ID:
246100), cancer/testis antigen 1B (CTAG1B; CT6.1, CTAG, CTAG1,
ESO1, LAGE-2, LAGE2B, NY-ESO-1; NCBI Gene ID: 1485), cancer/testis
antigen 2 (CTAG2; CAMEL, CT2, CT6.2, CT6.2a, CT6.2b, ESO2, LAGE-1,
LAGE2B; NCBI Gene ID: 30848), CCCTC-binding factor like (CTCFL;
BORIS, CT27, CTCF-T, HMGBIL, dJ579F20.2; NCBI Gene ID: 140690),
catenin alpha 2 (CTNNA2; CAP-R, CAPR, CDCBM9, CT114, CTNR; NCBI
Gene ID: 1496), cancer/testis antigen 83 (CT83; CXorf61, KK-LC-1,
KKLC1; NCBI Gene ID: 203413), cyclin A1 (CCNA1; CT146; NCBI Gene
ID: 8900), DEAD-box helicase 43 (DDX43; CT13, HAGE; NCBI Gene ID:
55510), developmental pluripotency associated 2 (DPPA2; CT100,
ECAT15-2, PESCRG1; NCBI Gene ID: 151871), fetal and adult testis
expressed 1 (FATE1; CT43, FATE; NCBI Gene ID: 89885), FMR1 neighbor
(FMR1NB; CT37, NY-SAR-35, NYSAR35; NCBI Gene ID: 158521), HORMA
domain containing 1 (HORMADI; CT46, NOHMA; NCBI Gene ID: 84072),
insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3; CT98,
IMP-3, IMP3, KOC, KOC1, VICKZ3; NCBI Gene ID: 10643), leucine
zipper protein 4 (LUZP4; CT-28, CT-8, CT28, HOM-TES-85; NCBI Gene
ID: 51213), lymphocyte antigen 6 family member K (LY6K; CT97,
HSJ001348, URLC10, ly-6K; NCBI Gene ID: 54742), maelstrom
spermatogenic transposon silencer (MAEL; CT128, SPATA35; NCBI Gene
ID: 84944), MAGE family member A1 (MAGEA1; CT1.1, MAGE1; NCBI Gene
ID: 4100); MAGE family member A3 (MAGEA3; CT1.3, HIP8, HYPD, MAGE3,
MAGEA6; NCBI Gene ID: 4102); MAGE family member A4 (MAGEA4; CT1.4,
MAGE-41, MAGE-X2, MAGE4, MAGE4A, MAGE4B; NCBI Gene ID: 4103); MAGE
family member A11 (MAGEA11; CT1.11, MAGE-11, MAGE11, MAGEA-11; NCBI
Gene ID: 4110); MAGE family member C1 (MAGEC1; CT7, CT7.1; NCBI
Gene ID: 9947); MAGE family member C2 (MAGEC2; CT10, HCA587,
MAGEE1; NCBI Gene ID: 51438); MAGE family member D1 (MAGED1;
DLXIN-1, NRAGE; NCBI Gene ID: 9500); MAGE family member D2 (MAGED2;
11B6, BARTS5, BCG-1, BCG1, HCA10, MAGE-D2; NCBI Gene ID: 10916),
kinesin family member 20B (KIF20B; CT90, KRMP1, MPHOSPH1, MPP-1,
MPP1; NCBI Gene ID: 9585), NUF2 component of NDC80 kinetochore
complex (NUF2; CDCA1, CT106, NUF2R; NCBI Gene ID: 83540), nuclear
RNA export factor 2 (NXF2; CT39, TAPL-2, TCP11X2; NCBI Gene ID:
56001), PAS domain containing repressor 1 (PASD1; CT63, CT64,
OXTES1; NCBI Gene ID: 139135), PDZ binding kinase (PBK; CT84,
HEL164, Nori-3, SPK, TOPK; NCBI Gene ID: 55872), piwi like
RNA-mediated gene silencing 2 (PIWIL2; CT80, HILI, PIWIL1L, mili;
NCBI Gene ID: 55124), preferentially expressed antigen in melanoma
(PRAME; CT130, MAPE, OIP-4, OIP4; NCBI Gene ID: 23532), sperm
associated antigen 9 (SPAG9; CT89, HLC-6, HLC4, HLC6, JIP-4, JIP4,
JLP, PHET, PIG6; NCBI Gene ID: 9043), sperm protein associated with
the nucleus, X-linked, family member A1 (SPANXAl; CT11.1, CT11.3,
NAP-X, SPAN-X, SPAN-Xa, SPAN-Xb, SPANX, SPANX-A; NCBI Gene ID:
30014), SPANX family member A2 (SPANXA2; CT11.1, CT11.3, SPANX,
SPANX-A, SPANX-C, SPANXA, SPANXC; NCBI Gene ID: 728712), SPANX
family member C (SPANXC; CT11.3, CTp11, SPANX-C, SPANX-E, SPANXE;
NCBI Gene ID: 64663), SPANX family member D (SPANXD; CT11.3,
CT11.4, SPANX-C, SPANX-D, SPANX-E, SPANXC, SPANXE, dJ171K16.1; NCBI
Gene ID: 64648), SSX family member 1 (SSX1; CT5.1, SSRC; NCBI Gene
ID: 6756), SSX family member 2 (SSX2; CT5.2, CT5.2A, HD21,
HOM-MEL-40, SSX; NCBI Gene ID: 6757), synaptonemal complex protein
3 (SYCP3; COR1, RPRGL4, SCP3, SPGF4; NCBI Gene ID: 50511), testis
expressed 14, intercellular bridge forming factor (TEX14; CT113,
SPGF23; NCBI Gene ID: 56155), transcription factor Dp family member
3 (TFDP3; CT30, DP4, HCA661; NCBI Gene ID: 51270), serine protease
50 (PRSS50; CT20, TSP50; NCBI Gene ID: 29122), TTK protein kinase
(TTK; CT96, ESK, MPH1, MPS1, MPS1L1, PYT; NCBI Gene ID: 7272) and
zinc finger protein 165 (ZNF165; CT53, LD65, ZSCAN7; NCBI Gene ID:
7718). T cell receptors (TCRs) and TCR-like antibodies that bind to
an epitope of a cancer testis antigen presented in a major
histocompatibility complex (MHC) molecule are known in the art and
can be used in the herein described heterodimers. Cancer testis
antigens associated with neoplasia are summarized, e.g., in Gibbs,
et al., Trends Cancer 2018 October; 4(10):701-712 and the CT
database website at cta.lncc.br/index.php. Illustrative TCRs and
TCR-like antibodies that bind to an epitope of NY-ESO-1 presented
in an MHC are described, e.g., in Stewart-Jones, et al., Proc Natl
Acad Sci USA. 2009 Apr. 7; 106(14):5784-8; WO2005113595,
WO2006031221, WO2010106431, WO2016177339, WO2016210365,
WO2017044661, WO2017076308, WO2017109496, WO2018132739,
WO2019084538, WO2019162043, WO2020086158 and WO2020086647.
Illustrative TCRs and TCR-like antibodies that bind to an epitope
of PRAME presented in an MHC are described, e.g., in WO2011062634,
WO2016142783, W2016191246, WO2018172533, W2018234319 and
W2019109821. Illustrative TCRs and TCR-like antibodies that bind to
an epitope of a MAGE variant presented in an MHC are described,
e.g., in WO2007032255, WO2012054825, WO2013039889, WO2013041865,
WO2014118236, WO2016055785, WO2017174822, WO2017174823,
WO2017174824, WO2017175006, WO2018097951, WO2018170338,
WO2018225732 and WO2019204683. Illustrative TCRs and TCR-like
antibodies that bind to an epitope of alpha fetoprotein (AFP)
presented in an MHC are described, e.g., in WO2015011450.
Illustrative TCRs and TCR-like antibodies that bind to an epitope
of SSX2 presented in an MHC are described, e.g., in W2020063488.
Illustrative TCRs and TCR-like antibodies that bind to an epitope
of KK-LC-1 (CT83) presented in an MHC are described, e.g., in
WO2017189254.
[0629] Examples of cell therapies include without limitation:
Algenpantucel-L, Sipuleucel-T, (BPX-501) rivogenlecleucel U.S. Pat.
No. 9,089,520, WO2016100236, AU-105, ACTR-087, activated allogeneic
natural killer cells CNDO-109-AANK, MG-4101, AU-101, BPX-601,
FATE-NK100, LFU-835 hematopoietic stem cells, Imilecleucel-T,
baltaleucel-T, PNK-007, UCARTCS1, ET-1504, ET-1501, ET-1502,
ET-190, CD19-ARTEMIS, ProHema, FT-1050-treated bone marrow stem
cell therapy, CD4CARNK-92 cells, CryoStim, AlloStim, lentiviral
transduced huCART-meso cells, CART-22 cells, EGFRt/19-28z/4-1BBL
CAR T cells, autologous 4H11-28z/fIL-12/EFGRt T cell,
CCR5-SBC-728-HSPC, CAR4-1BBZ, CH-296, dnTGFbRII-NY-ESOc259T,
Ad-RTS-IL-12, IMA-101, IMA-201, CARMA-0508, TT-18, CMD-501,
CMD-503, CMD-504, CMD-502,CMD-601,CMD-602, CSG-005.
[0630] Additional agents for targeting tumors include without
limitation: [0631] Alpha-fetoprotein, such as ET-1402, and AFP-TCR;
[0632] Anthrax toxin receptor 1, such as anti-TEM8 CAR T-cell
therapy; [0633] TNF receptor superfamily member 17 (TNFRSF17,
BCMA), such as bb-2121, UCART-BCMA, ET-140, KITE-585, MCM-998,
LCAR-B38M, CART-BCMA, SEA-BCMA, BB212, UCART-BCMA, ET-140,
P-BCMA-101, AUTO-2 (APRL-CAR); [0634] Anti-CLL-1 antibodies, such
as KITE-796; [0635] Anti-PD-LI-CAR tank cell therapy, such as
KD-045; [0636] B7 homolog 6, such as CAR-NKp30 and CAR-B7H6; [0637]
B-lymphocyte antigen CD19, such as TBI-1501, CTL-119 huCART-19 T
cells, JCAR-015 U.S. Pat. No. 7,446,190, JCAR-014, JCAR-017,
(WO2016196388, WO2016033570, WO2015157386), axicabtagene ciloleucel
(KTE-C19, Yescarta.RTM.), KTE-X19, U.S. Pat. Nos. 7,741,465,
6,319,494, UCART-19, EBV-CTL, T tisagenlecleucel-T (CTL019),
WO2012079000, WO2017049166, CD19CAR-CD28-CD3zeta-EGFRt-expressing T
cells, CD19/4-1BBL armored CAR T cell therapy, C-CAR-011,
CIK-CAR.CD19, CD19CAR-28-zeta T cells, PCAR-019, MatchCART,
DSCAR-01, IM19 CAR-T, TC-110; [0638] B-lymphocyte antigen CD20,
such as ACTR707 ATTCK-20; [0639] B-lymphocyte antigen
CD19/B-lymphocyte antigen 22, such as TC-310; [0640] B-lymphocyte
antigen 22 cell adhesion, such as UCART-22, JCAR-018 WO2016090190;
[0641] NY-ESO-1, such as GSK-3377794, TBI-1301; [0642] Carbonic
anhydrase, such as DC-Ad-GMCAIX; [0643] Caspase 9 suicide gene,
such as CaspaCIDe DLI, BPX-501; [0644] CCR5, such as SB-728; [0645]
CDw123, such as MB-102, UCART-123; [0646] CD4, such as ICG-122;
[0647] CD33, such as CIK-CAR.CD33; [0648] CD38, such as T-007,
UCART-38; [0649] CD40ligand, such as BPX-201; [0650] CEACAM protein
5 modulators, such as MG7-CART; [0651] Claudin 6, such as CSG-002;
[0652] EBV targeted, such as CMD-003; [0653] MUC16EGFR, such as
autologous 4H11-28z/fIL-12/EFGRt T cell; [0654] Endonuclease, such
as PGN-514, PGN-201; [0655] Epstein-Barr virus specific
T-lymphocytes, such as TT-10; [0656] Erbb2, such as CST-102,
CIDeCAR; [0657] Ganglioside (GD2), such as 4SCAR-GD2; [0658] folate
hydrolase 1 (FOLH1, Glutamate carboxypeptidase II, PSMA; NCBI Gene
ID. 2346), such as CIK-CAR.PSMA, CART-PSMA-TGFORDN, P-PSMA-101;
[0659] Glypican-3(GPC3), such as TT-16, GLYCAR; [0660] Hemoglobin,
such as PGN-236; [0661] Hepatocyte growth factor receptor, such as
anti-cMet RNA CAR T; [0662] Human papillomavirus E7 protein, such
as KITE-439; [0663] Immunoglobulin gamma Fc receptor III, such as
ACTR087; [0664] IL-12, such as DC-RTS-IL-12; [0665] IL-12
agonist/mucin 16, such as JCAR-020; [0666] IL-13 alpha 2, such as
MB-101; [0667] IL-2, such as CST-101; [0668] K-Ras GTPase, such as
anti-KRAS G12V mTCR cell therapy; [0669] Neural cell adhesion
molecule L1 LlCAM (CD171), such as JCAR-023; [0670] Latent membrane
protein 1/Latent membrane protein 2, such as
Ad5f35-LMPd1-2-transduced autologous dendritic cells; [0671]
Melanoma associated antigen 10, such as MAGE-A10C796T MAGE-A10 TCR;
[0672] Melanoma associated antigen 3/Melanoma associated antigen 6
(MAGE A3/A6) such as KITE-718; [0673] Mesothelin, such as CSG-MESO,
TC-210; [0674] NKG2D, such as NKR-2; [0675] Ntrkrl tyrosine kinase
receptor, such as JCAR-024; [0676] PRAMET cell receptor, such as
BPX-701; [0677] T-lymphocyte, such as TT-12; [0678] Tumor
infiltrating lymphocytes, such as LN-144, LN-145; and/or [0679]
Wilms tumor protein, such as JTCR-016, WT1-CTL.
[0680] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a gene or cell therapy regimen that can
target a cell infected with a virus (e.g., HIV). A gene or cell
therapy that can be combined with an agent disclosed herein
includes without limitation the genetic modification to silence a
gene; genetic approaches to directly kill the infected cells; the
infusion of immune cells designed to replace most of the patient's
own immune system to enhance the immune response to infected cells,
or activate the patient's own immune system to kill infected cells,
or find and kill the infected cells; genetic approaches to modify
cellular activity to further alter endogenous immune responsiveness
against the infection. Illustrative examples of a cell therapy that
can be combined with an agent disclosed herein include LB-1903,
ENOB-HV-01, GOVX-B01, and SupT1 cell-based therapy. Illustrative
examples of a dendritic cell therapy that can be combined with an
agent disclosed herein include AGS-004. An illustrative example of
a CCR5 gene editing agent that can be used in combination with an
agent disclosed herein is SB-728T. An illustrative example of a
CCR5 gene inhibitor that can be used in combination with an agent
disclosed herein is Cal-1. In some embodiments, C34-CCR5/C34-CXCR4
expressing CD4-positive T-cells are co-administered with an agent
disclosed herein. In some embodiments, the agents described herein
are co-administered with AGT-103-transduced autologous T-cell
therapy or AAV-eCD4-Ig gene therapy.
[0681] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, can be co-administered with a population of immune effector
cells engineered to express a chimeric antigen receptor (CAR),
wherein the CAR comprises an HIV antigen binding domain. The HIV
antigen include an HIV envelope protein or a portion thereof, gp120
or a portion thereof, a CD4 binding site on gp120, the CD4-induced
binding site on gpl20, N glycan on gpl20, the V2 of gpl20, the
membrane proximal region on gp41. The immune effector cell is a
T-cell or an NK cell. In some embodiments, the T-cell is a CD4+
T-cell, a CD8+ T-cell, or a combination thereof. Cells can be
autologous or allogeneic. Examples of HIV CAR-T include
convertibleCAR-T, VC-CAR-T, CMV-N6-CART, anti-CD4 CART-cell
therapy, CD4 CAR+C34-CXCR4+CCR5 ZFN T-cells, autologous
hematopoietic stem cells genetically engineered to express a CD4
CAR and the C46 peptide.
[0682] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a population of TCR-T-cells. TCR-T-cells
are engineered to target HIV derived peptides present on the
surface of virus-infected cells, for example ImmTAV.
[0683] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a population of B cells genetically
modified to express broadly neutralizing antibodies, such as
3BNC117 (Hartweger et al., J. Exp. Med. 2019, 1301; Moffett et al.,
Sci. Immunol. 4, eaax0644 (2019) 17 May 2019).
Gene Editors
[0684] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with gene editor. Illustrative gene editing
system that can be co-administered include without limitation a
CRISPR/Cas9 system, a zinc finger nuclease system, a TALEN system,
a homing endonucleases system (e.g., an ARCUS), and a homing
meganuclease system.
[0685] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein described herein are combined with an HIV targeted gene
editor. An illustrative HIV targeted gene editor includes without
limitation the CRISPR/Cas9 system EBT-101.
HIV Therapeutic Agents
[0686] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an HIV therapeutic agent.
[0687] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include an HIV protease
inhibitor. Illustrative HIV protease inhibitors that can be
co-administered include without limitation amprenavir, atazanavir,
brecanavir, darunavir, fosamprenavir, fosamprenavir calcium,
indinavir, indinavir sulfate, lopinavir, nelfinavir, nelfinavir
mesylate, ritonavir, saquinavir, saquinavir mesylate, tipranavir,
AEBL-2, DG-17, GS-1156, TMB-657 (PPL-100), T-169, BL-008, MK-8122,
TMB-607, GRL-02031, and TMC-310911.
[0688] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include an HIV ribonuclease
H inhibitor. Examples of HIV ribonuclease H inhibitors that can be
combined with an agent of this disclosure include NSC-727447.
[0689] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include an HIV Nef
inhibitor. Examples of HIV Nef inhibitors that can be combined with
an agent of this disclosure include FP-1.
[0690] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include a reverse
transcriptase inhibitor. In some embodiments the reverse
transcriptase inhibitor is a non-nucleoside/non-nucleotide
inhibitor. Illustrative examples of non-nucleoside/non-nucleotide
inhibitors that can be co-administered include without limitation
dapivirine, delavirdine, delavirdine mesylate, doravirine,
efavirenz, etravirine, lentinan, nevirapine, rilpivirine, ACC-007,
ACC-008, AIC-292, F-18, KM-023, PC-1005, VM-1500A-LAI, PF-3450074,
elsulfavirine (sustained release oral, HIV infection),
elsulfavirine (long-acting injectable nanosuspension, HIV
infection), and elsulfavirine (VM-1500). In some embodiments, the
non-nucleoside/non-nucleotide inhibitor is selected from
nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine. In
some embodiments the reverse transcriptase inhibitor that can be
combined with an agent of this disclosure is a nucleoside or
nucleotide inhibitor. Illustrative examples of nucleoside or
nucleotide inhibitors of reverse transcriptase inhibitor that can
be co-administered include without limitation adefovir, adefovir
dipivoxil, azvudine, emtricitabine, tenofovir, tenofovir
alafenamide, tenofovir alafenamide fumarate, tenofovir alafenamide
hemifumarate, tenofovir disoproxil, tenofovir disoproxil fumarate,
tenofovir octadecyloxyethyl ester (AGX-1009), tenofovir disoproxil
hemifumarate, VIDEX.RTM. and VIDEX EC.RTM. (didanosine, ddl),
abacavir, abacavir sulfate, alovudine, apricitabine, censavudine,
didanosine, elvucitabine, festinavir, fosalvudine tidoxil, CMX-157,
dapivirine, doravirine, etravirine, OCR-5753, tenofovir disoproxil
orotate, fozivudine tidoxil, lamivudine, phosphazid, stavudine,
zalcitabine, zidovudine, rovafovir et alafenamide (GS-9131),
GS-9148, MK-8504, MK-8591, MK-8583, VM-2500 and KP-1461. In some
embodiments the nucleoside or nucleotide inhibitors of reverse
transcriptase inhibitor is selected from zidovudine, didanosine,
zalcitabine, stavudine, lamivudine, abacavir, and
emtricitabine.
[0691] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include an HIV integrase
inhibitor. In some embodiments, the HIV integrase inhibitor that
can be combined with an agent of this disclosure is selected from
the group consisting of. elvitegravir, elvitegravir
(extended-release microcapsules), curcumin, derivatives of
curcumin, chicoric acid, derivatives of chicoric acid,
3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic
acid, aurintricarboxylic acid, derivatives of aurintricarboxylic
acid, caffeic acid phenethyl ester, derivatives of caffeic acid
phenethyl ester, tyrphostin, derivatives of tyrphostin, quercetin,
derivatives of quercetin, raltegravir, PEGylated raltegravir,
dolutegravir, JTK-351, bictegravir, AVX-15567, cabotegravir
(long-acting injectable), diketo quinolin-4-1 derivatives,
integrase-LEDGF inhibitor, ledgins, M-522, M-532, MK-0536,
NSC-310217, NSC-371056, NSC-48240, NSC-642710, NSC-699171,
NSC-699172, NSC-699173, NSC-699174, stilbenedisulfonic acid, T-169,
STP-0404, VM-3500 and cabotegravir. In some embodiments, the HIV
integrase inhibitor that can be combined with an agent of this
disclosure is an HIV non-catalytic site, or allosteric, integrase
inhibitor (NCINI). Illustrative examples of such HIV non-catalytic
site, or allosteric, integrase inhibitors (NCINI) include CX-05045,
CX-05168, and CX-14442.
[0692] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include HIV an entry
inhibitor. In some embodiments, the entry inhibitor is an HBV entry
inhibitor (e.g., Myrcludex B). Illustrative examples of entry
inhibitors that can be combined with an agent of this disclosure
include without limitation AAR-501, LBT-5001, cenicriviroc, a CCR5
inhibitor, a gp41 inhibitor, a CD4 attachment inhibitor, a gpl20
inhibitor, a gp160 inhibitor a, and a CXCR4 inhibitor.
[0693] In some embodiments the entry inhibitor that can be combined
with an agent of this disclosure is a CCR5 inhibitor selected from
the group consisting of. aplaviroc, vicriviroc, maraviroc,
maraviroc (long-acting injectable nanoemulsion), cenicriviroc,
leronlimab (PRO-140), adaptavir (RAP-101), nifeviroc (TD-0232),
anti-GP120/CD4 or CCR5 bispecific antibodies, B-07, MB-66,
polypeptide C25P, TD-0680, thioraviroc and vMIP (Haimipu).
[0694] In some embodiments the entry inhibitor that can be combined
with an agent of this disclosure is a gp41 inhibitor selected from
the group consisting of. albuvirtide, enfuvirtide, griffithsin
(gp41/gpl20/gpl60 inhibitor), BMS-986197, enfuvirtide biobetter,
enfuvirtide biosimilar, HIV-1 fusion inhibitors (P26-Bapc), ITV-1,
ITV-2, ITV-3, ITV-4, CPT-31, Cl3hmAb, PIE-12 trimer and
sifuvirtide.
[0695] In some embodiments the entry inhibitor that can be combined
with an agent of this disclosure is a CD4 attachment inhibitor
selected from ibalizumab and a CADA analog.
[0696] In some embodiments the entry inhibitor that can be combined
with an agent of this disclosure is a gpl20 inhibitor selected from
anti-HIV microbicide, Radha-108 (receptol) 3B3-PE38, BanLec,
bentonite-based nanomedicine, fostemsavir tromethamine, IQP-0831,
VVX-004, and BMS-663068.
[0697] In some embodiments the entry inhibitor that can be
co-administered is a gp160 inhibitor such as fangchinoline.
[0698] In some embodiments the entry inhibitor that can be combined
with an agent of this disclosure is a CXCR4 inhibitor selected from
plerixafor, ALT-1188, N15 peptide, and vMIP (Haimipu).
[0699] In some embodiments the entry inhibitor that can be combined
with an agent of this disclosure is selected from docosanol,
enfuvirtide, maraviroc, palivizumab, respiratory syncytial virus
immune globulin, intravenous [RSV-IGIV], varicella-zoster
immunoglobulin [VariZIG], and varicella-zoster immune globulin
[VZIG]).
[0700] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include an HIV maturation
inhibitor. In some embodiments, the HIV maturation inhibitor that
can be co-administered is selected from BMS-955176, GSK-3640254 and
GSK-2838232.
[0701] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include a latency reversing
agent. Illustrative examples of latency reversing agent that can be
combined with an agent of this disclosure include without
limitation toll-like receptor (TLR) agonists (including TLR7
agonists, e.g., GS-9620 (vesatolimod), vesatolimod analogs),
histone deacetylase (HDAC) inhibitors, proteasome inhibitors such
as velcade, protein kinase C (PKC) activators, Smyd2 inhibitors,
BET-bromodomain 4 (BRD4) inhibitors, ionomycin, IAP antagonists
(inhibitor of apoptosis proteins, such as APG-1387, LBW-242), SMAC
mimetics (including TL32711, LCL161, GDC-0917, HGS1029, AT-406,
Debio-1143), PMA, SAHA (suberanilohydroxamic acid, or suberoyl,
anilide, and hydroxamic acid), NIZ-985, IL-15 modulating antibodies
(including IL-15, IL-15 fusion proteins and IL-15 receptor
agonists), JQ1, disulfiram, amphotericin B, and ubiquitin
inhibitors such as largazole analogs, APH-0812, and GSK-343.
Examples of PKC activators that can be co-administered include
without limitation indolactam, prostratin, ingenol B, and
DAG-lactones.
[0702] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include a capsid inhibitor.
Illustrative examples of capsid inhibitors that can be combined
with an agent of this disclosure include without limitation capsid
polymerization inhibitors, capsid disrupting compounds, HIV
nucleocapsid p7 (NCp7) inhibitors (e.g., azodicarbonamide), HIV p24
capsid protein inhibitors (e.g., GS-6207, GS-CA1, AVI-621, AVI-101,
AVI-201, AVI-301, AVI-CAN1-15 series, and PF-3450074), and
compounds described in the International Patent Publication No.
WO2019/087016.
[0703] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include an HIV targeting
antibody. HIV targeting antibodies that can be combined with an
agent of this disclosure include bispecific antibodies, trispecific
antibodies, and "antibody-like" therapeutic proteins. In some
embodiments, the HIV targeting antibodies that can be
co-administered with an agent of this disclosrue are selected from
DARTs.RTM., DUOBODIES, BITES.RTM., XmAbs.RTM., TandAbs.RTM., Fab
derivatives, bNAbs (broadly neutralizing HIV-1 antibodies),
TMB-360, antibodies targeting HIV gp120 or gp41,
antibody-recruiting molecules targeting HIV, anti-CD63 monoclonal
antibodies, anti-GB virus C antibodies, anti-GP120/CD4, gpl20
bispecific monoclonal antibodies, CCR5 bispecific antibodies,
anti-Nef single domain antibodies, anti-Rev antibody, camelid
derived anti-CD18 antibodies, camelid-derived anti-ICAM-1
antibodies, DCVax-001, gpl40 targeted antibodies, gp41-based HIV
therapeutic antibodies, human recombinant mAbs (PGT-121),
PGT121.414.LS, ibalizumab, Immuglo, MB-66, and
VRC-HIVMAB091-00-AB.
[0704] In some embodiments the HIV targeting antibody that can be
combined with an agent of this disclosure is a bNAb, including
without limitation those described in U.S. Pat. Nos. 8,673,307,
9,493,549, and 9,783,594, and in International Patent Publication
Nos. WO2014/063059, WO2012/158948, WO2015/117008, WO/2016/014484,
and WO2017/09622. In some embodiments the bNAb that can be combined
with an agent of this disclosure is selected from 12A12, 12A21,
NI145-46, bANC131, 8ANC134, IB2530, INC9, 8ANC195. 8ANC196, 10-259,
10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146,
10-1341, 10-1369, and 10-1074GM. Additional examples of bNAbs that
can be co-administered with an agent of this disclosure include
those described in Klein et al., Nature, 492(7427): 118-22 (2012),
Horwitz et al., Proc Natl Acad Sci USA, 110(41): 16538-43 (2013),
Scheid, et al., Science, 333: 1633-1637 (2011), Scheid, et al.,
Nature, 458:636-640 (2009), Eroshkin et al, Nucleic Acids Res., 42
(Database issue):Dl 133-9 (2014), Mascola et al., Immunol Rev.,
254(1):225-44 (2013), such as 2F5, 4E10, M66.6, CAP206-CH12, 10E81
(all of which bind the MPER of gp41); PG9, PG16, CHO1-04 (all of
which bind V1V2-glycan), 2G12 (which binds to outer domain glycan);
b12, HJ16, CH103-106, VRCO1-03, VRC-PG04, 04b, VRC-CH30-34, 3BNC62,
3BNC89, 3BNC91, 3BNC95, 3BNC104, 3BNC176, and 8ANC131 (all of which
bind to the CD4 binding site). Additional illustrative examples of
bNAbs that can be co-administered with an agent of this disclosure
are described, e.g., in U.S. Pat. Nos. 8,673,307; 9,493,549;
9,783,594; and in International Patent Publication Nos. WO
2012/154312; WO2012/158948; WO 2013/086533; WO 2013/142324;
W2014/063059; WO 2014/089152, WO 2015/048462; WO 2015/103549; WO
2015/117008; W2016/014484; WO 2016/154003; WO 2016/196975; WO
2016/149710; WO2017/096221; WO 2017/133639; and WO 2017/133640.
Additional examples of bNAbs that can be combined with an agent of
this disclosure are described, e.g., in Sajadi, et al., Cell.
(2018) 173(7):1783-1795; Sajadi, et al., J Infect Dis. (2016)
213(1):156-64; Klein et al., Nature, 492(7427): 118-22 (2012),
Horwitz et al., Proc Natl Acad Sci USA, 110(41): 16538-43 (2013),
Scheid, et al., Science, 333: 1633-1637 (2011), Scheid, et al.,
Nature, 458:636-640 (2009), Eroshkin et al, Nucleic Acids Res., 42
(Database issue):D1 133-9 (2014), Mascola et al., Immunol Rev.,
254(1):225-44 (2013), such as 2F5, 4E10, M66.6, CAP206-CH12, 10E8,
10E8v4, 10E8-5R-100cF, DH511.11P, 7b2, and LN01 (all of which bind
the MPER of gp41).
[0705] In some embodiments, the HIV targeting antibody that can be
combined with an agent of this disclosure is selected from the
group consisting of: UB-421, BF520.1, CHO1, CH59, C2F5, C4E10,
C2F5+C2G12+C4E10, 3BNC117, 3BNC117-LS, 3BNC60, DH270.1, DH270.6,
D1D2, 10-1074-LS, Cl3hmAb, GS-9722 (elipovimab), DH411-2, BG18,
GS-9721, PGT145, PGT121, PGT-121.60, PGT-121.66, PGT122, PGT-123,
PGT-124, PGT-125, PGT-126, PGT-151, PGT-130, PGT-133, PGT-134,
PGT-135, PGT-128, PGT-136, PGT-137, PGT-138, PGT-139, MDXO10
(ipilimumab), DH511, DH511-2, N6, N6LS, N49P6, N49P7, N49P7.1,
N49P9, N49P11, N60P1.1, N60P25.1, N60P2.1, N60P31.1, N60P22, NIH
45-46, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGDM1400, PGDM12,
PGDM21, PCDN-33A, 2Dm2m, 4Dm2m, 6Dm2m, PGDM1400, MDXO10
(ipilimumab), VRCO1, VRC-01-LS, A32, 7B2, 10E8, VRC-07-523,
VRC07-523LS, VRC24, VRC41.01, 10E8VLS, 3810109, 10E8v4, IMC-HIV,
iMabm36, eCD4-Ig, IOMA, CAP256-VRC26.25,
DRVIA7,VRC-HIVMAB080-00-AB, VRC-HIVMAB060-00-AB, P2G12, VRC07,
354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411,
354BG426, VRC29.03, CAP256, CAP256-VRC26.08, CAP256-VRC26.09,
CAP256-VRC26.25, PCT64-24E and VRC38.01, PGT-151, CAP248-2B, 35022,
ACS202, VRC34 and VRC34.01, 10E8, 10E8v4, 10E8-5R-100cF, 4E10,
DH511.11P, 2F5, 7b2, and LN01.
[0706] In some embodiments, the HIV targeting antibody that can be
co-administered with an agent of this disclosure is a bispecific or
trispecific antibody such as MGD014, B12BiTe, BiIA-SG,
TMB-bispecific, SAR-441236, VRC-O1/PGDM-1400/10E8v4, 10E8.4/iMab,
or 10E8v4/PGT121-VRC01.
[0707] In some embodiments, an agent of this disclosure can be
combined with in vivo delivered bNAbs such as AAV8-VRC07; mRNA
encoding anti-HIV antibody VRC01; or engineered B-cells encoding
3BNC117 (Hartweger et al, J. Exp. Med. 2019, 1301).
[0708] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include an HIV vaccine.
Illustrative examples of HIV vaccines that can be combined with an
agent of this disclosure include without limitation peptide
vaccines, recombinant subunit protein vaccines, live vector
vaccines, DNA vaccines, HIV MAG DNA vaccine, CD4-derived peptide
vaccines, vaccine combinations, adenoviral vector vaccines (an
adenoviral vector such as Ad5, Ad26 or Ad35), simian adenovirus
(chimpanzee, gorilla, rhesus i.e., rhAd), adeno-associated virus
vector vaccines, Chimpanzee adenoviral vaccines (e.g., ChAdOXI,
ChAd68, ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan5, Pan6, Pan7,
Pan9), Coxsackieviruses based vaccines, enteric virus based
vaccines, Gorilla adenovirus vaccines, lentiviral vector based
vaccine, arenavirus vaccines (such as LCMV, Pichinde), bi-segmented
or tri-segmented arenavirus based vaccine, trimer-based HIV-1
vaccine, measles virus based vaccine, flavivirus vector based
vaccines, tobacco mosaic virus vector based vaccine,
Varicella-zoster virus based vaccine, Human parainfluenza virus 3
(PIV3) based vaccines, poxvirus based vaccine (modified vaccinia
virus Ankara (MVA), orthopoxvirus-derived NYVAC, and
avipoxvirus-derived ALVAC (canarypox virus) strains); fowlpox virus
based vaccine, rhabdovirus-based vaccines, such as VSV and
marabavirus; recombinant human CMV (rhCMV) based vaccine,
alphavirus-based vaccines, such as semliki forest virus, venezuelan
equine encephalitis virus and sindbis virus; (see Lauer, Clinical
and Vaccine Immunology, 2017, DOI: 10.1128/CVI.00298-16); a
lipoplex, such as an LNP, formulated mRNA based therapeutic
vaccines; and LNP-formulated self-replicating RNA/self-amplifying
RNA vaccines.
[0709] Additional illustrative examples of HIV vaccines that can be
combined with an agent of this disclosure include without
limitation anti-CD40.Env-gp140 vaccine, Ad4-EnvC150, BG505
SOSIP.664 gp140 adjuvanted vaccine, BG505 SOSIP.GT1.1 gp140
adjuvanted vaccine, Chimigen HIV vaccine, ConM SOSIP.v7 gpl40,
rgp120 (AIDSVAX), ALVAC HIV (vCP1521)/AIDSVAX B/E (gpl20) (RV144),
monomeric gp120 HIV-1 subtype C vaccine, MPER-656 liposome subunit
vaccine, Remune, ITV-1, Contre Vir, Ad5-ENVA-48, DCVax-001
(CDX-2401), Vacc-4x, Vacc-C5, VAC-3S, multiclade DNA recombinant
adenovirus-5 (rAd5), rAd5 gag-pol env A/B/C vaccine, Pennvax-G,
Pennvax-GP, Pennvax-G/MVA-CMDR, HIV-TriMix-mRNA vaccine,
HIV-LAMP-vax, Ad35, Ad35-GRIN, NAcGM3/VSSP ISA-51, poly-ICLC
adjuvanted vaccines, TatImmune, GTU-multiHIV (FIT-06),
ChAdV63.HIVconsv, gp140[delta]V2.TV1+MF-59, rVSVIN HIV-1 gag
vaccine, SeV-EnvF, SeV-Gag vaccine, AT-20, DNK-4, ad35-Grin/ENV,
TBC-M4, HIVAX, HIVAX-2, N123-VRC-34.01 inducing epitope-based HIV
vaccine, NYVAC-HIV-PT1, NYVAC-HIV-PT4, DNA-HIV-PT123, rAAV1-PG9DP,
GOVX-B11, GOVX-B21, GOVX-C55, TVI-HIV-1, Ad-4 (Ad4-env Clade
C+Ad4-mGag), Paxvax, EN41-UGR7C, EN41-FPA2, ENOB-HV-11, PreVaxTat,
AE-H, MYM-V101, CombiHIVvac, ADVAX, MYM-V201, MVA-CMDR, MagaVax,
DNA-Ad5 gag/pol/nef/nev (HVTN505), MVATG-17401, ETV-01, CDX-1401,
DNA and Sev vectors vaccine expressing SCaVII, rcAD26.MOS1.HIV-Env,
Ad26.Mod.HIV vaccine, Ad26.Mod.HIV+MVA mosaic vaccine+gp140,
AGS-004, AVX-101, AVX-201, PEP-6409, SAV-001, ThV-01, TL-01,
TUTI-16, VGX-3300, VIR-1111, IHV-001, and virus-like particle
vaccines such as pseudovirion vaccine, CombiVICHvac, LFn-p24 B/C
fusion vaccine, GTU-based DNA vaccine, HIV gag/pol/nef/env DNA
vaccine, anti-TAT HIV vaccine, conjugate polypeptides vaccine,
dendritic-cell vaccines (such as DermaVir), gag-based DNA vaccine,
GI-2010, gp41 HIV-1 vaccine, HIV vaccine (PIKA adjuvant), i-key/MHC
class II epitope hybrid peptide vaccines, ITV-2, ITV-3, ITV-4,
LIPO-5, multiclade Env vaccine, MVA vaccine, Pennvax-GP,
pp71-deficient HCMV vector HIV gag vaccine, rgp160 HIV vaccine,
RNActive HIV vaccine, SCB-703, Tat Oyi vaccine, TBC-M4, UBI HIV
gpl20, Vacc-4x+romidepsin, variant gp120 polypeptide vaccine, rAd5
gag-pol env A/B/C vaccine, DNA.HTI and MVA.HTI,
VRC-HIVDNA016-00-VP+VRC-HIVADV014-00-VP, INO-6145, JNJ-9220, gp145
C.6980; eOD-GT8 60mer based vaccine, PD-201401, env (A, B, C,
A/E)/gag (C) DNA Vaccine, gp120 (A,B,C,A/E) protein vaccine,
PDPHV-201401, Ad4-EnvCN54, EnvSeq-1 Envs HIV-1 vaccine (GLA-SE
adjuvanted), HIV p24gag prime-boost plasmid DNA vaccine, HIV-1
iglbl2 neutralizing VRC-01 antibody-stimulating anti-CD4 vaccine,
arenavirus vector-based vaccines (Vaxwave, TheraT), MVA-BN HIV-1
vaccine regimen, UBI HIV gpl20, mRNA based prophylactic vaccines,
VPI-211, or TBL-1203HI.
[0710] In some embodiments, the HIV therapeutic agents that can be
combined with an agent disclosed herein include a birth control or
contraceptive regimen. Illustrative examples of birth control or
contraceptive regimens that can be combined with an agent of this
disclosure include without limitation cyproterone acetate,
desogestrel, dienogest, drospirenone, estradiol valerate, ethinyl
estradiol, ethynodiol, etonogestrel, levomefolate, levonorgestrel,
lynestrenol, medroxyprogesterone acetate, mestranol, mifepristone,
misoprostol, nomegestrol acetate, norelgestromin, norethindrone,
noretynodrel, norgestimate, ormeloxifene, segestersone acetate,
ulipristal acetate, and any combinations thereof.
HBV Therapeutic Agents
[0711] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an HBV therapeutic agent.
[0712] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein are selected from
alpha-hydroxytropolones, amdoxovir, antroquinonol,
beta-hydroxycytosine nucleosides, ARB-199, CCC-0975, ccc-R08,
elvucitabine, ezetimibe, cyclosporin A, gentiopicrin
(gentiopicroside), HH-003, hepalatide, JNJ-56136379, nitazoxanide,
birinapant, NJK14047, NOV-205 (molixan, BAM-205), oligotide,
mivotilate, feron, GST-HG-131, levamisole, Ka Shu Ning, alloferon,
WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3,
BP-Inter-014, oleanolic acid, HepB-nRNA, cTP-5 (rTP-5), HSK-II-2,
HEISCO-106-1, HEISCO-106, Hepbarna, IBPB-0061A, Hepuyinfen,
DasKloster 0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210,
OB-AI-004, PF-06, picroside, DasKloster-0039, hepulantai, IMB-2613,
NCO-48 Fumarate, TCM-800B, reduced glutathione, RO-6864018,
RG-7834, QL-007sofosbuvir, ledipasvir, UB-551, PA-1010, HPN-BV1,
STSG-0002, and ZH-2N, and the compounds disclosed in US20150210682,
(Roche), US 2016/0122344 (Roche), WO2015173164 (Roche),
WO2016023877 (Roche), US2015252057A (Roche), WO16128335A1 (Roche),
WO16120186A1 (Roche), US2016237090A (Roche), W16107833A1 (Roche),
WO16107832A1 (Roche), US2016176899A (Roche), W16102438A1 (Roche),
WO16012470A1 (Roche), US2016220586A (Roche), and US2015031687A
(Roche).
[0713] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein are selected from include
an HBV vaccine. In some embodiments, the HBV vaccine is selected
from HBsAG-HBIG complex, ARB-1598, Bio-Hep-B, NASVAC, abi-HB
(intravenous), ABX-203, Tetrabhay, GX-110E, GS-4774, peptide
vaccine (epsilonPA-44), Hepatrol-07, NASVAC (NASTERAP), IMP-321,
BEVAC, Revac B mcf, Revac B+, MGN-1333, KW-2, CVI-HBV-002,
AltraHepB, VGX-6200, FP-02, FP-02.2 (HepTcell), NU-500, HBVax,
im/TriGrid/antigen vaccine, Mega-CD40L-adjuvanted vaccine, HepB-v,
RG7944 (INO-1800), recombinant VLP-based therapeutic vaccine (HBV
infection, VLP Biotech), hepatitis B therapeutic DNA vaccine,
AdTG-17909, AdTG-17910, AdTG-18202, ChronVac-B, TG-1050, VVX-001,
GSK-3528869A (ChAdl55-hli-HBV+MVA-HBV +Hbc-HBs/ASO1B-4), VBI-2601,
VTP-300 (ChAdOx1-SIi-HBV-CPmut-TPA-Ssh prime and
MVA-SIi-HBV-CPmut-TPA-Ssh boost), MVA-BN, AVA-2100, HBV-ADV311,
YS-HBV-002, and Lm HBV. HBV Arenavirus vaccines are described,
e.g., in WO2017076988 and WO2017198726.
[0714] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an HBV polymerase
inhibitor. In some embodiments, the HBV polymerase inhibitor is
selected from adefovir (HEPSERA.RTM.), emtricitabine
(EMTRIVA.RTM.), tenofovir disoproxil fumarate (VIREAD.RTM.),
tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir
alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir
dipivoxil, tenofovir dipivoxil fumarate, tenofovir
octadecyloxyethyl ester, CMX-157, tenofovir exalidex, besifovir,
entecavir (BARACLUDE.RTM.), entecavir maleate, telbivudine
(TYZEKA.RTM.), filocilovir, pradefovir, clevudine, ribavirin,
lamivudine (EPIVIR-HBV.RTM.), phosphazide, famciclovir, fusolin,
metacavir, ATI-2173, SNC-019754, FMCA, AGX-1009, AR-II-04-26,
HIP-1302, tenofovir disoproxil aspartate, tenofovir disoproxil
orotate, and HS-10234.
[0715] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an immunomodulator.
In some embodiments, the immunomodulator is selected from
rintatolimod, imidol hydrochloride, ingaron, dermaVir, plaquenil
(hydroxychloroquine), proleukin, hydroxyurea, mycophenolate mofetil
(MPA) and its ester derivative mycophenolate mofetil (MMF),
JNJ-440,WF-10,AB-452, ribavirin, IL-12, INO-9112, polymer
polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ-0535,
TG-1050, ABI-H2158, BMS-936559,GS-9688, RO-7011785, RG-7854,
RO-6871765, AIC-649, and IR-103.
[0716] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an interferon alpha
receptor ligand. In some embodiments the interferon alpha receptor
ligand is selected from interferon alpha-2b (INTRON A.RTM.),
pegylated interferon alpha-2a (PEGASYS.RTM.), PEGylated interferon
alpha-lb, interferon alpha 1b (HAPGEN.RTM.), Veldona, Infradure,
Roferon-A, YPEG-interferon alfa-2a (YPEG-rhIFNalpha-2a), P-1101,
Algeron, Alfarona, Ingaron (interferon gamma), rSIFN-co
(recombinant super compound interferon), Ypeginterferon alfa-2b
(YPEG-rhIFNalpha-2b), MOR-22, peginterferon alfa-2b
(PEG-INTRON.RTM.), Bioferon, Novaferon, Inmutag (Inferon),
MULTIFERON.RTM., interferon alfa-n1(HUMOFERON.RTM.), interferon
beta-ia (AVONEX.RTM.), Shaferon, interferon alfa-2b (Axxo),
Alfaferone, interferon alfa-2b (BioGeneric Pharma),
interferon-alpha 2 (CJ), Laferonum, VIPEG, BLAUFERON-A,
BLAUFERON-B, Intermax Alpha, Realdiron, Lanstion, Pegaferon,
PDferon-B PDferon-B, interferon alfa-2b (IFN, Laboratorios
Bioprofarma), alfainterferona 2b, Kalferon, Pegnano, Feronsure,
PegiHep, interferon alfa 2b (Zydus-Cadila), interferon alfa 2a,
Optipeg A, Realfa 2B, Reliferon, interferon alfa-2b (Amega),
interferon alfa-2b (Virchow), ropeginterferon alfa-2b, rHSA-IFN
alpha-2a (recombinant human serum albumin intereferon alpha 2a
fusion protein), PEG-IFN-alpha, rHSA-IFN alpha 2b, recombinant
human interferon alpha-(1b, 2a, 2b), peginterferon alfa-2b (Amega),
peginterferon alfa-2a, Reaferon-EC, Proquiferon, Uniferon, Urifron,
interferon alfa-2b (Changchun Institute of Biological Products),
Anterferon, Shanferon, Layfferon, Shang Sheng Lei Tai, INTEFEN,
SINOGEN, Fukangtai, Pegstat, rHSA-IFN alpha-2b, SFR-9216, and
Interapo (Interapa).
[0717] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a hyaluronidase
inhibitor. In some embodiments the hyaluronidase inhibitor is
astodrimer.
[0718] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a Hepatitis B
Surface Antigen (HBsAg) inhibitor. In some embodiments the HBsAg
inhibitor is selected from AK-074, HBF-0259, PBHBV-001, PBHBV-2-15,
PBHBV-2-1, REP-9AC, REP-9C, REP-9, REP-2139, REP-2139-Ca, REP-2055,
REP-2163, REP-2165, REP-2053, REP-2031, REP-006, and REP-9AC'. In
some embodiments, the HBsAg inhibitor is an HBsAg secretion
inhibitor such as BM601, GST-HG-131, AB-452.
[0719] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a cyclophilin
inhibitor. In some embodiments, the cyclophilin inhibitor is
selected from CPI-431-32, EDP-494, OCB-030, SCY-635, NVP-015,
NVP-018, NVP-019, STG-175, and the compounds disclosed in U.S. Pat.
No. 8,513,184 (Gilead Sciences), US20140030221 (Gilead Sciences),
US20130344030 (Gilead Sciences), and US20130344029 (Gilead
Sciences).
[0720] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an antisense
oligonucleotide targeting viral mRNA. In some embodiments, the
antisense oligonucleotide is selected from ISIS-HBVRx, IONIS-HBVRx,
IONIS-HBV-LRx, IONIS-GSK6-LRx, GSK-3389404, and RG-6004.
[0721] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a short interfering
RNA (siRNA) or DNA-directed RNA interference (ddRNAi). In some
embodiments, the siRNA is selected from TKM-HBV (TKM-HepB), ALN-HBV
(e.g., ALN-HBV02), SR-008, HepB-nRNA, ARC-520, ARC-521, ARB-1740,
ARB-1467, AB-729, DCR-HBVS, RG-6084 (PD-L1), RG-6217, ALN-HBV-02,
JNJ-3989 (ARO-HBV), STSG-0002, ALG-010133, ALG-ASO, LUNAR-HBV and
DCR-HBVS (DCR-5219). An illustrative example of ddRNAi is
BB-HB-331.
[0722] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an endonuclease
modulator (e.g., PGN-514).
[0723] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a ribonucleotide
reductase inhibitor (e.g., Trimidox).
[0724] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a non-nucleoside
reverse transcriptase inhibitor (NNRTI). In some embodiments, the
NNRTI is selected from the compounds disclosed in WO2018118826
(Merck), WO2018080903 (Merck), WO2018119013 (Merck), WO2017100108
(Idenix), WO2017027434 (Merck), WO2017007701 (Merck), and
WO2008005555 (Gilead).
[0725] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an HBV replication
inhibitor. In some embodiments, the HVB replication inhibitor is
selected from GP-31502, isothiafludine, IQP-HBV, RM-5038, and
Xingantie.
[0726] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a non-canonical RNA
polymerase PAPD5 and PAPD7 inhibitor. Illustrative examples of
non-canonical RNA polymerase PAPD5 and PAPD7 inhibitors include
PAPD5 and PAPD7 targeting locked nucleic acid antisense
oligonucleotides.
[0727] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a covalently closed
circular DNA inhibitor (cccDNA), such as BSBI-25, ccc-R08, and
CHR-101.
[0728] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a caspase 9
stimulator such as ENOB-HB-01.
[0729] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a CD3 modulator
such as IMC-109V.
[0730] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a Ffar2 and Ffar3
agonist, such as SFA-001.
[0731] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an additional HBV
antibody. In some embodiments, the additional HBV antibody targets
a surface antigen of hepatitis B virus. The additional HBV antibody
can include monoclonal and polyclonal antibodies. In some
embodiments, the additional HBV antibody is selected from
lenvervimab (GC-1102), XTL-17, XTL-19, KN-003, IV Hepabulin SN,
VIR-3434, and fully human monoclonal antibody therapy (hepatitis B
virus infection, Humabs BioMed). In some embodiments, the
additional HBV antibody is selected from Zutectra, Shang Sheng Gan
Di, Uman Big (Hepatitis B Hyperimmune), Omri-Hep-B, Nabi-HB,
Hepatect CP, HepaGam B, igantibe, Niuliva, CT-P24, hepatitis B
immunoglobulin (intravenous, pH4, HBV infection, Shanghai RAAS
Blood Products), and Fovepta (BT-088). In some embodiments, the
additional HBV antibody is a fully human monoclonal antibody such
as HBC-34. In some embodiments, the additional HBV antibody is an
antibody against HBV viral peptide/major histocompatibility complex
(MHC) class I (pMHC) complexes, e.g., as are described in Sastry,
et al., J Virol. 2011 March; 85(5):1935-42 or in WO2011062562.
[0732] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a CCR2 chemokine
antagonist such as propagermanium.
[0733] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a farnesoid x
receptor (FXR) agonists. In some embodiments, the FXR agonist is
selected from EYP-001, GS-9674, EDP-305, MET-409, Tropifexor,
AKN-083, RDX-023, BWD-100, LMB-763, INV-3, NTX-023-1, EP-024297 and
GS-8670.
[0734] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a thymosine
antagonist such as Thymalfasin, recombinant thymosin alpha 1
(GeneScience), NL-004 or PEGylated thymosin alpha-1.
[0735] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a nucleoprotein
modulator. In some embodiments, the nucleoprotein modulator is
selected from GS-4882, AB-423, AB-836, AT-130, ALG-001075,
ALG-001024, ALG-000184, EDP-514, GLS4, NVR-1221, NVR-3778, AL-3778,
BAY 41-4109, morphothiadine mesilate, ARB-168786, ARB-880,
ARB-1820, GST-HG-141, JNJ-379, JNJ-632, RG-7907, GST-HG-141,
HEC-72702, KL-060332, AB-506, ABI-H0731, ABI-H3733, JNJ-440,
ABI-H2158, CB-HBV-001, AK-0605, SOC-10, SOC-11 and DVR-23.
[0736] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a capsid inhibitor.
In some embodiments, the capsid inhibitor is selected from the
compounds disclosed in US20140275167 (Novira Therapeutics),
US20130251673 (Novira Therapeutics), US20140343032 (Roche),
WO2014037480 (Roche), US20130267517 (Roche), WO2014131847
(Janssen), WO2014033176 (Janssen), WO2014033170 (Janssen),
WO2014033167 (Janssen), WO2015/059212 (Janssen),
WO2015118057(Janssen), WO2015011281 (Janssen), W2014184365
(Janssen), WO2014184350 (Janssen), WO2014161888 (Janssen),
WO2013096744 (Novira), US20150225355 (Novira), US20140178337
(Novira), US20150315159 (Novira), US20150197533 (Novira),
US20150274652 (Novira), US20150259324, (Novira), US20150132258
(Novira), U.S. Pat. No. 9,181,288 (Novira), WO2014184350 (Janssen),
WO2013144129 (Roche), WO2017198744 (Roche), US 20170334882
(Novira), US 20170334898 (Roche), WO2017202798 (Roche),
WO2017214395 (Enanta), WO2018001944 (Roche), WO2018001952 (Roche),
WO2018005881 (Novira), WO2018005883 (Novira), W2018011100 (Roche),
WO2018011160 (Roche), WO2018011162 (Roche), WO2018011163 (Roche),
WO2018036941 (Roche), WO2018043747 (Kyoto Univ), US20180065929
(Janssen), WO2016168619 (Indiana University), WO2016195982 (The
Penn State Foundation), WO2017001655 (Janssen), WO2017048950
(Assembly Biosciences), WO2017048954 (Assembly Biosciences),
WO2017048962 (Assembly Biosciences), US20170121328 (Novira), and
US20170121329 (Novira).
[0737] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a transcript
inhibitor. In some embodiments, the transcript inhibitor is
selected from the compounds disclosed in WO2017013046 (Roche),
WO2017016960 (Roche), WO2017017042 (Roche), WO2017017043 (Roche),
WO2017061466 (Toyoma Chemicals), WO2016177655 (Roche), WO2016161268
(Enanta), WO2017001853 (Redex Pharma), WO2017211791 (Roche),
WO2017216685 (Novartis), WO2017216686 (Novartis), WO2018019297
(Ginkgo Pharma), WO2018022282 (Newave Pharma), US20180030053
(Novartis), and WO2018045911 (Zhejiang Pharma).
[0738] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a retinoic
acid-inducible gene stimulator 1. In some embodiments, the retinoic
acid-inducible gene stimulator 1 is selected from inarigivir
soproxil (SB-9200), SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537,
ORI-9020, ORI-9198, ORI-7170, and RGT-100.
[0739] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an arginase
inhibitor. In some embodiments, the arginase inhibitor is selected
from CB-1158, C-201, and resminostat.
[0740] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a CAR-T cell
therapy. CAR-T cell therapy includes a population of immune
effector cells engineered to express a chimeric antigen receptor
(CAR), wherein the CAR includes an HBV antigen-binding domain. In
certain embodiments, the antigen-binding domain is a domain
disclosed herein. In certain embodiments, the antigen-binding
domain is other than a domain disclosed herein. In certain
embodiments, the antigen is HBsAg (HbsAg-CART). The immune effector
cell is a T-cell or an NK cell. In certain embodiments, the T-cell
is a CD4+ T-cell, a CD8+ T-cell, a NK cell or a combination
thereof. Cells can be autologous or allogeneic. An example of a
CART directed to HBV is described in Cytotherapy. 2018 May;
20(5):697-705. doi: 10.1016/j.jcyt.2018.02.
[0741] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include a TCR-T cell
therapy. TCR-T cell therapy includes T cells expressing
HBV-specific T cell receptors. TCR-T cells are engineered to target
HBV derived peptides presented on the surface of virus-infected
cells. An example of a TCR directed to HBV is described in
Wisskirchen, K. et al. T cell receptor grafting allows virological
control of hepatitis B virus infection. JClinInvest. 2019;
129(7):2932-2945. In some embodiments, the TCR-T cell therapy
includes T-Cells expressing HBV surface antigen (HBsAg)-specific
TCR. In some embodiments, the TCR-T cell therapy includes TCR-T
therapy directed to treatment of HBV, such as LTCR-H2-1.
[0742] In some embodiments, the HBV therapeutic agents that can be
combined with an agent disclosed herein include an inhibitor of
certain HCV nonstructural proteins, such as a NS5A inhibitor, a
NS5B inhibitor, a NS3 inhibitor, or combinations thereof. In some
embodiments. In one embodiment, the NS5A inhibitor is ledipasvir or
velpatasvir. In one embodiment, the NS5B inhibitor is sofosbuvir or
mericitabine. In one embodiment, the NS5A inhibitor is ledipasvir
and the NS5B inhibitor is sofosbuvir. In one embodiment, the NS3
inhibitor is voxilaprevir.
HCV Therapeutic Agents
[0743] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an HCV therapeutic agent such as an HCV
NS5A/NS5B inhibitor, HCV NS3/4A protease inhibitor, HCV NS5A
protein inhibitor, HCV NS5B polymerase inhibitor of the
nucleoside/nucleotide type, or HCV NS5B polymerase inhibitor of the
nonnucleoside type. In various embodiments, the FLT3L-Fc fusion
proteins, homodimers, heterodimers, polynucleotides, vectors,
lipoplexes, such as LNPs, and/or pharmaceutical compositions, as
described herein, are combined with an additional therapeutic agent
selected from the group consisting of daclatasvir, ledipasvir,
ombitasvir, elbasvir, sofosbuvir, dasabuvir, ribavirin,
asunaprevir, simeprevir, paritaprevir, ritonavir, elbasvir, and
grazoprevir. In some embodiments, an additional therapeutic agent
is selected from the group consisting of daclatasvir, ledipasvir,
ombitasvir, elbasvir, sofosbuvir, and dasabuvir.
Influenza Virus Inhibitors
[0744] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an influenza virus inhibitor. In some
embodiments the influenza virus inhibitor that can be combined with
an agent of this disclosure includes a matrix 2 inhibitor (e.g.,
amantadine, rimantadine), a neuraminidase inhibitor (e.g.,
zanamivir, oseltamivir, peramivir, laninamivir octanoate), or a
polymerase inhibitor (e.g., ribavirin, favipiravir), or
combinations thereof. In some embodiments, the influenza virus
inhibitor is selected from amantadine, rimantadine, arbidol
(umifenovir), baloxavir marboxil, oseltamivir, peramivir,
ingavirin, laninamivir octanoate, zanamivir, favipiravir,
ribavirin, and combinations thereof. In some embodiments, the
influenza virus inhibitor is selected from amantadine, rimantadine,
zanamivir, oseltamivir, peramivir, laninamivir octanoate,
ribavirin, favipiravir, and combinations thereof.
RSV Therapeutic Agents
[0745] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an RSV therapeutic agent, such as
ribavirin, ALS-8112, presatovir, or combinations thereof.
Picornavirus Therapeutic Agents
[0746] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a picornavirus therapeutic agent, such as
hydantoin, guanidine hydrochloride, L-buthionine sulfoximine,
Py-11, rupintrivir, or combinations thereof.
Ebola Virus Therapeutic Agents
[0747] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an ebola virus therapeutic agent. In some
embodiments the ebola virus therapeutic agent that can be combined
with an agent of this disclosure is selected from ribavirin,
palivizumab, motavizumab, RSV-IGIV (RespiGam.RTM.), MEDI-557,
A-60444, MDT-637, BMS-433771, amiodarone, dronedarone, verapamil,
Ebola Convalescent Plasma (ECP), TKM-100201, BCX4430
((2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hyd-
roxymethyl)pyrrolidine-3,4-diol), favipiravir (also known as T-705
or Avigan), T-705 monophosphate, T-705 diphosphate, T-705
triphosphate, FGI-106
(1-N,7-N-bis[3-(dimethylamino)propyl]-3,9-dimethylquinolino[8,7-h-
]quinolone-1,7-diamine), JK-05, TKM-Ebola, ZMapp, rNAPc2,
VRC-EBOADC076-00-VP, OS-2966, MVA-BN filo, brincidofovir, Vaxart
adenovirus vector 5-based ebola vaccine, Ad26-ZEBOV, FiloVax
vaccine, GOVX-E301, GOVX-E302, ebola virus entry inhibitors (NPC1
inhibitors), rVSV-EBOV, and combinations thereof. In some
embodiments, the ebola virus therapeutic agent is selected from
ZMapp, mAB114, REGEN-EB3, and combinations thereof.
Coronavirus Therapeutic Agents
[0748] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a coronavirus therapeutic agent. In some
embodiments, the coronavirus therapeutic agent is a SARS
therapeutic agent. In some embodiments, the coronavirus therapeutic
agent is a MERS therapeutic agent. In some embodiments, the
coronavirus therapeutic agent is a COVID-19 therapeutic agent.
[0749] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a COVID-19 therapeutic agent.
Illustrative examples of COVID-19 therapeutic agents that can be
combined with an agent disclosed herein include without limitation,
an adjuvant, antiangiogenic, antibiotic, antiemetic, antifibrotic,
antioxidant, antiparasitic, antiproliferative, antithrombotic,
antiviral, convalescent plasma, epigenetic mofifier,
immunomodulator, immunostimulant, immunosuppressant, metabolic
modifier, mucolytic, neuromodulatory, neutralizing antibody, oxygen
delivery, proapoptotic, surfactant, thyromimetic, vaccine,
vasoconstrictor, or vasodilator.
[0750] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an adjuvant. Illustrative examples of
adjuvants that can be combined with an agent disclosed herein
include without limitation a TLR9 agonist (e.g., CpG 1018
(Dynavax)), Matrix-M (Novavax), CoVaccine HT (Boston Scientific),
7HP349 (7 Hills Pharma), FirmaVacc (PCI Biotech).
[0751] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antiangiogenic, such as an integrin
alpha 4 modulator (e.g., LY3127804 (Eli Lilly)).
[0752] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antibiotic. Illustrative examples of
antibiotics that can be combined with an agent disclosed herein
include without limitation azithromycin, olumiant baricitinib,
chloramphenicol, dactinomycin, linezolid, tigecycline, carrimycin,
incyclinide.
[0753] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antiemetic, such as a neurokinin 1
receptor (NK1R) modulator (e.g., tradipitant (Vanda
Pharmaceuticals)).
[0754] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antifibrotic. Illustrative examples of
antifibrotics that can be combined with an agent disclosed herein
include without limitation a platelet derived growth factor
receptor (PDGFR) modulator (e.g., nintedanib), tight junction
protein 1 modulator (e.g., aCTI), FK506 binding protein 4 (FKBP4)
modulator (e.g., RT1840), amphiregulin (AREG) modulator (e.g.,
amphisiran (siRNAgen Therapeutics), and angiotensin II receptor
type 2 (AGTR2) modulator (e.g., VPO1 (Vicore Pharma)).
[0755] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antifungal, such as inhaled
pentamidine or oral amphotericin B (e.g., ICO-019, iCo
Therapeutics).
[0756] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antimicrobial, such as NORS (Nitric
Oxide Releasing Solution) or brilacidin (PMX-30063).
[0757] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antioxidant, such as a sirtuin 1
(SIRT1) agonist (e.g., resveratrol, JOTROL (high oral
bioavailability resveratrol)), transglutaminase 2 modulator (e.g.,
TTI-0102 (cysteamine prodrug, Thiogenesis)), or bucillamine
(Revive).
[0758] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antiparasitic. Illustrative examples
of antiparasitics that can be combined with an agent disclosed
herein include without limitation chloraquine phosphate,
hydroxychloroquine, plasmodium cytochrome bcl modulator (e.g.,
atovaquone), Glutamate decarboxylase 2 (GAD2; GAD65) modulator
(e.g., LAI ivermectin), pyruvate ferredoxin oxidoreductase
modulator (e.g., nitazoxanide, NT-300 (nitazoxanide
extended-release tablets, Romark Pharmaceuticals)), nicotinic
acetylcholine receptor (nAchR) modulator (e.g., levamisole),
FW-1022 (oral niclosamide; First Wave), emetine (Acer
Therapeutics), or UNI911 (niclosamide) (Union Therapeutics).
[0759] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antiproliferative. Illustrative
examples of antiproliferatives that can be combined with an agent
disclosed herein include without limitation estrogen receptor 1
(ER1) modulator (e.g., toremifene), BRAF inhibitor (e.g., trafinlar
dabrafenib), Bruton's tyrosine kinase (Btk) inhibitor (e.g.,
brukinsa zanubrutinib (Beigene), acalabrutinib), eukaryotic
translation elongation factor 1 alpha 2 (EEF1A2) modulator (e.g.,
aplidin plitidepsin (PharmaMar)) NEDD8 activating enzyme (NAE)
modulator (e.g., pevonedistat), RAS inhibitor (e.g., rigosertib),
vimentin modulator (e.g., pritumumab), Mammalian target of
rapamycin (mTOR; FRAP; RAFT1) inhibitor (e.g., sapanisertib
(TAK-228); Dactolisib (RTB101)), MAP kinase interacting
serine-threonine kinase 1 (MKNK1; MNK1) or MKNK2 inhibitor (e.g.,
tomivosertib), Urokinase-type plasminogen activator (PLAU; uPA)
inhibitor (e.g., upamostat (RHB-107, WX-671)), tubulin inhibitor
(e.g., VERU-111), eukaryotic translation initiation factor 4A1
(EIF4A1; EIF4A) inhibitor (e.g., zotatifin), MAP kinase inhibitor
(e.g., KTH-222 (Kalos Oncology)), MAP kinase interacting
serine-threonine kinase 1 (MKNK1; MNK1)inhibitor, p53 (TP53);
mitogen-activated protein kinase kinase kinase kinase 1 (HPK1;
MAP4K1) inhibitor, c-jun N-terminal kinase (INK) inhibitor (e.g.,
Satcon), vascular endothelial growth factor (VEGF) inhibitor (e.g.,
avastin bevacizumab), or exportin 1 (XPO1; CRM1) inhibitor (e.g.,
selinexor).
[0760] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antithrombotic. Illustrative examples
of antithrombotics that can be combined with an agent disclosed
herein include without limitation plasmin inhibitor (e.g.,
tranexamic acid), Purinergic receptor P2Y G protein-coupled 12
(P2RY12; P2Y12) inhibitor (e.g., clopidogrel), factor Xa (e.g.,
rivaroxaban), fibrin inhibitor (e.g., alteplase), cathepsin G
inhibitor (e.g., defibrotide), or serine protease inhibitor (e.g.,
nafamostat).
[0761] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antiviral. Illustrative examples of
antivirals that can be combined with an agent disclosed herein
include without limitation HIV protease inhibitor (e.g.,
lopinavir/ritonavir, atazanavir, ritonavir), HIV reverse
transcriptase inhibitor (e.g., Emtricitabine/tenofovir disoproxil),
neuraminidase inhibitor (e.g., oseltamivir), HIV
protease/cytochrome P450 (e.g., darunavir/cobicistat), amantadine,
viral RNA polymerase PA subunit inhibitor (e.g., baloxavir
marboxil), RNA-dependent RNA polymerase inhibitor (e.g., ribavirin,
galidesivir, remdesivir), omega 3 viruxide, serine protease
inhibitor (e.g., camostat), viral RNA polymerase inhibitor (e.g.,
avigan favipiravir), arbidol umifenovir, HCV protease+HIV protease
inhibitors (e.g., Ganovo danoprevir+ritonavir), SARS-CoV-2 3C-like
protease (SARS-CoV-2 3CLpro; SARS-CoV-2 NSP5; SARS-CoV-2 Mpro;
SARS-CoV-2 main protease) inhibitor, AT-H201 (Atossa Therapeutics),
xylometazoline hydrochloride and iota-carrageenan, sialic acid,
berdazimer sodium, dihydroorotate dehydrogenase (DHODH) inhibitor
(e.g., IU-838), Inosine monophosphate dehydrogenase (IMPDH)
inhibitor (e.g., merimepodib), viral reverse transcriptase
inhibitor (e.g., azvudine), checkpoint kinase 1 (Chkl; CHEK1)
inhibitor (e.g., prexasertib), heat shock 70 kDa protein 5
(glucose-regulated protein, 78 kDa; GRP78; HspA5) inhibitor (e.g.,
BOLD-100), exportin 1 (XPO1; CRM1) inhibitor (e.g., verdinexor),
CD147 inhibitor (e.g., meplzumab), bacterial 30S ribosomal subunit
protein S5 (RpsE) inhibitor (e.g., eravacycline), or Calpain 1
(CAPN1), CAPN2 or CAPN9 inhibitor (e.g., BLD-2660, Blade
Therapeutics).
[0762] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with convalescent plasma, including without
limitation intravenous immunoglobulin (Grifols) or hyperimmune
plasma.
[0763] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an epigenetic modifier, such as a BET
bromodomain protein inhibitor (e.g., apabetalone, CPI-0610,
ABBV-744).
[0764] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an immunomodulator. Illustrative examples
of immunomodulators that can be combined with an agent disclosed
herein include without limitation BCR-ABL tyrosine kinase (BCR-ABL)
inhibitor, stem cell factor (SCF) receptor tyrosine kinase (c-Kit;
KIT; CD117) inhibitor (e.g., imatinib, masitinib), spleen tyrosine
kinase (SYK) inhibitor (e.g., fostamatinib), Toll-like receptor 3
(TLR3) agonist (e.g., rintatolimod, polyIC), signal transducer and
activator of transcription 3 (STAT3) or STAT6 inhibitor 9 (e.g.,
mosedipimod), neuropilin 2 (NRP2) inhibitor (e.g., ATYR1923 (aTyr
Pharma)), natural killer (NK) cell (e.g., ex vivo expanded
allogeneic MRx-4DP0004, 4D Pharma), or type I interferon (IFN)
receptor agonist (e.g., interferon beta-la, novaferon, pegylated
interferon alpha-2b, interferon-I a, interferon (IFN) lambda
receptor (IL28RA; IFNLR1) agonist (e.g., peginterferon lambda-la,
interferon-a). Programmed cell death 1 (PD-1; PDCD1; CD279)
inhibitor (e.g., pembrolizumab, camrelizumab), granulocyte
macrophage colony-stimulating factor (GM-CSF) receptor (CSF2RA)
(GMR) (CD116) (GMCSFR) agonist (e.g., sargramostim), LAG-31g fusion
protein (e.g., eftilagimod alpha), interleukin-15 (IL-15) receptor
alpha chain (IL-15RA) agonist (e.g., IL-15 mutant (IL-15N72D) bound
to an IL-15 receptor a/IgG1 Fc fusion protein (N-803,
ImmunityBio)), Toll-like receptor (TLR) 2 (TLR2), TLR6, or TLR9
agonist, humanized virus suppressing factor variant 13 (e.g.,
hzVSF-v13 (ImmuneMed)), intercellular adhesion molecule-3 (ICAM-3;
CD50) modulator (e.g., AVM0703 (AVM Biotechnology)), glucocorticoid
receptor modulator (e.g., dexamethasone, methylprednisolone,
hydrocortisone, fluasterone), histamine H1 receptor (HRH1),
interleukin-6 (IL-6) receptor (CD126) antagonist (e.g.,
tocilizumab, sarilumab), tubulin inhibitor (e.g., colchicine),
interleukin-1 (IL-1) beta antagonist (e.g., canakinumab), Janus
kinase-1 (JAK-1), JAK-2 or JAK-3 inhibitor (e.g., ruxolitinib,
tofacitinib, pacritinib), cyclooxygenase (COX) inhibitor (e.g.,
naproxen, aspirin, ibuprofen), FK506 binding protein 1A 12 kDa
(FKBP12; FKBPlA) modulator (e.g., tacrolimus, rapamycin),
sphingosine 1-phosphate receptor 1 (S1PR1; SlPl; EDG1), S1PR3,
SlPR4, or S1PR5 modulator (e.g., fingolimod), complement 5
antagonist (eculizumab, ravulizumab-cwvz, avdoralimab), neutophil
elastase inhibitor (e.g., alpha-1 antitrypsin), phosphodiesterase-4
(PDE-4) inhibitor (e.g., apremilast, ibudilast), IL-6 antagonist
(e.g., siltuximab, olokizumab), interleukin-1 (IL-1) receptor 1
(IL1R1) antagonist (e.g., anakinra), interferon gamma antagonist
(e.g., emapalumab), vasoactive intestinal peptide receptor 1
(VPAC1) or VPAC2 agonist (e.g., Aviptadil (NeuroRx)), telomerase
reverse transcriptase (TERT) inhibitor (e.g., tertomotide), stem
cell therapy (e.g., mesenchymal stem cells (MSC), human amniotic
fluid, PLX cells (Pluristem)), granulocyte macrophage
colony-stimulating factor (GM-CSF; CSF2) antagonist (e.g.,
otilimab, lenzilumab, namilumab, gimsilumab), CC chemokine receptor
5 (CCR5) antagonist (e.g., leronlimab (PRO 140)), CD24-Fc fusion
protein, adenosine A3 receptor (ADORA3) antagonist (e.g.,
piclidenoson), transforming growth factor (TGF) beta 2 (TGFB2)
antagonist (e.g., OT-101 (siRNA)), CD14 antagonist (OT-101), matrix
metalloproteinase 12 (MMP12) inhibitor, VE-cadherin (CD144;
cadherin-5) modulator (FX06), dipeptidyl peptidase-1 (DPP-1; CTSC)
(e.g., brensocatib), Chemokine CXC motif ligand 10 (CXCL10; IP-10)
inhibitor (e.g., NI-0101), Galectin-3 (LGALS3) modulator,
Microsomal prostaglandin E synthase-1 (PTGES; mPGES-1) inhibitor
(e.g., Sonlicromanol), neutrophil elastase (ELANE; NE; HLE)
inhibitor (e.g., lonodelestat), complement receptor 5A (C5AR1;
C5AR; CD88) antagonist (avdoralimab), cyclin dependent kinase 2
(CDK2) or CDK9 inhibitor (e.g., fadraciclib, seliciclib), Transient
receptor potential vanilloid 1 (TRPV1; VR1) modulator (Ser-014
(e.g., Serentrix)), pirfenidone, dociparstat, or Sphingosine kinase
2 (SPHK2) inhibitor (e.g., opaganib).
[0765] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a metabolic agent. Illustrative examples
of metabolic agents that can be combined with an agent disclosed
herein include without limitation Dipeptidyl peptidase-4 (DPP-4;
CD26) inhibitor (linagliptin), sodium-glucose cotransporter 2
(SGLT2) inhibitor (dapagliflozin), H+/K ATPase pump inhibitor
(e.g., omeprazole), aldose reductase inhibitor (AT-100),
a-glucosidase inhibitor (e.g., IHVR-19029), or protein tyrosine
phosphatase 1B (PTP-1B; PTPN1) inhibitor (e.g., trodusquemine).
[0766] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a vaccine. Illustrative examples of
metabolic agents that can be combined with an agent disclosed
herein include without limitation BCG vaccine, MMR vaccine,
SARS-CoV-2 spike protein (SARS-CoV-2 S) vaccine (e.g., Ad5-nCoV
(CanSino Biologics), ChAdOxI nCoV-19 (e.g., ChAdOxI Consortium),
INO-4800 (Inovio Pharmaceuticals), bacTRL-Spike (Symvivo
Corporation), mRNA-1273 (Moderna Therapeutics), NVX-CoV2373
(Novavax; Emergent BioSolutions)), SARS-CoV-2 3C-like protease
(SARS-CoV-2 3CLpro; SARS-CoV-2 NSP5; SARS-CoV-2 Mpro; SARS-CoV-2
main protease)(e.g., BNT162 (BioNTech; Pfizer; Fosun Pharma)); or
new coronavirus inactivated vaccine (Vero cells; Sinopharm). In
some embodiments the vaccine is Ad-nCoV (CanSino Biologics). In
some embodiments, the vaccine is mRNA-1273 (Moderna Therapeutics).
In some embodiments, the vaccine is BNT-162 (CanSino Biologics,
Biontech). In some embodiments, the vaccine is AZD-1222 (ChAdOx1
nCoV-19, AstraZeneca). In some embodiments the vaccine is INO-4800
(Inovio Pharmaceuticals). In some embodiments, the vaccine is
NVX-CoV2373 (Novavax).
[0767] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a vasoconstrictor or vasodilator.
Illustrative examples of vasoconstrictors or vasodilators that can
be combined with an agent disclosed herein include without
limitation angiotensin II type 1 (AT1) receptor (AGTR1) agonist
(e.g., angiotensin II); angiotensin II type 1 (AT1) receptor
(AGTR1) antagonist (e.g., valsartan, losartan); cGMP specific
phosphodiesterase type 5 (PDE5) inhibitor (e.g., sildenafil),
Angiotensin-converting enzyme (ACE) inhibitor (e.g., captopril,
lisonopril), endothelin receptor antagonist (e.g., iloprost),
calcitonin gene-related peptide (CGRP) receptor antagonist (e.g.,
vazegepant), or MAS receptor agonist (e.g., angiotensin-(1-7)).
[0768] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a 2019-nCoV virus antibody. Illustrative
examples of a 2019-nCoV virus antibody that can be combined with an
agent disclosed herein include without limitation LY-CoV555, S309,
SAB-185, CB6, STI-1499, JS016, VNAR, VIR-7832, VIR-7831, REGN-COV2,
BAT2020, BAT2019, 47D11, or COVI-SHIELD.
[0769] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a COVID-19 therapeutic agent.
Illustrative examples of COVID-19 therapeutic agents that can be
combined with an agent disclosed herein include without limitation,
a COVID-19 vaccine (e.g., BN162, Ad5-nCoV, INO-4800, mRN1273), an
anti-IL6 antibody, an anti-IL6 receptor antibody (e.g.,
tocilizumab, sarilumab, TZLS-501), an anti-IL6 antibody (e.g.,
siltuximab), an RNA dependent RNA polymerase (RdRp) inhibitor
(e.g., favipravir, remdesivir), an anti-CCR5 antibody (e.g.,
leronlimab (PRO 140)), a broadly neutralizing antibody (e.g., an
anti-ACE2 receptor antibody, SAB-185, COVID-HIG, COVID-EIG),
including monoclonal or polyclonal neutralizing antibodies, an ACE2
(angiotensin-converting enzyme 2)-Fc fusion protein (COVIDTRAP) or
recombinant human ACE2 protein (APN1), ACE-MAB.TM. bi-specific
fusion protein designed to bind to the spike protein of
coronaviruses including SARS-CoV-2 and SARS-CoV (STI-4920,
CMAB020), a Janus kinase (JAK1/JAK2) inhibitor (e.g., ruxolitinib,
baricitinib), an siRNA (e.g., targeting angiotensin converting
enzyme-2 (ACE2) or transmembrane protease, serine 2 (TMPRSS2)),
HIV-1 protease inhibitor (e.g., lopinavir/ritonavir), a complement
inhibitor (e.g., eculizumab), a recombinant human
angiotensin-converting enzyme 2 (rhACE2; e.g., APN01), an HCV
protease inhibitor (e.g., danoprevir), a stem cell therapy (e.g.,
MultiStem.RTM., Remestemcel-L, CYNK-001) or NK cell therapy
(NKG2D-ACE2 CAR-NK celIs), a neutralizing antibody against human
granulocyte-macrophage colony stimulating factor (GM-CSF) (e.g.,
IZN-101, gimsilumab), a vasoconstrictor (e.g., angiotensin II), or
a selective inhibitor of nuclear export (SINE), such as XPO1
inhibitor (e.g., selinexor), NSAID, including COX inhibitors (e.g.,
ibuprofen, aspirin (acetylsalicylate), diclofenac, and naproxen)
and selective COX2 inhibitors (e.g., celecoxib, rofecoxib,
etoricoxib, lumiracoxib, and valecoxib), or other antiviral agents
(e.g., ENU200, lopinavir/ritonavir combination). In some
embodiments, the COVID-19 vaccine is an mRNA vaccine (e.g., BN162),
including a lipid-nanoparticle (LNP) encapsulated vaccine (e.g.,
mRNA1273). In some embodiments, the COVID-19 vaccine is a DNA
vaccine (e.g., INO-4800). In some embodiments, the COVID-19 vaccine
encodes for a prefusion stabilized form of the Spike (S) protein
(e.g., mRNA1273). In some embodiments, the COVID-19 vaccine is a
recombinant protein-based vaccine consisting of the receptor
binding domain (RBD) of the spike protein of the coronavirus. In
some embodiments, the COVID-19 vaccine uses a Ligand Antigen
Epitope Presentation Systern (LEAPS) peptide including conserved
regions of coronavirus proteins to stimulate protective cell
mediated T cell responses and reduce viral load. In some
embodiments, the COVID-19 vaccine is a microneedle array
(MNA)--delivered recombinant protein subunit delivered vaccine. In
some embodiments, the vaccine is based on a flu vector expressing
the surface antigen of SARS-CoV-2. In some embodiments, the
COVID-19 vaccine is an intranasal vaccine (e.g., AdCOVID). In some
embodiments, the COVID-19 vaccine is NVX-CoV2373, IN04800, or
BNT-162. Additional illustrative examples of COVID-19 therapeutic
agents that can be combined with an agent disclosed herein include
without limitation a PIKfyve kinase inhibitor (e.g., apilimod), an
immunemodulator (e.g., rintatolimod), a T-cell immunotherapy, a
recombinant sialidase (e.g., DAS181), a CRAC channel inhibitor
(e.g., CM-4620-IE), a cardiac cell therapy using allogeneic
cardiosphere-derived cells (e.g., CAP-1002), a cardioprotective
drug (e.g., aspirin, plavix, lipitor, opremazole), an SIP receptor
antagonist (e.g., fingolimod), a cyclooxygenase-2 (COX-2) inhibitor
(e.g., celecoxib), a phosphodiesterase-5 (PDE5) inhibitor (e.g.,
sildenafil citrate), a serine protease TMPRSS2 inhibitor (camostat
mesylate), an anti-human complement 5a antibody (e.g., IFX-1), a
macrophage migration inhibitory factor (MIF) inhibitor and
phosphodiesterase (PDE)-4 and -10 inhibitor (e.g., ibudilast), an
eEF1A2 inhibitor (e.g., plitidepsin), a sphingosine kinase 2 (SK2)
inhibitor (e.g., ABC294640, RHB-107), a galectin inhibitor (e.g.,
BXT-10), an HIV-1 protease inhibitor (e.g., darunavir alone or in
combination with cobicistat), a membrane fusion inhibitor (e.g.,
umifenovir), an anti-PD1 antibody, thymosine, or other antiviral
therapeutics (e.g., HTCC (N-(2-hydroxypropyl)-3-rimethylammonium 47
chitosan chloride, OYA1). Additional illustrative examples of
COVID-19 therapeutic agents that can be combined with an agent
disclosed herein include without limitation chloroquine or
hydroxychloroquine. In some embodiments, the COVID-19 therapeutic
agent is selected from ifenprodil (Algernon Therapeutics),
recombinant sialidase (DAS-181, Ansun Biopharma), ruxolitinib,
angiotensin II, and lenzilumab. In some embodiments the COVID-19
therapeutic agent is tocilizumab (Actemra).
[0770] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an anti-SARS-CoV-2 hyperimmune globulin
therapy (plasma from convalescent COVID-19 patients, e.g.,
processed into a hyperimmune globulin) (e.g., TAK-888).
[0771] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with and RNA polymerase inhibitor (e.g.,
remdesivir, galidesivir). In various embodiments, the FLT3L-Fc
fusion proteins, homodimers, heterodimers, polynucleotides,
vectors, lipoplexes, such as LNPs, and/or pharmaceutical
compositions, as described herein, are combined with remdesivir
(GS-5734).
Antimalarial Agents
[0772] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an antimalarial agent. In some
embodiments, the antimalarial agent that can be combined with an
agent disclosed herein is selected from hydroxychloroquine,
chloroquine, artemether, lumefantrine, atovaquone, proguanil,
tafenoquine, pyronaridine, artesunate, artenimol, piperaquine,
artesunate, amodiaquine, pyronaridine, artesunate, halofantrine,
quinine sulfate, mefloquine, solithromycin, pyrimethamine,
MMV-390048, ferroquine, artefenomel mesylate, ganaplacide, DSM-265,
cipargamin, artemisone, and combinations thereof.
CDK Inhibitors
[0773] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a CDK inhibitor such as VS2-370.
STING Agonists, RIG-I and NOD2 Modulators
[0774] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a stimulator of interferon genes (STING)
agonist or activator, a RIG-I modulator (e.g., RGT-100), or a NOD2
modulator (e.g., SB-9200, IR-103). In some embodiments, the STING
receptor agonist or activator that can be co-administered with an
agent of this disclosure is selected from ADU-S100 (MIW-815),
SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532, SYN-STING, MSA-1,
SR-8291, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), cyclic-GAMP
(cGAMP) and cyclic-di-AMP. In some embodiments, the STING agonist
is selected from the compounds disclosed in WO 2018065360 ("Biolog
Life Science Institute Forschungslabor und Biochemica-Vertrieb
GmbH, Germany), WO 2018009466 (Aduro Biotech), WO 2017186711
(InvivoGen), WO 2017161349 (Immune Sensor), WO 2017106740 (Aduro
Biotech), US 20170158724 (Glaxo Smithkiline), WO 2017075477 (Aduro
Biotech), US 20170044206 (Merck), WO 2014179760 (University of
California), WO2018098203 (Janssn), WO2018118665 (Merck),
WO2018118664 (Merck), WO2018100558 (Takeda), WO2018067423 (Merck),
and WO2018060323 (Boehringer).
LAG-3 and TIM-3 Inhibitors
[0775] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a LAG-3 inhibitor or a TIM-3 inhibitor.
In some embodiments, the LAG-3 inhibitor that can be
co-administered with an agent of this disclosure is selected from
relatlimab (ONO-4482), LAG-525, MK-4280, REGN-3767, and INCAGN2385.
In some embodiments, the TIM-3 inhibitor that can be
co-administered with an agent of this disclosure is an anti-TIM-3
antibody, such as TSR-022, LY-3321367, MBG-453, or INCAGN-2390.
Interleukine Agonists
[0776] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an interleukin agonist, such as an IL-2,
IL-7, IL-15, IL-10, or IL-12 agonist. Illustrative examples of IL-2
agonists that can be combined with an agent of this disclosure
include without limitation proleukin (aldesleukin, IL-2); pegylated
IL-2 (e.g., NKTR-214); modified variants of IL-2 (e.g., THOR-707),
bempegaldesleukin, AIC-284, ALKS-4230, CUI-101, and Neo-2/15.
Illustrative examples of IL-15 agonists that can be combined with
an agent of this disclosure include without limitation ALT-803,
NKTR-255, hetIL-15, interleukin-15/Fc fusion protein, AM-0015,
NIZ-985, SO-C101, IL-15 Synthorin (pegylated Il-15), P-22339, and
IL-15-PD-1 fusion protein N-809. An illustrative example of an IL-7
agonist that can be combined with an agent of this disclosure is
CYT-107.
Pharmacokinetic Enhancers
[0777] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a pharmacokinetic enhancer such as
cobicistat and ritonavir.
5-Substituted 2'-Deoxyuridine Analogues
[0778] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a 5-substituted 2'-deoxyuridine analogue.
Illustrative examples of 5-substituted 2'-deoxyuridine analogues
that can be combined with an agent disclosed herein include without
limitation idoxuridine, trifluridine, brivudine [BVDU], and
combinations thereof.
Nucleoside Analogue
[0779] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a nucleoside analogue. Illustrative
examples of nucleoside analogues that can be combined with an agent
disclosed herein include without limitation vidarabine, entecavir
(ETV), telbivudine, lamivudine, adefovir dipivoxil, tenofovir
disoproxil fumarate (TDF), and combinations thereof.
Pyrophosphate Analogue
[0780] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a pyrophosphate analogue. Illustrative
examples of pyrophosphate analogues that can be combined with an
agent disclosed herein include without limitation foscarnet or
phosphonoacetic acid. In some embodiments, the pyrophosphate
analogue includes foscarnet.
Acyclic Guanosine Analogue
[0781] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an acyclic guanosine analogue.
Illustrative examples of acyclic guanosine analogue that can be
combined with an agent disclosed herein include without limitation
acyclovir, ganciclovir, valacyclovir (also known as valaciclovir),
valganciclovir, penciclovir, and famciclovir.
Acyclic Nucleoside Phosphonate Analogue
[0782] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an acyclic nucleoside phosphonate
analogue. Illustrative examples of acyclic nucleoside phosphonate
analogue that can be combined with an agent disclosed herein
include without limitation cidofovir, adefovir, adefovir dipivoxil,
tenofovir, TDF, emtricitabine, efavirenz, rilpivirine, and
elvitegravir. In some embodiments, the acyclic nucleoside
phosphonate analogue is selected from cidofovir, adefovir, adefovir
dipivoxil, tenofovir, TDF, emtricitabine, efavirenz, rilpivirine,
and elvitegravir. In some embodiments, the acyclic nucleoside
phosphonate analogue is selected from cidofovir, adefovir, adefovir
dipivoxil, tenofovir, TDF. In some embodiments, the acyclic
nucleoside phosphonate analogue is selected from cidofovir,
adefovir dipivoxil, TDF.
Interferons
[0783] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an interferon. In some embodiments, the
interferon that can be combined with an agent of this disclosure is
selected from interferon alfacon 1, interferon alfa 1b, interferon
alfa 2a, interferon alfa 2b, pegylated interferon alfacon 1,
pegylated interferon alfa 1b, pegylated interferon alfa 2a
(PegIFNa-2a), and PegIFNa-2b, and combinations thereof. In some
embodiments, the interferon that can be combined with an agent of
this disclosure is selected from interferon alfacon 1, pegylated
interferon alfa 2a (PegIFNa-2a), PegIFNa-2b, ribavirin, and
combinations thereof. In some embodiments, the interferon that can
be combined with an agent of this disclosure is selected from
pegylated interferon alfa-2a, pegylated interferon alfa-2b, and
combinations thereof.
Immunostimulatory Agents
[0784] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an immunostimulatory agent, such as an
oligonucleotide or an antimitotic inhibitor. In some embodiments,
the immunostimulatory agent that can be combined with an agent of
this disclosure is selected from fomivirsen, podofilox, imiquimod,
sinecatechins, and combinations thereof.
Additional Therapeutic Agents
[0785] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an additional therapeutic agent selected
from the compounds disclosed in WO 2004/096286 (Gilead Sciences),
WO 2006/015261 (Gilead Sciences), WO 2006/110157 (Gilead Sciences),
WO 2012/003497 (Gilead Sciences), WO 2012/003498 (Gilead Sciences),
WO 2012/145728 (Gilead Sciences), WO 2013/006738 (Gilead Sciences),
WO 2013/159064 (Gilead Sciences), WO 2014/100323 (Gilead Sciences),
US 2013/0165489 (University of Pennsylvania), US 2014/0221378
(Japan Tobacco), US 2014/0221380 (Japan Tobacco), WO 2009/062285
(Boehringer Ingelheim), WO 2010/130034 (Boehringer Ingelheim), WO
2013/006792 (Pharma Resources), US 20140221356 (Gilead Sciences),
US 20100143301 (Gilead Sciences) and WO 2013/091096 (Boehringer
Ingelheim).
[0786] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an additional therapeutic agent selected
from besifovir, nitazoxanide, REGN2222, doravirine, sofosbuvir,
velpatasvir, daclatasvir, asunaprevir, beclabuvir, FV100, and
letermovir, and combinations thereof.
[0787] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with an additional therapeutic agent selected
from IFX-1, FM-201, CYNK-001, DPP4-Fc, ranpirnase, nafamostat,
LB-2, AM-1, anti-viroporins, and combinations thereof.
[0788] Exemplified Combination Therapies
Lymphoma or Leukemia Combination Therapy
[0789] Some chemotherapy agents are suitable for treating lymphoma
or leukemia. These agents include aldesleukin, alvocidib,
amifostine trihydrate, aminocamptothecin, antineoplaston A10,
antineoplaston AS2-1, anti-thymocyte globulin, arsenic trioxide,
Bcl-2 family protein inhibitor ABT-263, beta alethine, BMS-345541,
bortezomib (VELCADE.RTM.), bortezomib (VELCADE.RTM., PS-341),
bryostatin 1, bulsulfan, campath-1H, carboplatin, carfilzomib
(Kyprolis.RTM.), carmustine, caspofungin acetate, CC-5103,
chlorambucil, CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone), cisplatin, cladribine, clofarabine, curcumin, CVP
(cyclophosphamide, vincristine, and prednisone), cyclophosphamide,
cyclosporine, cytarabine, denileukin diftitox, dexamethasone,
docetaxel, dolastatin 10, doxorubicin, doxorubicin hydrochloride,
DT-PACE (dexamethasone, thalidomide, cisplatin, doxorubicin,
cyclophosphamide, and etoposide), enzastaurin, epoetin alfa,
etoposide, everolimus (RAD001), FCM (fludarabine, cyclophosphamide,
and mitoxantrone), FCR (fludarabine, cyclophosphamide, and
rituximab), fenretinide, filgrastim, flavopiridol, fludarabine, FR
(fludarabine and rituximab), geldanamycin (17 AAG), hyperCVAD
(hyperfractionated cyclophosphamide, vincristine, doxorubicin,
dexamethasone, methotrexate, and cytarabine), ICE (iphosphamide,
carboplatin, and etoposide), ifosfamide, irinotecan hydrochloride,
interferon alpha-2b, ixabepilone, lenalidomide (REVLIMID, CC-5013),
lymphokine-activated killer cells, MCP (mitoxantrone, chlorambucil,
and prednisolone), melphalan, mesna, methotrexate, mitoxantrone
hydrochloride, motexafin gadolinium, mycophenolate mofetil,
nelarabine, obatoclax (GX15-070), oblimersen, octreotide acetate,
omega-3 fatty acids, Omr-IgG-am (WNIG, Omrix), oxaliplatin,
paclitaxel, palbociclib (PD0332991), pegfilgrastim, PEGylated
liposomal doxorubicin hydrochloride, perifosin, prednisolone,
prednisone, recombinant flt3 ligand, recombinant human
thrombopoietin, recombinant interferon alfa, recombinant
interleukin-11, recombinant interleukin-12, rituximab, R-CHOP
(rituximab and CHOP), R-CVP (rituximab and CVP), R-FCM (rituximab
and FCM), R-ICE (rituximab and ICE), and R MCP (rituximab and MCP),
R-roscovitine (seliciclib, CYC202), sargramostim, sildenafil
citrate, simvastatin, sirolimus, styryl sulphones, tacrolimus,
tanespimycin, temsirolimus (CC-779), thalidomide, therapeutic
allogeneic lymphocytes, thiotepa, tipifarnib, vincristine,
vincristine sulfate, vinorelbine ditartrate, SAHA
(suberanilohydroxamic acid, or suberoyl, anilide, and hydroxamic
acid), vemurafenib (Zelboraf @), venetoclax (ABT-199).
[0790] One modified approach is radioimmunotherapy, wherein a
monoclonal antibody is combined with a radioisotope particle, such
as indium-111, yttrium-90, and iodine-131. Examples of combination
therapies include, but are not limited to, iodine-131 tositumomab
(BEXXAR.RTM.), yttrium-90 ibritumomab tiuxetan (ZEVALIN.RTM.), and
BEXXAR.RTM. with CHOP.
[0791] The abovementioned therapies can be supplemented or combined
with stem cell transplantation or treatment. Therapeutic procedures
include peripheral blood stem cell transplantation, autologous
hematopoietic stem cell transplantation, autologous bone marrow
transplantation, antibody therapy, biological therapy, enzyme
inhibitor therapy, total body irradiation, infusion of stem cells,
bone marrow ablation with stem cell support, in vitro-treated
peripheral blood stem cell transplantation, umbilical cord blood
transplantation, immunoenzyme technique, low-LET cobalt-60 gamma
ray therapy, bleomycin, conventional surgery, radiation therapy,
and nonmyeloablative allogeneic hematopoietic stem cell
transplantation.
Non-Hodgkin's Lymphomas Combination Therapy
[0792] Treatment of non-Hodgkin's lymphomas (NHL), especially those
of B cell origin, includes using monoclonal antibodies, standard
chemotherapy approaches (e.g., CHOP (cyclophosphamide, doxorubicin,
vincristine, and prednisone), CVP (cyclophosphamide, vincristine,
and prednisone), FCM (fludarabine, cyclophosphamide, and
mitoxantrone), MCP (Mitoxantrone, Chlorambucil, Prednisolone), all
optionally including Rituximab.RTM. and the like),
radioimmunotherapy, and combinations thereof, especially
integration of an antibody therapy with chemotherapy.
[0793] Examples of unconjugated monoclonal antibodies for the
treatment of NHL/B-cell cancers include rituximab, alemtuzumab,
human or humanized anti-CD20 antibodies, lumiliximab,
anti-TNF-related apoptosis-inducing ligand (anti-TRAIL),
bevacizumab, galiximab, epratuzumab, SGN-40, and anti-CD74.
[0794] Examples of experimental antibody agents used in treatment
of NHL/B-cell cancers include ofatumumab, ha20, PRO131921,
alemtuzumab, galiximab, SGN-40, CHIR-12.12, epratuzumab,
lumiliximab, apolizumab, milatuzumab, and bevacizumab.
[0795] Examples of standard regimens of chemotherapy for NHL/B-cell
cancers include CHOP, FCM, CVP, MCP, R-CHOP (rituximab,
cyclophosphamide, doxorubicin, vincristine, and prednisone), R-FCM,
R-CVP, and R MCP.
[0796] Examples of radioimmunotherapy for NHL/B-cell cancers
include yttrium-90 ibritumomab tiuxetan (ZEVALIN.RTM.) and
iodine-131 tositumomab (BEXXAR.RTM.).
Mantle Cell Lymphoma Combination Therapy
[0797] Therapeutic treatments for mantle cell lymphoma (MCL)
include combination chemotherapies such as CHOP, hyperCVAD, and
FCM. These regimens can also be supplemented with the monoclonal
antibody rituximab to form combination therapies R-CHOP,
hyperCVAD-R, and R-FCM. Any of the abovementioned therapies may be
combined with stem cell transplantation or ICE in order to treat
MCL.
[0798] An alternative approach to treating MCL is immunotherapy.
One immunotherapy uses monoclonal antibodies like rituximab.
Another uses cancer vaccines, such as GTOP-99, which are based on
the genetic makeup of an individual patient's tumor.
[0799] A modified approach to treat MCL is radioimmunotherapy,
wherein a monoclonal antibody is combined with a radioisotope
particle, such as iodine-131 tositumomab (BEXXAR.RTM.) and
yttrium-90 ibritumomab tiuxetan (ZEVALIN.RTM.). In another example,
BEXXAR.RTM. is used in sequential treatment with CHOP.
[0800] Other approaches to treating MCL include autologous stem
cell transplantation coupled with high-dose chemotherapy,
administering proteasome inhibitors such as bortezomib
(VELCADE.RTM. or PS-341), or administering antiangiogenesis agents
such as thalidomide, especially in combination with rituximab.
[0801] Another treatment approach is administering drugs that lead
to the degradation of Bcl-2 protein and increase cancer cell
sensitivity to chemotherapy, such as oblimersen, in combination
with other chemotherapeutic agents.
[0802] A further treatment approach includes administering mTOR
inhibitors, which can lead to inhibition of cell growth and even
cell death. Non-limiting examples are sirolimus, temsirolimus
(TORISEL.RTM., CCI-779), CC-115, CC-223, SF-1126, PQR-309
(bimiralisib), voxtalisib, GSK-2126458, and temsirolimus in
combination with RITUXAN.RTM., VELCADE.RTM., or other
chemotherapeutic agents.
[0803] Other recent therapies for MCL have been disclosed. Such
examples include flavopiridol, palbociclib (PD0332991),
R-roscovitine (selicicilib, CYC202), styryl sulphones, obatoclax
(GX15-070), TRAIL, Anti-TRAIL death receptors DR4 and DR5
antibodies, temsirolimus (TORISEL.RTM., CCl-779), everolimus
(RAD001), BMS-345541, curcumin, SAHA, thalidomide, lenalidomide
(REVLIMID, CC-5013), and geldanamycin (17 AAG).
Waldenstrom's Macroglobulinemia Combination Therapy
[0804] Therapeutic agents used to treat Waldenstrom's
Macroglobulinemia (WM) include aldesleukin, alemtuzumab, alvocidib,
amifostine trihydrate, aminocamptothecin, antineoplaston A10,
antineoplaston AS2-1, anti-thymocyte globulin, arsenic trioxide,
autologous human tumor-derived HSPPC-96, Bcl-2 family protein
inhibitor ABT-263, beta alethine, bortezomib (VELCADE.RTM.),
bryostatin 1, busulfan, campath-1H, carboplatin, carmustine,
caspofungin acetate, CC-5103, cisplatin, clofarabine,
cyclophosphamide, cyclosporine, cytarabine, denileukin diftitox,
dexamethasone, docetaxel, dolastatin 10, doxorubicin hydrochloride,
DT-PACE, enzastaurin, epoetin alfa, epratuzumab (hLL2-anti-CD22
humanized antibody), etoposide, everolimus, fenretinide,
filgrastim, fludarabine, ibrutinib, ifosfamide, indium-111
monoclonal antibody MN-14, iodine-131 tositumomab, irinotecan
hydrochloride, ixabepilone, lymphokine-activated killer cells,
melphalan, mesna, methotrexate, mitoxantrone hydrochloride,
monoclonal antibody CD19 (such as tisagenlecleucel-T, CART-19,
CTL-019), monoclonal antibody CD20, motexafin gadolinium,
mycophenolate mofetil, nelarabine, oblimersen, octreotide acetate,
omega-3 fatty acids, oxaliplatin, paclitaxel, pegfilgrastim,
PEGylated liposomal doxorubicin hydrochloride, pentostatin,
perifosine, prednisone, recombinant flt3 ligand, recombinant human
thrombopoietin, recombinant interferon alfa, recombinant
interleukin-11, recombinant interleukin-12, rituximab,
sargramostim, sildenafil citrate (VIAGRA.RTM.), simvastatin,
sirolimus, tacrolimus, tanespimycin, thalidomide, therapeutic
allogeneic lymphocytes, thiotepa, tipifarnib, tositumomab,
ulocuplumab, veltuzumab, vincristine sulfate, vinorelbine
ditartrate, vorinostat, WT1 126-134 peptide vaccine, WT-1 analog
peptide vaccine, yttrium-90 ibritumomab tiuxetan, yttrium-90
humanized epratuzumab, and any combination thereof.
[0805] Examples of therapeutic procedures used to treat WM include
peripheral blood stem cell transplantation, autologous
hematopoietic stem cell transplantation, autologous bone marrow
transplantation, antibody therapy, biological therapy, enzyme
inhibitor therapy, total body irradiation, infusion of stem cells,
bone marrow ablation with stem cell support, in vitro-treated
peripheral blood stem cell transplantation, umbilical cord blood
transplantation, immunoenzyme techniques, low-LET cobalt-60 gamma
ray therapy, bleomycin, conventional surgery, radiation therapy,
and nonmyeloablative allogeneic hematopoietic stem cell
transplantation.
Diffuse Large B-cell Lymphoma Combination Therapy
[0806] Therapeutic agents used to treat diffuse large B-cell
lymphoma (DLBCL) include cyclophosphamide, doxorubicin,
vincristine, prednisone, anti-CD20 monoclonal antibodies,
etoposide, bleomycin, many of the agents listed for WM, and any
combination thereof, such as ICE and R ICE.
Chronic Lymphocytic Leukemia Combination Therapy
[0807] Examples of therapeutic agents used to treat chronic
lymphocytic leukemia (CLL) include chlorambucil, cyclophosphamide,
fludarabine, pentostatin, cladribine, doxorubicin, vincristine,
prednisone, prednisolone, alemtuzumab, many of the agents listed
for WM, and combination chemotherapy and chemoimmunotherapy,
including the following common combination regimens: CVP, R-CVP,
ICE, R-ICE, FCR, and FR.
Myelofibrosis Combination Therapy
[0808] Myelofibrosis inhibiting agents include, but are not limited
to, hedgehog inhibitors, histone deacetylase (HDAC) inhibitors, and
tyrosine kinase inhibitors. Non-limiting examples of hedgehog
inhibitors are saridegib and vismodegib. Examples of HDAC
inhibitors include, but are not limited to, pracinostat and
panobinostat. Non-limiting examples of tyrosine kinase inhibitors
are lestaurtinib, bosutinib, imatinib, gilteritinib, radotinib, and
cabozantinib.
Hyperproliferative Disorder Combination Therapy
[0809] Gemcitabine, nab-paclitaxel, and gemcitabine/nab-paclitaxel
may be used with a JAK inhibitor and/or PI3K6 inhibitor to treat
hyperproliferative disorders.
Bladder Cancer Combination Therapy
[0810] Therapeutic agents used to treat bladder cancer include
atezolizumab, carboplatin, cisplatin, docetaxel, doxorubicin,
fluorouracil (5-FU), gemcitabine, idosfamide, Interferon alfa-2b,
methotrexate, mitomycin, nab-paclitaxel, paclitaxel, pemetrexed,
thiotepa, vinblastine, and any combination thereof.
Breast Cancer Combination Therapy
[0811] Therapeutic agents used to treat breast cancer include
albumin-bound paclitaxel, anastrozole, capecitabine, carboplatin,
cisplatin, cyclophosphamide, docetaxel, doxorubicin, epirubicin,
everolimus, exemestane, fluorouracil, fulvestrant, gemcitabine,
Ixabepilone, lapatinib, Letrozole, methotrexate, mitoxantrone,
paclitaxel, pegylated liposomal doxorubicin, pertuzumab, tamoxifen,
toremifene, trastuzumab, vinorelbine, and any combinations
thereof.
Triple Negative Breast Cancer Combination Therapy
[0812] Therapeutic agents used to treat triple negative breast
cancer include cyclophosphamide, docetaxel, doxorubicin,
epirubicin, fluorouracil, paclitaxel, and combinations thereof.
Colorectal Cancer Combination Therapy
[0813] Therapeutic agents used to treat colorectal cancer include
bevacizumab, capecitabine, cetuximab, fluorouracil, irinotecan,
leucovorin, oxaliplatin, panitumumab, ziv-aflibercept, and any
combinations thereof.
Castration-Resistant Prostate Cancer Combination Therapy
[0814] Therapeutic agents used to treat castration-resistant
prostate cancer include abiraterone, cabazitaxel, docetaxel,
enzalutamide, prednisone, sipuleucel-T, and any combinations
thereof.
Esophageal and Esophagogastric Junction Cancer Combination
Therapy
[0815] Therapeutic agents used to treat esophageal and
esophagogastric junction cancer include capecitabine, carboplatin,
cisplatin, docetaxel, epirubicin, fluoropyrimidine, fluorouracil,
irinotecan, leucovorin, oxaliplatin, paclitaxel, ramucirumab,
trastuzumab, and any combinations thereof.
Gastric Cancer Combination Therapy
[0816] Therapeutic agents used to treat gastric cancer include
capecitabine, carboplatin, cisplatin, docetaxel, epirubicin,
fluoropyrimidine, fluorouracil, Irinotecan, leucovorin, mitomycin,
oxaliplatin, paclitaxel, ramucirumab, trastuzumab, and any
combinations thereof.
Head and Neck Cancer Combination Therapy
[0817] Therapeutic agents used to treat head & neck cancer
include afatinib, bleomycin, capecitabine, carboplatin, cetuximab,
cisplatin, docetaxel, fluorouracil, gemcitabine, hydroxyurea,
methotrexate, nivolumab, paclitaxel, pembrolizumab, vinorelbine,
and any combinations thereof.
Hepatobiliary Cancer Combination Therapy
[0818] Therapeutic agents used to treat hepatobiliary cancer
include capecitabine, cisplatin, fluoropyrimidine, 5-fluorourcil,
gemecitabine, oxaliplatin, sorafenib, and any combinations
thereof.
Hepatocellular Carcinoma Combination Therapy
[0819] Therapeutic agents used to treat hepatocellular carcinoma
include capecitabine, doxorubicin, gemcitabine, sorafenib, and any
combinations thereof.
Non-Small Cell Lung Cancer Combination Therapy
[0820] Therapeutic agents used to treat non-small cell lung cancer
(NSCLC) include afatinib, albumin-bound paclitaxel, alectinib,
bevacizumab, bevacizumab, cabozantinib, carboplatin, cisplatin,
crizotinib, dabrafenib, docetaxel, erlotinib, etoposide,
gemcitabine, nivolumab, paclitaxel, pembrolizumab, pemetrexed,
ramucirumab, trametinib, trastuzumab, vandetanib, vemurafenib,
vinblastine, vinorelbine, and any combinations thereof.
Small Cell Lung Cancer Combination Therapy
[0821] Therapeutic agents used to treat small cell lung cancer
(SCLC) include bendamustime, carboplatin, cisplatin,
cyclophosphamide, docetaxel, doxorubicin, etoposide, gemcitabine,
ipillimumab, irinotecan, nivolumab, paclitaxel, temozolomide,
topotecan, vincristine, vinorelbine, and any combinations
thereof.
Melanoma Combination Therapy
[0822] Therapeutic agents used to treat melanoma cancer include
albumin bound paclitaxel, carboplatin, cisplatin, cobiemtinib,
dabrafenib, dacrabazine, IL-2, imatinib, interferon alfa-2b,
ipilimumab, nitrosourea, nivolumab, paclitaxel, pembrolizumab,
pilimumab, temozolomide, trametinib, vemurafenib, vinblastine, and
any combinations thereof.
Ovarian Cancer Combination Therapy
[0823] Therapeutic agents used to treat ovarian cancer include
5-flourouracil, albumin bound paclitaxel, altretamine, anastrozole,
bevacizumab, capecitabine, carboplatin, cisplatin,
cyclophosphamide, docetaxel, doxorubicin, etoposide, exemestane,
gemcitabine, ifosfamide, irinotecan, letrozole, leuprolide acetate,
liposomal doxorubicin, megestrol acetate, melphalan, olaparib,
oxaliplatin, paclitaxel, Pazopanib, pemetrexed, tamoxifen,
topotecan, vinorelbine, and any combinations thereof.
Pancreatic Cancer Combination Therapy
[0824] Therapeutic agents used to treat pancreatic cancer include
5-fluorourcil, albumin-bound paclitaxel, capecitabine, cisplatin,
docetaxel, erlotinib, fluoropyrimidine, gemcitabine, irinotecan,
leucovorin, oxaliplatin, paclitaxel, and any combinations
thereof.
Renal Cell Carcinoma Combination Therapy
[0825] Therapeutic agents used to treat renal cell carcinoma
include axitinib, bevacizumab, cabozantinib, erlotinib, everolimus,
levantinib, nivolumab, pazopanib, sorafenib, sunitinib,
temsirolimus, and any combinations thereof.
HBV Combination Therapy
[0826] Therapeutic agents used to treat an infection caused by HBV
include compounds such as those disclosed in U.S. Publication No.
2010/0143301 (Gilead Sciences), U.S. Publication No. 2011/0098248
(Gilead Sciences), U.S. Publication No. 2009/0047249 (Gilead
Sciences), U.S. Pat. No. 8,722,054 (Gilead Sciences), U.S.
Publication No. 2014/0045849 (Janssen), U.S. Publication No.
2014/0073642 (Janssen), WO2014/056953 (Janssen), WO2014/076221
(Janssen), WO2014/128189 (Janssen), U.S. Publication No.
2014/0350031 (Janssen), WO2014/023813 (Janssen), U.S. Publication
No. 2008/0234251 (Array Biopharma), U.S. Publication No.
2008/0306050 (Array Biopharma), U.S. Publication No. 2010/0029585
(Ventirx Pharma), U.S. Publication No. 2011/0092485 (Ventirx
Pharma), US2011/0118235 (Ventirx Pharma), U.S. Publication No.
2012/0082658 (Ventirx Pharma), U.S. Publication No. 2012/0219615
(Ventirx Pharma), U.S. Publication No. 2014/0066432 (Ventirx
Pharma), U.S. Publication No. 2014/0088085 (Ventirx Pharma), U.S.
Publication No. 2014/0275167 (Novira Therapeutics), U.S.
Publication No. 2013/0251673 (Novira Therapeutics), U.S. Pat. No.
8,513,184 (Gilead Sciences), U.S. Publication No. 2014/0030221
(Gilead Sciences), U.S. Publication No. 2013/0344030 (Gilead
Sciences), U.S. Publication No. 2013/0344029 (Gilead Sciences),
US20140275167 (Novira Therapeutics), US20130251673 (Novira
Therapeutics),U.S. Publication No. 2014/0343032 (Roche),
WO2014037480 (Roche), U.S. Publication No. 2013/0267517 (Roche),
WO2014131847 (Janssen), WO2014033176 (Janssen), WO2014033170
(Janssen), WO2014033167 (Janssen), WO2015/059212 (Janssen),
WO2015118057(Janssen), WO2015011281 (Janssen), WO2014184365
(Janssen), WO2014184350 (Janssen), WO2014161888 (Janssen),
WO2013096744 (Novira), US20150225355 (Novira), US20140178337
(Novira), US20150315159 (Novira), US20150197533 (Novira),
US20150274652 (Novira), US20150259324, (Novira), US20150132258
(Novira), U.S. Pat. No. 9,181,288 (Novira), WO2014184350 (Janssen),
WO2013144129 (Roche), US20100015178 (Incyte), US2016137652 (Flexus
Biosciences, Inc.), WO2014073738 (Flexus Biosciences, Inc.),
WO2015188085(Flexus Biosciences, Inc.), U.S. Publication No.
2014/0330015 (Ono Pharmaceutical), U.S. Publication No.
2013/0079327 (Ono Pharmaceutical), U.S. Publication No.
2013/0217880 (Ono pharmaceutical), WO2016057924
(Genentech/Constellation Pharmaceuticals), US20140275092
(Genentech/Constellation Pharmaceuticals), US20140371195
(Epitherapeutics) and US20140371214 (Epitherapeutics),
US20160102096 (Epitherapeutics), US20140194469 (Quanticel),
US20140171432, US20140213591 (Quanticel), US20160039808
(Quanticel), US20140275084 (Quanticel), WO2014164708 (Quanticel),
U.S. Pat. No. 9,186,337B2 (Oryzon Genomics), and other drugs for
treating HBV, and combinations thereof.
[0827] In certain embodiments, an agent disclosed herein is
combined with 5 mg-30 mg tenofovir alafenamide fumarate, tenofovir
alafenamide hemifumarate, or tenofovir alafenamide. In certain
embodiments, an agent disclosed herein, or a pharmaceutically
acceptable salt thereof, is combined with 5 mg-10 mg; 5 mg-15 mg; 5
mg-20 mg; 5 mg-25 mg; 25 mg-30 mg; 20 mg-30 mg; 15 mg-30 mg; or 10
mg-30 mg tenofovir alafenamide fumarate, tenofovir alafenamide
hemifumarate, or tenofovir alafenamide. In certain embodiments, an
agent disclosed herein, or a pharmaceutically acceptable salt
thereof, is combined with 10 mg tenofovir alafenamide fumarate,
tenofovir alafenamide hemifumarate, or tenofovir alafenamide. In
certain embodiments, an agent disclosed herein, or a
pharmaceutically acceptable salt thereof, is combined with 25 mg
tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate,
or tenofovir alafenamide. An agent as disclosed herein may be
combined with the agents provided herein in any dosage amount of
the compound (e.g., from 50 mg to 500 mg of compound) the same as
if each combination of dosages were specifically and individually
listed.
[0828] In certain embodiments, an agent disclosed herein is
combined with a combination of either ledipasvir or velpatasvir,
together with sofosbuvir and voxilaprevir. In certain embodiments,
an agent disclosed herein is combined with a combination of either
ledipasvir or velpatasvir, together with sofosbuvir and
tenofovir.
HIV Combination Therapy
[0829] Therapeutic agents used to treat an infection caused by HIV
include ATRIPLA.RTM. (efavirenz, tenofovir disoproxil fumarate, and
emtricitabine); COMPLERA.RTM. (EVIPLERA.RTM.; rilpivirine,
tenofovir disoproxil fumarate, and emtricitabine); STRIBILD.RTM.
(elvitegravir, cobicistat, tenofovir disoproxil fumarate, and
emtricitabine); TRUVADA.RTM. (tenofovir disoproxil fumarate and
emtricitabine; TDF+FTC); DESCOVY.RTM. (tenofovir alafenamide and
emtricitabine); ODEFSEY.RTM. (tenofovir alafenamide, emtricitabine,
and rilpivirine); GENVOYA.RTM. (tenofovir alafenamide,
emtricitabine, cobicistat, and elvitegravir); BIKTARVY
(bictegravir+emtricitabine+tenofovir alafenamide), adefovir;
adefovir dipivoxil; cobicistat; emtricitabine; tenofovir; tenofovir
alafenamide and elvitegravir; tenofovir disoproxil; tenofovir
disoproxil fumarate; tenofovir alafenamide; tenofovir alafenamide
hemifumarate; TRIUMEQ.RTM. (dolutegravir, abacavir, and
lamivudine); dolutegravir, abacavir sulfate, and lamivudine;
raltegravir; PEGylated raltegravir; raltegravir and lamivudine;
maraviroc; tenofovir+emtricitabine+maraviroc, enfuvirtide;
ALUVIA.RTM. (KALETRA.RTM.; lopinavir and ritonavir); COMBIVIR.RTM.
(zidovudine and lamivudine; AZT+3TC); EPZICOM.RTM. (LIVEXA.RTM.;
abacavir sulfate and lamivudine; ABC+3TC); TRIZIVIR.RTM. (abacavir
sulfate, zidovudine, and lamivudine; ABC+AZT+3TC); rilpivirine;
rilpivirine hydrochloride; atazanavir sulfate and cobicistat;
atazanavir and cobicistat; darunavir and cobicistat; atazanavir;
atazanavir sulfate; dolutegravir; elvitegravir; ritonavir;
atazanavir sulfate and ritonavir; darunavir; lamivudine; prolastin;
fosamprenavir; fosamprenavir calcium efavirenz; etravirine;
nelfinavir; nelfinavir mesylate; interferon; didanosine; stavudine;
indinavir; indinavir sulfate; tenofovir and lamivudine; zidovudine;
nevirapine; saquinavir; saquinavir mesylate; aldesleukin;
zalcitabine; tipranavir; amprenavir; delavirdine; delavirdine
mesylate; Radha-108 (receptol); lamivudine and tenofovir disoproxil
fumarate; efavirenz, lamivudine, and tenofovir disoproxil fumarate;
phosphazid; lamivudine, nevirapine, and zidovudine; abacavir; and
abacavir sulfate.
[0830] Additional examples of therapeutic agents treating an
infection caused by HIV include aspernigrin C, acemannan,
alisporivir, BanLec, deferiprone, Gamimune, metenkefalin,
naltrexone, Prolastin, REP 9, RPI-MN, VSSP, Hlviral, SB-728-T,
1,5-dicaffeoylquinic acid, rHIV7-shl-TAR-CCR5RZ, AAV-eCD4-Ig gene
therapy, MazF gene therapy, BlockAide, bevirimat derivatives,
ABX-464, AG-1105, APH-0812, bryostatin analogs, BIT-225, CYT-107,
CS-TATI-1, fluoro-beta-D-arabinose nucleic acid (FANA)-modified
antisense oligonucleotides, FX-101, griffithsin, HGTV-43, HPH-116,
HS-10234, hydroxychloroquine, IMB-10035, IMO-3100, IND-02,
JL-18008, LADAVRU, MK-1376, MK-2048, MK-4250, MK-8507, MK-8558,
MK-8591 (islatravir), NOV-205, OB-002H, ODE-Bn-TFV, M1-TFV,
PA-1050040 (PA-040), PC-707, PGN-007, QF-036, S-648414, SCY-635,
SB-9200, SCB-719, TR-452, TEV-90110, TEV-90112, TEV-90111,
TEV-90113, RN-18, DIACC-1010, Fasnall, Immuglo, 2-CLIPS peptide,
HRF-4467, thrombospondin analogs, TBL-1004HI, VG-1177, xl-081,
rfhSP-D, [18F]-MC-225, URMC-099-C, RES-529, VIR-576, and any
combinations thereof.
[0831] In various embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a an HIV nucleoside or nucleotide
inhibitor of reverse transcriptase and an HIV non-nucleoside
inhibitor of reverse transcriptase. In another specific embodiment,
an agent disclosed herein, or a pharmaceutical composition thereof,
is combined with an HIV nucleoside or nucleotide inhibitor of
reverse transcriptase, and an HIV protease inhibiting compound. In
an additional embodiment, an agent disclosed herein, or a
pharmaceutical composition thereof, is combined with an HIV
nucleoside or nucleotide inhibitor of reverse transcriptase, an HIV
non-nucleoside inhibitor of reverse transcriptase, and a
pharmacokinetic enhancer. In certain embodiments, an agent
disclosed herein, or a pharmaceutical composition thereof, is
combined with at least one HIV nucleoside inhibitor of reverse
transcriptase, an integrase inhibitor, and a pharmacokinetic
enhancer. In another embodiment, an agent disclosed herein, or a
pharmaceutical composition thereof, is combined with two HIV
nucleoside or nucleotide inhibitors of reverse transcriptase.
[0832] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with abacavir sulfate, tenofovir, tenofovir
disoproxil, tenofovir disoproxil fumarate, tenofovir disoproxil
hemifumarate, tenofovir alafenamide, or tenofovir alafenamide
hemifumarate.
[0833] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with tenofovir, tenofovir disoproxil,
tenofovir disoproxil fumarate, tenofovir alafenamide, or tenofovir
alafenamide hemifumarate.
[0834] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a first additional therapeutic agent
selected from the group consisting of abacavir sulfate, tenofovir,
tenofovir disoproxil, tenofovir disoproxil fumarate, tenofovir
alafenamide, and tenofovir alafenamide hemifumarate, and a second
additional therapeutic agent selected from the group consisting of
emtricitabine and lamivudine.
[0835] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a first additional therapeutic agent
selected from the group consisting of tenofovir, tenofovir
disoproxil, tenofovir disoproxil fumarate, tenofovir alafenamide,
and tenofovir alafenamide hemifumarate, and a second additional
therapeutic agent, wherein the second additional therapeutic agent
is emtricitabine.
[0836] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with a first additional therapeutic agent (a
contraceptive) selected from the group consisting of cyproterone
acetate, desogestrel, dienogest, drospirenone, estradiol valerate,
ethinyl Estradiol, ethynodiol, etonogestrel, levomefolate,
levonorgestrel, lynestrenol, medroxyprogesterone acetate,
mestranol, mifepristone, misoprostol, nomegestrol acetate,
norelgestromin, norethindrone, noretynodrel, norgestimate,
ormeloxifene, segestersone acetate, ulipristal acetate, and any
combinations thereof.
[0837] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with one or more additional therapeutic agents
selected from compounds such as those disclosed in U.S. Publication
No. 2010/0143301 (Gilead Sciences), U.S. Publication No.
2011/0098248 (Gilead Sciences), U.S. Publication No. 2009/0047249
(Gilead Sciences), U.S. Pat. No. 8,722,054 (Gilead Sciences), U.S.
Publication No. 2014/0045849 (Janssen), U.S. Publication No.
2014/0073642 (Janssen), WO2014/056953 (Janssen), WO2014/076221
(Janssen), WO2014/128189 (Janssen), U.S. Publication No.
2014/0350031 (Janssen), WO2014/023813 (Janssen), U.S. Publication
No. 2008/0234251 (Array Biopharma), U.S. Publication No.
2008/0306050 (Array Biopharma), U.S. Publication No. 2010/0029585
(Ventirx Pharma), U.S. Publication No. 2011/0092485 (Ventirx
Pharma), US2011/0118235 (Ventirx Pharma), U.S. Publication No.
2012/0082658 (Ventirx Pharma), U.S. Publication No. 2012/0219615
(Ventirx Pharma), U.S. Publication No. 2014/0066432 (Ventirx
Pharma), U.S. Publication No. 2014/0088085 (Ventirx Pharma), U.S.
Publication No. 2014/0275167 (Novira Therapeutics), U.S.
Publication No. 2013/0251673 (Novira Therapeutics), U.S. Pat. No.
8,513,184 (Gilead Sciences), U.S. Publication No. 2014/0030221
(Gilead Sciences), U.S. Publication No. 2013/0344030 (Gilead
Sciences), U.S. Publication No. 2013/0344029 (Gilead Sciences),
US20140275167 (Novira Therapeutics), US20130251673 (Novira
Therapeutics),U.S. Publication No. 2014/0343032 (Roche),
WO2014037480 (Roche), U.S. Publication No. 2013/0267517 (Roche),
WO2014131847 (Janssen), WO2014033176 (Janssen), WO2014033170
(Janssen), WO2014033167 (Janssen), WO2015059212 (Janssen),
WO2015118057(Janssen), WO2015011281 (Janssen), WO2014184365
(Janssen), WO2014184350 (Janssen), WO2014161888 (Janssen),
WO2013096744 (Novira), US20150225355 (Novira), US20140178337
(Novira), US20150315159 (Novira), US20150197533 (Novira),
US20150274652 (Novira), US20150259324, (Novira), US20150132258
(Novira), U.S. Pat. No. 9,181,288 (Novira), WO2014184350 (Janssen),
WO2013144129 (Roche), US20100015178 (Incyte), US2016137652 (Flexus
Biosciences, Inc.), WO2014073738 (Flexus Biosciences, Inc.),
WO2015188085 (Flexus Biosciences, Inc.), U.S. Publication No.
2014/0330015 (Ono Pharmaceutical), U.S. Publication No.
2013/0079327 (Ono Pharmaceutical), U.S. Publication No.
2013/0217880 (Ono Pharmaceutical), WO2016057924
(Genentech/Constellation Pharmaceuticals), US20140275092
(Genentech/Constellation Pharmaceuticals), US20140371195
(Epitherapeutics) and US20140371214 (Epitherapeutics),
US20160102096 (Epitherapeutics), US20140194469 (Quanticel),
US20140171432 (Quanticel), US20140213591 (Quanticel), US20160039808
(Quanticel), US20140275084 (Quanticel), WO2014164708 (Quanticel),
U.S. Pat. No. 9,186,337B2 (Oryzon Genomics), and other drugs for
treating HBV, and combinations thereof.
Coronavirus Combination Therapy
[0838] In some embodiments, the FLT3L-Fc fusion proteins,
homodimers, heterodimers, polynucleotides, vectors, lipoplexes,
such as LNPs, and/or pharmaceutical compositions, as described
herein, are combined with remdesivir.
9. Kits
[0839] Further provided are kits comprising one or more containers
comprising one or more unitary doses of a FLT3L-Fc fusion protein,
as described herein, a homodimer or heterodimer comprising such
fusion protein, a polynucleotide encoding such fusion protein, a
vector or lipoplex, such as a lipid nanoparticle (LNP) comprising
such polynucleotide, or pharmaceutical composition comprising such
fusion protein or polynucleotide. In some embodiments, the kits
comprise two or more unitary doses of the FLT3L-Fc fusion protein,
the homodimer or heterodimer comprising such fusion protein, the
polynucleotide encoding such fusion protein, the vector or
lipoplex, such as a lipid nanoparticle (LNP) comprising such
polynucleotide, or pharmaceutical composition comprising such
fusion protein or polynucleotide, in two or more containers. In
some embodiments, the kit comprises one or more unitary doses of
the FLT3L-Fc fusion protein, the homodimer or heterodimer
comprising such fusion protein, the polynucleotide encoding such
fusion protein, the vector or lipoplex, such as a lipid
nanoparticle (LNP) comprising such polynucleotide, or
pharmaceutical composition comprising such fusion protein or
polynucleotide and one or more (e.g., one, two, three, one or two,
or one to three) additional therapeutic agents in separate
containers. The one or more additional therapeutic agents (e.g.,
for vaccination and/or for treating cancer or a viral infection)
are as described above and herein. In some embodiments, the kits
comprise two or more unitary doses wherein the unitary doses are
the same. In some embodiments, the kits comprise two or more
unitary doses, wherein the unitary doses are different.
[0840] In one embodiment, the kit comprises one or more
pharmaceutical packs comprising one or more containers (e.g.,
vials, ampules, pre-loaded syringes) containing one or more of the
ingredients of the pharmaceutical compositions described herein,
such as the FLT3L-Fc fusion protein, the homodimer or heterodimer
comprising such fusion protein, the polynucleotide encoding such
fusion protein, the vector or lipoplex, such as a lipid
nanoparticle (LNP), comprising such polynucleotide, or
pharmaceutical composition comprising such fusion protein or
polynucleotide, as provided herein. In some instances, the kits
contain a pharmaceutical composition described herein. In some
embodiments, the kit comprises one or more containers comprising
the FLT3L-Fc fusion protein, the homodimer or heterodimer
comprising such fusion protein, the polynucleotide encoding such
fusion protein, the vector or lipoplex, such as a lipid
nanoparticle (LNP), comprising such polynucleotide, or
pharmaceutical composition comprising such fusion protein or
polynucleotide, in an aqueous solution. In some embodiments, the
aqueous solution comprises the FLT3L-Fc fusion protein, the
homodimer or heterodimer comprising such fusion protein, or
pharmaceutical composition comprising such fusion protein, at a
concentration in the range of about 1 mg/ml to about 2 mg/ml, 3
mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10
mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml,
17 mg/ml, 18 mg/ml, 19 mg/ml or 20 mg/ml. In some embodiments, the
kit comprises one or more containers comprising the FLT3L-Fc fusion
protein, the homodimer or heterodimer comprising such fusion
protein, the polynucleotide encoding such fusion protein, the
vector or lipoplex, such as a lipid nanoparticle (LNP), comprising
such polynucleotide, or pharmaceutical composition comprising such
fusion protein or polynucleotide, in lyophilized form.
[0841] Optionally associated with such container(s) can be a notice
in the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
EXAMPLES
[0842] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
In Vitro Potency of Different FLT3 Agonist Modalities
[0843] In this example, we compared the in vitro potency of FLT3
agonists of different modalities, including recombinant ligand,
FLT3 ligand-Fc fusion protein, and anti-mouse FLT3 agonist antibody
(comparator 1). We tested in vitro potency employing a M1 L-6
Secretion Assay.
Methods
[0844] M1 IL-6 Secretion Assay: Murine myeloid leukemic M1 cells
(American Type Culture Collection (ATCC), TIB-192) were collected
from culture, counted, and resuspended to 0.5.times.10.sup.6
cells/ml with serum-free RPMI1640. In a 96-well U-bottom tissue
culture plate, 100 .mu.l of resuspended cells (50,000 cells) was
added to each well, then 50 .mu.l of 4x test article was added to
each well and 50 .mu.l of serum-free RPMI was added into the wells
for a final volume of 200 .mu.l per well. Cells were incubated
overnight at 37.degree. C. The next day, cells were spun down at
500 g for 5 min at ambient temperatures. Supernatants were then
collected for mouse IL-6 quantification (Meso Scale Discovery
(MSD), Cat: K152AKB-1) which was performed by following the
manufacturer's protocol. EC50 and Emax values of each sample were
determined by plotting the concentration of the FLT3 agonist
compound against the L-6 supernatant concentration (pg/mL) and fit
to a four parameter logistic (4PL) regression curve.
Results
[0845] The data demonstrated that recombinant FLT3-ligand and
recombinant FLT3-ligand Fc fusion protein were superior to FLT3
agonist antibody (comparator 1) in activating M1 cells to produce
IL-6 in a dose dependent manner. These data also demonstrated that
human FLT3-ligand proteins can potently activate murine FLT3. These
results are summarized in Table 1 and depicted in FIG. 1.
TABLE-US-00008 TABLE 1 EC50 and Emax values for Recombinant
huFlt3L, Recombinant huFLT3L-Fc and Comparator 1 in the M1 IL-6
release assay anti-mouse FLT3 agonist antibody Recombinant
Recombinant (Comparator 1) huFlt3L-Fc huFLT3L EC50(nM) 4.188 0.117
0.024 Emax(pg/ml) 244.1 516.6 392.3
[0846] The results guided us to further pursue a FLT3-ligand Fc
fusion protein as a FLT3 agonist.
Example 2
In Vitro Potency of FLT3L-Fc Fusion Proteins Having Different IgG
Backbones
[0847] In this example, we compared the in vitro potency of
different FLT3-Ligand Fc fusion protein variants: one having a
hingeless human IgG1 backbone (SEQ ID NO:1) and a second having a
human IgG1 backbone (SEQ ID NO:21). For this comparison, we tested
the in vitro potency employing an AML5 Proliferation Assay.
Methods
[0848] AML5 Proliferation Assay: AML5 cells (Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), ACC247) were starved O/N
(18-24h) with serum free MEM-.alpha.. The next day, using a 96-well
white opaque half area flat bottom TC plates (Costar, 3688), 25,000
cells per well were stimulated with the various compounds for 72h.
Plates were sealed with a breathable plate sealer (Sigma,
Z380059-1PAK) during the incubation. After incubation,
proliferation was assessed through CellTiter Glo assay (Promega,
G7571) using manufacturer's recommendations. Luminescence signals
were measured using a SpectraMax plate reader. EC50 value of each
sample was determined by plotting the concentration of the compound
against the luminescence signal and fit to a 4PL curve.
Results
[0849] The data demonstrated that the potency of FLT3-ligand Fc
fusion with hingeless IgG1 (SEQ ID NO:1) and FLT3-ligand Fc fusion
with IgG1 (SEQ ID NO:21) was similar to that of recombinant human
FLT3 ligand in inducing FLT3-dependent proliferation in AML5 cells,
with EC50 values ranging between 0.035-0.04 nM. The results are
summarized in Table 2 and depicted in FIG. 2.
TABLE-US-00009 TABLE 2 EC50 values for Inducing Proliferation of
AML5 Cells by FLT3L-Fc Variants Having Different IgG Backbones EC50
(nM) recombinant hingeless IgG1 IgG1 huFLT3L SEQ ID NO: 1 SEQ ID
NO: 21 0.039 0.040 0.035
Example 3
In Vitro Potency of FLT3L-Fc Variants Having Mutations in the FLT3L
EC Domain
[0850] In this example, we compared the in vitro potency of human
FLT3-ligand hingeless human IgG1 fusion proteins containing
different FLT3-ligand gain-of-fusion mutations. We tested the in
vitro potency FLT3L-Fc fusion protein variants having mutations in
the FLT3L extracellular (EC) domain (H8Y and/or K84E) by employing
an AML5 Proliferation Assay. The methods are as described above in
Example 2.
Results
[0851] The data demonstrated that the potency of FLT3-ligand Fc
fusion protein variants with gain-of-function mutations in the
FLT3L extracellular domain (H8Y and/or K84E; SEQ ID NOs: 22, 23 and
24) in inducing FLT3-dependent proliferation in AML5 cells was
approximately 2.5x fold higher than that of human FLT3-ligand Fc
fusion protein (SEQ ID NO:1). The results are summarized in Table 3
and depicted in FIG. 3.
TABLE-US-00010 TABLE 3 EC50 values for Inducing Proliferation of
AML5 Cells by FLT3L-Fc Variants Having Mutations in the FLT3L EC
Domain EC50 (nM) hFLT3L ECD hFLT3L ECD hFLT3L ECD hFLT3L ECD (H8Y)
(K84E) (H8Y/K84E) SEQ ID NO: 1 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID
NO: 24 0.034 0.014 0.013 0.013
Example 4
In Vitro Potency of Murine Surrogate FLT3-Ligand Fc Fusion
Variants
[0852] In this example, we compared the in vitro potency of human
FLT3-ligand hingeless human IgG1 fusion protein with two murine
surrogate FLT3-ligand Fc fusion proteins. These murine surrogate
proteins contained the wild type murine FLT3-ligand extracellular
region fused to a L234A/L235A/P329G variant of murine IgG2a Fc
region (IgG2a-LALA-PG), or, a C136S variant of murine FLT3-ligand
extracellular region fused to the same Fc, where the C136S mutation
was incorporated to eliminate an unpaired cysteine liability. We
tested the in vitro potency employing an AML5 Proliferation Assay.
The methods are as described above in Example 2.
Results
[0853] The data demonstrated that the potency of murine surrogate
FLT3-ligand Fc fusion proteins (SEQ ID NOs: 19 and 20) in inducing
human FLT3-dependent proliferation in AML5 cells is similar to that
of human FLT3-ligand Fc fusion protein (SEQ ID NO:1), with EC50
values ranging between 0.171-0.078 nM. The results are summarized
in Table 4 and depicted in FIG. 4.
TABLE-US-00011 TABLE 4 EC50 values for Inducing Proliferation of
AML5 Cells by Murine Surrogate FLT3-Ligand Fc Fusion Variants EC50
(nM) SEQ ID NO: 1 SEQ ID NO: 19 SEQ ID NO: 20 0.078 0.171 0.115
[0854] Based on these assay results and reduced risk of
disulfide-mediated aggregation, we proceeded with using the murine
surrogate FLT3-ligand Fc fusion variant of SEQ ID NO:20 in mouse
preclinical models.
Example 5
In Vitro Potency of Different FLT3L-Fc Proteins
[0855] In this example, we compared the in vitro potency of eight
different human FLT3-ligand human Fc fusion proteins. We tested the
in vitro potency employing an AML5 Proliferation Assay. The methods
are as described above in Example 2.
Results
[0856] We tested the potency in inducing FLT3-dependent
proliferation in AML5 cells of the eight human FLT3-ligand Fc
fusion protein variants with different Fc regions, or containing
modifications in the FLT3-ligand derived sequence (SEQ ID NOs:
1-8). The eight FLT3L-Fc variants tested are as follows: human
FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1),
human FLT3-ligand (.DELTA.5 amino acid) human hingeless IgG1 fusion
protein (SEQ ID NO:2), human FLT3-ligand human IgG4 (S228P/L235E)
fusion protein (SEQ ID NO:3), human FLT3-ligand human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:4), human FLT3-ligand
(S128A/S151A) human hingeless IgG1 fusion protein (SEQ ID NO:5),
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:6), human FLT3-ligand
(.DELTA.10 amino acid) human hingeless IgG1 fusion protein (SEQ ID
NO:7), or human FLT3-ligand (.DELTA.10 amino acid) human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:8). The resulting
EC50 values ranged between 0.071-0.088 nM. The results are
summarized in Table 5 and depicted in FIG. 5.
TABLE-US-00012 TABLE 5 EC50 Values for SEQ ID NOs: 1-8 in an AML5
Proliferation Assay FLT3L-Fc Variant SEQ ID NO: EC50 (nM) 1 0.080 2
0.083 3 0.088 4 0.073 5 0.078 6 0.078 7 0.071 8 0.075
Example 6
In Vitro FLT3 Binding of Different FLT3L-Fc Fusion Proteins
[0857] In this example, we compared the in vitro binding to human
recombinant FLT3 of eight different human FLT3-ligand human Fc
fusion protein variants. We tested the in vitro FLT3 binding
employing an enzyme-linked immunosorbent assay (ELISA).
Methods
[0858] Flt3L-Fc fusion protein constructs were serially diluted and
added to 96-well nickel plates (Pierce) coated with his-tagged
recombinant human Flt3 receptor (Sino Biologicals). Bound Flt3L-Fc
was detected using a goat anti-human (H+L) polyclonal antibody
conjugated to horseradish peroxidase (Jackson Immunoresearch).
Signal was developed using TMB substrate then quenched prior to
reading absorbance at 450 nm on a SpectraMax plate reader. Flt3L-Fc
concentration was plotted against signal and fit to a 4PL curve to
determine the EC50 value of each construct.
Results
[0859] These data demonstrated that binding to human FLT3 receptor
was similar for FLT3-ligand Fc fusion proteins of the same Fc
isotype (IgG1, SEQ ID NOs: 1, 2, 5, 7; or IgG4, SEQ ID NOs: 3, 4,
6, 8). EC50 values ranged between 0.11-0.13 nM for IgG constructs,
0.18-0.22 nM for IgG4 constructs. These data also demonstrated that
short truncations at the C-terminus of the FLT3-ligand portion (SEQ
ID NOs: 2, 6, 7, 8) or mutations that eliminate N-linked glycans in
FLT3-ligand (SEQ ID NO: 5) also had negligible effect on binding to
FLT3. The results are summarized in Table 6 and depicted in FIG.
6.
TABLE-US-00013 TABLE 6 EC50 Values for FLT3L-Fc SEQ ID NOs: 1-8
Binding to Human Recombinant FLT3 FLT3L-Fc Variant SEQ ID NO: EC50
(nM) 1 0.12 2 0.13 3 0.18 4 0.18 5 0.11 6 0.22 7 0.12 8 0.20
Example 7
In Vitro FcRn Binding of Different FLT3L-Fc Fusion Proteins
[0860] In this example, we compared the in vitro binding of eight
different human FLT3-ligand human Fc fusion proteins to human
recombinant FcRn. We tested the in vitro FLT3 binding employing an
enzyme-linked immunosorbent assay (ELISA).
Methods
[0861] Flt3L-Fc constructs were serially diluted and added to
96-well plates coated with recombinant human FcRn. The bound
Flt3L-Fc was detected using a donkey anti-human (H+L) antibody
conjugated to horseradish peroxidase (Jackson Immunoresearch).
Signal was developed using TMB substrate then quenched prior to
reading absorbance at 450-650 nm on a SpectraMax plate reader.
Flt3L-Fc concentration was plotted against signal and fit to a 4PL
curve. A full length IgG1 and IgG4 isotype was included in the
initial experiment as Fc isotype controls.
Results
[0862] These data demonstrated that binding to human FcRn in this
assay was weaker for the eight human FLT3-ligand Fc fusion proteins
with different Fc variants (SEQ ID NOs: 1-8) compared to the human
IgG1 and IgG4 isotype antibody controls, but relatively similar to
each other. EC50 values for the FLT3L-Fc protein variant samples
summarized in Table 7 are only estimates, as none of the FLT3L-Fc
variants of SEQ ID NOs: 1-8 demonstrated saturating signal at the
highest concentration tested. The results are also depicted in FIG.
7.
TABLE-US-00014 TABLE 7 Estimated EC50 values for FLT3L-Fc Variants
SEQ ID NOs: 1-8 Binding to Human Recombinant FcRn FLT3L-Fc Variant
SEQ ID NO: EC50 (nM) 1 150.70 2 300.40 3 183.30 4 68.76 5 132.70 6
136.50 7 157.90 8 139.90 hIgG1 Isotype 4.46 hIgG4 Isotype 25.86
Example 8
Ability of FLT3L-Fc Variants to Compete for Binding to Human
Fc.gamma.RI
[0863] In this example, we compared the in vitro ability of eight
different human FLT3-ligand human Fc fusion proteins to compete
with a human IgG molecule for binding to human recombinant
Fc.gamma.RI. To evaluate the ability to compete for binding to
Fc.gamma.RI, we employed an amplified luminescent proximity
homogeneous assay (AlphaScreen.RTM. by Perkin Elmer).
Methods
[0864] Serial dilutions of the Flt3L-Fc constructs were added to
96-well plates containing biotinylated Fc.gamma.RI protein (Sino
Biological). Human IgG acceptor beads (Perkin Elmer) were added to
the plate, followed by streptavidin donor beads (Perkin Elmer).
Acceptor beads contain thioxene derivatives. Donor beads contain a
photosensitizer, phthalocyanine, which converts ambient oxygen to
an excited and reactive form of 02, singlet oxygen (molecular
oxygen with a single excited electron), upon illumination at 680
nm. If an acceptor bead is within 200 nm of a donor bead, energy is
transferred from the singlet oxygen to thioxene derivatives within
the acceptor bead, subsequently culminating in light production at
520-620 nm. Signal was measured on an EnVision.TM. plate reader
(Perkin Elmer). Flt3L-Fc concentration was plotted against signal
and fit to a 4PL curve. Full length IgG1 and IgG4 molecules were
included on each plate as Fc isotype controls.
Results
[0865] These data demonstrated that none of the eight human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID
NOs: 1-8) could fully compete with human IgG for binding to
Fc.gamma.RI at the highest concentrations tested. Both the human
IgG1 and IgG4 isotype antibody controls demonstrated complete
dose-response curves, with the IgG4 isotype showing reduced
competition compared to the IgG1. The results are summarized in
Table 8 and depicted in FIG. 8.
TABLE-US-00015 TABLE 8 EC50 Values for the Ability of FLT3L-Fc
Variants to Compete for Binding to Fc.gamma.RI FLT3L-Fc Variant SEQ
ID NO: EC50 (nM) 1 NA 2 NA 3 NA 4 NA 5 NA 6 NA 7 NA 8 NA hIgG1
Isotype 4.44 hIgG4 Isotype 13.62
Example 9
Ability of FLT3L-Fc Variants to Compete for Binding to Human
Fc.gamma.RIIIa
[0866] In this example, we compared the in vitro ability of eight
different human FLT3-ligand human Fc fusion proteins to compete for
binding of human recombinant Fc.gamma.RIIIa (V-variant) with a
human IgG molecule. To evaluate the ability to compete for binding
to Fc.gamma.RIIIa, we employed an AlphaScreen.RTM. by Perkin Elmer.
The methods are analogous to those described in Example 8.
Methods
[0867] Serial dilutions of the Flt3L-Fc constructs were added to
96-well plates containing biotinylated FeyRIIIa (Val176 variant)
protein (Sino Biological). Human IgG acceptor beads (Perkin Elmer)
were added to the plate, followed by streptavidin donor beads
(Perkin Elmer), and signal was measured on an EnVision.TM. plate
reader. Flt3L-Fc concentration was plotted against signal and fit
to a 4PL curve. Full length IgG and IgG4 molecules were included on
each plate as Fc isotype controls.
Results
[0868] These data demonstrated that none of the eight human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID
NOs: 1-8) could fully compete with human IgG for binding to
Fc.gamma.RIIIa (Val176 variant) at the highest concentrations
tested. Only human IgG1 isotype control demonstrated a complete
dose-response curve. The results are summarized in Table 9 and
depicted in FIG. 9.
TABLE-US-00016 TABLE 9 EC50 Values for the Ability of FLT3L-Fc
Variants to Compete for Binding to Fc.gamma.RIIIa FLT3L-Fc Variant
SEQ ID NO: EC50 (nM) 1 NA 2 NA 3 NA 4 NA 5 NA 6 NA 7 NA 8 NA hIgG1
Isotype 32.09 hIgG4 Isotype 325.90
Example 10
In Vitro Binding of FLT3L-Fc Variants to Human C1q
[0869] In this example, we compared the in vitro binding of eight
different human FLT3-ligand human Fc fusion proteins to human
recombinant complement protein, C1q. To evaluate binding to C1q, we
employed an ELISA.
Methods
[0870] Serial dilutions of the Flt3L-Fc constructs were immobilized
onto 96-well plates, followed by incubation with the recombinant
human C1q protein (Fitzgerald). Binding was detected using a sheep
anti-C1q antibody conjugated to horseradish peroxidase (BioRad).
Signal was developed using TMB substrate then quenched prior to
reading absorbance at 450-650 nm on a SpectraMax plate reader.
Flt3L-Fc concentration was plotted against signal and fit to a 4PL
curve. Full length IgG and IgG4 molecules were included on each
plate as Fc isotype controls.
Results
[0871] These data demonstrated that the eight human FLT3-ligand Fc
fusion proteins with different Fc variants (SEQ ID NOs: 1-8) were
devoid of C1q binding ability. Both the human IgG1 and IgG4 isotype
controls demonstrated binding to C1q, with the IgG4 isotype showing
reduced binding compared to the IgG1. The results are summarized in
Table 10 and depicted in FIG. 10.
TABLE-US-00017 TABLE 10 EC50 Values for FLT3L-Fc Variants Binding
to Human C1q FLT3L-Fc Variant SEQ ID NO: EC50 (nM) 1 NA 2 NA 3 NA 4
NA 5 NA 6 NA 7 NA 8 NA hIgG1 Isotype 8.40 hIgG4 Isotype 16.69
Example 11
In Vivo Pharmacokinetics of FLT3L-Fc Variants in Mice
[0872] In this example, we compared the single dose
pharmacokinetics of eight different human FLT3-ligand human Fc
fusion proteins in C57Bl/6 mice.
Methods
[0873] FLT3L-Fc Variants (SEQ ID NOs:1-8) were administered to male
C57Bl/6 mice n=4/group (Covance, WI) at 5 mg/kg via a single
intraperitoneal (P) injection to characterize their basic
pharmacokinetic (PK) profiles. Serial serum samples collected from
mice were analyzed using U-PLEX FLT3L assay (Mesos Scale Discovery,
MSD) according to the manufacturer's instructions. The calibration
curve used the respective individual FLT3-ligand fusion proteins as
reference standards in spiked mouse matrix fit to a 4-parameter
logistic model with 1/Y2 weighting. Analyte concentrations were
determined from the electrochemiluminescence signals back-fitted to
the calibration curve. Serum concentration-time profiles were used
to calculate the mean SD serum PK parameters by non-compartmental
PK analysis. Area under the curve (AUC0-7d) was determined through
day 7 due to development of immunogenicity. Clearance (Cl/F) and
half-life values reported in Table 11 are considered estimates due
to the incomplete terminal extrapolation.
Results
[0874] Pharmacokinetic analysis demonstrated that all eight
Fc-fusions significantly enhanced the AUC exposure by approximately
5-12-fold in comparison to native human Flt3L, resulting in reduced
FLT3-ligand clearance and prolonging the half-life (Table 11). We
further observed a potential role of N-linked glycosylation on the
pharmacokinetics because the aglycosylated FLT3L-Fc variant (SEQ ID
NO: 5) had the highest AUC exposure. Additionally, we observed cell
line dependent differences in PK evident by comparing data for
samples corresponding to SEQ ID NO:1 produced in either Expi293 or
ExpiCHO cells. The results are summarized in Table 11 and depicted
in FIGS. 11A-11B.
TABLE-US-00018 TABLE 11 Single-Dose Pharmacokinetic Values for SEQ
ID NOs: 1-8 in C57Bl/6 mice. AUC.sub.0-7 d Cl/F* C.sub.max
Half-life* SEQ ID NO (.mu.g*d/mL) (mL/d/kg) (.mu.g/mL) (d) 1 -
Expi293 209 .+-. 28.2 13.3 .+-. 2.26 46.8 .+-. 9.72 6.54 .+-. 0.653
1 - ExpiCHO 159 .+-. 29.6 20.9 .+-. 7.07 34.8 .+-. 7.45 5.15 .+-.
1.40 2 171 .+-. 45.7 18.9 .+-. 6.11 38.9 .+-. 8.13 5.34 .+-. 0.795
3 131 .+-. 9.60 28.9 .+-. 2.83 29.7 .+-. 4.00 3.64 .+-. 0.313 4 128
.+-. 37.4 33.3 .+-. 9.38 33.3 .+-. 8.80 3.34 .+-. 0.162 5 287 .+-.
71.4 8.72 .+-. 2.38 54.1 .+-. 13.4 8.02 .+-. 3.24 6 205 .+-. 8.77
15.9 .+-. 3.10 42.3 .+-. 1.57 5.10 .+-. 1.54 7 143 .+-. 26.1 25.1
.+-. 6.38 33.0 .+-. 7.63 4.63 .+-. 1.00 8 158 .+-. 52.3 24.4 .+-.
8.02 31.9 .+-. 11.4 4.16 .+-. 0.238 Recombinant 23.5 .+-. 3.84 215
.+-. 35.4 17.1 .+-. 4.51 0.785 .+-. 0.0441 huFLT3L *CL/F and
Half-life are estimates for Fc-fusions due to incomplete terminal
extrapolation.
Example 12
Ability of FLT3L-Fc Variants to Promote Proliferation and Expansion
of cDC1
[0875] In this example, we compared the ability of eight different
human FLT3-ligand human Fc fusion proteins (SEQ ID NOs: 1-8) to
induce proliferation of and expand conventional dendritic cell
subtype 1(cDC1) in C57Bl/6.
Methods
[0876] Spleens were harvested from C57BL/6 mice from FIG. 11 at Day
11 post injection at 4.degree. C. in HypoThermosol solution
(BioLife Solutions). Spleens were then dissociated by using the
gentleMACS Dissociator (Miltenyi Biotec) with heaters, following
manufacturer's protocol. After enzymatic digestion, cell suspension
was filtered through a 70 .mu.m cell strainer. The remaining tube
and strainer were rinsed 1.times. with 15-20 ml of RPMI and
collected with the rest of the sample. Cells were centrifuged at
500 g for 5 min at room temperature. Supernatant was discarded and
cells were washed 1.times. with PBS. Residual red blood cells were
lysed by adding 2 ml of ACK lysis to each sample for 1-2 min at
room temperature. FACS staining Buffer (BD Bioscience) was added to
the samples to stop the ACK lysis activity. Cells were spun down
and washed additionally with PBS. Samples were then stained with
Live/Dead Fixable Aqua Dead Cell Stain Kit (ThermoFisher) at 1:750
dilution for 15 min at 4.degree. C. Cells were washed 2x with FACS
staining buffer, then Fc blocked for 30 min at 4.degree. C. FACS
antibodies (Biolegend) were directly added to the blocked samples
and incubated at 4.degree. C. for 30 min without spinning down or
washing out the Fc block. Cells were washed 2x, resuspended in
Staining Buffer, and analyzed by LSR Fortessa FACS analyzer. Raw
data were analyzed by FlowJo X (BD Bioscience).
Results
[0877] The data demonstrated that the ability of the eight human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID
NOs: 1-8) to expand splenic conventional dendritic cell subtype 1
(cDC1) in vivo at day 11 in mice was greater than that of the
recombinant FLT3-ligand after a single dose administration at day
0. The results are summarized in Table 12 and depicted in FIG.
12.
TABLE-US-00019 TABLE 12 Average Frequency of Splenic cDC1 at day 11
in C57Bl/6 Mice Injected with FLT3L-Fc Variants SEQ ID NO: 1-8 at
Day 0 FLT3L-Fc Variant % cDC1 in total SEQ ID NO: MNCs 1 - Expi293
19.8 1 - ExpiCHO 17.9 2 17.85 3 14.8 4 11.62 5 20.2 6 15.05 7 14.62
8 12.87 Recombinant huFLT3L 2.8 Baseline 1.36
Example 13
In Vitro Potency of FLT3L-Fc Variants in Cell Proliferation
Assay
[0878] In this example, we compared the in vitro potency of four
different human FLT3-ligand human Fc fusion proteins: human
FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO:1),
human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A) fusion protein (SEQ ID NO:6), human FLT3-ligand
human hingeless IgG1 (M252Y/S254T/T256E) fusion protein (SEQ ID
NO:9), or human FLT3-ligand (.DELTA.5 amino acid) human IgG4
(S228P/F234A/L235A/M252Y/S254T/T256E) fusion protein (SEQ ID
NO:14). To evaluate the in vitro potency, we employed an AML5 cell
proliferation assay. The methods are as described above in Example
5.
Results
[0879] These data demonstrated that the potency of the four human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID
NOs: 1, 6, 9 and 14) in inducing FLT3-dependent proliferation in
AML5 cells was similar, with EC50 values ranging between
0.037-0.050 nM. The results are summarized in Table 13 and depicted
in FIG. 13.
TABLE-US-00020 TABLE 13 EC50 values for FLT3L-Fc Variant SEQ ID
NOs: 1, 6, 9 and 14 in AML5 Proliferation Assay FLT3L-Fc Variant
SEQ ID NO: EC50 (nM) 1 0.050 6 0.048 9 0.037 14 0.046
Example 14
In Vitro Potency of FLT3L-Fc Variants in cDC1 Differentiation
Assay
[0880] In this example, we compared the in vitro potency of four
different human FLT3-ligand human Fc fusion proteins to
differentiate human bone marrow CD34+ stem cells into conventional
dendritic cell subtype 1 (cDC1).
Methods
[0881] 96-well flat-bottom tissue culture plates (Falcon, 353072)
were coated with recombinant DLL1 (R&D Systems, 1818-DL-050) as
follows. DLL1 was reconstituted in PBS to create a stock solution
of 500 pg/ml. The stock solution was diluted in DPBS (Corning,
21-030-CV) to a final working concentration of 5 .mu.g/ml, and 100
.mu.l of this was plated into each well. Plates were sealed and
placed on a flat surface at 4.degree. C. overnight.
[0882] Bone marrow CD34+ stem cells from 13 healthy donors were
thawed in a 37.degree. C. water bath and transferred into complete
media (Alpha-Mem (Gibco, 12561056), 10% heat-inactivated FCS, 1x
Pen/Strep). To recover the cells, 20,000 cells per well were plated
into a 96-well round bottom tissue culture plate.
[0883] The next day, the DLL1 coated plates were washed 3.times.
with DBPS, then 10,000 recovered cells per well were cultured with
20 ng/ml human GM-CSF, 20 ng/ml human SCF, 2.5 ng/ml human TL-4 and
various test articles. On day 6, half the media was removed and
fresh cytokines and compounds were added to the cells. On day 14,
cells were collected. Staining antibodies were then added to the
cells and incubated for 30 min at 4.degree. C. Cells were then
washed twice with FACS staining buffer and analyzed LSR Fortessa
FACS analyzer (BD Bioscience). Raw data were analyzed by FlowJo X
(BD Bioscience).
Results
[0884] These data demonstrated that the potency of the four human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID
NOs: 1, 6, 9 and 14) in inducing cDC1 differentiation in vitro from
primary human CD34+ bone marrow stem cells was similar, with EC50
values ranging between 0.788-1.252 nM. The results are summarized
in Table 14 and depicted in FIG. 14.
TABLE-US-00021 TABLE 14 EC50 values for differentiation of human
CD34+ stem cells into cDC1 by proteins corresponding to FLT3L-Fc
Variant SEQ ID NOs: 1, 6, 9 and 14 FLT3L-Fc Variant SEQ ID NO: EC50
(nM) 1 1.252 6 1.031 9 0.915 14 0.788
Example 15
In Vitro Potency of FLT3L-Fc Variants in Promoting cDC1
Survival
[0885] In this example, we compared the in vitro potency of four
different human FLT3-ligand human Fc fusion proteins to enhance the
survival of human PBMC-derived conventional dendritic cell subtype
1 (cDC1).
Methods
[0886] Sixteen fresh human healthy donor PBMCs were obtained from
PPA Research Group Inc. Pan-DCs were isolated from the PBMCs
following the manufacturer's protocol for EasySep Human Pan-DC
Pre-Enrichment Kit (Stemcell Technologies, Inc, 19251). Pan-DCs
were then stained with eBioscience Cell Proliferation Dye efluor
450 (Invitrogen, 65-0842-85) for 8 min in a 37.degree. C. water
bath. After staining with the cell proliferation dye, cells were
washed 2.times. and resuspended with complete RPMI. 100,000 cells
per well were plated into a 96-well round bottom tissue culture
plate and titrations of the compounds were added to the cells for a
final volume of 200 ul per well. Plates were sealed with a
breathable plate sealer (Breathe Easy Sealing Membrane, Millipore
Sigma, Z380059-1Pak) and then incubated for 4 days at 37.degree. C.
After incubation, cells were washed with FACS staining buffer and
Fc blocked (Human TruStain FcX Biolegend, 422302) for at least 10
min at 4.degree. C. Staining antibodies were then directly added to
the cells and incubated for 30 min at 4.degree. C. Cells were then
washed twice with FACS staining buffer and analyzed LSR Fortessa
FACS analyzer (BD Bioscience). Raw data were analyzed by FlowJo X
(BD Bioscience).
Results
[0887] The data demonstrated that the potency of the 4 human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID
NOs: 1, 6, 9 and 14) in enhancing primary human cDC1 survival in
vitro was similar, with EC50 values ranging between 0.067-0.102 nM.
The results are summarized in Table 15 and depicted in FIG. 15.
TABLE-US-00022 TABLE 15 EC50 values for promoting cDC1 survival by
proteins corresponding to FLT3L-Fc Variant SEQ ID NOs: 1, 6, 9 and
14 FLT3L-Fc Variant SEQ ID NO: EC50 (nM) 1 0.067 6 0.102 9 0.102 14
0.087
Example 16
In Vitro Binding of FLT3L-Fc Variants to FLT3
[0888] In this example, we compared the in vitro binding of four
different human FLT3-ligand human Fc fusion proteins to human
recombinant FLT3 by ELISA. The methods are as described above in
Example 6.
Results
[0889] These data demonstrated that binding to human FLT3 receptor
was similar among the four human FLT3-ligand Fc fusion proteins
with different Fc variants (SEQ ID NOs: 1, 6, 9 and 14), with EC50
values ranged between 0.70 to 0.92 nM. The results are summarized
in Table 16 and depicted in FIG. 16.
TABLE-US-00023 TABLE 16 EC50 values for FLT3L-Fc Variant SEQ ID
NOs: 1, 6, 9 and 14 Binding to Human Recombinant FLT3 FLT3L-Fc
Variant SEQ ID NO: EC50 (nM) 1 0.81 6 0.70 9 0.88 14 0.92
Example 17
In Vitro Binding of FLT3L-Fc Variants to FcRn
[0890] In this example, we compared the in vitro binding of four
different human FLT3-ligand human Fc fusion proteins to human
recombinant FcRn by ELISA. The methods are as described above in
Example 7.
Results
[0891] The data demonstrated that M252Y/S254T/T256E mutations in
the human IgG Fc region leads to improved FcRn binding for SEQ ID
NOs: 9 and 14, compared to their counterparts without these
mutations (SEQ ID NOs: 1 and 6). A 38-fold increase in FcRn binding
was observed for Flt3L-Fc in the hingeless IgG1 format (SEQ ID NOs:
1 and 9), compared to a 2-fold increase in the IgG4 format (SEQ ID
NOs: 6 and 14). The results are summarized in Table 17 and depicted
in FIG. 17.
TABLE-US-00024 TABLE 17 EC50 Values for FLT3L-Fc Variant SEQ ID
NOs: 1, 6, 9 and 14 Binding to Human Recombinant FcRn FLT3L-Fc
Variant SEQ ID NO: EC50 (nM) 1 2332 6 365.00 9 61 14 178
Example 18
Ability of FLT3L-Fc Variants to Compete for Binding to Human
Fc.gamma.RI
[0892] In this example, we compared the in vitro ability of four
different human FLT3-ligand human Fc fusion proteins to compete for
binding of human recombinant Fc.gamma.RI with a human IgG molecule.
To evaluate the ability to compete for binding to Fc.gamma.RI, we
employed an amplified luminescent proximity homogeneous assay
(AlphaScreen.RTM. by Perkin Elmer). The methods are as described
above in Example 8.
Results
[0893] These data demonstrated that none of the 4 human FLT3-ligand
Fc fusion proteins with different Fc variants (SEQ ID NOs: 1, 6, 9
and 14) could fully compete with human IgG for binding to
Fc.gamma.RI at the highest concentrations tested. Both the human
IgG1 and IgG4 isotype antibody controls demonstrated complete
dose-response curves, with the IgG4 isotype showing reduced
competition compared to the IgG1. The results are summarized in
Table 18 and depicted in FIG. 18.
TABLE-US-00025 TABLE 18 EC50 values for the ability of FLT3L-Fc
Variant SEQ ID NOs: 1, 6, 9 and 14 to compete with a human IgG
molecule for binding to human recombinant Fc.gamma.RI FLT3L-Fc
Variant SEQ ID NO: EC50 (nM) 1 NA 6 NA 9 NA 14 NA hIgG1 Isotype
5.25 hIgG4 Isotype 14.54
Example 19
Ability of FLT3L-Fc Variants to Compete for Binding to Human
Fc.gamma.RIIIa
[0894] In this example, we compared the in vitro ability of four
different human FLT3-ligand human Fc fusion proteins to compete for
binding of human recombinant Fc.gamma.RIIIa (Val176 variant) with a
human IgG molecule. To evaluate the ability to compete for binding
to Fc.gamma.RIIIa, we employed an amplified luminescent proximity
homogeneous assay (AlphaScreen.RTM. by Perkin Elmer). The methods
are as described above in Example 9.
Results
[0895] The results demonstrated that none of the 4 human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID
NOs: 1, 6, 9 and 14) could fully compete with human IgG for binding
to Fc.gamma.RIIIa (Val176 variant). Only human IgG1 isotype
antibody control demonstrated a complete dose-response curve. The
results are summarized in Table 19 and depicted in FIG. 19.
TABLE-US-00026 TABLE 19 EC50 values for the ability of FLT3L-Fc
Variant SEQ ID NOs: 1, 6, 9 and 14 to compete with a human IgG for
binding to human recombinant Fc.gamma.RIIIa (V-variant) FLT3L-Fc
Variant SEQ ID NO: EC50 (nM) 1 NA 6 NA 9 NA 14 NA hIgG1 Isotype
38.80 hIgG4 Isotype 368.30
Example 20
In Vitro Binding of FLT3L-Fc Variants to Human C1q
[0896] In this example, we compared the in vitro binding of four
different human FLT3-ligand human Fc fusion proteins to human
recombinant C1q. To evaluate the in vitro binding to C1q, we
employed an ELISA. The methods are as described above in Example
10.
Results
[0897] These data demonstrated that the four human FLT3-ligand Fc
fusion proteins with different Fc variants (SEQ ID NOs: 1, 6, 9 and
14) are devoid of C1q binding ability. Both the human IgG1 and IgG4
isotype controls demonstrated binding to C1q, with the IgG4 isotype
showing reduced binding compared to the IgG1. The results are
summarized in Table 20 and depicted in FIG. 20.
TABLE-US-00027 TABLE 20 EC50 values for FLT3L-Fc Variants SEQ ID
NOs: 1, 6, 9 and 14 Binding to Human Recombinant C1q FLT3L-Fc
Variant SEQ ID NO: EC50 (nM) 1 NA 6 NA 9 NA 14 NA hIgG1 Isotype
8.44 hIgG4 Isotype 13.71
Example 21
Single Dose Pharmacokinetics of FLT3L-Fc Variants in Cynomolgus
Macaque
[0898] In this example, we compared the single dose
pharmacokinetics of four different human FLT3-ligand human Fc
fusion proteins in Cynomolgus macaque.
Methods
[0899] Four FLT3L-Fc variants (SEQ ID NOs: 1, 6, 9, and 14) were
administered to cynomologus macaques n=3/group (Covance, TX) at 0.5
mg/kg via a single intravenous (IV) and subcutaneous (SC) injection
to characterize their basic PK profiles. Serial serum samples
collected from macaques were analyzed using U-PLEX FLT3L assay
(Meso Scale Discovery, MSD) according to the manufacturer's
instructions. The calibration curve used the respective individual
FLT3-ligand fusion proteins as reference standards in spiked
macaque matrix fit to a 4-parameter logistic model with 1/Y2
weighting. Analyte concentrations were determined from the
electrochemiluminescence signals back-fitted to the calibration
curve. Serum concentration-time profiles were used to calculate the
mean SD serum PK parameters by non-compartmental PK analysis.
Results
[0900] Pharmacokinetic (PK) analysis demonstrated that all 4
Fc-fusions had IgG-like pharmacokinetics with C1 values range from
4.71-7.74 mL/d/kg (Table 21) following IV administration in
cynomologus macaques. Similar pharmacokinetics were observed
following subcutaneous administration with bioavailability ranging
from 66.8-91.4%. SEQ ID NOs: 9 and 14 containing the
M252Y/S254T/T256E modifications had reduced clearance relative to
unmodified sequence, consistent with improved FcRn binding. The
results are summarized in Table 21 and depicted in FIGS.
21A-21B.
TABLE-US-00028 TABLE 21 Single dose PK values for FLT3L-Fc Variants
SEQ ID NOs: 1, 6, 9 and 14 in Cynomolgus macaques FLT3L-Fc
Intravenous Subcutaneous Variant AUC.sub.0-last Cl AUC.sub.0-last
Cl/F SEQ ID NO: (.mu.g*d/mL) (mL/d/kg) (.mu.g*d/mL) (mL/d/kg) 1
59.3 .+-. 5.29 7.74 .+-. 1.35 54.2 .+-. 5.04 9.29 .+-. 0.91 6 82.4
.+-. 15.6 6.23 .+-. 1.31 66.2 .+-. 20.1 7.98 .+-. 2.10 9 73.8 .+-.
12.2 6.89 .+-. 1.08 49.3 .+-. 3.36 9.16 .+-. 1.01 14 107 .+-. 11.6
4.71 .+-. 0.502 79.4 .+-. 30.4 7.04 .+-. 3.27
Example 22
Ability of FLT3L-Fc Variants to Promote cDC1 Proliferation in
Cynomolgus Macaque
[0901] In this example, we compared the ability of four different
FLT3L-Fc variants (SEQ ID NOs: 1, 6, 9 and 14) to induce
proliferation and expansion of conventional dendritic cell subtype
1 (cDC1) in Cynomolgus macaque.
Methods
[0902] Whole blood samples from cynomolgus macaques administered
one of SEQ ID NO: 1, 6, 9, or 14 at day 0 were drawn into Sodium
Heparin blood collection tubes at indicated time points. One
hundred microliters of each sample were transferred to FACS tubes
containing FACS antibodies and Fc blocker. Cells were incubated at
room temperature for 20 min, then washed twice with
1.times.DPBS-CMF. Residual red blood cells were then lysed for 8-12
minutes in the dark at room temperature with 1 mL 1.times.FACS Lyse
(BD Biosciences). After incubation, the samples were centrifuged
and washed once by adding 1.times.DPBS-CMF (1 mL). Samples were
then resuspended in 125 .mu.L 1.times.DPBS-CMF and 100 .mu.L
CountBright Beads for acquisition on the Canto FACS analyzer (BD
Biosciences). Raw data were analyzed by FlowJo X (BD
Bioscience).
Results
[0903] These data demonstrated a similar ability of the four human
FLT3-ligand Fc fusion proteins with different Fc variants (SEQ ID:
1, 6, 9, and 14) to expand conventional dendritic cell subtype 1
(cDC1) in peripheral blood of Cynomolgus macaque from day 0 to day
14 after single-dose intravenous or subcutaneous administration.
The cDC1 number of each group gradually declined to baseline levels
between days 14 to 38 after test article administration, at a rate
which paralleled the corresponding decline in serum levels of
FLT3-ligand Fc fusion proteins as shown in FIG. 21. These results
are depicted in FIGS. 22A-22B.
Example 23
Anti-Tumor Activity of Murine Surrogate FLT3L-Fc Fusion Protein
[0904] In this example, we demonstrated the anti-tumor activity of
a murine surrogate FLT3L-Fc fusion protein (SEQ ID NO:20) in a
mouse tumor model.
Methods
[0905] Eight-week old C57Bl/6 mice (Jackson Laboratory) were
inoculated with 2.5.times.10.sup.5 MC38 cells subcutaneously. Mice
were then randomized when tumor volume reached 45-55 mm.sup.3 on
day 0 and dosed intraperitoneally with SEQ ID NO: 20 or an
Fc-silent (N297A) mouse IgG2a isotype control at indicated
concentration within the same day. Tumors were measured 3 times a
week using calipers. Tumor volumes were calculated using the
following equation: (longest diameter* shortest
diameter.sup.2)/2.
Results
[0906] The data demonstrated a dose-dependent tumor growth
inhibition of MC38 tumors in C57BL/6 mice after a single-dose
intraperitoneal administration by the mouse FLT3-ligand Fc-fusion
protein (SEQ ID NO:20) at day 0. Groups dosed with 150 .mu.g/kg,
750 .mu.g/kg and 3750 .mu.g/kg of SEQ ID NO:20 show significantly
slower tumor growth rates compared to the isotype control group.
The results are summarized in Table 22 and depicted in FIG. 23.
TABLE-US-00029 TABLE 22 Tumor Growth Inhibition (TGI) at Day 14
Post-Dose for SEQ ID NO: 20 Relative to Isotype Control in MC38
Mouse Tumor Model Dose TGI (%) p-value 3750 .mu.g/kg 52.67
<0.0001 750 .mu.g/kg 38.68 <0.0001 150 .mu.g/kg 14.51
<0.0035 30 .mu.g/kg -2.35; not significant
Example 24
Intratumoral and Peripheral Expansion of cDC1 in Tumor Bearing
Mice
[0907] In this example, we demonstrated the intratumoral and
peripheral expansion of conventional dendritic cell subtype 1
(cDC1) in tumor bearing mice by FLT3L-Fc fusion protein using a
murine surrogate (SEQ ID NO:20).
Methods
[0908] Tumors and spleens were harvested at Day 7 post
administration and shipped O/N at 4.degree. C. in HypoThermosol
solution (BioLife Solutions) from the CROs. Tumors and spleens were
then dissociated using the gentleMACS Dissociator (Miltenyi Biotec)
with heaters, following manufacturer's protocol. After enzymatic
digestion, cell suspension was filtered through a 70 .mu.m cell
strainer. The remaining tube and strainer were rinsed 1.times. with
15-20m1 of RPMI and collected with the rest of the sample. Cells
were centrifuged at 500.times.g for 5 min at room temperature.
Supernatant was discarded and cells were washed 1.times. with PBS.
Residual red blood cells from spleens were lysed by adding 2m1 of
ACK lysis buffer to each sample for 1-2 min at room temperature.
FACS staining Buffer (BD Bioscience) was added to the samples to
stop the ACK lysis activity. Cells were spun down and washed
additionally with PBS. Samples were then stained with Live/Dead
Fixable Aqua Dead Cell Stain Kit (ThermoFisher) at 1:750 dilution
for 15 min at 4.degree. C. Ten microliters of cells from each
sample were taken for count by 123count eBeads (eBiosciences)
following manufacturer's protocol. Cells were washed 2.times. with
FACS staining buffer, then Fc blocked for 30 min at 4.degree. C.
FACS antibodies (Biolegend) were directly added to the blocked
samples and incubated at 4.degree. C. for 30 min without spinning
down or washing out the Fc block. Cells were washed 2x, resuspended
in Staining Buffer, and analyzed by LSR Fortessa FACS analyzer. Raw
data were analyzed by FlowJo X (BD Bioscience).
Results
[0909] The results demonstrated a dose-dependent increase of
conventional dendritic cell subtype 1 (cDC1) number in tumors (FIG.
24A) or in spleens (FIG. 24B) in the MC38 tumor model after a
single-dose intraperitoneal administration by the mouse surrogate
FLT3-ligand Fc-fusion protein (SEQ ID NO:20) at day 0. Groups dosed
750 .mu.g/kg and 3750 .mu.g/kg of SEQ ID NO:20 showed a significant
increase of cDC1 number in tumors compared to the isotype group,
while groups dosed 750 .mu.g/kg and 3750 .mu.g/kg of SEQ ID NO:20
show significant increase of cDC1 number in spleens compared to the
isotype group. Similar increase of cDC1 number in tumors and
spleens was observed in groups dosed with 750 .mu.g/kg and 3750
.mu.g/kg of SEQ ID NO:20. The results are summarized in Table 23
and depicted in FIG. 24.
TABLE-US-00030 TABLE 23 cDC1 Fold-Change in Tumor and Spleen at day
7 Post-Dose for SEQ ID NO: 20 Relative to Isotype Control in MC38
Mouse Tumor Model cDC1 fold change in cDC1 fold change in Dose
tumor spleen 3750 .mu.g/kg 65.57 50.7 750 .mu.g/kg 44.21 81.76 150
.mu.g/kg 4.72 6.84 30 .mu.g/kg -0.17 4.18
Example 25
Sialic Acid Content Evaluation of Eight FLT3L-Fc Variants
[0910] In this example, we determined the total sialic acid content
of eight Flt3L-Fc constructs. To evaluate sialic acid content, we
performed chemical release of sialic acids followed by fluorescence
labeling and reversed-phase separation with fluorescence
detection.
Methods
[0911] Proteins from Flt3L-Fc constructs SEQ ID NOs: 1-8 were
diluted to 10 or 50 .mu.g/mL in water. Sialic acids were released
by mild acid hydrolysis with acetic acid, separated from the
protein by filtration, and fluorescently labeled with
1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) by a
reductive amination reaction. The labeled sialic acid products were
then separated by reversed-phase chromatography using a C18 column
with fluorescence detection at 373 nm (excitation) and 448 nm
(emission). The concentrations of N-acetylneuraminic acid (NANA)
and N-Glycolylneuraminic acid (NGNA) present were determined from
an identically labeled 6-point standard curve and expressed as a
mole to mole ratio of sialic acid to protein content.
Results
[0912] The results are summarized in Table 24.
TABLE-US-00031 TABLE 24 Sialic Acid Content of SEQ ID NO. 1-8
FLT3L-Fc Variant mol/mol mol/mol SEQ ID NO: NANA NGNA 1 16.5 ND* 2
13.7 ND 3 15.2 ND 4 17.4 ND 5 14.3 ND 6 15.4 ND 7 8.1 ND 8 13.0 ND
*ND = not detected
[0913] The data obtained for SEQ ID NOs: 1-8 showed similar amounts
of N-acetylneuraminic acid (NANA) in each sample, at an average of
15 mol/mol. One exception was noted, in which SEQ ID NO: 7
contained only 8 mol/mol sialic acid. A subtle trend was also
observed in which sialic acid content decreased in constructs
containing C-terminal truncations of the Flt3L portion of the
fusion proteins (constructs SEQ ID NO: 2 and 7 relative to SEQ ID
NO:1; constructs SEQ ID NO: 6 and 8 relative to SEQ ID NO: 4),
indicating this region contained the majority of sialic acid.
Furthering this idea was the observation that N-glycan removal from
the ligand domain did not result in a major loss of sialic acid
content, shown by comparison of SEQ ID NO: 5 and SEQ ID NO: 1.
[0914] Total sialic acid content was shown to have a positive
correlation to PK in mAbs and Fc-fusion proteins, consistent with
the reporting of, e.g., Li, et al., J Pharm Sci (2015)
104:1866-1884; and Liu, et al., Protein Cell. (2018)
9(1):15-32.
Example 26
Sialic Acid Content Evaluation of Four FLT3L-Fc Variants
[0915] In this example, we determined the sialic acid content on
the two FLT3L N-glycans at positions 100 and 123 of SEQ ID NOs: 1,
6, 9, and 14 ("Asn100" and "Asn123," respectively).
Methods
[0916] Protein digest followed by reversed-phase separation with
detection by mass spectrometry was performed.
[0917] Following denaturation, reduction and cysteine
carboxymethylation, proteins from SEQ ID NOs: 1, 6, 9, and 14 were
digested for 6 hours at 37.degree. C. using a 1:10 enzyme:substrate
(w:w) ratio of Lys-C and Glu-C Mix. Digestion was then quenched by
adding trifluoroacetic acid to 0.1% final concentration. The
resulting Lys-C/Glu-C peptides were separated by reverse phase
gradient UPLC on a C18 column. The separation of the peptides was
monitored at 214 nm prior to elution into the inlet of a Thermo
Scientific QE HF Orbitrap mass spectrometer operating in positive,
data-dependent acquisition mode. N-glycan peptide assignments were
based on matching the observed masses of the intact peptides in the
LC/MS analysis to the masses predicted based on a theoretical Lys-C
and Glu-C digest of SEQ ID NOs: 1,6,9,and 14.
Results
[0918] The results are presented in Table 25.
TABLE-US-00032 TABLE 25 Sialylalation Analysis of FLT3L-Fc Variants
SEQ ID NOs: 1, 6, 9, and 14 SEQ ID NO. 1 SEQ ID NO. 6 SEQ ID NO. 9
SEQ ID NO. 14 Sialylated Sialylated Sialylated Sialylated
Peptides.sup.1 Occupancy.sup.2 Peptides.sup.1 Occupancy.sup.2
Peptides.sup.1 Occupancy.sup.2 Peptides.sup.1 Occupancy.sup.2 (%)
(%) (%) (%) (%) (%) (%) (%) Asn 43 66 43 64 33 61 46 63 100 Asn 63
70 57 68 39 63 57 67 123 Relative abundance of sialylated FLT3L
N-glycans corrected by occupancy (%) Asn 65 68 54 72 100 Asn 90 84
62 94 123 Average across the two sites (%) 77 76 58 83 .sup.1FLT3L
N-glycans with one or more sialic acid unit. Percentage determined
including asialylated peptides .sup.2N-glycan occupancy at the two
sites was determined by peptide mapping
[0919] The molecular masses of the N-glycosylated peptides were
consistent with the predicted masses based on the amino acid
sequences of the FTL3L-Fc constructs. FTL3L-Fc SEQ ID NOs 1, 6, and
14 contained a consistent level of species with one or more sialic
acid unit (43-63%, Table 25). FTL3L-Fc SEQ ID NO: 9 showed a
decrease of approximately 10% at Asn 100 and 24% at Asn 123 (Table
25). Ligand N-glycans site occupancy for SEQ ID NOs: 1, 6, 9 and 14
was consistent across all four molecules tested and ranged from 61%
to 70% across the two sites (Table 25).
[0920] The percent of ligand N-glycans species containing at least
one sialic acid unit were corrected by the percent occupancy at
each site and averaged across the two sites to allow a direct
comparison across the constructs (Table 25). After correction and
averaging, FTL3L-Fc SEQ ID NOs: 1, 6, and 14 showed a level of
overall ligand sialylated N-glycans ranging from 76 to 83%; while
FTL3L-Fc SEQ ID NO: 9 showed a lower overall level at 58%.
Example 27
Conformational Stability of FLT3L-Fc Variants
[0921] In this example, we evaluated the conformational stability
of FLT3L-Fc variants SEQ ID NOs: 1 to 9 and SEQ ID NO: 14.
[0922] Each FLT3L-Fc construct was prepared in a matrix of 20 mM
sodium phosphate, 9% sucrose, 0.02% PS80 pH 6.5 and loaded into a
capillary. Intrinsic fluorescence was measured by a Nano
differential scanning fluorimetry (NanoTemper) instrument as the
samples were heated from 25-95.degree. C. The ratio of fluorescence
signal at 350/330 nm was plotted versus temperature to compare
melting profiles of each sample. Duplicate measurements were
performed for each construct.
[0923] The data obtained for FLT3L-Fc variants SEQ ID NOs: 1 to 8
show similar conformational stability, with onset temperatures
(T.sub.on) greater than 55.degree. C. for each FLT3L-Fc variant.
FLT3L-Fc SEQ ID NO 3 has a lower T.sub.m1 compared to the three
other IgG4 FLT3L-Fc constructs, indicating that the L235E mutation
is slightly destabilizing resulting in lower conformational
stability. Results of the IgG1 FLT3L-Fc constructs also show that
FLT3L-Fc SEQ ID NO 5 has a decreased T.sub.on and T.sub.m1 compared
with the three other IgG1 constructs (Table 26).
[0924] The half-life enhancing YTE mutation lowers the T.sub.m1
value of FLT3L-Fc variants SEQ ID NOs: 9 and 14. Onset temperatures
remain well above physiological temperature despite this shift
(Table 26).
TABLE-US-00033 TABLE 26 Conformational Stability by Nano
Differential Scanning Fluorimetry FLT3L-Fc Variant SEQ ID NO:
T.sub.on (.degree. C.) T.sub.m1 (.degree. C.) 1 60.3 67.4 2 60.6
67.6 3 59.5 65.3 4 63.5 68.8 5 55.0 66.3 6 64.2 68.4 7 59.5 71.3 8
64.3 68.9 9 51.0 55.2 14 49.4 52.3
Example 28
FLT3L-Fc and Anti-PD1 Combination Study
[0925] In this example, we demonstrated the combined effects of
anti-PD1 antibody (clone RMP1-14) and a FLT3L-Fc fusion protein
using SEQ ID NO:20 (a murine surrogate) in a syngeneic mouse tumor
model.
Methods:
[0926] Eight-week old C57BL/6 mice (Jackson Laboratory) were
inoculated with 2.5.times.10.sup.5 MC38 cells subcutaneously. Mice
were then randomized when tumor volume reached 45-55 mm.sup.3 on
day 0 and dosed intraperitoneally with FTL3L-Fc SEQ ID NO:20,
anti-mouse PD-1 (clone RMP1-14) or an isotype control at the
indicated concentrations and dosing frequencies. Tumors were
measured at least 2 times a week using calipers. Tumor volumes were
calculated using the following equation: (longest diameter*shortest
diameter.sup.2)/2.
Results
[0927] The results demonstrated a modest growth inhibition of MC38
tumors in C57BL/6 mice after single agent treatment with either one
of the mouse FLT3-ligand Fc-fusion protein (SEQ ID NO:20) or
anti-mouse PD-1, while combined treatment with the anti-PD1
antibody (clone RMP1-14) and the FLT3L-Fc fusion protein yielded
strong tumor growth inhibition. The results are depicted in FIG.
25.
Example 29
FLT3L-Fc and Anti-CTLA4 Combination Study
[0928] In this example, we demonstrated the combined effects of
anti-CTLA4 antibody (clone 9D9) and a FLT3L-Fc fusion protein using
SEQ ID NO:20 (a murine surrogate) in a syngeneic mouse tumor
model.
Methods:
[0929] Nine-week old BALB/c mice (Taconic) were inoculated with
8.times.10.sup.5 CT26 cells subcutaneously. Mice were then
randomized when tumor volume reached 60-70 mm.sup.3 on day 0 and
dosed intraperitoneally with FTL3L-Fc SEQ ID NO:20, anti-mouse
CTLA4 (clone 9D9) or an isotype control at the indicated
concentrations and dosing frequencies. Tumors were measured at
least 2 times a week using calipers. Tumor volumes were calculated
using the following equation: (longest diameter*shortest
diameter.sup.2)/2.
Results:
[0930] The results demonstrated a modest growth inhibition of CT26
tumors in BALB/c mice after single agent treatment with either one
of the mouse FLT3-ligand Fc-fusion protein (SEQ ID NO:20) or
anti-mouse CTLA4, while combined treatment with the anti-CTLA4
antibody (clone 9D9) and the FLT3L-Fc fusion protein yielded strong
tumor growth inhibition. The results are depicted in FIG. 26.
Example 30
Effect of FLT3L on the Immunogenicity of an HBV Vaccine in a Mouse
Model of Chronic HBV
[0931] We evaluated the potential effect of FLT3L on the
immunogenicity of an HBV vaccine in the context of chronic HBV
using an Adeno-Associated Virus (AAV)-HBV mouse model (Dion, et
al., J Virol. (2013) 87(10):5554-63; and Yang, et al., Cell Mol
Immunol. (2014) 11(1):71-8). Other immunomodulators including
antibodies targeting PD-1, CTLA-4 and CD137 were also tested.
Methods
[0932] In this model, C57BL/6 mice were transduced with an AAV
vector encoding a 1.2.times. length HBV genome (AAV-HBV mice),
resulting in persistent HBV protein and virion production in
hepatocytes, accompanied by antigenemia and viremia in serum.
AAV-HBV mice were administered 3 doses of an HBV vaccine that is an
arenavirus vector expressing HBV antigens including HBsAg, core and
polymerase. Mice were treated with saline, mouse FLT3L, anti-mouse
inhibitory PD-1, anti-mouse inhibitory CTLA-4 or anti-mouse
stimulatory CD137 antibodies. A control group of mice received the
HBV vaccine alone but no AAV-HBV to determine how the
immunogenicity of the HBV vaccine was affected in the context of
chronic HBV. HBV-specific IFN-.gamma. ELISPOT was performed using
spleens of all animals at the end of the study (day 105 post first
vaccination). A diagram of this AAV-HBV immunogenicity study is
shown in FIG. 27 and treatment groups are shown in Table 27. Data
are expressed after subtraction of background signal in no-peptide
control wells. Statistical analysis was performed using
Mann-Whitney non-parametric test.
Results
[0933] IFN-.gamma. ELISPOT responses specific for HBsAg, HBV core
and HBV polymerase are summarized in FIGS. 28A-C. Robust ELISPOT
responses were observed for all 3 HBV antigens in mice without
persistent HBV. In contrast, the ELISPOT responses obtained from
AAV-HBV mice that received the HBV vaccine alone were significantly
reduced, demonstrating T cell tolerance against the HBV proteins in
AAV-HBV mice. In these mice, combined administration of FLT3L and
HBV vaccine significantly increased the HBV-specific IFN-.gamma.
ELISPOT responses for all 3 HBV antigens. A comparable effect
(except for HBV pol-specific responses) was observed with the other
immunomodulators anti PD-1, anti CTLA-4 and anti CD137 antibodies
although with lower magnitude.
TABLE-US-00034 TABLE 27 Study Groups in AAV-HBV Immunogenicity
Study AAV- HBV Immuno- Group N HBV vaccine modulator Molecule and
Dose 1 11 Yes Yes Vehicle Saline 2 12 Yes Yes .alpha.-PD-1 Clone
RMP1-14 8 mg/kg/dose 3 12 Yes Yes .alpha.-CTLA-4 Clone 9D9 10
mg/kg/dose 4 12 Yes Yes .alpha.-CD137 Clone mAb8 2.5 mg/kg/dose 5
12 Yes Yes FLT3L Mouse surrogate of FLT3L-Fc (SEQ ID NO: 20) 1
mg/kg/dose 6 5 No Yes Vehicle Saline
[0934] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
1101369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile
Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu
Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu
Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys
Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala
Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Gly
Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 165 170 175Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 180 185 190Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 195 200 205Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 210 215 220Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu225 230 235
240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 260 265 270Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 275 280 285Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu305 310 315 320Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 325 330 335Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 340 345 350Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 355 360
365Lys2364PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile
Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu
Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu
Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys
Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala
Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Gly Gly Pro Ser Val Phe
Leu Phe145 150 155 160Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 165 170 175Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe 180 185 190Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 195 200 205Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 210 215 220Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val225 230 235
240Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
245 250 255Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 260 265 270Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 275 280 285Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 290 295 300Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser305 310 315 320Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 325 330 335Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 340 345 350Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355
3603386PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile
Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu
Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu
Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys
Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala
Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Glu
Ser Lys145 150 155 160Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly 165 170 175Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 180 185 190Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu 195 200 205Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 210 215 220Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg225 230 235
240Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
245 250 255Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu 260 265 270Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 275 280 285Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu 290 295 300Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp305 310 315 320Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 325 330 335Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 340 345 350Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 355 360
365Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
370 375 380Gly Lys3854386PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Thr Gln Asp Cys Ser Phe
Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu
Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val
Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser
Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75
80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro
Thr Ala Pro Gln Glu Ser Lys145 150 155 160Tyr Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 165 170 175Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 180 185 190Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 195 200
205Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
210 215 220Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg225 230 235 240Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 245 250 255Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu 260 265 270Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 275 280 285Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 290 295 300Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp305 310 315
320Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
325 330 335Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp 340 345 350Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His 355 360 365Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu 370 375 380Gly Lys3855369PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5
10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ala Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ala Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Gly Gly Pro145 150 155
160Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
165 170 175Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 180 185 190Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 195 200 205Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 210 215 220Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu225 230 235 240Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 245 250 255Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 260 265 270Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 275 280
285Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
290 295 300Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu305 310 315 320Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 325 330 335Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 340 345 350Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 355 360 365Lys6381PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5
10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Glu Ser Lys Tyr Gly Pro Pro Cys145 150 155
160Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
165 170 175Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu 180 185 190Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val Gln 195 200 205Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys 210 215 220Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu225 230 235 240Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280
285Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
290 295 300Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln305 310 315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly 325 330 335Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln 340 345 350Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys 370 375 3807359PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5
10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys145 150 155 160Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 165 170 175Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp 180 185 190Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr 195 200 205Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 210 215 220Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu225 230 235
240Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys 260 265 270Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 275 280 285Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 290 295 300Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser305 310 315 320Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 325 330 335Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 340 345 350Leu
Ser Leu Ser Pro Gly Lys 3558376PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Thr Gln Asp Cys Ser Phe
Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu
Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val
Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser
Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75
80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala145 150 155 160Pro Glu Ala Ala Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 165 170 175Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 180 185 190Val Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 195 200
205Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
210 215 220Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln225 230 235 240Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly 245 250 255Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro 260 265 270Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr 275 280 285Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 290 295 300Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr305 310 315
320Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
325 330 335Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe 340 345 350Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 355 360 365Ser Leu Ser Leu Ser Leu Gly Lys 370
3759369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile
Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu
Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu
Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys
Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala
Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Gly
Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Tyr Ile Thr 165 170 175Arg Glu Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 180 185 190Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 195 200 205Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 210 215 220Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu225 230 235
240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 260 265 270Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 275 280 285Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu305 310 315 320Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 325 330 335Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 340 345 350Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 355 360
365Lys10364PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile
Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu
Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu
Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys
Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala
Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Gly Gly Pro Ser Val Phe
Leu Phe145 150 155 160Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr
Arg Glu Pro Glu Val 165 170 175Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe 180 185 190Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 195 200 205Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 210 215 220Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val225 230 235
240Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
245 250 255Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 260 265 270Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 275 280 285Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 290 295 300Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser305 310 315 320Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 325 330 335Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 340 345 350Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355
36011386PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile
Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu
Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu
Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys
Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala
Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Glu
Ser Lys145 150 155 160Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly 165 170 175Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Tyr Ile 180 185 190Thr Arg Glu Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu 195 200 205Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 210 215 220Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg225 230 235
240Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
245 250 255Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu 260 265 270Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 275 280 285Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu 290 295 300Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp305 310 315 320Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 325 330 335Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 340 345 350Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 355 360
365Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
370 375 380Gly Lys38512386PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Thr Gln Asp Cys Ser Phe
Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu
Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val
Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser
Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75
80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro
Thr Ala Pro Gln Glu Ser Lys145 150 155 160Tyr Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 165 170 175Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile 180 185 190Thr Arg
Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 195 200
205Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
210 215 220Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg225 230 235 240Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 245 250 255Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu 260 265 270Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 275 280 285Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 290 295 300Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp305 310 315
320Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
325 330 335Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp 340 345 350Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His 355 360 365Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu 370 375 380Gly Lys38513369PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ala Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ala Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Gly Gly Pro145 150 155
160Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr
165 170 175Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 180 185 190Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 195 200 205Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 210 215 220Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu225 230 235 240Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 245 250 255Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 260 265 270Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 275 280
285Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
290 295
300Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu305 310 315 320Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 325 330 335Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 340 345 350Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 355 360 365Lys14381PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Glu Ser Lys Tyr Gly Pro Pro Cys145 150 155
160Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
165 170 175Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
Pro Glu 180 185 190Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val Gln 195 200 205Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys 210 215 220Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu225 230 235 240Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280
285Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
290 295 300Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln305 310 315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly 325 330 335Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln 340 345 350Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys 370 375 38015359PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys145 150 155
160Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val
165 170 175Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp 180 185 190Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr 195 200 205Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 210 215 220Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu225 230 235 240Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 245 250 255Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 260 265 270Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 275 280
285Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser305 310 315 320Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 325 330 335Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser 340 345 350Leu Ser Leu Ser Pro Gly Lys
35516376PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile
Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu
Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu
Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys
Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala
Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala145 150 155 160Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 165 170 175Lys Asp Thr Leu Tyr Ile Thr Arg Glu
Pro Glu Val Thr Cys Val Val 180 185 190Val Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val 195 200 205Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 210 215 220Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln225 230 235
240Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
245 250 255Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro 260 265 270Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr 275 280 285Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser 290 295 300Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr305 310 315 320Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 325 330 335Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 340 345 350Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 355 360
365Ser Leu Ser Leu Ser Leu Gly Lys 370 37517376PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ala Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ala Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala145 150 155
160Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
165 170 175Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val 180 185 190Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val 195 200 205Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln 210 215 220Phe Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln225 230 235 240Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 245 250 255Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 260 265 270Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 275 280
285Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
290 295 300Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr305 310 315 320Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr 325 330 335Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe 340 345 350Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 355 360 365Ser Leu Ser Leu Ser
Leu Gly Lys 370 37518376PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 18Thr Gln Asp Cys Ser Phe
Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu
Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val
Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser
Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75
80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95Val Gln Thr Asn Ile Ala Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ala
Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala145 150 155 160Pro Glu Ala Ala Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 165 170 175Lys Asp Thr Leu
Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val 180 185 190Val Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 195 200
205Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
210 215 220Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln225 230 235 240Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly 245 250 255Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro 260 265 270Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr 275 280 285Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 290 295 300Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr305 310 315
320Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
325 330 335Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe 340 345 350Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 355 360 365Ser Leu Ser Leu Ser Leu Gly Lys 370
37519389PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Thr Pro Asp Cys Tyr Phe Ser His Ser Pro Ile
Ser Ser Asn Phe Lys1 5 10 15Val Lys Phe Arg Glu Leu Thr Asp His Leu
Leu Lys Asp Tyr Pro Val 20 25 30Thr Val Ala Val Asn Leu Gln Asp Glu
Lys His Cys Lys Ala Leu Trp 35 40 45Ser Leu Phe Leu Ala Gln Arg Trp
Ile Glu Gln Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Thr Leu
Leu Glu Asp Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Ser Cys
Thr Phe Gln Pro Leu Pro Glu Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser His Leu Leu Lys Asp Thr Cys Thr Gln Leu 100 105 110Leu Ala
Leu Lys Pro Cys Ile Gly Lys Ala Cys Gln Asn Phe Ser Arg 115 120
125Cys Leu Glu Val Gln Cys Gln Pro Asp Ser Ser Thr Leu Leu Pro Pro
130 135 140Arg Ser Pro Ile Ala Leu Glu Ala Thr Glu Leu Pro Glu Pro
Arg Gly145 150 155 160Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
Pro Ala Pro Asn Ala 165 170 175Ala Gly Gly Pro Ser Val Phe Ile Phe
Pro Pro Lys Ile Lys Asp Val 180 185 190Leu Met Ile Ser Leu Ser Pro
Ile Val Thr Cys Val Val Val Asp Val 195 200 205Ser Glu Asp Asp Pro
Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val 210 215 220Glu Val His
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser225 230 235
240Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met
245 250 255Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu
Gly Ala 260 265 270Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser
Val Arg Ala Pro 275 280 285Gln Val Tyr Val Leu Pro Pro Pro Glu Glu
Glu Met Thr Lys Lys Gln 290 295 300Val Thr Leu Thr Cys Met Val Thr
Asp Phe Met Pro Glu Asp Ile Tyr305 310 315 320Val Glu Trp Thr Asn
Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr 325 330 335Glu Pro Val
Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu 340 345 350Arg
Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser 355 360
365Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser
370 375 380Arg Thr Pro Gly Lys38520389PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
20Thr Pro Asp Cys Tyr Phe Ser His Ser Pro Ile Ser Ser Asn Phe Lys1
5 10 15Val Lys Phe Arg Glu Leu Thr Asp His Leu Leu Lys Asp Tyr Pro
Val 20 25 30Thr Val Ala Val Asn Leu Gln Asp Glu Lys His Cys Lys Ala
Leu Trp 35 40 45Ser Leu Phe Leu Ala Gln Arg Trp Ile Glu Gln Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Thr Leu Leu Glu Asp Val
Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Ser Cys Thr Phe Gln Pro Leu
Pro Glu Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser His Leu Leu
Lys Asp Thr Ser Thr Gln Leu 100 105 110Leu Ala Leu Lys Pro Cys Ile
Gly Lys Ala Cys Gln Asn Phe Ser Arg 115 120 125Cys Leu Glu Val Gln
Cys Gln Pro Asp Ser Ser Thr Leu Leu Pro Pro 130 135 140Arg Ser Pro
Ile Ala Leu Glu Ala Thr Glu Leu Pro Glu Pro Arg Gly145 150 155
160Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala
165 170 175Ala Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys
Asp Val 180 185 190Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val
Val Val Asp Val 195 200 205Ser Glu Asp Asp Pro Asp Val Gln Ile Ser
Trp Phe Val Asn Asn Val 210 215 220Glu Val His Thr Ala Gln Thr Gln
Thr His Arg Glu Asp Tyr Asn Ser225 230 235 240Thr Leu Arg Val Val
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met 245 250 255Ser Gly Lys
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Gly Ala 260 265 270Pro
Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro 275 280
285Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln
290 295 300Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp
Ile Tyr305 310 315 320Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
Asn Tyr Lys Asn Thr 325 330 335Glu Pro Val Leu Asp Ser Asp Gly Ser
Tyr Phe Met Tyr Ser Lys Leu 340 345 350Arg Val Glu Lys Lys Asn Trp
Val Glu Arg Asn Ser Tyr Ser Cys Ser 355 360 365Val Val His Glu Gly
Leu His Asn His His Thr Thr Lys Ser Phe Ser 370 375 380Arg Thr Pro
Gly Lys38521369PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 21Thr Gln Asp Cys Ser Phe Gln His
Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser
Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala
Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met
Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val
Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val
Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105
110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro
Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
Gln Gly Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 165 170 175Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 180 185 190Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 195 200 205Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 210 215 220Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu225 230
235 240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 260 265 270Lys Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Ser 275 280 285Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Val Pro Val Leu305 310 315 320Asp Ser Asp Gly
Ser Phe Arg Leu Ala Ser Tyr Leu Thr Val Asp Lys 325 330 335Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 340 345
350Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
355 360 365Lys22369PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 22Thr Gln Asp Cys Ser Phe Gln Tyr
Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser
Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala
Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met
Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val
Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val
Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105
110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro
Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
Gln Gly Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 165 170 175Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 180 185 190Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 195 200 205Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 210 215 220Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu225 230
235 240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 260 265 270Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 275 280 285Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu305 310 315 320Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 325 330 335Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 340 345
350Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
355 360 365Lys23369PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 23Thr Gln Asp Cys Ser Phe Gln His
Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser
Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala
Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met
Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val
Thr Glu Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val
Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105
110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro
Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
Gln Gly Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 165 170 175Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 180 185 190Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 195 200 205Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 210 215 220Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu225 230
235 240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 260 265 270Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 275 280 285Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu305 310 315 320Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 325 330 335Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 340 345
350Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
355 360 365Lys24369PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 24Thr Gln Asp Cys Ser Phe Gln Tyr
Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser
Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala
Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met
Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val
Thr Glu Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val
Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105
110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro
Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
Gln Gly Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 165 170 175Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 180 185 190Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 195 200 205Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 210 215 220Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu225 230
235 240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 260 265 270Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 275 280 285Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu305 310 315 320Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 325 330 335Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 340 345
350Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
355 360 365Lys25381PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 25Thr Gln Asp Cys Ser Phe Gln His
Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser
Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala
Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met
Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val
Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val
Gln Thr Asn Ile Ala Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105
110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ala Arg Cys Leu
115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro
Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Glu Ser Lys Tyr
Gly Pro Pro Cys145 150 155 160Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu 165 170 175Phe Pro Pro Lys Pro Lys Asp
Thr Leu Tyr Ile Thr Arg Glu Pro Glu 180 185 190Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 195 200 205Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 210 215 220Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu225 230
235 240Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys 245 250 255Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys 260 265 270Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser 275 280 285Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys 290 295 300Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln305 310 315 320Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 325 330 335Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 340 345
350Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
355 360 365His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 370
375 38026381PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 26Thr Gln Asp Cys Ser Phe Gln His
Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser
Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala
Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met
Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val
Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val
Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105
110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125Glu Leu Gln Cys Gln Pro Asp Ala Ala Ala Leu Pro Pro Pro
Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Glu Ser Lys Tyr
Gly Pro Pro Cys145 150 155 160Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu 165 170 175Phe Pro Pro Lys Pro Lys Asp
Thr Leu Tyr Ile Thr Arg Glu Pro Glu 180 185 190Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 195 200 205Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 210 215 220Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val Val Ser Val Leu225 230 235 240Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280 285Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 290 295
300Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln305 310 315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly 325 330 335Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg Trp Gln 340 345 350Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys 370 375 38027381PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ala Ala Ala Leu Pro Pro Pro Trp Ala 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Glu Ala Lys Tyr Gly Pro Pro Cys145 150 155
160Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
165 170 175Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
Pro Glu 180 185 190Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val Gln 195 200 205Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys 210 215 220Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu225 230 235 240Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 245 250 255Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 260 265 270Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280
285Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
290 295 300Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln305 310 315 320Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly 325 330 335Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln 340 345 350Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn 355 360 365His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys 370 375 380281107DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
28acacaggatt gcagcttcca gcacagcccc atcagcagcg atttcgccgt gaagatcaga
60gagctgagcg actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tctggaagcc acagctccca ccgctcctca
aggcggaccg 480tcagtcttcc tcttcccccc aaaacccaag gacaccctca
tgatctcccg gacccctgag 540gtcacatgcg tggtggtgga cgtgagccac
gaagaccctg aggtcaagtt caactggtac 600gtggacggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gtacaacagc 660acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
720tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 780gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggaggagatg 840accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 900gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 960gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1020caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 1080aagagcctct ccctgtctcc gggtaaa
1107291107DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 29acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggcct
gtggcgactg gtgttggctc agagatggat ggaacggctg 180aaaaccgtgg
ccggctctaa gatgcagggc ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatccgag cagctggtgg
ctctgaagcc ctggatcacc 360cggcagaact tctctcggtg tctggaactg
cagtgtcagc ccgactcttc taccctgcct 420ccaccttgga gccccagacc
tttggaagct accgctccaa cagctcctca aggcggccct 480tccgtgtttc
tgttccctcc aaagcctaag gacaccctga tgatctctcg gacccctgaa
540gtgacctgcg tggtggtgga tgtgtctcac gaggatcccg aagtgaagtt
caattggtac 600gtggacggcg tggaagtgca caacgccaag accaagccta
gagaggaaca gtacaactcc 660acctacagag tggtgtccgt gctgaccgtg
ctgcaccagg attggctgaa cggcaaagag 720tacaagtgca aggtgtccaa
caaggccctg cctgctccta tcgaaaagac catctccaag 780gccaagggcc
agcctaggga accccaggtt tacaccctgc cacctagccg ggaagagatg
840accaagaacc aggtgtccct gacctgcctg gtcaagggct tctacccctc
tgatatcgcc 900gtggaatggg agagcaatgg ccagcctgag aacaactaca
agaccacacc tcctgtgctg 960gactccgacg gctcattctt cctgtactcc
aagctgacag tggacaagtc cagatggcag 1020cagggcaacg tgttctcctg
ctccgtgatg cacgaggctc tgcacaacca ctacacccag 1080aagtccctgt
ctctgtcccc tggcaaa 1107301092DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 30acacaggatt
gcagcttcca gcacagcccc atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg
actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tctggaagcc acagctgggg gaccgtcagt
cttcctcttc 480cccccaaaac ccaaggacac cctcatgatc tcccggaccc
ctgaggtcac atgcgtggtg 540gtggacgtga gccacgaaga ccctgaggtc
aagttcaact ggtacgtgga cggcgtggag 600gtgcataatg ccaagacaaa
gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 660agcgtcctca
ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc
720tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa
agggcagccc 780cgagaaccac aggtgtacac cctgccccca tcccgggagg
agatgaccaa gaaccaggtc 840agcctgacct gcctggtcaa aggcttctat
cccagcgaca tcgccgtgga gtgggagagc 900aatgggcagc cggagaacaa
ctacaagacc acgcctcccg tgctggactc cgacggctcc 960ttcttcctct
acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc
1020tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag
cctctccctg 1080tctccgggta aa 1092311092DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
31acccaggact gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga
60gagctgtccg actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atctcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360cggcagaact
tctctcggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gccctagacc tttggaagct acagctggcg gcccaagcgt
gttcctgttt 480cctccaaagc ctaaggacac cctgatgatc tctcggaccc
ctgaagtgac ctgcgtggtg 540gtggatgtgt ctcacgagga tcccgaagtg
aagttcaatt ggtacgtgga cggcgtggaa 600gtgcacaacg ccaagaccaa
gcctagagag gaacagtaca actccaccta cagagtggtg 660tccgtgctga
ccgtgctgca ccaggattgg ctgaacggca aagagtacaa gtgcaaggtg
720tccaacaagg ccctgcctgc tcctatcgaa aagaccatct ccaaggccaa
gggccagcct 780agggaacctc aggtttacac cctgccacct agccgggaag
agatgaccaa aaaccaggtg 840tccctgacct gcctggtcaa gggcttctac
ccatccgata tcgccgtgga atgggagtct 900aacggccagc ctgagaacaa
ctacaagacc acacctcctg tgctggactc cgacggctca 960ttcttcctgt
actccaagct gacagtggac aagtctcggt ggcagcaggg caacgtgttc
1020tcctgttctg tgatgcacga ggccctgcac aaccactaca cccagaagtc
cctgtctctg 1080tcccctggca aa 1092321158DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
32acacaggatt gcagcttcca gcacagcccc atcagcagcg atttcgccgt gaagatcaga
60gagctgagcg actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tctggaagcc acagctccca ccgctcctca
agaatctaag 480tacggccctc cctgccctcc ttgcccagcc cctgaatttg
agggcggacc ctccgtgttc 540ctgttccccc caaagcccaa ggacaccctg
atgatcagcc ggacccccga agtgacctgc 600gtggtggtgg atgtgtccca
ggaagatccc gaggtgcagt tcaattggta cgtggacggc 660gtggaagtgc
acaacgccaa gaccaagccc agagaggaac agttcaacag cacctaccgg
720gtggtgtccg tgctgacagt gctgcaccag gactggctga acggcaaaga
gtacaagtgc 780aaggtgtcca acaagggcct gcccagctcc atcgagaaaa
ccatcagcaa ggccaagggc 840cagccccgcg aaccccaggt gtacacactg
cctccaagcc aggaagagat gaccaagaac 900caggtgtccc tgacctgtct
cgtgaaaggc ttctacccct ccgatatcgc cgtggaatgg 960gagagcaacg
gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac
1020ggctcattct tcctgtacag cagactgacc gtggacaaga gccggtggca
ggaaggcaac 1080gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc
actacaccca gaagtccctg 1140tctctgtccc tgggcaaa
1158331158DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 33acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactat
cctgtgaccg tggccagcaa cctgcaggat 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaaagactg 180aaaaccgtgg
ccggctccaa gatgcaggga ctgctggaaa gagtgaacac agagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatctgag cagctggtgg
ccctgaagcc ttggatcacc 360cggcagaact tctctcggtg cctggaactg
cagtgtcagc ctgattcttc taccctgcct 420ccaccttgga gccctagacc
tttggaggct acagctccta ccgctcctca agagtctaag 480tacggccctc
cttgtcctcc atgtcctgct ccagaatttg aaggcggccc aagcgtgttc
540ctgtttcctc caaagcctaa ggacaccctg atgatctctc ggacccctga
agtgacctgc 600gtggtggtgg atgtgtctca agaggacccc gaggtgcagt
tcaattggta cgtggacggc 660gtggaagtgc acaacgccaa gaccaagcct
agagaggaac agttcaactc cacctacaga 720gtggtgtccg tgctgaccgt
gctgcaccag gattggctga acggcaaaga gtacaagtgc 780aaggtgtcca
acaagggcct gcctagctcc atcgaaaaga ccatctccaa ggccaagggc
840cagccaagag aacctcaggt gtacacactg cctccaagcc aagaggaaat
gaccaagaac 900caggtgtccc tgacctgcct ggtcaagggc ttctacccat
ccgatatcgc cgtggaatgg 960gagtctaacg gccagcctga gaacaactac
aagaccacac ctcctgtgct ggactccgac 1020ggctccttct ttctgtactc
tcgcctgacc gtggacaagt ctagatggca agagggcaac 1080gtgttctcct
gctctgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
1140tctctgtccc tgggcaaa 1158341158DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 34acacaggatt
gcagcttcca gcacagcccc atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg
actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tctggaagcc acagctccca ccgctcctca
agagtctaag 480tacggccctc cttgtcctcc atgtcctgct ccagaagctg
ctggcggccc ttccgtgttt 540ctgttccctc caaagcctaa ggacaccctg
atgatctctc ggacccctga agtgacctgc 600gtggtggtgg atgtgtccca
agaggatccc gaggtgcagt tcaattggta cgtggacggc 660gtggaagtgc
acaacgccaa gaccaagcct agagaggaac agttcaactc cacctacaga
720gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga
gtacaagtgc 780aaggtgtcca acaagggcct gccttccagc atcgaaaaga
ccatctccaa ggccaagggc 840cagcctaggg aaccccaggt ttacaccctg
cctccaagcc aagaggaaat gaccaagaac 900caggtgtccc tgacctgcct
ggtcaagggc ttctaccctt ccgatatcgc cgtggaatgg 960gagagcaatg
gccagcctga gaacaactac aagaccacac ctcctgtgct ggactccgac
1020ggctccttct ttctgtactc ccgcctgacc gtggacaagt ccagatggca
agagggcaac 1080gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc
actacaccca gaagtccctg 1140tctctgtccc tgggcaaa
1158351158DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 35acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactat
cctgtgaccg tggccagcaa cctgcaggat 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaaagactg 180aaaaccgtgg
ccggctccaa gatgcaggga ctgctggaaa gagtgaacac agagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatctgag cagctggtgg
ccctgaagcc ttggatcacc 360cggcagaact tctctcggtg cctggaactg
cagtgtcagc ctgattcttc taccctgcct 420ccaccttgga gccctagacc
tttggaggct acagctccta ccgctcctca agagtctaag 480tacggccctc
cttgtcctcc atgtcctgct ccagaagctg ctggcggacc aagcgttttc
540ctgtttcctc caaagcctaa ggacaccctg atgatctctc ggacccctga
agtgacctgc 600gtggtggtgg atgtgtctca agaggacccc gaggtgcagt
tcaattggta cgtggacggc 660gtggaagtgc acaacgccaa gaccaagcct
agagaggaac agttcaactc cacctacaga 720gtggtgtccg tgctgaccgt
gctgcaccag gattggctga acggcaaaga gtacaagtgc 780aaggtgtcca
acaagggcct gcctagctcc atcgaaaaga ccatctccaa ggccaagggc
840cagccaagag aacctcaggt gtacacactg cctccaagcc aagaggaaat
gaccaagaac 900caggtgtccc tgacctgcct ggtcaagggc ttctacccat
ccgatatcgc cgtggaatgg 960gagtctaacg gccagcctga gaacaactac
aagaccacac ctcctgtgct ggactccgac 1020ggctccttct ttctgtactc
tcgcctgacc gtggacaagt ctagatggca agagggcaac 1080gtgttctcct
gctctgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
1140tctctgtccc tgggcaaa 1158361185DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 36atgacagttt
tggctccagc ttggtcccct acaacctacc tgctgctgct gttgctgctc 60tcctctggcc
tgtctggcac ccaggactgt tccttccagc actcccctat ctccagcgac
120ttcgccgtga agatcagaga gctgtccgac tatctgctgc aggactaccc
tgtgaccgtg 180gccagcaatc tgcaggacga agaactgtgt ggtggcctgt
ggcgactggt gttggctcag 240agatggatgg aacggctgaa aaccgtggcc
ggctctaaga tgcagggcct gctggaaaga 300gtgaacaccg agatccactt
cgtgaccaag tgcgcctttc agcctcctcc atcctgcctg 360agattcgtgc
agaccaatat cgcccggctg ctgcaagaga catccgagca gctggtggct
420ctgaagccct ggatcaccag acagaacttc gcccggtgtc tggaactgca
gtgtcagcct 480gacagctcta ccctgcctcc accttggagc cctagacctc
tggaagctac cgctccaacc 540gctcctcaag ggggaccgtc agtcttcctc
ttccccccaa aacccaagga caccctcatg 600atctcccgga cccctgaggt
cacatgcgtg gtggtggacg tgagccacga agaccctgag 660gtcaagttca
actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg
720gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct
gcaccaggac 780tggctgaatg gcaaggagta caagtgcaag gtctccaaca
aagccctccc agcccccatc 840gagaaaacca tctccaaagc caaagggcag
ccccgagaac cacaggtgta caccctgccc 900ccatcccggg aggagatgac
caagaaccag gtcagcctga cctgcctggt caaaggcttc 960tatcccagcg
acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag
1020accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa
gctcaccgtg 1080gacaagagca ggtggcagca ggggaacgtc ttctcatgct
ccgtgatgca tgaggctctg 1140cacaaccact acacgcagaa gagcctctcc
ctgtctccgg gtaaa 1185371107DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 37acccaggact
gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga 60gagctgtccg
actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaat 300atcgcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcgcccggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gccctagacc tttggaggct acagctccta ctgctcctca
aggcggccca 480agcgttttcc tgtttcctcc aaagcctaag gacaccctga
tgatctctcg gacccctgaa 540gtgacctgcg tggtggtgga tgtgtctcac
gaggatcccg aagtgaagtt caattggtac 600gtggacggcg tggaagtgca
caacgccaag accaagccta gagaggaaca gtacaactcc 660acctacagag
tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag
720tacaagtgca aggtgtccaa caaggccctg cctgctccta tcgaaaagac
catctccaag 780gccaagggcc agcctaggga acctcaggtt tacaccctgc
cacctagccg ggaagagatg 840accaaaaacc aggtgtccct gacctgcctg
gtcaagggct tctacccatc cgatatcgcc 900gtggaatggg agtctaacgg
ccagcctgag aacaactaca agaccacacc tcctgtgctg 960gactccgacg
gctcattctt cctgtactcc aagctgacag tggacaagtc tcggtggcag
1020cagggcaacg tgttctcctg ttctgtgatg cacgaggccc tgcacaacca
ctacacccag 1080aagtccctgt ctctgtcccc tggcaaa
1107381143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 38acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tctggaagcc acagctgagt ctaagtacgg ccctccttgt 480cctccatgtc
ctgctccaga agctgctggc ggcccttccg tgtttctgtt ccctccaaag
540cctaaggaca ccctgatgat ctctcggacc cctgaagtga cctgcgtggt
ggtggatgtg 600tcccaagagg atcccgaggt gcagttcaat tggtacgtgg
acggcgtgga agtgcacaac 660gccaagacca agcctagaga ggaacagttc
aactccacct acagagtggt gtccgtgctg 720accgtgctgc accaggattg
gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 780ggcctgcctt
ccagcatcga aaagaccatc tccaaggcca agggccagcc tagggaaccc
840caggtttaca ccctgcctcc aagccaagag gaaatgacca agaaccaggt
gtccctgacc 900tgcctggtca agggcttcta cccttccgat atcgccgtgg
aatgggagag caatggccag 960cctgagaaca actacaagac cacacctcct
gtgctggact ccgacggctc cttctttctg 1020tactcccgcc tgaccgtgga
caagtccaga tggcaagagg gcaacgtgtt ctcctgctcc 1080gtgatgcacg
aggccctgca caatcactac acccagaagt ccctgtctct gtccctgggc 1140aaa
1143391143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 39acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactat
cctgtgaccg tggccagcaa cctgcaggat 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaaagactg 180aaaaccgtgg
ccggctccaa gatgcaggga ctgctggaaa gagtgaacac agagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatctgag cagctggtgg
ccctgaagcc ttggatcacc 360cggcagaact tctctcggtg cctggaactg
cagtgtcagc ctgattcttc taccctgcct 420ccaccttgga gccctagacc
tctggaagct accgccgagt ctaagtacgg acctccttgt 480cctccatgtc
ctgctccaga agctgctggc ggaccaagcg ttttcctgtt tcctccaaag
540cctaaggaca ccctgatgat ctctcggacc cctgaagtga cctgcgtggt
ggtggatgtg 600tctcaagagg accccgaggt gcagttcaat tggtacgtgg
acggcgtgga agtgcacaac 660gccaagacca agcctagaga ggaacagttc
aactccacct acagagtggt gtccgtgctg 720accgtgctgc accaggattg
gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 780ggcctgccta
gctccatcga aaagaccatc tccaaggcca agggccagcc aagagaacct
840caggtgtaca cactgcctcc aagccaagag gaaatgacca agaaccaggt
gtccctgacc 900tgcctggtca agggcttcta cccatccgat atcgccgtgg
aatgggagtc taacggccag 960cctgagaaca actacaagac cacacctcct
gtgctggact ccgacggctc cttctttctg 1020tactctcgcc tgaccgtgga
caagtctaga tggcaagagg gcaacgtgtt ctcctgctct 1080gtgatgcacg
aggccctgca caaccactac acccagaagt ccctgtctct gtccctgggc 1140aaa
1143401077DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 40acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tgggggaccg tcagtcttcc tcttcccccc aaaacccaag 480gacaccctca
tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac
540gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca
taatgccaag 600acaaagccgc gggaggagca gtacaacagc acgtaccgtg
tggtcagcgt cctcaccgtc 660ctgcaccagg actggctgaa tggcaaggag
tacaagtgca aggtctccaa caaagccctc 720ccagccccca tcgagaaaac
catctccaaa gccaaagggc agccccgaga accacaggtg 780tacaccctgc
ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg
840gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg
gcagccggag 900aacaactaca agaccacgcc tcccgtgctg gactccgacg
gctccttctt cctctacagc 960aagctcaccg tggacaagag caggtggcag
caggggaacg tcttctcatg ctccgtgatg 1020catgaggctc tgcacaacca
ctacacgcag aagagcctct ccctgtctcc gggtaaa 1077411077DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
41acccaggact gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga
60gagctgtccg actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atctcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360cggcagaact
tctctcggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gtcctagacc tggcggacca agcgtgttcc tgtttcctcc
aaagcctaag 480gacaccctga tgatctctcg gacccctgaa gtgacctgcg
tggtggtgga tgtgtctcac 540gaggatcccg aagtgaagtt caattggtac
gtggacggcg tggaagtgca caacgccaag 600accaagccta gagaggaaca
gtacaactcc acctacagag tggtgtccgt gctgaccgtg 660ctgcaccagg
attggctgaa cggcaaagag tacaagtgca aggtgtccaa caaggccctg
720cctgctccta tcgaaaagac catctccaag gccaagggcc agcctaggga
acctcaggtt 780tacaccctgc cacctagccg ggaagagatg accaaaaacc
aggtgtccct gacctgcctg 840gtcaagggct tctacccatc cgatatcgcc
gtggaatggg agtctaacgg ccagcctgag 900aacaactaca agaccacacc
tcctgtgctg gactccgacg gctcattctt cctgtactcc 960aagctgacag
tggacaagtc tcggtggcag cagggcaacg tgttctcctg ttctgtgatg
1020cacgaggccc tgcacaacca ctacacccag aagtccctgt ctctgtcccc tggcaaa
1077421128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 42acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tgagtctaag tacggccctc cttgtcctcc atgtcctgct 480ccagaagctg
ctggcggccc ttccgtgttt ctgttccctc caaagcctaa ggacaccctg
540atgatctctc ggacccctga agtgacctgc gtggtggtgg atgtgtccca
agaggatccc 600gaggtgcagt tcaattggta cgtggacggc gtggaagtgc
acaacgccaa gaccaagcct 660agagaggaac agttcaactc cacctacaga
gtggtgtccg tgctgaccgt gctgcaccag 720gattggctga acggcaaaga
gtacaagtgc aaggtgtcca acaagggcct gccttccagc 780atcgaaaaga
ccatctccaa ggccaagggc cagcctaggg aaccccaggt ttacaccctg
840cctccaagcc aagaggaaat gaccaagaac caggtgtccc tgacctgcct
ggtcaagggc 900ttctaccctt ccgatatcgc cgtggaatgg gagagcaatg
gccagcctga gaacaactac 960aagaccacac ctcctgtgct ggactccgac
ggctccttct ttctgtactc ccgcctgacc 1020gtggacaagt ccagatggca
agagggcaac gtgttctcct gctccgtgat gcacgaggcc 1080ctgcacaatc
actacaccca gaagtccctg tctctgtccc tgggcaaa 1128431128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
43acccaggact gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga
60gagctgtccg actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atctcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360cggcagaact
tctctcggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gccctcggcc tgaatctaag tatggccctc cttgtcctcc
atgtcctgct 480ccagaagctg ctggcggacc aagcgttttc ctgtttcctc
caaagcctaa ggacaccctg 540atgatctctc ggacccctga agtgacctgc
gtggtggtgg atgtgtctca agaggacccc 600gaggtgcagt tcaattggta
cgtggacggc gtggaagtgc acaacgccaa gaccaagcct 660agagaggaac
agttcaactc cacctacaga gtggtgtccg tgctgaccgt gctgcaccag
720gattggctga acggcaaaga gtacaagtgc aaggtgtcca acaagggcct
gcctagctcc 780atcgaaaaga ccatctccaa ggccaagggc cagccaagag
aacctcaggt gtacacactg 840cctccaagcc aagaggaaat gaccaagaac
caggtgtccc tgacctgcct ggtcaagggc 900ttctacccat ccgatatcgc
cgtggaatgg gagtctaacg gccagcctga gaacaactac 960aagaccacac
ctcctgtgct ggactccgac ggctccttct ttctgtactc tcgcctgacc
1020gtggacaagt ctagatggca agagggcaac gtgttctcct gctctgtgat
gcacgaggcc 1080ctgcacaacc actacaccca gaagtccctg tctctgtccc tgggcaaa
1128441107DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 44acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tctggaagcc acagctccca ccgctcctca aggcggaccg 480tcagtcttcc
tcttcccccc aaaacccaag gacaccctct acatcacccg ggaacctgag
540gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt
caactggtac 600gtggacggcg tggaggtgca taatgccaag acaaagccgc
gggaggagca gtacaacagc 660acgtaccgtg tggtcagcgt cctcaccgtc
ctgcaccagg actggctgaa tggcaaggag 720tacaagtgca aggtctccaa
caaagccctc ccagccccca tcgagaaaac catctccaaa 780gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg
840accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag
cgacatcgcc 900gtggagtggg agagcaatgg gcagccggag aacaactaca
agaccacgcc tcccgtgctg 960gactccgacg gctccttctt cctctacagc
aagctcaccg tggacaagag caggtggcag 1020caggggaacg tcttctcatg
ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1080aagagcctct
ccctgtctcc gggtaaa 1107451107DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 45acccaggact
gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga 60gagctgtccg
actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atctcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360cggcagaact
tctctcggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gccctagacc tttggaggct acagctccta ctgctcctca
aggcggccca 480agcgttttcc tgtttcctcc aaagcctaag gacaccctgt
acatcacccg cgagcctgaa 540gtgacatgcg tggtggtgga tgtgtcccac
gaggaccccg aagtgaagtt caattggtac 600gtggacggcg tggaagtgca
caacgccaag accaagccta gagaggaaca gtacaactcc 660acctacagag
tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag
720tacaagtgca aggtgtccaa caaggccctg cctgctccta tcgaaaagac
catctccaag 780gccaagggcc agcctaggga acctcaggtt tacaccctgc
cacctagccg ggaagagatg 840accaaaaacc aggtgtccct gacctgcctg
gtcaagggct tctacccatc cgatatcgcc 900gtggaatggg agtctaacgg
ccagcctgag aacaactaca agaccacacc tcctgtgctg 960gactccgacg
gctcattctt cctgtactcc aagctgacag tggacaagtc tcggtggcag
1020cagggcaacg tgttctcctg ttctgtgatg cacgaggccc tgcacaacca
ctacacccag 1080aagtccctgt ctctgtcccc tggcaaa
1107461092DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 46acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tctggaagcc acagctgggg gaccgtcagt cttcctcttc 480cccccaaaac
ccaaggacac cctctacatc acccgggaac ctgaggtcac atgcgtggtg
540gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga
cggcgtggag 600gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
acagcacgta ccgtgtggtc 660agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca aggagtacaa gtgcaaggtc 720tccaacaaag ccctcccagc
ccccatcgag aaaaccatct ccaaagccaa agggcagccc 780cgagaaccac
aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc
840agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga
gtgggagagc 900aatgggcagc cggagaacaa ctacaagacc acgcctcccg
tgctggactc cgacggctcc 960ttcttcctct acagcaagct caccgtggac
aagagcaggt ggcagcaggg gaacgtcttc 1020tcatgctccg tgatgcatga
ggctctgcac aaccactaca cgcagaagag cctctccctg 1080tctccgggta aa
1092471092DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 47acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactat
cctgtgaccg tggccagcaa cctgcaggat 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaaagactg 180aaaaccgtgg
ccggctccaa gatgcaggga ctgctggaaa gagtgaacac agagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatctgag cagctggtgg
ccctgaagcc ttggatcacc 360cggcagaact tctctcggtg cctggaactg
cagtgtcagc ctgattcttc taccctgcct 420ccaccttgga gccctagacc
tttggaagct acagctggcg gcccaagcgt gttcctgttt 480cctccaaagc
ctaaggacac cctgtacatc acccgcgagc ctgaagtgac atgcgtggtg
540gtggatgtgt cccacgagga ccccgaagtg aagttcaatt ggtacgtgga
cggcgtggaa 600gtgcacaacg ccaagaccaa gcctagagag gaacagtaca
actccaccta cagagtggtg 660tccgtgctga ccgtgctgca ccaggattgg
ctgaacggca aagagtacaa gtgcaaggtg 720tccaacaagg ccctgcctgc
tcctatcgaa aagaccatct ccaaggccaa gggccagcct 780agggaacctc
aggtttacac cctgccacct agccgggaag agatgaccaa aaaccaggtg
840tccctgacct gcctggtcaa gggcttctac ccatccgata tcgccgtgga
atgggagtct 900aacggccagc ctgagaacaa ctacaagacc acacctcctg
tgctggactc cgacggctca 960ttcttcctgt actccaagct gacagtggac
aagtctcggt ggcagcaggg caacgtgttc 1020tcctgttctg tgatgcacga
ggccctgcac aaccactaca cccagaagtc cctgtctctg 1080tcccctggca aa
1092481158DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 48acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tctggaagcc acagctccca ccgctcctca agaatctaag 480tacggccctc
cctgccctcc ttgcccagcc cctgaatttg agggcggacc ctccgtgttc
540ctgttccccc caaagcccaa ggacaccctg tacatcaccc gggaacccga
agtgacctgc 600gtggtggtgg atgtgtccca ggaagatccc gaggtgcagt
tcaattggta cgtggacggc 660gtggaagtgc acaacgccaa gaccaagccc
agagaggaac agttcaacag cacctaccgg 720gtggtgtccg tgctgacagt
gctgcaccag gactggctga acggcaaaga gtacaagtgc 780aaggtgtcca
acaagggcct gcccagctcc atcgagaaaa ccatcagcaa ggccaagggc
840cagccccgcg aaccccaggt gtacacactg cctccaagcc aggaagagat
gaccaagaac 900caggtgtccc tgacctgtct cgtgaaaggc ttctacccct
ccgatatcgc cgtggaatgg 960gagagcaacg gccagcccga gaacaactac
aagaccaccc cccctgtgct ggacagcgac 1020ggctcattct tcctgtacag
cagactgacc gtggacaaga gccggtggca ggaaggcaac 1080gtgttcagct
gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
1140tctctgtccc tgggcaaa 1158491158DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 49acccaggact
gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga 60gagctgtccg
actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atctcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360cggcagaact
tctctcggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gccctagacc tttggaggct acagctccta ccgctcctca
agagtctaag 480tacggccctc cttgtcctcc atgtcctgct
ccagaatttg aaggcggccc aagcgtgttc 540ctgtttcctc caaagcctaa
ggacaccctg tacatcaccc gcgagcctga agtgacatgc 600gtggtggtgg
atgtgtccca agaggacccc gaggtgcagt tcaattggta cgtggacggc
660gtggaagtgc acaacgccaa gaccaagcct agagaggaac agttcaactc
cacctacaga 720gtggtgtccg tgctgaccgt gctgcaccag gattggctga
acggcaaaga gtacaagtgc 780aaggtgtcca acaagggcct gcctagctcc
atcgaaaaga ccatctccaa ggccaagggc 840cagccaagag aacctcaggt
gtacacactg cctccaagcc aagaggaaat gaccaagaac 900caggtgtccc
tgacctgcct ggtcaagggc ttctacccat ccgatatcgc cgtggaatgg
960gagtctaacg gccagcctga gaacaactac aagaccacac ctcctgtgct
ggactccgac 1020ggctccttct ttctgtactc tcgcctgacc gtggacaagt
ctagatggca agagggcaac 1080gtgttctcct gctctgtgat gcacgaggcc
ctgcacaacc actacaccca gaagtccctg 1140tctctgtccc tgggcaaa
1158501158DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 50acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tctggaagcc acagctccca ccgctcctca agagtctaag 480tacggccctc
cttgtcctcc atgtcctgct ccagaagctg ctggcggccc ttccgtgttt
540ctgttccctc caaagcctaa ggacaccctg tacatcaccc gggaacccga
agtgacctgc 600gtggtggtgg atgtgtccca ggaagatccc gaggtgcagt
tcaattggta cgtggacggc 660gtggaagtgc acaacgccaa gaccaagccc
agagaggaac agttcaacag cacctaccgg 720gtggtgtccg tgctgacagt
gctgcaccag gactggctga acggcaaaga gtacaagtgc 780aaggtgtcca
acaagggcct gcccagctcc atcgagaaaa ccatcagcaa ggccaagggc
840cagccccgcg aaccccaggt gtacacactg cctccaagcc aggaagagat
gaccaagaac 900caggtgtccc tgacctgtct cgtgaaaggc ttctacccct
ccgatatcgc cgtggaatgg 960gagagcaacg gccagcccga gaacaactac
aagaccaccc cccctgtgct ggacagcgac 1020ggctcattct tcctgtacag
cagactgacc gtggacaaga gccggtggca ggaaggcaac 1080gtgttcagct
gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
1140tctctgtccc tgggcaaa 1158511158DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 51acccaggact
gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga 60gagctgtccg
actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atctcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360cggcagaact
tctctcggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gccctagacc tttggaggct acagctccta ccgctcctca
agagtctaag 480tacggccctc cttgtcctcc atgtcctgct ccagaagctg
ctggcggacc aagcgttttc 540ctgtttcctc caaagcctaa ggacaccctg
tacatcaccc gcgagcctga agtgacatgc 600gtggtggtgg atgtgtccca
agaggacccc gaggtgcagt tcaattggta cgtggacggc 660gtggaagtgc
acaacgccaa gaccaagcct agagaggaac agttcaactc cacctacaga
720gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga
gtacaagtgc 780aaggtgtcca acaagggcct gcctagctcc atcgaaaaga
ccatctccaa ggccaagggc 840cagccaagag aacctcaggt gtacacactg
cctccaagcc aagaggaaat gaccaagaac 900caggtgtccc tgacctgcct
ggtcaagggc ttctacccat ccgatatcgc cgtggaatgg 960gagtctaacg
gccagcctga gaacaactac aagaccacac ctcctgtgct ggactccgac
1020ggctccttct ttctgtactc tcgcctgacc gtggacaagt ctagatggca
agagggcaac 1080gtgttctcct gctctgtgat gcacgaggcc ctgcacaacc
actacaccca gaagtccctg 1140tctctgtccc tgggcaaa
1158521185DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 52atgacagttt tggctccagc ttggtcccct
acaacctacc tgctgctgct gttgctgctc 60tcctctggcc tgtctggcac ccaggactgt
tccttccagc actcccctat ctccagcgac 120ttcgccgtga agatcagaga
gctgtccgac tatctgctgc aggactaccc tgtgaccgtg 180gccagcaatc
tgcaggacga agaactgtgt ggtggcctgt ggcgactggt gttggctcag
240agatggatgg aacggctgaa aaccgtggcc ggctctaaga tgcagggcct
gctggaaaga 300gtgaacaccg agatccactt cgtgaccaag tgcgcctttc
agcctcctcc atcctgcctg 360agattcgtgc agaccaatat cgcccggctg
ctgcaagaga catccgagca gctggtggct 420ctgaagccct ggatcaccag
acagaacttc gcccggtgtc tggaactgca gtgtcagcct 480gacagctcta
ccctgcctcc accttggagc cctagacctc tggaagctac cgctccaacc
540gctcctcaag ggggaccgtc agtcttcctc ttccccccaa aacccaagga
caccctctac 600atcacccggg aacctgaggt cacatgcgtg gtggtggacg
tgagccacga agaccctgag 660gtcaagttca actggtacgt ggacggcgtg
gaggtgcata atgccaagac aaagccgcgg 720gaggagcagt acaacagcac
gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 780tggctgaatg
gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc
840gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta
caccctgccc 900ccatcccggg aggagatgac caagaaccag gtcagcctga
cctgcctggt caaaggcttc 960tatcccagcg acatcgccgt ggagtgggag
agcaatgggc agccggagaa caactacaag 1020accacgcctc ccgtgctgga
ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1080gacaagagca
ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg
1140cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa
1185531107DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 53acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactat
cctgtgaccg tggccagcaa cctgcaggat 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaaagactg 180aaaaccgtgg
ccggctccaa gatgcaggga ctgctggaaa gagtgaacac agagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaat 300atcgcccggc tgctgcaaga gacatctgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcgcccggtg cctggaactg
cagtgtcagc ctgattcttc taccctgcct 420ccaccttgga gccctagacc
tttggaggct acagctccta ctgctcctca aggcggccca 480agcgttttcc
tgtttcctcc aaagcctaag gacaccctgt acatcacccg cgagcctgaa
540gtgacatgcg tggtggtgga tgtgtcccac gaggaccccg aagtgaagtt
caattggtac 600gtggacggcg tggaagtgca caacgccaag accaagccta
gagaggaaca gtacaactcc 660acctacagag tggtgtccgt gctgaccgtg
ctgcaccagg attggctgaa cggcaaagag 720tacaagtgca aggtgtccaa
caaggccctg cctgctccta tcgaaaagac catctccaag 780gccaagggcc
agcctaggga acctcaggtt tacaccctgc cacctagccg ggaagagatg
840accaaaaacc aggtgtccct gacctgcctg gtcaagggct tctacccatc
cgatatcgcc 900gtggaatggg agtctaacgg ccagcctgag aacaactaca
agaccacacc tcctgtgctg 960gactccgacg gctcattctt cctgtactcc
aagctgacag tggacaagtc tcggtggcag 1020cagggcaacg tgttctcctg
ttctgtgatg cacgaggccc tgcacaacca ctacacccag 1080aagtccctgt
ctctgtcccc tggcaaa 1107541143DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 54acacaggatt
gcagcttcca gcacagcccc atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg
actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tctggaagcc acagctgagt ctaagtacgg
ccctccttgt 480cctccatgtc ctgctccaga agctgctggc ggcccttccg
tgtttctgtt ccctccaaag 540cccaaggaca ccctgtacat cacccgggaa
cccgaagtga cctgcgtggt ggtggatgtg 600tcccaggaag atcccgaggt
gcagttcaat tggtacgtgg acggcgtgga agtgcacaac 660gccaagacca
agcccagaga ggaacagttc aacagcacct accgggtggt gtccgtgctg
720acagtgctgc accaggactg gctgaacggc aaagagtaca agtgcaaggt
gtccaacaag 780ggcctgccca gctccatcga gaaaaccatc agcaaggcca
agggccagcc ccgcgaaccc 840caggtgtaca cactgcctcc aagccaggaa
gagatgacca agaaccaggt gtccctgacc 900tgtctcgtga aaggcttcta
cccctccgat atcgccgtgg aatgggagag caacggccag 960cccgagaaca
actacaagac caccccccct gtgctggaca gcgacggctc attcttcctg
1020tacagcagac tgaccgtgga caagagccgg tggcaggaag gcaacgtgtt
cagctgcagc 1080gtgatgcacg aggccctgca caaccactac acccagaagt
ccctgtctct gtccctgggc 1140aaa 1143551143DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
55acccaggact gctccttcca gcactcccct atctcttccg acttcgccgt gaagatcaga
60gagctgtccg actacctgct gcaggactat cctgtgaccg tggccagcaa cctgcaggat
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaaagactg 180aaaaccgtgg ccggctccaa gatgcaggga ctgctggaaa
gagtgaacac agagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atctcccggc tgctgcaaga
gacatctgag cagctggtgg ccctgaagcc ttggatcacc 360cggcagaact
tctctcggtg cctggaactg cagtgtcagc ctgattcttc taccctgcct
420ccaccttgga gccctagacc tctggaagct accgccgagt ctaagtacgg
acctccttgt 480cctccatgtc ctgctccaga agctgctggc ggaccaagcg
ttttcctgtt tcctccaaag 540cctaaggaca ccctgtacat cacccgcgag
cctgaagtga catgcgtggt ggtggatgtg 600tcccaagagg accccgaggt
gcagttcaat tggtacgtgg acggcgtgga agtgcacaac 660gccaagacca
agcctagaga ggaacagttc aactccacct acagagtggt gtccgtgctg
720accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt
gtccaacaag 780ggcctgccta gctccatcga aaagaccatc tccaaggcca
agggccagcc aagagaacct 840caggtgtaca cactgcctcc aagccaagag
gaaatgacca agaaccaggt gtccctgacc 900tgcctggtca agggcttcta
cccatccgat atcgccgtgg aatgggagtc taacggccag 960cctgagaaca
actacaagac cacacctcct gtgctggact ccgacggctc cttctttctg
1020tactctcgcc tgaccgtgga caagtctaga tggcaagagg gcaacgtgtt
ctcctgctct 1080gtgatgcacg aggccctgca caaccactac acccagaagt
ccctgtctct gtccctgggc 1140aaa 1143561077DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
56acacaggatt gcagcttcca gcacagcccc atcagcagcg atttcgccgt gaagatcaga
60gagctgagcg actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tgggggaccg tcagtcttcc tcttcccccc
aaaacccaag 480gacaccctct acatcacccg ggaacctgag gtcacatgcg
tggtggtgga cgtgagccac 540gaagaccctg aggtcaagtt caactggtac
gtggacggcg tggaggtgca taatgccaag 600acaaagccgc gggaggagca
gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 660ctgcaccagg
actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc
720ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga
accacaggtg 780tacaccctgc ccccatcccg ggaggagatg accaagaacc
aggtcagcct gacctgcctg 840gtcaaaggct tctatcccag cgacatcgcc
gtggagtggg agagcaatgg gcagccggag 900aacaactaca agaccacgcc
tcccgtgctg gactccgacg gctccttctt cctctacagc 960aagctcaccg
tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg
1020catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa
1077571077DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 57acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactat
cctgtgaccg tggccagcaa cctgcaggat 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaaagactg 180aaaaccgtgg
ccggctccaa gatgcaggga ctgctggaaa gagtgaacac agagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatctgag cagctggtgg
ccctgaagcc ttggatcacc 360cggcagaact tctctcggtg cctggaactg
cagtgtcagc ctgattcttc taccctgcct 420ccaccttgga gtcctagacc
tggcggacca agcgtgttcc tgtttcctcc aaagcctaag 480gacaccctgt
acatcacccg cgagcctgaa gtgacatgcg tggtggtgga tgtgtcccac
540gaggaccccg aagtgaagtt caattggtac gtggacggcg tggaagtgca
caacgccaag 600accaagccta gagaggaaca gtacaactcc acctacagag
tggtgtccgt gctgaccgtg 660ctgcaccagg attggctgaa cggcaaagag
tacaagtgca aggtgtccaa caaggccctg 720cctgctccta tcgaaaagac
catctccaag gccaagggcc agcctaggga acctcaggtt 780tacaccctgc
cacctagccg ggaagagatg accaaaaacc aggtgtccct gacctgcctg
840gtcaagggct tctacccatc cgatatcgcc gtggaatggg agtctaacgg
ccagcctgag 900aacaactaca agaccacacc tcctgtgctg gactccgacg
gctcattctt cctgtactcc 960aagctgacag tggacaagtc tcggtggcag
cagggcaacg tgttctcctg ttctgtgatg 1020cacgaggccc tgcacaacca
ctacacccag aagtccctgt ctctgtcccc tggcaaa 1077581128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
58acacaggatt gcagcttcca gcacagcccc atcagcagcg atttcgccgt gaagatcaga
60gagctgagcg actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tgagtctaag tacggccctc cttgtcctcc
atgtcctgct 480ccagaagctg ctggcggccc ttccgtgttt ctgttccctc
caaagcctaa ggacaccctg 540tacatcaccc gggaacccga agtgacctgc
gtggtggtgg atgtgtccca ggaagatccc 600gaggtgcagt tcaattggta
cgtggacggc gtggaagtgc acaacgccaa gaccaagccc 660agagaggaac
agttcaacag cacctaccgg gtggtgtccg tgctgacagt gctgcaccag
720gactggctga acggcaaaga gtacaagtgc aaggtgtcca acaagggcct
gcccagctcc 780atcgagaaaa ccatcagcaa ggccaagggc cagccccgcg
aaccccaggt gtacacactg 840cctccaagcc aggaagagat gaccaagaac
caggtgtccc tgacctgtct cgtgaaaggc 900ttctacccct ccgatatcgc
cgtggaatgg gagagcaacg gccagcccga gaacaactac 960aagaccaccc
cccctgtgct ggacagcgac ggctcattct tcctgtacag cagactgacc
1020gtggacaaga gccggtggca ggaaggcaac gtgttcagct gcagcgtgat
gcacgaggcc 1080ctgcacaacc actacaccca gaagtccctg tctctgtccc tgggcaaa
1128591128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 59acccaggact gctccttcca gcactcccct
atctcttccg acttcgccgt gaagatcaga 60gagctgtccg actacctgct gcaggactat
cctgtgaccg tggccagcaa cctgcaggat 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaaagactg 180aaaaccgtgg
ccggctccaa gatgcaggga ctgctggaaa gagtgaacac agagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatctgag cagctggtgg
ccctgaagcc ttggatcacc 360cggcagaact tctctcggtg cctggaactg
cagtgtcagc ctgattcttc taccctgcct 420ccaccttgga gccctcggcc
tgaatctaag tatggccctc cttgtcctcc atgtcctgct 480ccagaagctg
ctggcggacc aagcgttttc ctgtttcctc caaagcctaa ggacaccctg
540tacatcaccc gcgagcctga agtgacatgc gtggtggtgg atgtgtccca
agaggacccc 600gaggtgcagt tcaattggta cgtggacggc gtggaagtgc
acaacgccaa gaccaagcct 660agagaggaac agttcaactc cacctacaga
gtggtgtccg tgctgaccgt gctgcaccag 720gattggctga acggcaaaga
gtacaagtgc aaggtgtcca acaagggcct gcctagctcc 780atcgaaaaga
ccatctccaa ggccaagggc cagccaagag aacctcaggt gtacacactg
840cctccaagcc aagaggaaat gaccaagaac caggtgtccc tgacctgcct
ggtcaagggc 900ttctacccat ccgatatcgc cgtggaatgg gagtctaacg
gccagcctga gaacaactac 960aagaccacac ctcctgtgct ggactccgac
ggctccttct ttctgtactc tcgcctgacc 1020gtggacaagt ctagatggca
agagggcaac gtgttctcct gctctgtgat gcacgaggcc 1080ctgcacaacc
actacaccca gaagtccctg tctctgtccc tgggcaaa 1128601128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
60acccaggact gttccttcca gcactcccct atctccagcg acttcgccgt gaagatcaga
60gagctgtccg actatctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggcct gtggcgactg gtgttggctc agagatggat
ggaacggctg 180aaaaccgtgg ccggctctaa gatgcagggc ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt tcagcctcct
ccatcctgcc tgagattcgt gcagaccaat 300atcgcccggc tgctgcaaga
gacatccgag cagctggtgg ctctgaagcc ctggatcacc 360agacagaact
tcgcccggtg tctggaactg cagtgtcagc ctgacagctc taccctgcct
420ccaccttgga gccctagacc tgagtctaag tacggccctc cttgtcctcc
atgtcctgct 480ccagaagctg ctggcggccc ttccgtgttt ctgttccctc
caaagcctaa ggacaccctg 540atgatctctc ggacccctga agtgacctgc
gtggtggtgg atgtgtccca agaggatccc 600gaggtgcagt tcaattggta
cgtggacggc gtggaagtgc acaacgccaa gaccaagcct 660agagaggaac
agttcaactc cacctacaga gtggtgtccg tgctgaccgt gctgcaccag
720gattggctga acggcaaaga gtacaagtgc aaggtgtcca acaagggcct
gccttccagc 780atcgaaaaga ccatctccaa ggccaagggc cagcctaggg
aaccccaggt ttacaccctg 840cctccaagcc aagaggaaat gaccaagaac
caggtgtccc tgacctgcct ggtcaagggc 900ttctaccctt ccgatatcgc
cgtggaatgg gagagcaatg gccagcctga gaacaactac 960aagaccacac
ctcctgtgct ggactccgac ggctccttct ttctgtactc ccgcctgacc
1020gtggacaagt ccagatggca agagggcaac gtgttctcct gctccgtgat
gcacgaggcc 1080ctgcacaatc actacaccca gaagtccctg tctctgtccc tgggcaaa
1128611128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 61acccaggact gttccttcca gcactcccct
atctccagcg acttcgccgt gaagatcaga 60gagctgtccg actatctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggcct
gtggcgactg gtgttggctc agagatggat ggaacggctg 180aaaaccgtgg
ccggctctaa gatgcagggc ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaat 300atcgcccggc tgctgcaaga gacatccgag cagctggtgg
ctctgaagcc ctggatcacc 360agacagaact tcgcccggtg tctggaactg
cagtgtcagc ctgacagctc taccctgcct 420ccaccttgga gccctagacc
tgagtctaag tacggccctc cttgtcctcc atgtcctgct 480ccagaagctg
ctggcggccc ttccgtgttt ctgttccctc caaagcccaa ggacaccctg
540tacatcaccc gggaacccga agtgacctgc
gtggtggtgg atgtgtccca ggaagatccc 600gaggtgcagt tcaattggta
cgtggacggc gtggaagtgc acaacgccaa gaccaagccc 660agagaggaac
agttcaacag cacctaccgg gtggtgtccg tgctgacagt gctgcaccag
720gactggctga acggcaaaga gtacaagtgc aaggtgtcca acaagggcct
gcccagctcc 780atcgagaaaa ccatcagcaa ggccaagggc cagccccgcg
aaccccaggt gtacacactg 840cctccaagcc aggaagagat gaccaagaac
caggtgtccc tgacctgtct cgtgaaaggc 900ttctacccct ccgatatcgc
cgtggaatgg gagagcaacg gccagcccga gaacaactac 960aagaccaccc
cccctgtgct ggacagcgac ggctcattct tcctgtacag cagactgacc
1020gtggacaaga gccggtggca ggaaggcaac gtgttcagct gcagcgtgat
gcacgaggcc 1080ctgcacaacc actacaccca gaagtccctg tctctgtccc tgggcaaa
1128621167DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 62acccctgact gctacttcag ccactctcct
atctccagca acttcaaagt gaagttccgc 60gagctgaccg accatctgct gaaggactat
cctgtgaccg tggccgtgaa cctgcaggac 120gaaaagcact gcaaggccct
gtggtccctg tttctggccc agagatggat cgagcagctg 180aaaaccgtgg
ctggctccaa gatgcagacc ctgctggaag atgtgaacac cgagatccac
240ttcgtgacca gctgcacctt ccagcctctg cctgagtgcc tgagattcgt
gcagaccaac 300atctcccacc tgttgaagga cacatgcacc cagctgctgg
ccctgaagcc ttgtatcggc 360aaggcctgcc agaacttctc ccggtgtctg
gaagtgcagt gccagcctga ctcctccaca 420ctgctgccac ctagaagccc
tatcgctctg gaagctaccg agctgcctga gcctagaggc 480cctaccatca
agccttgtcc tccatgcaag tgccccgctc ctaatgctgc tggtggccct
540tccgtgttca tcttcccacc taagatcaag gacgtgctga tgatctccct
gtctcctatc 600gtgacctgcg tggtggtgga cgtgtccgag gatgatcctg
acgtgcagat cagttggttc 660gtgaacaacg tggaagtgca caccgctcag
acccagacac acagagagga ctacaacagc 720accctgagag tggtgtctgc
cctgcctatc cagcaccagg attggatgtc cggcaaagaa 780ttcaagtgca
aagtgaacaa caaggacctg ggcgctccca tcgagcggac catctctaag
840cctaagggat ccgtcagagc ccctcaggtg tacgttctgc ctccacctga
ggaagagatg 900accaagaaac aagtgaccct gacctgcatg gtcaccgact
tcatgcccga ggacatctac 960gtggaatgga ccaacaacgg caagaccgag
ctgaactaca agaacaccga gcctgtgctg 1020gactccgacg gctcctactt
catgtactcc aagctgcgcg tcgagaagaa gaactgggtc 1080gagagaaact
cctactcctg ctccgtggtg cacgagggcc tgcacaatca ccacaccacc
1140aagtccttct ctcggacccc tggcaaa 1167631167DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
63acccctgact gctacttcag ccactctcct atctccagca acttcaaagt gaagttccgc
60gagctgaccg accatctgct gaaggactat cctgtgaccg tggccgtgaa cctgcaggac
120gaaaagcact gcaaggccct gtggtccctg tttctggccc agagatggat
cgagcagctg 180aaaaccgtgg ctggctccaa gatgcagacc ctgctggaag
atgtgaacac cgagatccac 240ttcgtgacca gctgcacctt ccagcctctg
cctgagtgcc tgagattcgt gcagaccaac 300atctcccacc tgttgaagga
cacatccacc cagctgctgg ccctgaagcc ttgtatcggc 360aaggcctgcc
agaacttctc ccggtgtctg gaagtgcagt gccagcctga ctcctccaca
420ctgctgccac ctagaagccc tatcgctctg gaagctaccg agctgcctga
gcctagaggc 480cctaccatca agccttgtcc tccatgcaag tgccccgctc
ctaatgctgc tggtggccct 540tccgtgttca tcttcccacc taagatcaag
gacgtgctga tgatctccct gtctcctatc 600gtgacctgcg tggtggtgga
cgtgtccgag gatgatcctg acgtgcagat cagttggttc 660gtgaacaacg
tggaagtgca caccgctcag acccagacac acagagagga ctacaacagc
720accctgagag tggtgtctgc cctgcctatc cagcaccagg attggatgtc
cggcaaagaa 780ttcaagtgca aagtgaacaa caaggacctg ggcgctccca
tcgagcggac catctctaag 840cctaagggat ccgtcagagc ccctcaggtg
tacgttctgc ctccacctga ggaagagatg 900accaagaaac aagtgaccct
gacctgcatg gtcaccgact tcatgcccga ggacatctac 960gtggaatgga
ccaacaacgg caagaccgag ctgaactaca agaacaccga gcctgtgctg
1020gactccgacg gctcctactt catgtactcc aagctgcgcg tcgagaagaa
gaactgggtc 1080gagagaaact cctactcctg ctccgtggtg cacgagggcc
tgcacaatca ccacaccacc 1140aagtccttct ctcggacccc tggcaaa
1167641107DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 64acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tctggaagcc acagctccca ccgctcctca aggcggaccg 480tcagtctttc
tgttccctcc aaagcctaag gacaccctga tgatcagcag aacccctgaa
540gtgacctgcg tggtggtgga tgtgtcccac gaggatcccg aagtgaagtt
caattggtac 600gtggacggcg tggaagtgca caacgccaag accaagccta
gagaggaaca gtacaacagc 660acctacagag tggtgtccgt gctgaccgtg
ctgcaccagg attggctgaa cggcaaagag 720tacaagtgca aggtgtccaa
caaggccctg cctgctccta tcgagaaaac catcagcaag 780gccaagggcc
agcctaggga accccaggtg tacacaaagc ctccaagccg ggaagagatg
840accaagaacc aggtgtccct gagctgcctg gtcaagggct tttaccccag
cgacattgcc 900gtggaatggg agagcaatgg ccagcctgag aacaactaca
agaccaccgt gcctgtgctg 960gacagcgacg gctcttttag actggccagc
tacctgaccg tggacaagag cagatggcag 1020cagggcaacg tgttcagctg
cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1080aagtccctgt
ctctgagccc cggcaaa 1107651107DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 65acacaggatt
gcagcttcca gtacagcccc atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg
actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgacca agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tctggaagcc acagctccca ccgctcctca
aggcggaccg 480tcagtcttcc tcttcccccc aaaacccaag gacaccctca
tgatctcccg gacccctgag 540gtcacatgcg tggtggtgga cgtgagccac
gaagaccctg aggtcaagtt caactggtac 600gtggacggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gtacaacagc 660acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
720tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 780gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggaggagatg 840accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 900gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 960gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1020caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 1080aagagcctct ccctgtctcc gggtaaa
1107661107DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 66acacaggatt gcagcttcca gcacagcccc
atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg actacctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggact
gtggcgactg gtgctggctc agagatggat ggaacggctg 180aaaacagtgg
ccggcagcaa gatgcaggga ctgctggaaa gagtgaacac cgagatccac
240ttcgtgaccg agtgcgcctt ccagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atcagcagac tgctgcaaga gacaagcgag cagctggtgg
ccctgaagcc ttggatcacc 360agacagaact tcagccggtg cctggaactg
cagtgtcagc ccgatagcag cacactgcct 420ccgccttgga gtcctagacc
tctggaagcc acagctccca ccgctcctca aggcggaccg 480tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag
540gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt
caactggtac 600gtggacggcg tggaggtgca taatgccaag acaaagccgc
gggaggagca gtacaacagc 660acgtaccgtg tggtcagcgt cctcaccgtc
ctgcaccagg actggctgaa tggcaaggag 720tacaagtgca aggtctccaa
caaagccctc ccagccccca tcgagaaaac catctccaaa 780gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg
840accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag
cgacatcgcc 900gtggagtggg agagcaatgg gcagccggag aacaactaca
agaccacgcc tcccgtgctg 960gactccgacg gctccttctt cctctacagc
aagctcaccg tggacaagag caggtggcag 1020caggggaacg tcttctcatg
ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1080aagagcctct
ccctgtctcc gggtaaa 1107671107DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 67acacaggatt
gcagcttcca gtacagcccc atcagcagcg atttcgccgt gaagatcaga 60gagctgagcg
actacctgct gcaggactac cctgtgaccg tggccagcaa tctgcaggac
120gaagaactgt gtggtggact gtggcgactg gtgctggctc agagatggat
ggaacggctg 180aaaacagtgg ccggcagcaa gatgcaggga ctgctggaaa
gagtgaacac cgagatccac 240ttcgtgaccg agtgcgcctt ccagcctcct
ccatcctgcc tgagattcgt gcagaccaac 300atcagcagac tgctgcaaga
gacaagcgag cagctggtgg ccctgaagcc ttggatcacc 360agacagaact
tcagccggtg cctggaactg cagtgtcagc ccgatagcag cacactgcct
420ccgccttgga gtcctagacc tctggaagcc acagctccca ccgctcctca
aggcggaccg 480tcagtcttcc tcttcccccc aaaacccaag gacaccctca
tgatctcccg gacccctgag 540gtcacatgcg tggtggtgga cgtgagccac
gaagaccctg aggtcaagtt caactggtac 600gtggacggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gtacaacagc 660acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
720tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 780gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggaggagatg 840accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 900gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 960gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1020caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 1080aagagcctct ccctgtctcc gggtaaa
1107681143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 68acccaggact gttccttcca gcactcccct
atctccagcg acttcgccgt gaagatcaga 60gagctgtccg actatctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggcct
gtggcgactg gtgttggctc agagatggat ggaacggctg 180aaaaccgtgg
ccggctctaa gatgcagggc ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaat 300atcgcccggc tgctgcaaga gacatccgag cagctggtgg
ctctgaagcc ctggatcacc 360agacagaact tcgcccggtg tctggaactg
cagtgtcagc ctgacagctc taccctgcct 420ccaccttgga gccctagacc
tctggaagct accgctgagt ctaagtacgg ccctccttgt 480cctccatgtc
ctgctccaga agctgctggc ggcccttccg tgtttctgtt ccctccaaag
540cccaaggaca ccctgtacat cacccgggaa cccgaagtga cctgcgtggt
ggtggatgtg 600tcccaggaag atcccgaggt gcagttcaat tggtacgtgg
acggcgtgga agtgcacaac 660gccaagacca agcccagaga ggaacagttc
aacagcacct accgggtggt gtccgtgctg 720acagtgctgc accaggactg
gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 780ggcctgccca
gctccatcga gaaaaccatc agcaaggcca agggccagcc ccgcgaaccc
840caggtgtaca cactgcctcc aagccaggaa gagatgacca agaaccaggt
gtccctgacc 900tgtctcgtga aaggcttcta cccctccgat atcgccgtgg
aatgggagag caacggccag 960cccgagaaca actacaagac caccccccct
gtgctggaca gcgacggctc attcttcctg 1020tacagcagac tgaccgtgga
caagagccgg tggcaggaag gcaacgtgtt cagctgcagc 1080gtgatgcacg
aggccctgca caaccactac acccagaagt ccctgtctct gtccctgggc 1140aaa
1143691143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 69acccaggact gttccttcca gcactcccct
atctccagcg acttcgccgt gaagatcaga 60gagctgtccg actatctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggcct
gtggcgactg gtgttggctc agagatggat ggaacggctg 180aaaaccgtgg
ccggctctaa gatgcagggc ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatccgag cagctggtgg
ctctgaagcc ctggatcacc 360cggcagaact tctctcggtg tctggaactg
cagtgtcagc ctgatgctgc cgctttgcct 420ccaccttgga gccctagacc
tctggaagct accgccgagt ctaagtacgg acctccttgt 480cctccatgtc
ctgctccaga agctgctggc ggcccttccg tgtttctgtt ccctccaaag
540cccaaggaca ccctgtacat cacccgggaa cccgaagtga cctgcgtggt
ggtggatgtg 600tcccaggaag atcccgaggt gcagttcaat tggtacgtgg
acggcgtgga agtgcacaac 660gccaagacca agcccagaga ggaacagttc
aacagcacct accgggtggt gtccgtgctg 720acagtgctgc accaggactg
gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 780ggcctgccca
gctccatcga gaaaaccatc agcaaggcca agggccagcc ccgcgaaccc
840caggtgtaca cactgcctcc aagccaggaa gagatgacca agaaccaggt
gtccctgacc 900tgtctcgtga aaggcttcta cccctccgat atcgccgtgg
aatgggagag caacggccag 960cccgagaaca actacaagac caccccccct
gtgctggaca gcgacggctc attcttcctg 1020tacagcagac tgaccgtgga
caagagccgg tggcaggaag gcaacgtgtt cagctgcagc 1080gtgatgcacg
aggccctgca caaccactac acccagaagt ccctgtctct gtccctgggc 1140aaa
1143701143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 70acccaggact gttccttcca gcactcccct
atctccagcg acttcgccgt gaagatcaga 60gagctgtccg actatctgct gcaggactac
cctgtgaccg tggccagcaa tctgcaggac 120gaagaactgt gtggtggcct
gtggcgactg gtgttggctc agagatggat ggaacggctg 180aaaaccgtgg
ccggctctaa gatgcagggc ctgctggaaa gagtgaacac cgagatccac
240ttcgtgacca agtgcgcctt tcagcctcct ccatcctgcc tgagattcgt
gcagaccaac 300atctcccggc tgctgcaaga gacatccgag cagctggtgg
ctctgaagcc ctggatcacc 360cggcagaact tctctcggtg tctggaactg
cagtgtcagc ctgatgctgc cgctttgcct 420cctccttggg ctcctcgacc
tctggaagct acagccgagg ctaagtatgg ccctccttgt 480cctccatgtc
ctgctccaga agctgctggc ggcccttccg tgtttctgtt ccctccaaag
540cccaaggaca ccctgtacat cacccgggaa cccgaagtga cctgcgtggt
ggtggatgtg 600tcccaggaag atcccgaggt gcagttcaat tggtacgtgg
acggcgtgga agtgcacaac 660gccaagacca agcccagaga ggaacagttc
aacagcacct accgggtggt gtccgtgctg 720acagtgctgc accaggactg
gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 780ggcctgccca
gctccatcga gaaaaccatc agcaaggcca agggccagcc ccgcgaaccc
840caggtgtaca cactgcctcc aagccaggaa gagatgacca agaaccaggt
gtccctgacc 900tgtctcgtga aaggcttcta cccctccgat atcgccgtgg
aatgggagag caacggccag 960cccgagaaca actacaagac caccccccct
gtgctggaca gcgacggctc attcttcctg 1020tacagcagac tgaccgtgga
caagagccgg tggcaggaag gcaacgtgtt cagctgcagc 1080gtgatgcacg
aggccctgca caaccactac acccagaagt ccctgtctct gtccctgggc 1140aaa
114371147PRTHomo sapiens 71Thr Gln Asp Cys Ser Phe Gln His Ser Pro
Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr
Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp
Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg
Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly
Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys
Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr
Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val
Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro14572148PRTHomo sapiens 72Thr Gln Asp Cys Ser
Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg
Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala
Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu
Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly
Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75
80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Leu14573149PRTHomo
sapiens 73Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp
Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp
Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys
Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg
Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg
Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln
Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg
Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro
Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln
Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro
Arg Pro Leu Glu14574150PRTHomo sapiens 74Thr Gln Asp Cys Ser Phe
Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu
Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val
Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser
Lys Met Gln Gly
Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys
Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr
Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val
Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala145 15075151PRTHomo sapiens 75Thr
Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10
15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val
20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr
Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr
Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro
Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln
Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr
Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro
Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Leu
Glu Ala Thr145 15076152PRTHomo sapiens 76Thr Gln Asp Cys Ser Phe
Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu
Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val
Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser
Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75
80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala145
15077153PRTHomo sapiens 77Thr Gln Asp Cys Ser Phe Gln His Ser Pro
Ile Ser Ser Asp Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr
Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp
Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg
Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly
Leu Leu Glu Arg Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys
Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr
Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val
Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120
125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro145 15078154PRTHomo
sapiens 78Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp
Phe Ala1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp
Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys
Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg
Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg
Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln
Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg
Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro
Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln
Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro
Arg Pro Leu Glu Ala Thr Ala Pro Thr145 15079155PRTHomo sapiens
79Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Pro Thr Ala145 150 15580156PRTHomo sapiens
80Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Pro Thr Ala Pro145 150 15581157PRTHomo sapiens
81Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1
5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
Thr Glu Ile His65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro
Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu 100 105 110Val Ala Leu Lys Pro Trp Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln
Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro
Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln145 150
1558217PRTUnknownDescription of Unknown Serum albumin signal
peptide 82Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser
Ala Tyr1 5 10 15Ser8326PRTUnknownDescription of Unknown FLT3L
signal peptide 83Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr
Tyr Leu Leu Leu1 5 10 15Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly 20
258427PRTUnknownDescription of Unknown FLT3L signal peptide 84Met
Thr Val Leu Ala Pro Ala Trp Ser Pro Asn Ser Ser Leu Leu Leu1 5 10
15Leu Leu Leu Leu Leu Ser Pro Cys Leu Arg Gly 20 25855PRTHomo
sapiens 85Pro Thr Ala Pro Gln1 5866PRTHomo sapiens 86Ala Pro Thr
Ala Pro Gln1 5877PRTHomo sapiens 87Thr Ala Pro Thr Ala Pro Gln1
5888PRTHomo sapiens 88Ala Thr Ala Pro Thr Ala Pro Gln1 5899PRTHomo
sapiens 89Glu Ala Thr Ala Pro Thr Ala Pro Gln1 59010PRTHomo sapiens
90Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln1 5 10917PRTHomo sapiens
91Pro Thr Ala Pro Gln Pro Pro1 5928PRTHomo sapiens 92Ala Pro Thr
Ala Pro Gln Pro Pro1 5939PRTHomo sapiens 93Thr Ala Pro Thr Ala Pro
Gln Pro Pro1 59410PRTHomo sapiens 94Ala Thr Ala Pro Thr Ala Pro Gln
Pro Pro1 5 109511PRTHomo sapiens 95Glu Ala Thr Ala Pro Thr Ala Pro
Gln Pro Pro1 5 109612PRTHomo sapiens 96Leu Glu Ala Thr Ala Pro Thr
Ala Pro Gln Pro Pro1 5 10975PRTHomo sapiens 97Glu Ser Lys Tyr Gly1
598159PRTMus musculus 98Thr Pro Asp Cys Tyr Phe Ser His Ser Pro Ile
Ser Ser Asn Phe Lys1 5 10 15Val Lys Phe Arg Glu Leu Thr Asp His Leu
Leu Lys Asp Tyr Pro Val 20 25 30Thr Val Ala Val Asn Leu Gln Asp Glu
Lys His Cys Lys Ala Leu Trp 35 40 45Ser Leu Phe Leu Ala Gln Arg Trp
Ile Glu Gln Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Thr Leu
Leu Glu Asp Val Asn Thr Glu Ile His65 70 75 80Phe Val Thr Ser Cys
Thr Phe Gln Pro Leu Pro Glu Cys Leu Arg Phe 85 90 95Val Gln Thr Asn
Ile Ser His Leu Leu Lys Asp Thr Cys Thr Gln Leu 100 105 110Leu Ala
Leu Lys Pro Cys Ile Gly Lys Ala Cys Gln Asn Phe Ser Arg 115 120
125Cys Leu Glu Val Gln Cys Gln Pro Asp Ser Ser Thr Leu Leu Pro Pro
130 135 140Arg Ser Pro Ile Ala Leu Glu Ala Thr Glu Leu Pro Glu Pro
Arg145 150 1559920PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMISC_FEATURE(1)..(20)This sequence may
encompass 3-4 "Gly Gly Gly Gly Ser" repeating units 99Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly
Gly Ser 2010012PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 100Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Pro Cys Pro1 5 1010115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 101Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10 15102232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
102Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1
5 10 15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 20 25 30Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 100 105 110Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155
160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser Leu Ser Pro Gly
Lys225 230103212PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 103Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu1 5 10 15Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 20 25 30His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 35 40 45Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 50 55 60Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn65 70 75 80Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 85 90 95Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 100 105
110Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
115 120 125Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 130 135 140Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro145 150 155 160Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 165 170 175Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 180 185 190Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 195 200 205Ser Pro Gly
Lys 210104212PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 104Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu1 5 10 15Tyr Ile Thr Arg Glu Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 20 25 30His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 35 40 45Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 50 55 60Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn65 70 75 80Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 85 90 95Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 100 105
110Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
115 120 125Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 130 135 140Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro145 150 155 160Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 165 170 175Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 180 185 190Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 195 200 205Ser Pro Gly
Lys 210105229PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 105Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe1 5 10 15Glu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100
105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln 130 135 140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215
220Leu Ser Leu Gly Lys225106229PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 106Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala1 5 10 15Ala Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln 130 135 140Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220Leu Ser Leu Gly Lys225107229PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
107Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1
5 10 15Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 20 25 30Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val
Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150 155
160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys22510815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Arg Arg Cys Pro Leu Tyr Ile Ser Tyr Asp Pro
Val Cys Arg Arg1 5 10 1510919PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 109Arg Arg Arg Arg Cys Pro
Leu Tyr Ile Ser Tyr Asp Pro Val Cys Arg1 5 10 15Arg Arg
Arg11020PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu 20
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