CN105816866B

CN105816866B - Immunogenic anti-inflammatory compositions

Info

Publication number: CN105816866B
Application number: CN201610090355.3A
Authority: CN
Inventors: 哈罗尔·戴维·贡; 萨利姆·丹吉
Original assignee: Qu Biologics Inc
Current assignee: Qu Biologics Inc
Priority date: 2010-07-26
Filing date: 2011-07-26
Publication date: 2020-03-06
Anticipated expiration: 2031-07-26
Also published as: CN105816865B; CN105816864A; CN105816863A; CN105816865A; CN105816866A

Abstract

The present invention provides methods of formulating anti-inflammatory compositions for treating inflammatory disease states in specific organs or tissues. The method comprises selecting at least one pathogen that is pathogenic in the specific organ or tissue; preparing an antigenic composition comprising antigenic determinants which together are specific for a pathogen; and formulating the antigenic composition for administration as an anti-inflammatory composition capable of inducing an anti-inflammatory response in said specific organ or tissue, wherein said disease state characterized by inflammation is not cancer. In an embodiment of the invention, the cancer is located in said specific organ or tissue.

Description

Immunogenic anti-inflammatory compositions

Technical Field

In various aspects, the invention relates to immunological therapies for treating disease states characterized by inflammation. In alternative embodiments, the present invention provides methods of formulating antigenic compositions for the treatment of inflammatory disease states.

Background

More than one third of the population in developed countries is diagnosed with cancer. More than one quarter of the people die from it. Therapies for cancer rely primarily on treatments such as surgery, chemotherapy, and radiation. However, while these methods are beneficial for certain types and stages of cancer, they have proven to have limited efficacy in many common types and stages of cancer. For example, surgical treatment of tumors requires complete removal of cancerous tissue to prevent recurrence. Also, radiation therapy requires complete destruction of the cancer cells. This is difficult because, in theory, a single malignant cell can proliferate enough to cause cancer recurrence. In addition, surgical treatment and radiation therapy involve localized areas of cancer and are relatively ineffective when the cancer metastasizes. Typically, surgery or radiation or both are used in combination with systemic methods such as chemotherapy. However, chemotherapy has nonspecific problems, problems with the coexistence of harmful side effects, and the possibility that cancer cells develop resistance to drugs.

The inherent disadvantages of chemotherapy have led to different attempts to restore aspects of the immune system to treat cancer. A subset of this work involves immunization with microbial vaccines. Despite the long history of this approach, as discussed in more detail below, the field is a very confusing confounding state, sometimes with dramatic success mixed with numerous failures that do not produce a comprehensive treatment that survives extensive clinical adoption testing.

Alternative methods for treating cancer have included therapies involving enhancing immune system function, such as cytokine therapy (e.g., recombinant interleukin 2 and gamma interferon for kidney cancer), dendritic cell therapy, autologous tumor vaccine therapy, genetically engineered vaccine therapy, lymphocyte therapy, and microbial vaccine therapy, the latter of which is believed to act on the host system in a non-specific manner. Microbial vaccines have been used to vaccinate individuals against pathogens associated with cancer, such as human papilloma virus. Immunostimulatory microbial vaccines that do not target cancer-inducing organisms, i.e., non-specific immunostimulatory vaccines, such as pyrogenic vaccines, have a long clinical history, which includes reports of success and failure in treating various cancers. For example, the Coley vaccine (a combination of Streptococcus pyogenes and Serratia marcescens) has been reported to be useful in the treatment of sarcomas and lymphomas (see, e.g., Nauts HC, Fowlergaa, Bogato FH. A review of the infection of bacterial infection and bacterial products [ Coley's toxins ] on bacterial tumors in man. acta Med and 1953; 145[ P. 276]: 5-103). Clinical trials have reported the benefits of Coley vaccine therapy for lymphomas and melanomas (see, e.g., Kempin S, Cirrincone C, Myers J et al: Combined modificationtherapy of advanced nodularia lymphamas: the role of nonpeptic immunology [ MBV ] as an immunogenic detector of response and subvalval. Proc Am Soc Clinoncol 1983; 24: 56; Kolmel KF, Vehmeyer K. treatment of advanced mammalian tumor by a pathogenic bacterial lysate: a pilot.

It has been shown that the efficacy of certain non-specific bacterial cancer vaccines is due to components or products of specific bacteria, such as bacterial DNA or endotoxin (LPS), or because they induce the expression of specific factors, such as Tumor Necrosis Factor (TNF) or interleukin-12. A relatively broad range of physiological mechanisms have been attributed to such treatments, ranging from the broad effects of fever to anti-angiogenic mechanisms. Based on these various principles, many microbial vaccines have been tested as general immunostimulants for the treatment of cancer. While most have shown negative results, a few have shown some compelling positive results in some cases, as discussed below.

Intradermal BCG (Mycobacterium bovis) vaccine therapy has been reported to be effective in treating the following cancers: stomach cancer (see, e.g., Ochia T, Sato J, Hayashi R, et al: Postopertideja great intestinal immunology of gastric cancer with BCG-cell walenoskeleton.three-to six-layer folow-up of a randomized clinical laboratory 1983; 14: 167) and colon cancer (Smith RE, Colangelol, Wiean HS, Begovic M, Wolman N. randomized tertiary of epithelial cancer in collagen. 10-Yeast result of NSABP clinical C-01.J.NCI 2004; 96[15]: 1128-32; Uyl-GroocMT, Mg, cell J.22 J.M., Mg. 9. faecal J.23. variance of yeast strain J.23. and growth J.23. organism J.23. III. the first culture J.23. the second culture J.23. the first culture J.23. the second J.A. the fifth culture of the fourth culture J.A. the fifth culture J.23. the fifth sample J.A. the fifth sample J.23. the fifth sample J.A. the fifth sample J.A. A. represents the fifth sample J.A.A.A. represents distinct culture J.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A. represents.

Mycobacterium w (Mycobacterium w) vaccine therapy combined with chemotherapy and radiation was found to significantly improve the quality of life and response to treatment in patients with lung cancer (see, e.g., Sun P, Dastida A. roll of Mycobacterium w as adjuvant therapy of non-small cell lung cancer. J Indian Med Assic 2003 Feb; 101[2]: 118-. Similarly, Mycobacterium vaccae (Mycobacterium vaccae) vaccines have been found to improve quality of life in lung Cancer patients (see, e.g., O' Brien M, Anderson H, Kaukel E, et al, SRL172[ kill Mycobacterium vaccae ] in addition to diagnosis and therapy approach quality, in patient analysis and therapy not n-small-cell Cancer: phase III responses, Ann Oncol 2004 Jun; 15[6 ]; 906-14) and symptom control (Harper-Wynne C, SumpK, Ryan C, et al, edition of SRL172 to therapy cell Cancer [ Cancer C ] 76: 2. 76. about. tuberculosis [ 47 ] Cancer [ 76, 2).

For the treatment of melanoma, a Corynebacterium parvum (Corynebacterium parvum) vaccine is associated with a tendency towards improved survival (see, e.g., Balch CM, Smalley RV, Bartolucci AA, et al. A random synergistic active triple of adjuvant C. parvum immunological in 260 genes with clinical localized tumor [ stage I ] Cancer 1982Mar 15; 49[6]: 1079-84).

Streptococcus pyogenes vaccine therapy in the dermis has been found to be effective in the treatment of gastric cancer (see, e.g., Hanaue H, KimDY, Machimura T, et al, biological streptococcus preparation OK-432; bovine mucosal therapy in regenerative gastric cancer. Tokai J Exp Clin Med 1987 Nov; 12[4]: 209-14).

Nocardia rubra (Nocardia rubra) was found To be effective in treating lung cancer (see, e.g., Yasumoto K, Yamamura Y. random. transformed clinical trial of non-specific immunological therapy with cell-wall mask of Nocardia rubra. biomed Pharma 1984; 38[1]: 48-54; Ogura T. immunological therapy of controllable cancer using Nocardia rubra cell wall mask of Feb 10[2Pt 2]:366-72) and, for treating acute myelogenous leukemia, it was associated with a tendency To improve survival (Ohno R, Nakakara H, Kodera Y, Randia. random. transformed cancer of cell of cancer. 12. wild cancer of cancer cell of microorganism of AMD. 12. wild cancer of cancer. 12. wild cancer. 3. wild cancer of cancer. 12. wild cancer of microorganism of wild cancer. 12. wild cancer of microorganism of AMD. 12. A. C. and AMD. 7. C. A. wild cancer of wild cancer of the same kind of the same species of the.

Lactobacillus casei (Lactobacillus casei) vaccine therapy in combination with radiation was found to be more effective in treating cervical cancer than radiation alone. (see, e.g., Okawa T, Kita M, Arai T, et al. phase II random administered with radiation in the treatment of the tissue center, its effect on the tissue reduction and cancer 1989Nov 1; 64[9]: 1769-76).

Pseudomonas aeruginosa (Pseudomonas aeruginosa) vaccine treatment was found to increase the efficacy of chemotherapy in the treatment of lymphoma and lung cancer (see, e.g., Li Z, Hao D, Zhang H, Ren L, et al. A clinical study PA _ MSHA vaccine used for additional therapy of lymphoma and lung cancer. HuaXi Yi Ke Da Xue Xue Xue Bao 2000 Sep; 31[3]: 334-7).

Childhood vaccination with smallpox vaccine (i.e., vaccinia virus vaccine) was found to be related to: reduce the risk of melanoma later in life (see, e.g., Pfahlberg A, Kolmel KF, Grange JM. et al. Inversie association between melanema and preservation variables against death and smallpox: results of the FEBIM student. J Invest Dermatol 2002[119]: 570. 575) and in those patients who do not develop melanoma (see, e.g., Kolmel KF, Grange JM, Krone B, et al. Prior immunization of tissues with malignant melanema with preservation or BCG is associated with growth with death and preservation medium. Europeanisation for Research and recovery of Cancer family [ 2005. Europee. Europeanish Organization and Cancer family ] No. 118. J. J. Easter J. Observation of Cancer J. Observation [ 32. J. Objectile. Observation ] No. 2. J. Observation of Cancer family et al. Observation of Cancer family [ 32. J. Observation ] A. Observation of Cancer family of Cancer No. J. Observation No. 11. Observation of FIGS.

Treatment with rabies vaccine was found to result in temporary remission in 8 patients out of 30 patients with melanoma (see, e.g., Higgins G, Pack G. Virus therapy in the treatment of cancer Bull Hosp Joint Dis 1951; 12: 379-.

Despite the numerous attempts to engage the immune system in fighting cancer using non-specific immunostimulatory microbial vaccines, most of these attempts have failed, and there is only a few clinical and research evidences of widespread success in improving survival in cancer patient populations. While immunostimulatory microbial vaccine approaches have been recognized as promising, a significant challenge has also been recognized as characteristic of the art (see, e.g., Ralf Kleef, Mary Ann Richardson, NancyRussell, Cristina Ramirez. "Endotoxin and Exotoxin Induced Tumor regressions with specific Reference to Coley Toxins: A Survey of the lipid and regulatory Medicine August; DL Mager. Bacteria and Cancer: Cause, Coordinator core [14] view of Journal of transform and media [ 14: 3514: 5814: 3514: 14: 5814).

Inflammatory Bowel Disease (IBD) is the name given to a group of inflammatory disease states of the colon and small intestine that are commonly characterized by similar symptoms and uncertain etiology. The major subtypes of IBD are clinically considered crohn's disease and ulcerative colitis. In addition to crohn's disease and ulcerative colitis, IBD may also include disease states that are considered to be any of the following: collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome or indeterminate colitis. The differences between these disease states are primarily related to the location and nature of inflammatory changes between the gastrointestinal tract (GIT). For example, crohn's disease is generally thought to potentially affect any part of the gastrointestinal tract, from the mouth to the anus, and is manifested in most cases by recurrence and reduction of granulomatous inflammation of the digestive tract in the terminal ileum and colon. In contrast, ulcerative colitis is generally considered to be limited to the colon and rectum. The various regions of the gastrointestinal tract in which these inflammatory disease states may manifest symptoms include: the intestine (bowel) or the intestine (intestine), including the small intestine (which has three sections: the duodenum, jejunum, and ileum); the large intestine (which has three sections, the cecum; the colon, which includes the ascending colon, the transverse colon, the descending colon, and the sigmoid curve; and the rectum); and the anus.

An understanding of Inflammatory bowel disease is progressing, but in many respects is not yet complete (see, for example, Baumgart DC, clipping SR (2007) "inflammation bone disease: mouse and immunology" The Lancet369(9573): 1627-40; Baumgart DC, Sandborn WJ (2007) "inflammation bone disease: clinical observations and analysis and visualization heat" The Lancet369(9573): 1641-57; Xavier DK, Podolsky (2007) "irradiation The bone disease of inflammation bone disease" Nature "Immunity 7152 (427-34): 427-34; J.H.Cho (2008)" The gene therapy of tissue disease of Nature 466 "(2008) recovery of Nature).

Anti-inflammatory drugs and immune system inhibitors, such as sulfasalazine (Azulfidine), may be used in the treatment of IBD^TM) Mesalamine (Asacol)^TM、Rowasa^TM) Corticosteroids (e.g., prednisone)) Azathioprine (Imuran)^TM) Mercaptopurine (Purinethol)^TM) Yinfiei (Remicade)^TM) Adalimumab (Humira)^TM) Cytuzumab ozogamicin (Cimzia)^TM) Methotrexate (Rheumatrex)^TM) Cyclosporine (Gengraf)^TM、Neoral^TM、Sandimmune^TM) Or natalizumab (Tysabri)^TM)。

Alternative treatments for IBD have been proposed, including treatment with various biologicals or allegedly modulating the natural intestinal flora, sometimes referred to as probiotic treatment (US 2007/0258953; US 2008/0003207; WO 2007/076534; WO 2007/136719; WO 2010/099824) for example, it has been reported that IBD can be treated with deliberate infection by parasites, for example by consuming live eggs of pig whipworms (Summers et al (2003), "Trichoderma suresems to be safe and reactive in the treatment of the nematode in the canal of infection of the animal bone disease". am.J. Gastrontenol.98 (9): 2034-41; B ü g et al, (2008) "bones as antibiotics of animal bone disease" Gut 57: 1182:. IBD 3; insect tissue and 2009) "Helwingflours et 15".

Brief description of the invention

In one aspect, methods of formulating anti-inflammatory compositions for treating disease states characterized by inflammation in specific organs or tissues are provided. The method comprises selecting at least one pathogen that is pathogenic in the specific organ or tissue; preparing an antigenic composition comprising antigenic determinants which together are specific for a pathogen; and formulating the antigenic composition for administration as an anti-inflammatory composition capable of inducing an anti-inflammatory response in said specific organ or tissue, wherein said disease state characterized by inflammation is not cancer.

The method may further comprise a diagnostic step of determining a specific organ or tissue in which inflammation is symptomatic prior to preparing the antigenic composition. Optionally, the tumor or proliferative disease state may be located in a specific organ or tissue.

Optionally, the antigenic composition may be formulated for subcutaneous or intradermal injection. Optionally, the antigenic composition can be formulated for injection to generate a local skin immune response at the site of administration. Optionally, the methods detailed herein are provided such that when the specific tissue or organ is X, the pathogen is selected from Y. More specifically, the following combinations are considered to be within the scope of the method:

in addition, the methods detailed herein are provided such that when the specific tissue or organ is X, the pathogen is selected from Y. More specifically, the following combinations are considered to be within the scope of the method:

optionally, the antigenic composition may be formulated for repeated subcutaneous or intradermal administration. Optionally, the antigenic composition may be formulated for administration by parenteral route. Optionally, the pathogen detailed herein is a bacterium, virus, protozoan, fungus, or parasite. In addition, the method may include killing the pathogen to formulate the antigen composition as a whole kill (whole kill) pathogen composition. In addition, the pathogen may be a member of a species that is an endogenous flora of a particular organ or tissue. In addition, the pathogen may be a foreign species.

In another aspect, a method of treating a disease state characterized by inflammation in a particular organ or tissue in an individual is provided. The method comprises administering to the individual an anti-inflammatory composition comprising an antigenic determinant. The antigenic determinants are selected or formulated so that together they are specific for at least one pathogen that is pathogenic in the specific organ or tissue. Optionally, the anti-inflammatory composition may be administered at the site of administration in successive doses administered at an administration interval of one hour to one month over an administration duration of at least two weeks.

In another aspect, the use of an anti-inflammatory composition for treating a disease state characterized by inflammation in a specific organ or tissue in a subject is disclosed. The anti-inflammatory composition comprises antigenic determinants that are selected or formulated so that together they are specific for at least one microbial pathogen that is pathogenic in a particular organ or tissue.

In another aspect, the use of an anti-inflammatory composition for formulating a medicament for treating a disease state characterized by inflammation in a specific organ or tissue in a subject is disclosed. The anti-inflammatory composition comprises antigenic determinants that are selected or formulated so that together they are specific for at least one microbial pathogen that is pathogenic in a particular organ or tissue.

Optionally, uses disclosed herein include those wherein the individual has cancer located in the organ or tissue. Additionally and optionally, the anti-inflammatory composition can be administered at the site of administration in successive doses administered at an administration interval of one hour to one month over an administration duration of at least two weeks.

In one aspect, methods of comparing immune responses are provided. The method comprises administering to an animal having an organ or tissue a medicament having an antigenic composition comprising antigenic determinants, selecting or formulating the antigenic determinants such that the antigenic determinants are together specific for at least one microbial pathogen that is pathogenic in the organ or tissue, extracting a quantifiable immune sample from the organ or tissue, measuring a characteristic of the immune response within the organ or tissue in the quantifiable immune sample after administration of the medicament, and comparing the characteristic of the immune response of the quantifiable immune sample to a corresponding characteristic of the immune response of a reference immune sample obtained from the corresponding organ or tissue. Optionally, the reference immune sample can be obtained from the corresponding organ or tissue in the animal prior to the step of administering the drug. Optionally, the reference immune sample can be obtained from a corresponding organ or tissue in the second animal. Optionally, the animal may have a cancer located in an organ or tissue.

Comparing the characteristic of the immune response may comprise comparing numerical indications of any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages. In addition, comparing the characteristics of the immune response may include comparing changes in macrophage activation state. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages. Additionally and optionally, macrophages can change from M1-like macrophages to M2-like macrophages.

Optionally, comparing the characteristic of the immune response may comprise determining a cellular marker on any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages.

Optionally, comparing the characteristic of the immune response may comprise determining in the quantifiable and reference immune samples cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. As described herein, macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, the cytokine is produced as a result of a change in the activation state of the macrophage. Optionally, the macrophage is changed from an M2-like macrophage to an M1-like macrophage. Additionally and optionally, the macrophage changes from an M1-like macrophage to an M2-like macrophage.

Optionally, comparing the characteristic of the immune response may comprise determining differential gene expression produced by any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, the differential gene expression is produced as a result of a change in the activation state of the macrophage. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages. Additionally and optionally, the macrophage changes from an M1-like macrophage to an M2-like macrophage.

Optionally, the drug may be administered at the site of administration in consecutive doses administered at dosing intervals of one hour to one month over a dosing duration of at least one week. Optionally, the drug may be administered intradermally or subcutaneously. Optionally, the drug may be administered at a dose effective to induce a significant local inflammatory immune response at the site of administration for each dose. Optionally, the drug may be administered so that significant local inflammation occurs at the site of administration within 1 to 48 hours. Additionally and optionally, the animal can be a mammal. Optionally, the animal can be a human or a mouse.

In another aspect, a method of selecting a therapeutic agent suitable for treating cancer in a particular organ or tissue of an individual is provided. The method comprises providing an animal having a cancer localized to a particular organ or tissue; providing a test formulation having antigenic determinants of one or more microbial pathogens that are pathogenic in a corresponding specific organ or tissue of a healthy individual; measuring a characteristic of an immune response in a reference immune sample obtained from an organ or tissue of the animal; administering a test formulation to the animal; measuring a characteristic of an immune response of a quantifiable immune sample obtained from a corresponding organ or tissue of the animal; comparing the characteristics of the immune response in the reference and quantifiable immune samples; and processing the increased characteristic of the immune response of the quantifiable immune sample as compared to a reference immune sample as an indication of the suitability of the test preparation as a therapeutic preparation. Optionally, the animal is sacrificed prior to obtaining a quantifiable immune sample.

Optionally, comparing the characteristic of the immune response may comprise comparing numerical indications of any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may comprise comparing a change in macrophage activation state. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages. Additionally and optionally, macrophages can change from M1-like macrophages to M2-like macrophages.

Optionally, comparing the characteristic of the immune response may comprise determining a cellular marker on any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages.

Optionally, comparing the characteristic of the immune response may comprise determining in the quantifiable and reference immune samples cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, the cytokine is produced as a result of a change in the activation state of the macrophage. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages. In addition, macrophages can change from M1-like macrophages to M2-like macrophages.

Additionally and optionally, comparing the characteristics of the immune response may comprise determining differential gene expression produced by any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, differential gene expression may result from changes in the activation state of macrophages. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages. Additionally and optionally, macrophages can change from M1-like macrophages to M2-like macrophages.

In another aspect, methods of selectively targeting an immune response to a cancerous tissue or organ in a human individual are provided. The method comprises administering to the individual a medicament having an effective amount of a microbial pathogen antigenic composition, wherein the microbial pathogen may be pathogenic in a particular cancerous organ or tissue of the individual and the antigenic composition comprises antigenic determinants that together are specific for the microbial pathogen. Optionally, the antigenic composition may comprise a whole killed bacterial cell composition. Optionally, the medicament may be administered to the individual at a dose and for a duration effective to upregulate an immune response in a cancerous organ or tissue of the individual. Optionally, the method may further comprise measuring a characteristic of the immune response. The methods also include the treatment of precancerous lesions including, but not limited to, actinic keratosis, cervical dysplasia, and colonic adenomas.

In another aspect, a method for treating cancer located in a tissue or organ in a human subject is provided. The method comprises administering to the individual a medicament having an effective amount of a microbial pathogen antigen composition comprising a whole killed bacterial cell composition, wherein the microbial pathogen is pathogenic in the specific organ or tissue of the individual in which the cancer is located. The medicament may be administered to the individual in an amount and for a duration effective to modulate the immune response. Optionally, modulation of the immune response may include a change in macrophage activation state. Optionally, modulation of the immune response may include a change from an M2-like macrophage response to an M-1-like macrophage response. Modulation of the immune response may include a change from M1-like macrophages to M2-like macrophages, as the term is defined below. Optionally and without limitation, the method may further comprise measuring a characteristic of the immune response.

Optionally, comparing the characteristic of the immune response may comprise comparing numerical indications of any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may comprise comparing a change in macrophage activation state. Additionally and optionally, macrophages can change from M2-like macrophages to M1-like macrophages. Optionally, macrophages can be changed from M1-like macrophages to M2-like macrophages.

Further, and without limitation, comparing the characteristics of the immune response may include determining cellular markers on any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may comprise determining in the quantifiable and reference immune samples cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages. In addition, cytokines may be produced due to changes in the activation state of macrophages. Macrophages can vary from M2-like macrophages to M1-like macrophages. Optionally, macrophages can be changed from M1-like macrophages to M2-like macrophages.

Additionally and optionally, comparing the characteristics of the immune response may comprise determining differential gene expression produced by any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, differential gene expression may result from changes in the activation state of macrophages. Additionally and optionally, macrophages can change from M2-like macrophages to M1-like macrophages. Macrophages can vary from M1-like macrophages to M2-like macrophages.

In another aspect, a method of monitoring the efficacy of a treatment regimen in an individual being treated for a cancer of a particular organ or tissue is provided. The method comprises measuring a characteristic of an immune response of a post-treatment immune sample obtained from a specific organ or tissue after the individual has been subjected to a treatment regimen for a period of time, wherein the presence of an immune response characteristic that is greater in magnitude than the immune response characteristic expected for an individual not subjected to the treatment regimen is indicative of the efficacy of the treatment regimen; and the treatment regimen comprises administering a formulation comprising one or more antigenic determinants of a microbial pathogen that is pathogenic in a corresponding specific organ or tissue of a healthy individual.

The methods detailed herein further comprise measuring a characteristic of an immune response of a pre-treatment reference sample, wherein the pre-treatment reference sample is obtained from a specific organ or tissue before, at the same time as, or after the start of the treatment regimen but before obtaining the post-treatment immune sample, and comparing the characteristic of the immune response of the pre-treatment and post-treatment samples, wherein an increase in magnitude of the immune response of the post-treatment immune sample compared to the pre-treatment reference sample is indicative of the efficacy of the treatment regimen. Optionally, measuring a characteristic of the immune response may comprise determining an indication of the number of inflammatory monocytes in the sample of the organ or tissue. Optionally, measuring a characteristic of the immune response may comprise determining an indication of the number of macrophages in a sample of the organ or tissue. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages.

Optionally, measuring a characteristic of the immune response may comprise determining an indication of the number of CD11b + Gr-1+ cells in the sample of the organ or tissue, or determining an indication of the number of dendritic cells in the sample of the organ or tissue. Additionally and optionally, measuring a characteristic of the immune response may comprise determining an indication of the number of CD11c + MHC class II + cells in the sample of the organ or tissue, or determining an indication of the number of CD4+ T cells in the sample of the organ or tissue, or determining an indication of the number of CD8+ T cells in the sample of the organ or tissue.

Optionally, measuring the magnitude of the immune response may comprise determining an indication of the number of NK cells in the sample of the organ or tissue. Additionally and optionally, comparing the characteristics of the immune response may comprise determining cellular markers on any one or more of the following cells in the reference and immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages.

Additionally and optionally, comparing the characteristics of the immune response may comprise determining in the reference and immune samples cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, cytokines may be produced as a result of changes in the activation state of macrophages. Macrophages can vary from M2-like macrophages to M1-like macrophages. Additionally and optionally, macrophages can change from M1-like macrophages to M2-like macrophages.

Optionally, comparing the characteristic of the immune response may comprise determining differential gene expression produced by any one or more of the following cells in the reference and immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Differential gene expression may result from changes in the activation state of macrophages. Macrophages can vary from M2-like macrophages to M1-like macrophages. Optionally, macrophages can be changed from M1-like macrophages to M2-like macrophages.

As detailed herein in another aspect, the invention provides methods for formulating an immunogenic composition for treating cancer localized to a specific organ or tissue in a mammal, e.g., a human patient. The method may include selecting at least one microbial pathogen that is naturally pathogenic in an organ or tissue of the mammal in which the cancer is located. Antigenic compositions can be prepared that include antigenic determinants that together are specific for a characteristic of a microbial pathogen.

The diagnostic procedure can be used to determine the specific organ or tissue in which the cancer is located prior to generating the antigenic composition that targets the site of the cancer. The site of the cancer may be the primary site or a secondary site of metastasis. The antigenic composition may be sufficiently specific so that it can elicit an immune response in a mammal that is specific for the microbial pathogen. The antigenic composition may be a bacterial composition, for example derived from a bacterial species or a species endogenous to the patient's flora or from an exogenous species or species. In alternative embodiments, the antigenic composition may be from one virus or multiple viruses. Thus, the microbial pathogen from the antigenic composition may be a virus. Microbial pathogens can be killed. In alternative embodiments, the microbial pathogen may be live or attenuated. The immunogenic compositions of the invention may also be formulated or administered with anti-inflammatory medications, such as NSAIDs. The administration site may be a site remote from the site of cancer, for example in an organ or tissue that is not the organ or tissue in which the cancer is located, such as skin or subcutaneous tissue.

For example, the antigen composition may be formulated for subcutaneous injection, intradermal injection, or oral administration. In embodiments for subcutaneous or intradermal injection, the dosage or formulation of the antigen composition can be adjusted to produce a significant local immune response in the skin at the site of administration, such as an inflammatory region, e.g., 2mm to 100mm in diameter, that occurs 2to 48 hours after administration and lasts, e.g., 2to 72 hours or more. The antigen composition may be formulated for repeated subcutaneous or intradermal administration, e.g., at alternating sequential sites.

In some embodiments, the invention relates to methods of treating cancer located in a tissue or organ in a mammal. In an alternative embodiment, the treatment may anticipate the development of cancer in a tissue, for example if the site of the primary tumor indicates a likelihood of metastasis to a particular tissue or organ, the patient may be treated prophylactically to prevent or reduce metastasis to that tissue or organ. The methods can include administering to the individual an effective amount of an antigenic composition comprising antigenic determinants that together are specific for at least one microbial pathogen. One aspect of the invention includes the use of microbial pathogens that are pathogenic in the specific organ or tissue of the mammal in which the cancer is located. The antigen composition may be administered, for example, by subcutaneous or intradermal injection at the site of administration, in successive doses administered at an administration interval of, for example, one hour to one month, over an administration duration of, for example, at least 1 week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or more. For example, each injection dose can be metered so as to be effective, for example, to cause significant local inflammation at the site of administration that occurs 1 to 48 hours after injection.

In another invention, a method is provided for treating cancer of a specific organ or tissue in an individual by administering one or more antigens of one or more microbial pathogens, such as bacterial or viral species that are pathogenic in the specific organ or tissue.

In alternative embodiments, the pathogenic microbial species is capable of naturally inducing an infection in a specific organ or tissue of a healthy individual (i.e., without human intervention), or may induce an infection in a specific organ or tissue of a healthy individual. In alternative embodiments, the antigen may be administered by administration to a whole microbial species. In alternative embodiments, the method may, for example, comprise administering at least two or more species of microorganisms or administering at least three or more species of microorganisms, and the microorganisms may be bacteria or viruses. In alternative embodiments, the method may further comprise administering a supplement or adjuvant. One aspect of the invention relates to administering an antigenic composition so as to elicit an immune response in said individual.

In an alternative embodiment, the microbial pathogen of the antigenic composition may be killed, thereby rendering it non-infectious. In some embodiments, the antigenic compositions are administered at a site remote from the site of cancer, and in selected such embodiments, the methods of the invention can be performed such that they do not produce infection at the site of cancer.

As detailed herein, various aspects of the invention include the treatment of cancer. Herein, treatment may be performed to provide various results. For example, the treatment may: stimulating an immune response effective to inhibit or reduce the growth or proliferation of the cancer; inhibiting the growth or proliferation of cancer cells or tumors; leading to remission of the cancer; improving the quality of life; reducing the risk of cancer recurrence; inhibiting metastasis of cancer; or improving patient survival in a patient population. In this context, extending the life expectancy of a patient or patient population refers to the number of patients that survive a given period of time after a particular diagnosis. In some embodiments, treatment may involve patients who do not respond to other treatments, such as patients for whom chemotherapy or surgery are not an effective treatment. Treatment in alternative embodiments may be, for example, before or after the onset of cancer. For example, prophylactic treatment may be performed on a patient, e.g., diagnosed as at risk for a particular cancer. For example, an immunogenic composition comprising an antigenic determinant of a pathogen that is pathogenic in an organ or tissue can be used to treat a patient who is genetically susceptible or lifestyle susceptible to cancer of that organ or tissue. Likewise, prophylactic treatment of metastases may be performed so that immunogenic compositions comprising antigenic determinants of pathogens that are pathogenic in a particular organ or tissue may be used to treat patients with primary cancer that have a predisposition to metastasis of that tissue or organ.

In another aspect, a method of treating cancer located in a lung of an individual is provided. The method comprises administering to the individual an effective amount of an antigen of one or more species of microorganism that is pathogenic in the lung, and administering to the individual an effective amount of a platinum-containing chemotherapeutic agent. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen.

Viral pathogens may be, but are not limited to: influenza virus, adenovirus, respiratory syncytial virus or parainfluenza virus. Bacterial pathogens can be, but are not limited to: streptococcus pneumoniae (Streptococcus pneumoniae), Moraxella catarrhalis (Moraxella catarrhalis), Mycoplasma pneumoniae (Mycoplasma pneumoniae), Klebsiella pneumoniae (Klebsiella pneumoniae), Haemophilus influenzae (Haemophilus influenza), Staphylococcus aureus (Staphylococcus aureus), Chlamydia pneumoniae (Chlamydia pneumoniae) or Legionella pneumophila (Legionella pneumoniae). Fungal pathogens may be, but are not limited to: aspergillus fumigatus (Aspergillus fumigatus), Blastomyces (Blastomyces sp.), Coccidioides (Coccidiodes immitis), Coccidiodessporosidium (Cryptococcus neoformans), Cryptococcus gracilis (Cryptococcus gatii), Fusarium (Fusarium sp.), Histoplasma capsulatum (Histoplasma capsulatum), Penicillium Paecilomyces (Paecilomyces sp.), Paracoccidioides brasiliensis (Paracoccus bractensis), Penicillium marneffei (Penicillium marneffei), Chrysosporium (Pneumocystis jirius), Pseudomyces boidinii (Pseudomyceliophyces boidinii), Pseudomonas aeruginosa (Pseudomonas cepacia), Polyporus tip (Staphylococcus sp.), Trichoderma (Aspergillus sp.), Trichoderma strain (Trichoderma sp.), Trichoderma sp., or Trichoderma sp. In addition, platinum-containing chemotherapeutic agents may be, but are not limited to: cisplatin, carboplatin, or oxaliplatin.

In another aspect, there is provided use of an effective amount of an antigen of one or more microbial species that are pathogenic in the lung to formulate a medicament for use in treating lung cancer in an individual in combination with a platinum-containing chemotherapeutic agent. In another aspect, there is provided the use of an effective amount of an antigen from one or more microbial species that is pathogenic in the lung for use with a platinum-containing chemotherapeutic agent to treat lung cancer in an individual. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen.

Viral pathogens may be, but are not limited to: influenza virus, adenovirus, respiratory syncytial virus or parainfluenza virus. Bacterial pathogens may be, but are not limited to: streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Chlamydia pneumoniae or Legionella pneumophila. Fungal pathogens may be, but are not limited to: aspergillus fumigatus, Blastomyces, Coccidiodes posadasii, Cryptococcus neoformans, Cryptococcus gatherens, Fusarium, Histoplasma capsulatum, Paecilomyces variotii, paracoccidioides brasiliensis, Penicillium marneffei, Pneumocystis jejuni, Pseudomyceliophthora boydii, Podospora tiphylla, Rhizopus, Mucor, Absidia, Ginkrella cunninghamii, Synechocystis polytrichum, Stachybotrys cinerea, Trichoderma longibrachiatum, or Sporomyces. In addition, platinum-containing chemotherapeutic agents may be, but are not limited to: cisplatin, carboplatin, or oxaliplatin.

In another aspect, an effective amount of an antigen of one or more species of microorganism that is pathogenic in the lung is provided to formulate a medicament for use with a platinum-containing chemotherapeutic agent for treating lung cancer in an individual. In another aspect, an antigen from one or more microbial species that is pathogenic in the lung is provided in an amount effective for use with a platinum-containing chemotherapeutic agent to treat lung cancer in an individual. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen.

In another aspect, a kit is provided. The kit comprises an antigen of one or more microbial species that are pathogenic in the lung, a platinum-containing chemotherapeutic agent; and instructions for providing the antigen and the platinum-containing chemotherapeutic agent to a patient in need thereof. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen.

Viral pathogens may be, but are not limited to, the foregoing: influenza virus, adenovirus, respiratory syncytial virus or parainfluenza virus. Bacterial pathogens may be, but are not limited to: streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Chlamydia pneumoniae or Legionella pneumophila. Fungal pathogens may be, but are not limited to: aspergillus fumigatus, Blastomyces, Coccidiodes posadasii, Cryptococcus neoformans, Cryptococcus gatherens, Fusarium, Histoplasma capsulatum, Paecilomyces variotii, paracoccidiobolus brasiliensis, Penicillium marneffei, Pneumocystis jejuni, Pseudomyceliophthora boydii, Podospora tiphylla, Rhizopus, Mucor, Absidia, Ginkrella cunninghamii, Synechocystis polytrichum, Stachybotrys cinerea, Trichoderma longibrachiatum, or Sporomyces. In addition, platinum-containing chemotherapeutic agents may be, but are not limited to: cisplatin, carboplatin, or oxaliplatin.

In various aspects, the present invention provides immunogenic compositions for the formulation and use in the treatment of IBD. IBD may for example be IBD that is symptomatic in one or more regions of the GIT of a human patient, such as crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, behcet's syndrome, or indeterminate colitis. Treatment of a patient may include a diagnostic step to determine the region of the GIT where IBD is symptomatic. The formulation may comprise an antigenic composition comprising antigenic determinants together specific for at least one pathogen that is pathogenic in the infected GIT region, such as a bacterium, virus, protozoan or parasite. The formulation may be prepared for administration as an immunogenic composition capable of eliciting an immune response, thereby treating IBD. For example, the composition may be formulated for parenteral routes, such as subcutaneous or intradermal injection, e.g., to generate a local cutaneous immune response, such as an inflammatory response, at the site of administration.

Brief Description of Drawings

Fig. 1 shows survival curves for some cumulative patients (all patients) diagnosed with stage 3B or 4 inoperable lung cancer, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 1: survival of stage 3B or 4 lung & bronchial carcinoma.

Fig. 2 shows survival curves for some cumulative patients diagnosed with stage 3B or 4 inoperable lung cancer (patients treated with MRV for at least 2 months), comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 2: survival of stage 3B or 4 lung & bronchial carcinoma.

Fig. 3 shows survival curves for some cumulative patients diagnosed with stage 3B or 4 lung cancer, showing the benefit of treatment with an MRV composition of the invention, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 3: combinatorial analysis (MRV versus non-MRV lung 2).

Fig. 4 shows survival curves for some cumulative patients diagnosed with stage 3B or 4 lung cancer, showing the effect of at least 2 months of treatment, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 4: combined analysis (MRV for more than 2 months versus non-MRV lung 2).

Fig. 5 shows survival curves for some cumulative patients diagnosed with stage 3B or 4 lung cancer, showing the effect of treatment for a duration of at least 6 months, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 5: combined analysis (MRV for greater than 6 months versus non-MRV lung 2).

Fig. 6 shows survival curves for some cumulative 52 breast cancer patients with metastasis to bone and/or lung, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 6: stage 4 breast cancer survival curves for bone/lung metastases.

Fig. 7 is a comparison of the survival of metastatic prostate cancer patients whose prostate was destroyed by some cumulative surgery or radiation (and thus, a primary cancer) and detectable cancer was limited to bone metastases, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 7: survival of stage 4 prostate cancer patients with bone metastases (surgical or radiation damaged prostate).

Figure 8 shows survival curves for some cumulative preliminary diagnoses of patients with stage 4 colorectal cancer, comparing patients treated with PVF, patients treated with MRV, patients not treated with the antigenic composition, and standard SEER survival curves. FIG. 8: comparison of colorectal cancer treatment at stage 4.

Fig. 9 shows survival curves for some cumulative preliminary diagnoses of patients with stage 4 colorectal cancer, and data from patients treated within 3 months of diagnosis, comparing patients treated with PVF, patients treated with MRV, patients not treated with the antigenic composition, and standard SEER survival curves. FIG. 9: within 3 months of the colorectal cancer metastasis day at stage 4 and at first visit.

Figure 10 shows the survival curves for some cumulative phase 3B lung cancer patients treated with oral antigen therapy (i.e., Respivax) compared to patients not using the antigen composition. FIG. 10: comparative survival of lung 2 in stage 3B lung cancer patients (vaccine-free versus Respivax).

Fig. 11 shows survival curves for some cumulative patients diagnosed with stage 3B lung cancer, showing the benefit of treatment with MRV compositions of the invention, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 11: survival of stage 3B &4 lung cancer in the diagnostic cases included in 1992-2000.

Fig. 12 shows survival curves measured from data obtained from a preliminary visit to some cumulative patients diagnosed with stage 3B lung cancer, showing the benefit of treatment with an MRV composition of the invention, comparing patients treated with MRV, patients not treated with MRV, and standard SEER survival curves. FIG. 12: stage 3B &4 lung cancer survival from CIH initial visit.

Figure 13 shows survival curves for some cumulative initial visits for patients diagnosed with stage 3B lung cancer within 3 months of diagnosis, showing the benefit of early treatment with an MRV composition of the invention, comparing patients treated with MRV with patients not treated with MRV. FIG. 13: CIH follow-up of stage 3B lung cancer within 3 months after the stage 3B period was diagnosed.

Figure 14 shows photographs of the lungs of mice treated with klebsiella pneumoniae vaccine only (row 1) and not treated with it (row 2) after being tried with Lewis lung cancer cells, as described in example 4A herein. The bottom row (not numbered) describes the lungs from mice that were not exposed to the lung cancer mouse model.

Figure 15 shows the average tumor volumes of mice treated with bacterial vaccine [ AB1-AB6] and untreated with it [ AB-7] after being tried with B16 melanoma cells, as described in example 4B herein.

Fig. 16 shows survival curves for a group of colon cancer model mice treated with or without various bacterial vaccines, as described in example 4C herein. FIG. 16: survival curves of colon cancer model mouse groups treated with various killed bacterial vaccines.

Figure 17 shows the number of inflammatory monocytes and dendritic cells in draining lymph nodes, lungs and spleen following treatment with a klebsiella pneumoniae antigen composition or PBS as described in example 5A herein.

Figure 18 shows the total amount of monocytes and dendritic cells in the lungs, peritoneum and spleen of mice after treatment with a klebsiella pneumoniae antigen composition, an escherichia coli antigen composition or PBS as described in example 5B herein.

Figure 19 shows the total amount of CD4+ T cells, CD8+ T cells, and NK cells from mice after treatment with a klebsiella pneumoniae antigen composition, an escherichia coli antigen composition, or PBS, as described in example 5B herein.

Figure 20 shows the total amount of (a) inflammatory monocytes and (B) CD4+ T cells, CD8+ T cells, and NK cells at day 9 or 16 from mice treated with heat inactivated klebsiella pneumoniae antigen composition, phenol inactivated klebsiella pneumoniae antigen composition, or PBS, as described in example 5C herein.

Figure 21 shows the total amount of (a) inflammatory monocytes and dendritic cells and (B) CD4+ T cells, CD8+ T cells, and NK cells from mice treated with heat-inactivated klebsiella pneumoniae antigen composition, phenol-inactivated klebsiella pneumoniae antigen composition, or PBS, as described in example 5D herein.

Figure 22 shows the total amount of tumor nodules in mice treated with PBS or different doses of a klebsiella pneumoniae antigen composition as described herein.

Figure 23 shows photographs of the lungs from mice treated with PBS or different doses of a klebsiella pneumoniae antigen composition as described herein.

Figure 24 shows the total amount of tumor nodules in mice treated with PBS or different doses of a klebsiella pneumoniae antigen composition as described herein.

Figure 25 shows the total amount of tumor nodules in mice treated with PBS or different doses of a klebsiella pneumoniae antigen composition in combination (or not) with cisplatin as described herein.

Figure 26 shows injection with Lewis lung cancer cells and control with (i) vehicle; (ii) cisplatin; (iii) (iii) the survival curves of the antigenic composition or (iv) the antigenic composition and cisplatin treated mice.

Figure 27 shows the percentage of CD11b + myeloid cells in blood at day 13 of the relevant experiment detailed herein.

FIG. 28 shows (left panel) the frequency of CD11b + NK 1.1-cells from the lungs of mice treated with various doses of the Klebsiella pneumoniae antigen composition or with the PBS control, or (right panel) the frequency of CD11b + NK1.1+ cells from the lungs of mice treated with various doses of the Klebsiella pneumoniae antigen composition or with the PBS control.

Figure 29 shows the amount of cytokines measured (in pg/g) from lung tissue from mice treated with various doses of the klebsiella pneumoniae antigen composition or with the PBS control.

Figure 30 shows the amount of cytokines determined (in pg/ml) from BAL fluid from mice treated with various doses of klebsiella pneumoniae antigen compositions or with PBS control.

Figure 31 shows the relative gene expression ratios of NOS 2to Arg1 from mice treated with various doses of klebsiella pneumoniae antigen compositions or with PBS control.

Figure 32 shows the relative frequency of CD206 expression from pulmonary macrophages from mice treated with various doses of the klebsiella pneumoniae antigen composition or with the PBS control.

FIG. 33 shows the relative frequency of F4/80 expression from pulmonary macrophages of mice treated with various doses of the Klebsiella pneumoniae antigen composition or with the PBS control.

FIG. 34 shows the relative frequency of CD11b + Gr-1+ cells determined from the colon of mice treated with Klebsiella pneumoniae or E.coli antigen compositions or with PBS control.

FIG. 35 shows the relative frequency of CD11b + Gr-1+ cells determined from the lungs of mice treated with Klebsiella pneumoniae or E.coli antigen compositions or with PBS control.

Fig. 36 shows the relative frequency of M1-like CD11b + cells isolated from subQ 4T1 tumor (left panel) and the relative frequency of M2-like CD11b + cells isolated from subQ 4T1 tumor (right panel).

Figure 37 shows results from treatment with indomethacin and PBS; or indomethacin and staphylococcus aureus antigen compositions; or EtOH and PBS; or tumor volume (mm) in mice treated with EtOH and Staphylococcus aureus antigen composition³) [ left picture ]]. The right panel of the figure also shows the relative frequency and composition of CD11b cells in the tumors at day 11 of the relevant experiments described herein.

Figure 38 shows the relative frequency and composition of CD11b + cells in tumors at day 22 of the relevant experiments described herein.

Figure 39 shows tumor volumes in a contemporaneous group of animals treated with indomethacin, indomethacin + antigen composition, vehicle alone, or antigen composition alone.

Figure 40 shows the percentage of CD11b + cells in tumors of animals treated with indomethacin, indomethacin + antigen composition, vehicle alone, or antigen composition alone (data on day 11).

Figure 41 shows the percentage of CD11b + cells in tumors of animals treated with indomethacin, indomethacin + antigen composition, vehicle alone, or antigen composition alone (data on day 22). FIG. 41: CD11b + cells in tumors.

Figure 42 shows the percentage of CD11b + CD94+ cells in tumors of animals treated with indomethacin, indomethacin + antigen composition, vehicle alone, or antigen composition alone (data on day 22).

Fig. 43 shows the relative expression of (a) Fizz1 and (B) Ym1 in resected tumors described herein.

FIG. 44 shows the relative expression of (A) Arg1 and (B) Fizz1 from tumors and spleens [ (C) and (D) ] described herein.

Figure 45 shows the relative expression of Nos2 and Ym1 from the tumors and spleens described herein.

Figure 46 shows the amount of IFN- γ produced in the lungs of mice with or without tumors and treated with or without the antigenic compositions described herein.

FIG. 47 shows the amount of IL-10 produced by bone marrow macrophages cultured overnight with Klebsiella pneumoniae antigen composition, LPS, and medium alone.

FIG. 48 shows the relative expression of IL-10 produced in tumors in mice under the conditions described herein.

Detailed Description

In various aspects, embodiments of the present invention relate to the surprising discovery that administration of a microbial pathogen that is pathogenic, e.g., killed, in a particular tissue or organ, e.g., at a site remote from the cancer, effectively treats the cancer located in the particular tissue or organ. Accordingly, the present invention provides antigenic compositions from these microbial pathogens, including whole killed bacterial or viral species or components thereof, for use in the treatment of cancer and methods of use of the antigenic compositions.

Based on observations from patients receiving treatment, it was found that administration of a bactericidal composition comprising a number of bacterial species that commonly cause lung infections was surprisingly and unexpectedly effective in improving the clinical course of lung cancer. Also, it was found that administration of a composition comprising killed staphylococcus aureus (one of the most common causes of bone, breast, skin, perineal and lymph node infections and sepsis) was surprisingly and unexpectedly effective in improving the clinical course of bone cancer, breast cancer, skin cancer, perineal cancer and lymphoma (cancer of the lymph glands) and multiple myeloma (a type of hematological cancer). Also, it was surprisingly and unexpectedly found that administration of a composition comprising escherichia coli, which is a common cause of colon, kidney, peritoneum, liver, abdominal, pancreatic and ovarian infections, was effective in improving the clinical course of cancer in colon, kidney, peritoneum, liver, abdominal lymph nodes, pancreas and ovaries.

These results indicate that compositions comprising antigens of pathogenic microbial species that induce infections in a particular tissue or organ would be effective agents for treating cancer in that tissue or organ. For example, cancer of the lungs is effectively treated with a microbial composition comprising one or more pathogenic species that normally induce lung infections, while cancer of the colon is effectively treated with a composition comprising one or more pathogenic microbial species that normally induce colon infections.

Antigenic compositions of the invention can be prepared that include antigenic determinants together that are specific for a characteristic of a microbial pathogen. In this context, "specificity" means that the antigenic determinants are sufficient to be characteristic of the pathogen, and if administered in an appropriate manner to have this effect, they can be used to generate an immune response, e.g., an adaptive immune response, against the pathogen of the patient. It is recognized that antigenic determinants need not be specific in that they are characteristic of only one particular strain or species of pathogen, as specific immune responses against a particular pathogen may also cross-respond with other closely related organisms that are naturally pathogenic in the tissue or organ in which the cancer is located and the tissue or organ targeted by the formulated or selected antigenic composition.

In some embodiments, the composition of pathogenic microorganisms may be used to treat a primary cancer site and/or a metastatic site. Thus, for example, the microbial composition may be used to treat cancer at a particular site, regardless of whether the cancer is a primary cancer or a metastasis. The compositions may be directed to the treatment of each cancer site, or may be combined compositions for use in primary cancers and metastatic sites. For example, to treat kidney cancer that has metastasized to the lung and bone, three different compositions or combinations thereof of one or more species known as renal pathogens, one or more species known as pulmonary pathogens, and one or more species known as osteopathogens may be included. In some embodiments, the compositions may be administered at different locations, either simultaneously or non-simultaneously.

For example, for lung cancer with metastasis to bone, in alternative embodiments, a microbial composition comprising one or more bacterial species (or viruses) that typically induce lung infection and a microbial composition comprising one or more bacterial species (or viruses) that typically induce bone infection may be used. Also, for colon cancers with metastasis to the lung, pathogenic bacterial (or viral) compositions comprising one or more bacterial species (or viruses) that typically induce colonic infection and microbial compositions comprising one or more bacterial species (or viruses) that typically induce pulmonary infection; for prostate cancer with metastasis to the bone, pathogenic bacterial (or viral) compositions comprising one or more bacterial species (or viruses) that typically induce prostate infection and pathogenic bacterial (or viral) compositions comprising one or more bacterial species (or viruses) that typically induce bone infection may be used.

The following list provides non-limiting examples of primary cancers and their common sites of secondary dissemination (metastasis):

in some embodiments, the antigenic composition can be used to treat or prevent cancer at the primary site or to treat or prevent metastasis. For example, in long-term smokers, an antigenic composition specific for lung cancer (e.g., comprising antigenic determinants of one or more bacterial species or viruses that normally induce lung infections) can be used to appropriately stimulate the immune system to defend against cancer development within lung tissue. As another example, an antigenic composition specific for breast cancer (e.g., comprising antigenic determinants of one or more bacterial species that typically cause breast infection) can be used to prevent breast cancer in women with a strong family history or genetic susceptibility to breast cancer. In alternative embodiments, antigenic compositions comprising one or more bacterial species that normally induce bone infections may be used to prevent or treat bone metastases in patients with prostate cancer. In other embodiments, antigenic compositions comprising one or more bacterial species or viruses that normally induce pulmonary infections may be used to prevent or treat pulmonary metastasis in patients with malignant melanoma.

Various alternative embodiments and examples of the invention are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

Cancer treatment

Most cancers fall into three broad histological categories: carcinomas (carcinomas), which are the major cancers and are cancers of epithelial cells or cells that cover the external or internal surfaces of organs, glands or other body structures (e.g., skin, uterus, lung, breast, prostate, stomach, intestine) and which are prone to metastasis; sarcomas, which are derived from connective or supportive tissue (e.g., bone, cartilage, tendon, ligament, fat, muscle); and hematological tumors, which originate from bone marrow and lymphoid tissues. The cancer may be an adenocarcinoma (which typically develops in an organ or gland capable of secretion, such as the breast, lung, colon, prostate or bladder), or may be a squamous cell carcinoma (which occurs in squamous epithelium and typically develops in most regions of the body). The sarcoma may be osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma or mesothelioma (membranous lining of the body cavity), fibrosarcoma (fibrous tissue), angiosarcoma or angioendothelioma (blood vessels), liposarcoma (adipose tissue), glioma or astrocytoma (neural connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue) or interstitial (mesenchymes) or mixed mesodermal tumors (mixed connective tissue type). A hematological tumor can be a myeloma, which occurs in plasma cells of the bone marrow; leukemias, which can be "liquid cancers" and are cancers of the bone marrow, and can be myeloid or myelocytic leukemias (myeloid and granulocytic white blood cells), lymphoid leukemias or lymphoblastic leukemias (lymphoid and lymphocytic blood cells), or polycythemia vera or erythrocytosis (various blood cell products, but with erythrocytes predominating); or a lymphoma, which may be a solid tumor and may develop in glands or nodules of the lymphatic system, and which may be a hodgkin's or non-hodgkin's lymphoma. In addition, mixed cancers also exist, such as adenosquamous carcinoma, mixed mesoblastic tumors, carcinosarcoma, or umbilical cord carcinoma.

Cancers named based on primary site may be associated with histological classification. For example, lung cancer is typically small cell lung cancer or non-small cell lung cancer, which can be squamous cell carcinoma, adenocarcinoma, or large cell carcinoma; skin cancer is typically basal cell carcinoma, squamous cell carcinoma, or melanoma. Lymphomas can occur in lymph nodes associated with the head, neck and chest as well as in the abdominal lymph nodes or in axillary or inguinal lymph nodes. The determination and classification of the type and stage of cancer can be done using information provided, for example, by the national cancer institute's monitoring, epidemiology, and end result (SEER) program, which is an authoritative source of information about cancer incidence and survival in the united states and is recognized worldwide. The SEER program currently collects and publishes cancer incidence and survival data from 14 population-based cancer registrations and 3 supplementary registrations, covering approximately 26% of the us population. Plans generally collect data based on patient demographics, primary tumor site, histology, diagnostic stage, primary course of treatment, and follow-up for important states, and are the only comprehensive source of population-based information in the united states, including cancer stage at diagnosis and survival within each stage. Information based on more than 3 million cases of orthotopic and aggressive cancer is included in the SEER database, and about 170,000 new cases are added annually within SEER coverage. The incidence and survival data of the SEER program can be used to assess the standard survival for specific cancer sites and stages. For example, to ensure an optimal comparison set, specific criteria may be selected from a database, including data for diagnosis and accurate stage (e.g., in the case of the lung cancer example herein, year is selected to match the time limit of the period of a retrospective survey, and stage 3B and 4 lung cancers are selected; and in the case of the colon cancer example herein, year is also selected to match the time limit of the period of a retrospective survey, and stage 4 colon cancer is selected).

Cancers may also be named based on the organ in which they appear, i.e., the "primary site," such as breast cancer, brain cancer, lung cancer, liver cancer, skin cancer, prostate cancer, testicular cancer, bladder cancer, colorectal cancer, cervical cancer, uterine cancer, and the like. This nomenclature is maintained even if the cancer metastasizes to another part of the body than the primary site. For the present invention, the treatment involves a cancer site, not a cancer type, so that any type of cancer located, for example, in the lungs can be treated based on this localization of the lungs.

A "cancer" or "neoplasm" is any undesired cell growth that is not used for physiological function. Typically, cancer cells are released from their normal control of cell division, i.e., cells whose growth is not regulated by the usual biochemical and physical influences in the cellular environment. Thus, "cancer" is a generic concept of a disease characterized by abnormal uncontrolled cell growth. In most cases, cancer cells proliferate to form malignant clonal cells. Lumps or clumps of cells, i.e., "tumors" or "neoplasms," can often invade and destroy surrounding normal tissue. As used herein, "malignancy" refers to abnormal growth of any cell type or tissue that has a deleterious effect in an organism having abnormal growth. The term "malignant tumor" or "cancer" includes cell growth that is technically benign but has the risk of becoming malignant. Cancer cells can spread from their primary site to other parts of the body through the lymphatic system or the bloodstream in a process called "metastasis". Many cancers are refractory to treatment and prove fatal. Examples of cancers or neoplasms include, but are not limited to: transformed and immortalized cells, tumors, cancers in various organs and tissues described herein or known to those skilled in the art.

A "cell" is the basic structural and functional unit of a living organism. In higher organisms, such as animals, cells with similar structures and functions are often aggregated into "tissues" that perform specific functions. Thus, a tissue includes a collection of similar cells and surrounding intercellular matter, e.g., epithelial tissue, connective tissue, muscle, nerves. An "organ" is a fully differentiated structural and functional unit in higher organisms that may consist of different types of tissues, and is specific to certain specific functions, such as kidney, heart, brain, liver, etc. Thus, reference herein to "a particular organ, tissue or cell" is intended to include any particular organ, and includes cells and tissues found in that organ.

A "pathogenic" agent is an agent such as a microorganism, e.g. a bacterium or virus, which is known to naturally induce infection in a host, and in this sense "pathogenic" as used in the present invention means "naturally pathogenic". Although many microorganisms are capable of inducing infection under artificial conditions, such as artificial inoculation of the microorganism into tissue, the range of naturally infection-inducing microorganisms must be limited and well established by medical practice.

An "infection" is a condition or disease state in which a pathogenic agent (e.g. a microorganism such as a bacterium) invades the body or a part thereof, which under favorable conditions multiplies and produces deleterious effects (Taber's cyclopedic medical Dictionary,14th ed., c.l. thomas, ed., f.a. davis Company, PA, USA). The infection may not be clinically dominant in general and may result in only local cell damage. If the body's defense mechanisms are effective, the infection can remain subclinical and transient. Infections can spread locally to become clinically dominant as acute, subacute or chronic clinical infections or disease states. Local infections may also become systemic when pathogenic agents increase access to the lymphatic or vascular system (On-Line Medical Dictionary, http:// cancerweb. nc. ac. uk/omd /). Infection is often accompanied by inflammation, but inflammation can occur in the absence of infection.

"inflammation" is a typical tissue response to injury (represented by swelling, redness, heat, and pain) and includes the continuous changes that occur in liver tissue when it is damaged. Infection and inflammation are distinct disease states, although one may be caused by the other (Taber's circulating Medical Dictionary, supra). Thus, inflammation may occur in the absence of infection, and infection may occur in the absence of inflammation (although inflammation is typically caused by infection with pathogenic bacteria or viruses). Inflammation is characterized by the following symptoms: redness (redness), heat (burning), swelling (mass), pain (pain). The locally visible inflammation on the skin is evident from the composition of these symptoms, in particular the redness at the site of administration.

Various individuals may be treated according to alternative aspects of the invention. As used herein, an "individual" is an animal, e.g., a mammal, to which a particular pathogenic bacterium, bacterial antigen, virus, viral antigen, or composition thereof of the invention can be administered. Thus, the subject may be a patient, e.g. a human, suffering from or suspected to be suffering from cancer or at risk of developing cancer. The subject may be an experimental animal, such as an animal model of cancer, as described in example 5. In some embodiments, the terms "individual" and "patient" are used interchangeably and can include humans, non-human mammals, non-human primates, rats, mice, dogs, and the like. A healthy individual may be a human that has not experienced cancer or is not suspected of having cancer or a human that has not experienced a chronic condition or disease state. A "healthy individual" may also be an individual with an immune insufficiency. Immune insufficiency refers to any disease state in which the immune system functions in an abnormal or incomplete manner. The insufficiency of the immune system can be attributed to disease, certain drugs or disease states that exist at birth. Immunocompromised individuals are more frequently found in infants, the elderly, and individuals undergoing extensive drug or radiation therapy.

An "immune response" includes, but is not limited to, one or more of the following responses in a mammal: induction or activation of antibodies, neutrophils, monocytes, macrophages (including M1-like macrophages and M2-like macrophages as described herein), B cells, T cells (including helper T cells, natural killer cells, cytotoxic T cells, γ δ T cells), for example by antigen in a composition or vaccine following administration of the composition or vaccine. Thus, an immune response to a composition or vaccine typically includes a cellular and/or antibody-mediated response to the composition or vaccine of interest that develops in the host animal. In certain embodiments, the immune response is as follows: it will also cause a slowing or cessation of the progression of the cancer in the animal. The immune response includes both cellular and humoral immune responses, as understood by those skilled in the art.

Bacteria and bacterial colonization and infection

Most animals are colonized to some extent by other organisms, such as bacteria, that normally exist in a symbiotic or symbiotic relationship with the host animal. Thus, many species of bacteria are found in healthy animals, which are generally harmless, and are often located on the surface of specific organs and tissues. Often, these bacteria contribute to the normal functioning of the body. For example, in humans, commensal e.coli can be found in the intestine, where they promote immunity and reduce the risk of infection by more virulent pathogens.

Bacteria that are normally harmless, such as e.g. e.coli, can induce infections in healthy individuals, and as a result range from mild to severe infections to death. Whether a bacterium is pathogenic (i.e., induces an infection) depends to some extent on factors such as the route to and reaching a particular host cell, tissue or organ; inherent toxicity of bacteria; the amount of bacteria present at the site of potential infection; or the health of the host animal. Thus, bacteria that are generally harmless can be pathogenic under the given favorable conditions for infection, and even most virulent bacteria require a specific environment to induce infection. Thus, microbial species that are members of the normal flora can be pathogens when they exceed their normal endogenous role in the endogenous flora. For example, endogenous species can induce infection outside their niches in anatomical vicinity, e.g., by serial dissemination. When this occurs, these normally harmless endogenous bacteria are considered pathogenic.

Specific bacterial species and viruses are known to induce infection in specific cells, tissues or organs of otherwise healthy individuals. Examples of bacteria and viruses that normally induce infections in specific organs and tissues of the body are listed below: it is to be understood that these examples are not intended to be limiting and that the skilled person will be able to readily recognise and determine infectious or pathogenic bacteria (and recognise the relative frequency of infection with each bacterial species) that induce infection or generally induce infection in various organs and tissues of healthy adults based on knowledge in the fields shown, for example, in the following disclosures: manual of Clinical Microbiology 8th edition, Patrick Murray, Ed.,2003, ASM Press American Society for Microbiology, Washington DC, USA; the disclosures of Principles and practices of Mandell, Douglas, and Bennett of Infectious Diseases5th Edition, G.L.Mandell, J.E.Bennett, R.Dolin, eds.,2000, Churchill Livingstone, Philadelphia, PA, USA, are incorporated herein by reference.

Infection of the skin is usually induced by bacterial species staphylococcus aureus, β hemolytic streptococcus A, B, C or group G, Corynebacterium diphtheriae (Corynebacterium dipetheriae), Corynebacterium ulcerans (Corynebacterium ulcerans) or pseudomonas aeruginosa, or viral pathogens measles virus, rubella virus, varicella zoster virus, echovirus, coxsackie virus, adenovirus, vaccinia virus, herpes simplex virus or parvovirus B19.

Infection of soft tissues (e.g., fat and muscle) is typically induced by: bacterial species: streptococcus pyogenes, staphylococcus aureus, Clostridium perfringens, or other Clostridium (Clostridium spp.); or a viral pathogen: influenza virus or coxsackievirus.

Infection of the mammary gland is usually induced by the following bacterial species: staphylococcus aureus or streptococcus pyogenes.

Head and neck lymph node infections are commonly induced by: bacterial species: staphylococcus aureus or streptococcus pyogenes; or a viral pathogen: epstein-barr virus, cytomegalovirus, adenovirus, measles virus, rubella virus, herpes simplex virus, coxsackie virus, or varicella-zoster virus.

Infection of the brachial/axillary lymph nodes is usually induced by: bacterial species: staphylococcus aureus or streptococcus pyogenes; or a viral pathogen: measles virus, rubella virus, epstein barr virus, cytomegalovirus, adenovirus or varicella-zoster virus.

Infection of mediastinal lymph nodes is usually induced by: bacterial species: viridans streptococci (viridans streptococci), Peptococcus spp (Peptococcus spp.), peptostridia spp (Peptostreptococcus spp.), Bacteroides spp (Bacteroides spp.), clostridium spp (Fusobacterium spp.) or mycobacterium tuberculosis; or a viral pathogen: measles virus, rubella virus, epstein barr virus, cytomegalovirus, varicella-zoster virus or adenovirus.

Infection of the pulmonary portal lymph nodes is usually induced by: bacterial species: streptococcus pneumoniae, moraxella catarrhalis, mycoplasma pneumoniae, klebsiella pneumoniae, haemophilus influenzae, chlamydophila pneumoniae, bordetella pertussis or mycobacterium tuberculosis; or a viral pathogen: influenza virus, adenovirus, rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus or coxsackie virus.

Infection of the intra-abdominal lymph nodes is usually induced by: bacterial species: yersinia enterocolitica, yersinia pseudotuberculosis, salmonella, streptococcus pyogenes, escherichia coli, staphylococcus aureus, or mycobacterium tuberculosis; or a viral pathogen; measles virus, rubella virus, epstein barr virus, cytomegalovirus, varicella-zoster virus, adenovirus, influenza virus or coxsackie virus.

Infection of lymph nodes in the leg/inguinal region is usually induced by: bacterial species: staphylococcus aureus or streptococcus pyogenes; or a viral pathogen: measles virus, rubella virus, epstein barr virus, cytomegalovirus or herpes simplex virus.

Infection of the blood (i.e. sepsis) is typically induced by: bacterial species: staphylococcus aureus, streptococcus pyogenes, coagulase-negative staphylococcus (coccococci), enterococcus (enterococcus pp.), escherichia coli, Klebsiella (Klebsiella spp.), Enterobacter (Enterobacter spp.), Proteus (Proteus spp.), pseudomonas aeruginosa, Bacteroides fragilis (Bacteroides fragilis), streptococcus pneumoniae, or group B streptococcus; or a viral pathogen: measles virus, rubella virus, varicella-zoster virus, echovirus, coxsackievirus, adenovirus, epstein barr virus, herpes simplex virus or cytomegalovirus.

Infection of bone is usually induced by: bacterial species: staphylococcus aureus, coagulase-negative staphylococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae (Streptococcus agalactiae), other Streptococcus; escherichia coli, Pseudomonas, Enterobacter, Proteus, or Serratia; or a viral pathogen: parvovirus B19, rubella virus or hepatitis B virus.

Infections of the meninges are usually induced by: bacterial species: haemophilus influenzae, diplococcus encephalitis (neisseriameningidis), streptococcus pneumoniae, streptococcus agalactiae, or listeria monocytogenes; or a viral pathogen: echovirus, coxsackievirus, other enteroviruses or mumps virus.

Infections of the brain are typically induced by: bacterial species: streptococci (including streptococcus pharyngis (s. angusticus), streptococcus constellatus (s. constellatus), streptococcus intermedius (s. intermedia)), staphylococcus aureus, bacteroides, Prevotella (Prevotella spp.), proteus, escherichia coli, klebsiella, pseudomonas, enterobacter, or borrelia burgdorferi; or a viral pathogen: coxsackievirus, echovirus, poliovirus, other enteroviruses, mumps virus, herpes simplex virus, varicella-zoster virus, arbovirus or bunyavirus.

Infection of the spinal cord is typically induced by: bacterial species: haemophilus influenzae, diplococcus encephalitis, streptococcus pneumoniae, streptococcus agalactiae, listeria monocytogenes, or borrelia burgdorferi; or a viral pathogen: coxsackievirus, echovirus, poliovirus, other enteroviruses, mumps virus, herpes simplex virus, varicella-zoster virus, arbovirus or bunyavirus.

Infections of the eye/orbit are typically induced by: bacterial species: staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mulleri (Streptococcus milleri), Escherichia coli, Bacillus cereus, Chlamydia trachomatis, Haemophilus influenzae, Pseudomonas, Klebsiella or Treponema pallidum; or a viral pathogen: adenovirus, herpes simplex virus, varicella-zoster virus or cytomegalovirus.

Infection of salivary glands is usually induced by: bacterial species: staphylococcus aureus, Streptococcus viridans (e.g., Streptococcus salivarius, Streptococcus sanguinis, Streptococcus mutans), Streptococcus digesta, or bacteroides, or other oral anaerobes; or a viral pathogen: mumps virus, influenza virus, enterovirus or rabies virus.

Infections of the oral cavity are typically induced by: bacterial species: prevotella melanogenes, Streptococcus anaerobicus, Streptococcus viridans, Actinomyces spp, Streptococcus digestus, or Bacteroides, or other oral anaerobes; or a viral pathogen: herpes simplex virus, coxsackie virus or epstein-barr virus.

Infections of the tonsils are typically induced by: bacterial species: streptococcus pyogenes or group C or G hemolytic streptococcus b; or a viral pathogen: rhinovirus, influenza virus, coronavirus, adenovirus, parainfluenza virus, respiratory syncytial virus or herpes simplex virus.

Infection of the sinuses is usually induced by a bacterial species, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, α -streptococci, anaerobes (e.g. Prevotella) or Staphylococcus aureus, or a viral pathogen, rhinovirus, influenza virus, adenovirus or parainfluenza virus.

Nasopharyngeal infection is usually induced by: bacterial species: streptococcus pyogenes or group C or G hemolytic streptococcus b; or a viral pathogen: rhinovirus, influenza virus, coronavirus, adenovirus, parainfluenza virus, respiratory syncytial virus or herpes simplex virus.

Infection of the thyroid gland is usually induced by: bacterial species: staphylococcus aureus, streptococcus pyogenes, or streptococcus pneumoniae; or a viral pathogen: mumps virus or influenza virus.

Infections of the larynx are typically induced by: mycoplasma pneumoniae, chlamydophila pneumoniae, or streptococcus pyogenes; or a viral pathogen: rhinovirus, influenza virus, parainfluenza virus, adenovirus, coronavirus, or human metapneumovirus.

Infections of the trachea are typically induced by: cell species: mycoplasma pneumoniae; or a viral pathogen: parainfluenza virus, influenza virus, respiratory syncytial virus or adenovirus.

Bronchial infections are usually induced by: mycoplasma pneumoniae, chlamydophila pneumoniae, bordetella pertussis, streptococcus pneumoniae, or haemophilus influenzae; or a viral pathogen: influenza virus, adenovirus, rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus or coxsackie virus.

Infections of the lung are usually induced by: cell species: streptococcus pneumoniae, moraxella catarrhalis, mycoplasma pneumoniae, klebsiella pneumoniae, or haemophilus influenzae; or a viral pathogen: influenza virus, adenovirus, respiratory syncytial virus or parainfluenza virus.

Pleural infections are usually induced by: cell species: staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides fragilis, Prevotella, Fusobacterium nucleatum, Streptococcus digestions or Mycobacterium tuberculosis; or a viral pathogen: influenza virus, adenovirus, respiratory syncytial virus or parainfluenza virus.

Infection of the longitudinal septum is typically induced by: bacterial species: viridans streptococci, peptococci, peptostreptococci, bacteroides or mycobacterium tuberculosis; or a viral pathogen: measles virus, rubella virus, epstein barr virus, or cytomegalovirus.

Infection of the heart is typically induced by: bacterial species: streptococci (including streptococcus mitis (s.mitior), streptococcus bovis (s.bovis), streptococcus sanguinis (s.sanguis), streptococcus mutans (s.mutans), streptococcus valacis), enterococci, Staphylococcus (staphyloccocus spp.), corynebacterium diphtheriae, clostridium perfringens, diplococcus encephalitis, or salmonella; or a viral pathogen: enteroviruses, coxsackieviruses, echoviruses, polioviruses, adenoviruses, mumps viruses, measles viruses or influenza viruses.

Infections of the esophagus are typically induced by: bacterial species: actinomycetes, mycobacterium avium, mycobacterium tuberculosis, or streptococcus; or a viral pathogen: cytomegalovirus, herpes simplex virus or varicella-zoster virus.

Infections of the stomach are typically induced by: bacterial species: streptococcus pyogenes or helicobacter pylori; or a viral pathogen: cytomegalovirus, herpes simplex virus, epstein-barr virus, rotavirus, norwalk virus or adenovirus.

Infection of the small intestine is usually induced by: bacterial species: escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus (Bacteroides vulgatus), Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron), Clostridium aeroginosum, Salmonella enteritidis (Salmonella enteridis), Yersinia enterocolitica, or Shigella flexneri; or a viral pathogen: adenovirus, astrovirus, calicivirus, norwalk virus, rotavirus, or cytomegalovirus.

Infections of the colon/rectum are usually induced by: bacterial species: escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium aeroginosum, Salmonella enteritidis, Yersinia enterocolitica, or Shigella flexneri; or a viral pathogen: adenovirus, astrovirus, calicivirus, norwalk virus, rotavirus, or cytomegalovirus.

Infection of the anus is usually induced by: bacterial species: streptococcus pyogenes, bacteroides, clostridium, anaerobic streptococcus, clostridium, escherichia coli, enterobacter, pseudomonas aeruginosa or treponema pallidum; or a viral pathogen: herpes simplex virus.

Perineal infections are usually induced by: bacterial species: escherichia coli, Klebsiella, Enterobacter, Bacteroides, Clostridium, Pseudomonas aeruginosa, Streptococcus anaerobicus, Clostridium, or Enterobacter; or a viral pathogen: herpes simplex virus.

Infections of the liver are typically induced by: bacterial species: coli, klebsiella, streptococcus (pharyngeal group), enterococcus, other viridans streptococci or bacteroides; or a viral pathogen: hepatitis A virus, Epstein-Barr virus, herpes simplex virus, mumps virus, rubella virus, measles virus, varicella-zoster virus, coxsackie virus or adenovirus.

Infection of the gallbladder is usually induced by: bacterial species: escherichia coli, Klebsiella, Enterobacter, enterococcus, Bacteroides, Clostridium, Salmonella enteritidis, Yersinia enterocolitica, or Shigella flexneri.

Infections of the biliary tract are typically induced by: bacterial species: escherichia coli, Klebsiella, Enterobacter, enterococcus, Bacteroides, Clostridium, Salmonella enteritidis, Yersinia enterocolitica, or Shigella flexneri; or a viral pathogen: hepatitis A virus, Epstein-Barr virus, herpes simplex virus, mumps virus, rubella virus, measles virus, varicella-zoster virus, coxsackie virus or adenovirus.

Infection of the pancreas is usually induced by: bacterial species: escherichia coli, klebsiella, enterococcus, pseudomonas, staphylococcus, Mycoplasma (Mycoplasma spp.), Salmonella typhi (Salmonella typhi), leptospira or legionella; or a viral pathogen: mumps virus, coxsackie virus, hepatitis B virus, cytomegalovirus, herpes simplex virus type 2 or varicella-zoster virus.

Infection of the spleen is usually induced by: bacterial species: streptococcus, staphylococcus, salmonella, pseudomonas, escherichia coli, or enterococcus; or a viral pathogen: epstein-barr virus, cytomegalovirus, adenovirus, measles virus, rubella virus, coxsackie virus, or varicella-zoster virus.

Infections of the adrenal glands are usually induced by: bacterial species: streptococcus, staphylococcus, salmonella, pseudomonas, escherichia coli, or enterococcus; or a viral pathogen: varicella-zoster virus.

Infections of the kidney are typically induced by: bacterial species: escherichia coli, Proteus mirabilis (Proteus mirabilis), Proteus vulgaris (Proteus vulgatus), providencia sp, Morganella sp, enterococcus faecalis, or Pseudomonas aeruginosa; or a viral pathogen: BK virus or mumps virus.

Urinary tract infections are usually induced by the following bacterial species: escherichia coli, Proteus mirabilis, Proteus vulgaris, providencia, Morganella or enterococcus.

Infections of the bladder are usually induced by: bacterial species: escherichia coli, Proteus mirabilis, Proteus vulgaris, providencia, Morganella, enterococcus, or Corynebacterium jekeum; or a viral pathogen: adenovirus or cytomegalovirus.

Infection of the peritoneum is usually induced by: bacterial species: staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae (Streptococcus pneumoniae), Escherichia coli, Klebsiella, Proteus, enterococcus, Bacteroides fragilis, Prevotella melanogenes, Pediococcus digestans, Streptococcus digestus, Clostridium or Clostridium.

Infection of the retroperitoneal region is typically induced by the following bacterial species: escherichia coli or Staphylococcus aureus.

Infection of the prostate gland is usually induced by: bacterial species: escherichia coli, Klebsiella, Enterobacter, Proteus mirabilis, enterococcus, Pseudomonas, Corynebacterium or Neisseria gonorrhoeae; or a viral pathogen: herpes simplex virus.

Infection of the testis is usually induced by: bacterial species: escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, staphylococcus, streptococcus or salmonella enteritidis; or a viral pathogen: mumps virus, coxsackie virus or lymphocytic choriomeningitis virus.

Infections of the penis are usually induced by: bacterial species: staphylococcus aureus, streptococcus pyogenes, neisseria gonorrhoeae or treponema pallidum; or a viral pathogen: herpes simplex virus.

Infection of the ovary/adnexa is typically induced by: bacterial species: neisseria gonorrhoeae, Chlamydia trachomatis, Gardenerella vaginalis, Prevotella, Bacteroides, Pediococcus, Streptococcus or Escherichia coli.

Infection of the uterus is usually induced by the following bacterial species: neisseria gonorrhoeae, Chlamydia trachomatis, Gardnerella vaginalis, Prevotella, Bacteroides, Peptococcus, Streptococcus or Escherichia coli.

Infections of the cervix are typically induced by: bacterial species: neisseria gonorrhoeae, chlamydia trachomatis or treponema pallidum; or a viral pathogen: herpes simplex virus.

Vaginal infections are usually induced by: bacterial species: gardnerella vaginalis, Prevotella, Bacteroides, Peptococcus, Escherichia coli, Neisseria gonorrhoeae, Chlamydia trachomatis or Treponema pallidum; or a viral pathogen: herpes simplex virus.

Vulvar infections are usually induced by: bacterial species: staphylococcus aureus, streptococcus pyogenes, or treponema pallidum; or a viral pathogen: herpes simplex virus.

Strain/virus subtype

It will be appreciated by those skilled in the art that bacterial species are operationally classified as a collection of approximate strains (which typically involve a putative sibling population with determinable physiological differences but typically without morphological differences, and which differences can be determined using serological techniques against bacterial surface antigens). Thus, each bacterial species (e.g., streptococcus pneumoniae) has many strains (or serotypes) that may differ in their ability to induce an infection or in their ability to induce an infection at a particular organ/site. For example, while there are at least 90 serotypes of streptococcus pneumoniae, serotypes 1, 3, 4, 7, 8, and 12 are most common causes of streptococcus pneumoniae disease in humans.

As a second example, certain strains of escherichia coli, also known as enteropathogenic escherichia coli (ExPEC), are more likely to induce urinary tract infections or other extraintestinal infections, e.g., neonatal meningitis, while other strains, including enterotoxigenic escherichia coli (ETEC), enteropathogenic escherichia coli (EPEC), enterohemorrhagic escherichia coli (EHEC), shiga toxin-producing escherichia coli (STEC), enteroaggregative escherichia coli (EAEC), enteroinvasive escherichia coli (EIEC), and Diffusively Adherent Escherichia Coli (DAEC), are more likely to induce gastrointestinal infections/diarrhea. Even within the ExPEC strain subclass, certain virulence factors (e.g., production of type 1 pili) make certain strains more likely to induce infection of the bladder, while other virulence factors (e.g., production of P pili) make other strains more likely to induce infection in the kidney. According to the present invention, ExPEC strains more likely to induce infection in the bladder can be selected for formulations targeting bladder cancer, while ExPEC strains more likely to induce infection in the kidney can be selected for formulations targeting kidney cancer. Likewise, one or more of the ETEC, EPEC, EHEC, STEC, EAEC, EIEC or DAEC strains of the large intestine rod (i.e., the colonic inflammation-inducing strains) may be selected for use in formulations for treating colon cancer.

Also, there may be a large number of subtypes of a particular virus. For example, there are three types of influenza viruses, i.e., influenza a, influenza b, and influenza c, which differ in epidemiological, host range, and clinical characteristics. For example, influenza a viruses are more likely to be associated with viral lung infections, while influenza b viruses are more likely to be associated with myositis (i.e., muscle infections). In addition, each of these three types of influenza viruses has many subtypes, which may also differ in epidemiology, host range, and clinical characteristics. According to the present invention, the influenza a virus subtype most commonly associated with lung infection may be selected to target lung cancer, while the influenza b virus strain most commonly associated with myositis may be selected to treat muscle/soft tissue cancer.

It is to be understood that clinical microbiologists in the art are thus able to select strains of a particular bacterial species (or a particular viral subtype) to target a particular organ or tissue based on the present disclosure and the primary knowledge associated with the strains of each bacterial species (and viral subtypes of each type of virus).

Bacterial compositions, dosages and administrations

The compositions of the invention comprise antigens of pathogenic microbial (bacterial or viral) species that are pathogenic in a particular tissue or organ. The composition may comprise whole bacterial species, or may comprise an extract or preparation of the pathogenic bacterial species of the invention, such as cell wall and membrane extracts, or whole cells, or exotoxins, or whole cells and exotoxins. The compositions may also comprise one or more antigens isolated from one or more pathogenic bacterial species of the invention; in certain embodiments, such compositions may be used where it may be necessary to accurately administer a particular dose of a particular antigen, or where administration of its whole cell species or component (e.g., toxin) may be harmful, the composition may be used. Pathogenic bacterial species may be commercially available (e.g. from ATCC (Manassas, VA, USA)), or may be clinically isolated from individuals suffering from bacterial infections of tissues or organs (e.g. pneumonia).

The microbial compositions of the invention can be provided alone or in combination with other compounds (e.g., nucleic acid molecules, small molecules, peptides or peptide analogs) in a form suitable for administration to a mammal, such as a human, in the presence of liposomes, adjuvants or any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier can be adapted for any suitable form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalation, intratumoral, or buccal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound (i.e., a particular bacterium, bacterial antigen, or composition thereof, of the invention), its use in compositions of the invention is contemplated. Auxiliary active compounds can also be incorporated into the compositions.

If desired, treatment with the bacterial antigens of the present invention can be combined with a variety of conventional and existing therapies for cancer, such as chemotherapy, radiation therapy, surgical and like compositions, or with any other therapy (e.g., nutrients, vitamins, and supplements) intended to stimulate the immune system, reduce inflammation, or otherwise benefit the subject^TM[ celecoxib]Or Vioxx^TM[ rofecoxib])。

Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions for administration of the compounds to individuals suffering from cancer. Suitable routes of administration may be employed, for example, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalational, aerosol, topical, intratumoral, sublingual or buccal. The therapeutic formulation may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; for intradermal formulations, in the form of a powder, nasal drops, or aerosol; and for sublingual formulations, in the form of drops, aerosols or capsules.

Methods well known in the art for preparing formulations are obtained, for example, in "Remington's pharmaceutical sciences" (20th edition), ed.a. gennaro,2000, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may, for example, comprise excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyethylene oxide-polypropylene oxide copolymers may be used to control the release of the compounds. Other parenteral delivery systems that may be used include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily liquids for administration in the form of nasal drops or gels. For therapeutic or prophylactic compositions, depending on the condition, the pathogenic bacterial species is administered to the individual in an effective amount to stop or slow the progression or metastasis of the cancer, or to increase the survival of the individual (relative to prognosis, e.g., from the SEER database).

An "effective amount" of a pathogenic microbial species or antigen thereof of the invention includes a therapeutically effective amount or a prophylactically effective amount. By "therapeutically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., reduction or elimination of cancer cells or tumors, prevention of carcinogenic processes, slowing tumor growth, or increasing survival beyond that expected using, for example, the SEER database. An effective amount of a pathogenic microbial (bacterial or viral) species or antigen thereof may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. The dosage regimen may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be an amount in which the therapeutically beneficial effect is superior to the toxic or deleterious effects of any pathogenic bacterial species or viruses or their antigens. By "prophylactically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, e.g., prevention of cancer, prevention of metastasis, reduction of tumor growth, reduction or elimination of cancer cells, tissues, organs, or tumors, or increase in survival beyond that expected using, for example, the SEER database. Typically, a prophylactic dose is administered to an individual prior to or at the initial stage of cancer, such that the prophylactically effective amount may be less than the therapeutically effective amount.

For administration by subcutaneous or intradermal injection, an exemplary range of therapeutically or prophylactically effective amounts of one or more pathogenic bacterial species can be about 1 to 1 billion organisms per ml, or can be 1 to 70 billion organisms per ml, or can be 5 to 60 billion organisms per ml, or can be 10 to 50 billion organisms per ml, or can be 20 to 40 billion organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may be 1 million to 1 billion organisms per ml, or may be 5 million to 70 million organisms per ml, or 1 million to 60 million organisms per ml, or may be 5 hundred million to 50 hundred million organisms per ml, or may be 10 hundred million to 40 hundred million organisms per ml, or any integer within these ranges. A therapeutically or prophylactically effective amount of an antigen of a pathogenic bacterial species may range from 0.1nM to 0.1M, 0.1nM to 0.05M, 0.05nM to 15 μ M, or any integer from 0.01nM to 10 μ M.

It should be noted that the dosage concentration and range may vary with the severity of the disease state to be alleviated, or may vary with the immune response of the individual. Generally, the goal is to achieve an adequate immune response. For administration by subcutaneous or intradermal injection, the extent of the immune response can be determined, for example, by the size (e.g., from 0.25 inch to 4 inches in diameter) of the delayed local immune skin response at the injection site. The dosage required to achieve an appropriate immune response may vary depending on the individual (and their immune system) and the response desired. Standardized dosages may also be used. In the case of subcutaneous or intradermal administration, if the goal is to achieve a2 inch local skin response, the total bacterial composition dose can range, for example, from 2 million bacteria (e.g., 0.001ml of vaccine at a concentration of 20 million organisms per ml) to greater than 200 million bacteria (e.g., 1ml of vaccine at a concentration of 200 million organisms per ml). The concentration of the individual bacterial species or antigens thereof within the composition may also be considered. For example, if the concentration of a particular pathogenic bacterial species, the cell size of that species, or the antigenic load thereof is higher in a vaccine relative to other pathogenic bacterial species, the local immune skin response of an individual may be attributed to its response to that particular bacterial species. In some embodiments, the immune system of an individual may be more intense than one bacterial species within a vaccine, for example depending on the past history of infection exposure to a particular species, then the dose or composition may be adjusted accordingly for that individual. However, in some embodiments detailed herein, the immune response will not be monitored by a skin response. For example, in some mouse models used herein, effective treatment of such animals with an antigen composition may not result in a corresponding skin response. It will be appreciated by those skilled in the art that there are alternative ways in which the immune response can be monitored, as compared to relying on the presence or absence of a skin response.

The timing and dosage of treatment can be adjusted over time for a particular individual according to the individual's needs and the professional judgment of the person administering or instructing the administration of the composition (e.g., the timing can be daily, every other day, weekly, monthly). For example, in the case of subcutaneous or intradermal administration, the composition may be administered every other day. An initial dose of about 0.05ml can be administered subcutaneously, followed by an increase of 0.01-0.02ml every other day until a sufficient cutaneous response is administered at the injection site (e.g., a delayed response of visibly redness at the injection site of 1 inch to 2 inches in diameter). Once this sufficient immune response is achieved, the dose may be continued as a maintenance dose. The maintenance dose can be adjusted from time to achieve the desired apparent skin response (inflammation) at the injection site. Administration can be for an administration duration of, e.g., at least 1 week, 2 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years or longer.

The oral dose may be, for example, 1000 to 1 trillion organisms per administration, including one or more species of antigenic determinants. Oral doses may be prescribed, for example, 4 times daily, once daily, or once weekly. Administration can be for an administration duration of, e.g., at least 1 week, 2 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years or longer.

In some embodiments, the invention may comprise an antigenic composition administered sublingually or by inhalation, or administered simultaneously or sequentially to one or more epithelial tissues (i.e., dermally by intradermal or subcutaneous injection; pulmonary epithelial by inhalation; gastrointestinal mucosal by buccal uptake; oromucosal by sublingual administration). Thus, in some embodiments, the antigenic compositions of the present invention are administered to elicit an immune response in epithelial tissues. In some embodiments, one or more epithelial routes of administration may be combined with one or more other routes of administration, such as intratumoral, intramuscular, or intravenous administration.

In various aspects of the invention, the antigenic composition administered to a patient may be characterized as having an antigenic signature, i.e., a combination of antigens or epitopes sufficient to be specific, such that the antigenic composition is capable of eliciting an immune response, e.g., an adaptive immune response, specific for a particular pathogen. A surprising and unexpected aspect of the present invention is that the non-adaptive or non-specific activation of immune responses mediated by these specific antigenic compositions is effective in the treatment of cancer located in tissues where the particular pathogen is pathogenic.

The routes of administration and dosage ranges described herein are exemplary only, and are not limiting as to the routes of administration and dosage ranges that may be selected by a practitioner. The amount of active compound (e.g., pathogenic bacterial species or virus or antigen thereof) in the composition may vary depending on factors such as the disease state, age, sex and weight of the individual. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, a single pill may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be advantageously formulated in dosage unit form for ease of administration and uniformity of dosage.

In the case of an antigenic preparation (similar to a vaccine), an immunologically effective amount of a compound of the invention can be provided alone or in combination with other compounds, with immunological adjuvants. The compounds may also be linked to a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin, to increase immunogenicity. An antigenic composition ("vaccine") is a composition comprising materials that elicit a desired immune response. The antigenic composition may select, activate or expand, but is not limited to: the memory B, T cells, neutrophils, monocytes or macrophages of the immune system, for example, to reduce or eliminate growth or proliferation of cancer cells or tissues. In some embodiments, a specific pathogenic microorganism, virus, viral antigen, bacterium, bacterial antigen, or composition thereof of the invention is capable of eliciting a desired immune response in the absence of any other agent, and thus can be considered an antigenic composition. In some embodiments, the antigenic composition includes a suitable carrier, such as an adjuvant, which is an agent that acts in a non-specific manner to increase the immune response to a particular antigen or population of antigens, enabling a reduction in the quality of the antigen in any given vaccine administration, or a reduction in the frequency of doses required to produce a desired immune response. The bacterial antigenic composition may comprise live or dead bacteria capable of eliciting an immune response against antigenic determinants normally associated with the bacteria. In some embodiments, the antigenic composition may comprise live bacteria with less virulent strains (attenuated) and thereby induce less severe infections. In some embodiments, the antigenic composition may comprise a live, attenuated, or killed virus capable of eliciting an immune response against antigenic determinants normally associated with the virus.

A composition comprising a bactericidally-effective antigen for administration by injection may be prepared as follows. The bacteria can be grown in a suitable medium and washed with a physiological salt solution. The bacteria can then be centrifuged, resuspended in a re-saline solution, and killed with heat. The suspensions can be standardized by direct microscopic counting, mixed with the required amounts and stored in suitable containers, which can be tested in an approved manner for safety, shelf life and sterility. In addition to pathogenic bacterial species and/or antigens thereof, the killed bacteria suitable for administration to humans may comprise 0.4% phenol preservative and/or 0.9% sodium chloride. The bacterial vaccine may also contain traces of brain heart infusion (bovine), peptone, yeast extract, agar, sheep blood, dextrose, sodium phosphate, and/or other media components.

In some embodiments, the bacterial vaccine may be used in the form of a tablet or capsule, or in the form of drops for oral ingestion, in the form of an aerosol for inhalation, or in the form of drops, aerosol or tablets for sublingual administration.

In the antigen composition comprising bacteria, the concentration of a particular bacterial species in the composition for sublingual or intradermal injection may be about 1 to 1 billion organisms per ml, or may be 1 to 70 billion organisms per ml, or may be 5 to 60 billion organisms per ml, or may be 10 to 50 billion organisms per ml, or may be 20 to 40 billion organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may be 1 million to 1 billion organisms per ml, or may be 5 million to 70 million organisms per ml, or 1 million to 60 million organisms per ml, or may be 5 hundred million to 50 hundred million organisms per ml, or may be 10 hundred million to 40 hundred million organisms per ml, or any integer within these ranges.

In some embodiments, the selected bactericidal vaccine for lung tissue cancer will include common bacterial lung pathogens and may be, for example:

or:

in some selected embodiments, the selected bactericidal vaccine for lung tissue cancer will include only the more common bacterial lung pathogens, and may be, for example:

or:

in other selected embodiments, the selected bactericidal vaccine for lung tissue cancer will include only the most common bacterial lung pathogens and may be:

or

Or

In some embodiments, an anti-microbial composition for treating cancer at a particular site (e.g., cancer of lung tissue) may include pathogenic microorganisms that typically, more typically, or most typically induce an infection in that tissue or organ (e.g., an infection in lung tissue, i.e., pneumonia).

Generally, the pathogenic bacterial species of the present invention and their antigens should be used without substantially inducing toxicity. Toxicity of the compounds of the invention can be determined using standard methods, e.g., by testing in cell cultures or experimental animals, and determining the therapeutic index, i.e., the ratio between LD50 (the dose lethal to 50% of the population) and LD100 (the dose lethal to 100% of the population).

In some aspects, the invention includes the use of anti-inflammatory drugs in conjunction with vaccination. In these embodiments, a wide range of anti-inflammatory therapies may be employed, including effective amounts of non-steroidal anti-inflammatory drugs (NSAIDs), including but not limited to: diclofenac potassium, diclofenac sodium, etodolac, indomethacin, ketorolac tromethamine, sulindac, tolmetin sodium, celecoxib, meloxicam, valdecoxib, fluoroquinuprate, mefenamic acid, nabumetone, meloxicam, piroxicam, tenoxicam, fenoprofen calcium, flurbiprofen, ibuprofen, ketoprofen, naproxen sodium, oxaprozin, tiaprofenic acid, acetylsalicylic acid, diflunisal, choline magnesium trisalicylate, choline salicylate, triethanolamine salicylate, COX1 inhibitor, COX2 inhibitor (e.g., Vioxx. RTM. X. TM^TMAnd Celebrex^TM). A variety of herbs and natural health products may also be used to provide anti-inflammatory therapy, including but not limited to: green tea, fish oil, vitamin D, antioxidant vitamins and antibiotics (e.g., B carotene, vitamin a, vitamin C, vitamin D, vitamin E, coenzyme Q10, selenium, etc.), resveratrol, turmeric, bromelain, frankincense, feverfew, quercetin, ginger, rosemary, oregano, capsicum, clove, nutmeg, willow bark. Alternative anti-inflammatory forms also include lifestyle changes, such as: exercise, weight loss, smoking cessation, stress relief, seeking social support, treatment of depression, stress management, abdominal breathing, and dietary changes (e.g., with Mediterranean diet, low-glycemic diet, use of non-burnt foods, including foods with omega-3 fatty acids).

As detailed herein and in one aspect of the invention, methods of comparing immune responses are provided. The method comprises administering to an animal having an organ or tissue a medicament having an antigenic composition as defined herein. The antigenic composition may have antigenic determinants selected or formulated so that together they are specific for at least one microbial pathogen that is pathogenic in an organ or tissue, extracting a quantifiable immune sample from the organ or tissue, measuring a characteristic of the immune response within the organ or tissue in the quantifiable immune sample after administration of the drug, and comparing the characteristic of the immune response of the quantifiable immune sample to a corresponding characteristic of the immune response of a reference immune sample obtained from the corresponding organ or tissue. As used herein, an immune sample will contain sufficient biological material to determine the characteristics of the immune response. As used herein, a "characteristic" of an immune response can include, but is not limited to, a specific amount of a particular immune cell type (e.g., macrophage) or a particular cellular marker (e.g., upregulation of integrins) or gene product (e.g., cytokine). The foregoing are examples and are not limiting.

Optionally, the reference immune sample can be obtained from the corresponding organ or tissue in the animal prior to the step of administering the drug. In another aspect, the reference immune sample may be obtained from a corresponding organ or tissue of the second animal, such that it comprises in particular: at least two animals (i.e., an animal from which the reference sample was obtained and a second animal from which the quantifiable immune sample was obtained) can be used in the methods described herein. Optionally, the animal may have a cancer located in an organ or tissue.

Comparing the characteristics of the immune response may include comparing numerical indications of any one or more of the following cells in the quantifiable and reference immune samples (as such cells are known to those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages.

One skilled in the art will recognize that macrophages can be defined as "M1-like macrophages" or "M2-like macrophages". For example, M1-like macrophages are generally understood by those skilled in the art to promote Th1CD4+ T cell-mediated responses (see, e.g., Biswas and Mantovani (2010), Nature Immunology10: 889-96). In addition, M1-like macrophages are generally understood to have potent antigen presenting capabilities and are responsible for killing intracellular pathogens (e.g., viruses). Furthermore, M1-like macrophages are generally understood to play an immune role in tumor destruction at least in comparison to M2-like macrophages. One skilled in the art will recognize that there are many biomarkers that can be employed to differentiate between M1-like macrophages and M2-like macrophages. For example, and as detailed herein, expression of Nos2 is generally understood to be associated with M1-like macrophages, as compared to M2-like macrophages (see, e.g., Laskin et al (2010) annualrev. In addition and for example, M1-like macrophages are generally understood to produce IL-12 and to be efficiently activated by IFN- γ R (Biswas and Mantovain, supra).

In contrast to M1-like macrophages, those skilled in the art will generally understand that M2-like macrophages promote Th2CD4+ T cell-mediated responses (see, generally: Biswas and Mantovani (2010), Nature Immunology10: 889-96). Furthermore, M2-like macrophages are generally understood to be potent and to encapsulate and clear extracellular parasites and the like. In addition and in contrast to M1-like macrophages, M2-like macrophages are generally understood by those of skill in the art as being useful for T_regAnd B cells play a more significant role in immune regulation (Biswas and mantovan, supra.) one of skill in the art will recognize that there are many biomarkers that can be employed to differentiate between M2-like macrophages and M1-like macrophages for example and as described herein, attenuated expression of Nos2 will generally be understood to be associated with M2-like macrophages compared to the higher expression typically found in M1-like macrophages, also as detailed in the experiments herein incorporated, expression of CD206 is generally understood to be associated with M2-like macrophages (see, e.g., Choi et al (2010) Gastroenterology 138(7) 2399-409.) additionally and as detailed in the experiments herein, expression of F4/80 is generally understood to be associated with M2-like macrophages additionally and for example, M2-like macrophages are generally understood to be activated by IL-4 or by IL-13 through IL-4R α (bistovan, supra).

In addition, comparing the characteristics of the immune response may include comparing changes in macrophage activation state. Changes in macrophage activation state can optionally be characterized as a change from M2-like macrophages to M1-like macrophages or vice versa. One skilled in the art will recognize that there are many biomarkers that can be employed to monitor macrophage activation. As detailed herein, one skilled in the art will recognize that macrophages are defined to be activated towards either the M1-like phenotype or the M2-like phenotype, which can be accomplished by selecting markers known to be associated with each of the phenotypes described herein. Diseases that have been associated with M1 and M2 macrophages include at least the following: atherosclerosis (see, e.g., Hirata et al (2011) j.am. col. cardio.58 (3):248-, Pancreatitis (see, e.g., Gea-Sorli and Closa (2009) BMC Immunol.31:42), myocarditis (see, e.g., Li et al (2009) Circuit. Res.105(4):353-64), liver fibrosis (see, e.g., Heymann et al (2009) Inflamm. Allergy Drug Targets 8(4):307-18), cystic fibrosis (see, e.g., Meyer et al (2009) am.J.Respir.cell mol. biol.41(5):590-602), inflammatory nephritis (see, e.g., Wang. (2007) Kidney int.72(3):290-299), and lung (see, e.g., Misson et al (2004) J.Leoc.Biol.76 (5): silicon ion 232).

Optionally, comparing the characteristics of the immune response may comprise determining cellular markers on any one or more of the following cells in the quantifiable and reference immune samples (as such cells are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. One skilled in the art will recognize that there are many cellular markers (both extracellular and intracellular) that can be selected to determine the immune response. Also as detailed herein, expression of CD206 is generally understood to be associated with M2-like macrophages (see, e.g., Choi et al (2010) Gastroenterology 138(7) 2399-409).

Optionally, comparing the characteristics of the immune response may include determining in the quantifiable and reference immune samples a cellular marker produced by any one or more of inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells, or NK cells, as those skilled in the art will recognize that cytokines refer to small cell signaling protein molecules and there are many cytokines known in the art, for example, cytokines have been classified into type 1 and type 2 based on their role in the immune response, typical type 1 cytokines include IFN- γ and TGF- β typical type 2 cytokines include, but are not limited to, IL-4 and IL-13.

As described herein, macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages as defined herein. Optionally, the cytokine is produced as a result of a change in the activation state of the macrophage. Optionally, the macrophage is changed from an M2-like macrophage to an M1-like macrophage. Additionally and optionally, the macrophage changes from an M1-like macrophage to an M2-like macrophage.

Optionally, comparing the characteristics of the immune response may comprise determining differential gene expression in the quantifiable and reference immune samples produced by any one or more of the following cells (as such cells are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. The term "differential gene expression" is understood to mean a considerable difference between the expression of a specific gene of interest from at least two experimental conditions. For example, if a particular gene has a defined expression level under a first experimental condition, as defined by the gene expression method used by those skilled in the art, and if the same gene has a considerable difference in its expression level under a second experimental condition, there is differential expression of the gene of interest. One skilled in the art will appreciate that there are many methods available for detecting differential gene expression. For example, commercially available quantitative PCR techniques can be used as detailed herein in determining the relative Nos2/Arg1 ratio (see, e.g., fig. 29). Optionally, the differential gene expression is produced as a result of a change in the activation state of the macrophage. Optionally, macrophages can change from M2-like macrophages to M1-like macrophages as those terms are defined herein.

In another embodiment, the drug may be administered at the site of administration in consecutive doses given at dosing intervals of one hour to one month over a dosing duration of at least one week. Optionally, the drug may be administered intradermally or subcutaneously. Optionally, the drug may be administered at a dose effective to induce a significant local inflammatory immune response at the site of administration for each dose. Optionally, the drug may be administered so that significant local inflammation occurs at the site of administration within 1 to 48 hours. However, a pronounced local inflammatory immune response may not be present in all cases frequently, although the immune response has already begun. One skilled in the art will recognize that there are other methods that can be used to monitor an increasing immune response. For example, the immune cell profile (and the relative changes characterized) from an individual who has undergone an immune response can be compared to those from an individual who has not undergone an immune response.

In addition and optionally for the methods disclosed herein, the animal can be a mammal. Optionally, the animal can be a human or a mouse. The foregoing is provided in a tangible form and is not intended to be limiting.

In another aspect, a method of selecting a therapeutic agent suitable for treating cancer in a particular organ or tissue of an individual is provided. The method comprises providing an animal having a cancer localized to a particular organ or tissue; providing a test formulation having antigenic determinants of one or more microbial pathogens that are pathogenic in a corresponding specific organ or tissue of a healthy individual; measuring a characteristic of an immune response in a reference immune sample obtained from an organ or tissue of the animal; administering a test formulation to the animal; measuring a characteristic of an immune response of a quantifiable immune sample obtained from a corresponding organ or tissue of the animal; comparing the characteristics of the immune response in the reference and quantifiable immune samples; and processing the increased characteristic of the immune response of the quantifiable immune sample as compared to the reference immune sample as an indication of the suitability of the test preparation as a therapeutic preparation. Optionally, the animal is sacrificed prior to obtaining a quantifiable immune sample.

Optionally, comparing the characteristics of the immune response may comprise comparing the quantitative indicia of any one or more of the following cells in the quantifiable and reference immune samples (as such cells are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages, as those terms are defined herein. Optionally, comparing the characteristic of the immune response may comprise comparing a change in activation state of macrophages. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages. Additionally and optionally, macrophages can change from M1-like macrophages to M2-like macrophages.

Optionally, comparing the characteristics of the immune response may comprise determining cellular markers on any one or more of the following cells in the quantifiable and reference immune samples (as they are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages, as those terms are defined herein.

Optionally, comparing the characteristic of the immune response may comprise determining in the quantifiable and reference immune samples cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages, as those terms are defined herein. Optionally, the cytokine is produced as a result of a change in the activation state of the macrophage. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages.

Additionally and optionally, comparing the characteristics of the immune response may comprise determining differential gene expression produced by any one or more of the following cells in the quantifiable and reference immune samples: inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages, as those terms are defined herein. Optionally, differential gene expression may result from changes in the activation state of macrophages. Optionally, macrophages can be changed from M2-like macrophages to M1-like macrophages. Additionally and optionally, macrophages can change from M1-like macrophages to M2-like macrophages.

In another aspect, methods of selectively targeting an immune response to a cancerous tissue or organ in a human individual are provided. The method comprises administering to the individual a medicament having an effective amount of a microbial pathogen antigenic composition, wherein the microbial pathogen may be pathogenic in a particular cancerous organ or tissue of the individual and the antigenic composition comprises antigenic determinants that together are specific for the microbial pathogen. Optionally, the antigenic composition may comprise a whole killed bacterial cell composition. Optionally, the medicament may be administered to the individual at a dose and for a duration effective to upregulate an immune response in a cancerous organ or tissue of the individual. Optionally, the method may further comprise measuring a characteristic of the immune response.

In another aspect, a method for treating cancer located in a tissue or organ in a human subject is provided. The method comprises administering to the individual a medicament having an effective amount of a microbial pathogen antigen composition comprising a whole killed bacterial cell composition, wherein the microbial pathogen is pathogenic in the specific organ or tissue of the individual in which the cancer is located. The medicament may be administered to the individual in an amount and for a duration effective to modulate the immune response. Optionally, modulation of the immune response may include a change in the activation state of macrophages. Optionally, modulation of the immune response may include a change from an M2-like macrophage response to an M-1-like macrophage response. Modulation of the immune response may include a change from an M1-like macrophage response to an M-2-like macrophage response. Optionally, the method may further comprise measuring a characteristic of the immune response.

Optionally, comparing the characteristics of the immune response may comprise comparing the quantitative indicia of any one or more of the following cells in the quantifiable and reference immune samples (as such cells are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages, as those terms are defined herein. Optionally, comparing the characteristic of the immune response may comprise comparing a change in activation state of macrophages. Additionally and optionally, macrophages can change from M2-like macrophages to M1-like macrophages. Optionally, macrophages can be changed from M1-like macrophages to M2-like macrophages.

Additionally and optionally, comparing the characteristics of the immune response may include determining cellular markers on any one or more of the following cells in the quantifiable and reference immune samples (as they are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages, as those terms are defined herein. Optionally, comparing the characteristics of the immune response may comprise determining cellular markers produced by any one or more of the following cells in the quantifiable and reference immune samples (as they are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages. In addition, cytokines may be produced due to changes in the activation state of macrophages. Macrophages can vary from M2-like macrophages to M1-like macrophages. Optionally, macrophages can be changed from M1-like macrophages to M2-like macrophages.

Additionally and optionally, comparing the characteristics of the immune response may include determining differential gene expression produced by any one or more of the following cells in the quantifiable and reference immune samples (as they are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, differential gene expression may result from changes in the activation state of macrophages. Additionally and optionally, macrophages can change from M2-like macrophages to M1-like macrophages. Macrophages can vary from M1-like macrophages to M2-like macrophages.

In another aspect, a method of monitoring the efficacy of a treatment regimen in an individual being treated for cancer in a particular organ or tissue is provided. The method comprises measuring a characteristic of an immune response of a post-treatment immune sample obtained from a specific organ or tissue after the individual has been subjected to a treatment regimen for a period of time, wherein the presence of an immune response characteristic that is greater in magnitude than the immune response characteristic expected for the individual without the treatment regimen is indicative of the efficacy of the treatment regimen; and the treatment regimen comprises administering a formulation comprising one or more antigenic determinants of a microbial pathogen that is pathogenic in a corresponding specific organ or tissue of a healthy individual.

The methods detailed herein further comprise measuring a characteristic of an immune response of a pre-treatment reference sample, wherein the pre-treatment reference sample is obtained from a specific organ or tissue before, at the same time as, or after the start of the treatment regimen, but before obtaining the post-treatment immune sample, and comparing the characteristics of the immune response of the pre-treatment and post-treatment samples, wherein an increase in magnitude of the immune response of the post-treatment immune sample compared to the pre-treatment reference sample is indicative of the efficacy of the treatment regimen. Optionally, measuring a characteristic of the immune response may comprise determining an indication of the number of inflammatory monocytes in the sample of the organ or tissue. Optionally, measuring a characteristic of the immune response may comprise determining an indication of the number of macrophages in a sample of the organ or tissue. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages.

Optionally, measuring the magnitude of the immune response may comprise determining an indication of the number of NK cells in the sample of the organ or tissue. Additionally and optionally, comparing the characteristics of the immune response may include determining cellular markers on any one or more of the following cells in the reference and immune samples (as such cells are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. Optionally, the macrophage may include any one or more of the following: m1-like macrophages or M2-like macrophages.

Additionally and optionally, comparing the characteristics of the immune response may include determining in the reference and immune samples cytokines produced by any one or more of the following cells (as such cells are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Optionally, cytokines may be produced as a result of changes in the activation state of macrophages. Macrophages can vary from M2-like macrophages to M1-like macrophages. Additionally and optionally, macrophages can change from M1-like macrophages to M2-like macrophages.

Optionally, comparing the characteristics of the immune response may comprise determining differential gene expression in the reference and immune samples produced by any one or more of the following cells (as such cells are generally understood by those of skill in the art): inflammatory monocytes, macrophages, CD11b + Gr-1+ cells, dendritic cells, CD11c + MHC class II + cells, CD4+ T cells, CD8+ T cells or NK cells. The macrophages may include any one or more of the following: m1-like macrophages or M2-like macrophages. Differential gene expression may result from changes in the activation state of macrophages. Macrophages can vary from M2-like macrophages to M1-like macrophages. Optionally, macrophages can be changed from M1-like macrophages to M2-like macrophages.

In various aspects, embodiments of the invention relate to methods of treating cancer located in the lungs of an individual. The method comprises administering to the individual an effective amount of an antigen of one or more species of microorganism that is pathogenic in the lung, and administering to the individual an effective amount of a platinum-containing chemotherapeutic agent. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen.

As used herein, the phrase "treating cancer" may include, but is not limited to, reducing lung tumor burden in an individual or increasing the life expectancy of an individual with lung cancer. It should be understood that there are many biometric reading mechanisms available to one skilled in the art to determine whether a cancer is being treated.

Viral pathogens, as used herein, include, but are not limited to: influenza virus, adenovirus, respiratory syncytial virus, parainfluenza virus, monkeypox, herpes simplex virus (type 1 and 2), varicella zoster virus, cytomegalovirus, epstein barr virus, coronavirus, human metapneumovirus, hendra virus, nipah virus, hantavirus, lassa fever virus, human T-cell lymphotrophic virus, coxsackie virus, echovirus, enterovirus or rhinovirus, or any virus that is pathogenic in the lung.

Bacterial pathogens as used herein may be, but are not limited to: streptococcus pneumoniae, moraxella catarrhalis, mycoplasma pneumoniae, klebsiella pneumoniae, haemophilus influenzae, staphylococcus aureus, chlamydia pneumoniae, legionella pneumophila, or bordetella pertussis, or any bacteria that is pathogenic in the lung.

Fungal pathogens as used herein may be, but are not limited to: aspergillus fumigatus, Blastomyces, Coccidiodesposasii, Cryptococcus neoformans, Cryptococcus gatherens, Fusarium, Histoplasma capsulatum, Paecilomyces variotii, paracoccidioides brasiliensis, Penicillium marneffei, Pneumocystis jejuni, Pseudomyceliophthora boydii, Podospora tiphylla, Rhizopus, Mucor, Absidia, Cunninghamella parvulus, Stachybotrys chartarum, Trichoderma longibrachiatum, Sporospora polyspora, or any fungus pathogenic in the lung.

As used herein, the term "platinum-containing chemotherapeutic agent" includes, but is not limited to: cisplatin, carboplatin or ormaplatin, oxaliplatin, DWA2114R ((-) - (R) -2 aminomethylpyrrolidine (1, 1-cyclobutanedicarboxylato) platinum), zeniplatin, enloplatin, leplatin, CI-973(SP-43(R) -l, l-cyclobutane-dicarboxylato (2-) - (2-methyl-l, 4-65 butanediamine-N, N') platinum), 254-S nedaplatin, JM-216 (bis-acetyl-amine-dichloro-cyclohexylamine platinum (IV)) (see: Weiss, R.B. and Christian, M.C., "New collagen antibody in Development," drugs.46 (03)360-377 (1993)); CPA)2Pt [ DOLYM ] and (DACH) Pt [ DOLYM ] cisplatin (Choi et al, Arch. pharmaceutical Res.22(2):151-156, 1999); 254-S cisplatin analogs (Koga et al, neuron. Res.18(3):244-247, 1996); cis-l, 4-diaminocyclohexane cisplatin analogs (Shamsuddin et al, J.Inorg.biochem.61(4): 291-; MeOH cisplatin (Shamsuddin et al, Inorg. chem.36(25):59695971,1997); CI-973 cisplatin analogs (Yang et al int. J. Oncol.5(3): 597-; cis-diamminedichloroplatinum (II) and its analogs cis-l, l-cyclobutanedicarboxylato (2R) 2-methyl-l, 4-butanediamine platinum (II) and cis-diamineglycolated platinum (Claycamp & Zimbrick, J.Inorg.Biochem.26(4):257-, cis-spiroplatinum, carboplatin, iproplatin and JM40 platinum analogs (Schroyen et al, Eur.J.cancer Clin. Oncol.24(8): 1309-.

In another aspect, there is provided use of an effective amount of an antigen of one or more microbial species that are pathogenic in the lung to formulate a medicament for use in treating lung cancer in an individual in combination with a platinum-containing chemotherapeutic agent. In another aspect, there is provided the use of an effective amount of an antigen of one or more microbial species that is pathogenic in the lung for use with a platinum-containing chemotherapeutic agent as detailed herein to treat lung cancer in an individual. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen, as detailed herein.

In another aspect, an effective amount of an antigen of one or more species of microorganism that is pathogenic in the lung is provided to formulate a medicament for use with a platinum-containing chemotherapeutic agent for treating lung cancer in an individual. In another aspect, an antigen of one or more microbial species that is pathogenic in the lung is provided in an amount effective for use with a platinum-containing chemotherapeutic agent as detailed herein to treat lung cancer in an individual. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen, as detailed herein. Platinum-containing chemotherapeutic agents may be, but are not limited to: cisplatin, carboplatin, or oxaliplatin.

In another aspect, a kit is provided. The kit comprises an antigen of one or more microbial species that are pathogenic in the lung, a platinum-containing chemotherapeutic agent; and instructions for providing the antigen and the platinum-containing chemotherapeutic agent to a patient in need thereof. The microbial species may be a viral pathogen or a bacterial pathogen or a fungal pathogen, as detailed herein.

In various aspects, embodiments of the invention relate to compositions comprising components of an organism that can induce gastrointestinal infections, such that the organism can be characterized as a pathogen. However, organisms that are pathogenic in some cases may not frequently induce disease. Most animals are colonized to some extent by other organisms, such as bacteria, that normally exist in a symbiotic or symbiotic relationship with the host animal. Thus, many species of bacteria are found in healthy animals, which are generally harmless, and are often located on the surface of specific organs and tissues. Often, these bacteria contribute to the normal functioning of the body. For example, in humans, commensal e.coli can be found in the intestine, where they promote immunity and reduce the risk of infection by more lethal pathogens.

Bacteria that are normally harmless, such as E.coli, can induce infections in healthy individuals, and as a result range from mild to severe infections to death. Whether an organism, such as a bacterium, is pathogenic (i.e., induces an infection) depends to some extent on factors such as the route to and reaching a particular host cell, tissue or organ; inherent toxicity of bacteria; the amount of bacteria present at the site of potential infection; or the health of the host animal. Thus, organisms that are generally harmless can be pathogenic given the favorable conditions for infection, and even toxic organisms may require a particular environment to induce an infection. Thus, organisms that are members of the normal flora can be pathogens when they exceed their normal endogenous role in the endogenous flora. For example, endogenous species can induce infection outside their niches in anatomical vicinity, e.g., by serial dissemination. When this occurs, and in the present invention, these normally harmless endogenous organisms are considered pathogenic.

Specific organisms, such as bacterial species, viruses, worms and protozoa, are known to induce infections in specific areas of the GIT in otherwise healthy individuals. Examples of organisms that typically induce infection in specific regions of the GIT are listed below; it is to be understood that these examples are not intended to be limiting, and that the skilled person will be readily able to recognize and determine infectious or pathogenic bacteria that induce infection or generally induce infection in various regions of the GIT of healthy adults, e.g. based on knowledge about a specific patient population, e.g. as shown by the following disclosure: manual of Clinical Microbiology 8th edition, Patrick Murray, Ed.,2003, ASM Press American Society for Microbiology, Washington DC, USA; principles and practices of Mandell, Douglas and Bennett of infectiou Diseases, 5th edition, g.l.mandell, j.e.bennett, r.dolin, eds.,2000, churchlilllingstone, philiadelphia, PA, USA, all disclosures of which are incorporated herein by reference.

Infections of the oral cavity are typically induced by: bacterial species: melanogenesis prevotella, anaerobic streptococcus, viridans streptococcus, actinomycetes, digestive streptococcus or bacteroides, or other oral anaerobes; or a viral pathogen: herpes simplex virus, coxsackie virus or epstein-barr virus.

Infection of the small intestine is usually induced by: bacterial species: escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium aeroginosum, Salmonella enteritidis, Yersinia enterocolitica, or Shigella flexneri; or a viral pathogen: adenovirus, astrovirus, calicivirus, norwalk virus, rotavirus, or cytomegalovirus.

Organisms such as bacteria are often operationally classified as a collection of closely related strains (which typically involve a putative sibling population with determinable physiological differences but often without morphological differences, and which differences can be determined using serological techniques against bacterial surface antigens). Thus, each bacterial species (e.g., e.coli) has many strains (or serotypes) that may differ in their ability to induce infection or in their ability to induce infection of a particular organ/site. Certain strains of escherichia coli are more likely to induce gastrointestinal infection/diarrhea, including enterotoxigenic escherichia coli (ETEC), enteropathogenic escherichia coli (EPEC), enterohemorrhagic escherichia coli (EHEC), shiga toxin-producing escherichia coli (STEC), enteroaggregative escherichia coli (EAEC), enteroinvasive escherichia coli (EIEC), and Diffusively Adherent Escherichia Coli (DAEC). According to the present invention, one or more of the ETEC, EPEC, EHEC, STEC, EAEC, EIEC or DAEC strains of the large intestine rod (i.e. the colonic inflammation inducing strains) may be selected for use in an agent for treating IBD.

Likewise, there may be many subtypes of specific viruses, worms or protozoa that are associated with a particular population and thus suitable for use in the present invention.

The compositions of the present invention include antigens of organisms that are pathogenic in specific regions of the GIT. The composition may comprise whole organisms, whole cells or whole virion components, or may comprise an extract or preparation of an organism, such as a cell wall or cell membrane extract or an exotoxin. The compositions may also comprise one or more antigens isolated from these organisms. Pathogenic microorganisms may be commercially available (e.g., from the American Type Culture Collection, Manassas, VA, USA), or may be clinical isolates from individuals with an infection.

The compositions of the invention derived from a pathogen may be provided alone or in combination with other compounds (e.g., nucleic acid molecules, small molecules, peptides or peptide analogs) in a form suitable for administration to a mammal, such as a human, in the presence of liposomes, adjuvants or any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier can be adapted for any suitable form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalation, intratumoral, or buccal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound (i.e., a particular bacterium, bacterial antigen, or composition thereof, of the invention), its use in the compositions of the invention is contemplated. Auxiliary active compounds can also be incorporated into the compositions.

Methods well known in the art for preparing formulations are found, for example, in "Remington's pharmaceutical sciences" (20th edition), ed.A. Gennaro,2000, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, comprise excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyethylene oxide-polypropylene oxide copolymers may be used to control the release of the compounds. Other parenteral delivery systems that may be used include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily liquids for administration in the form of nasal drops or gels. For therapeutic or prophylactic compositions, the formulation can be administered to an individual in an amount effective to halt or slow the progression of IBD.

An "effective amount" of a pathogenic species or antigen thereof of the invention includes a therapeutically effective amount or a prophylactically effective amount. By "therapeutically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., to reduce or eliminate the symptoms of IBD. An effective amount of a pathogenic microbial species or antigen thereof can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. The dosage regimen may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be an amount wherein the therapeutically beneficial effect outweighs the toxic or deleterious effects of any pathogenic species or antigen thereof. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, e.g., prevention of IBD. Typically, a prophylactic dose is administered to an individual prior to or at the onset of IBD, so that the prophylactically effective amount may be less than the therapeutically effective amount.

For administration by subcutaneous or intradermal injection, an exemplary range of therapeutically or prophylactically effective amounts of one or more pathogenic bacterial species can be about 1 to 1 billion organisms per ml, or can be 1 to 70 million organisms per ml, or can be 5 to 60 million organisms per ml, or can be 10 to 50 million organisms per ml, or can be 20 to 40 million organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may be 1 million to 1 billion organisms per ml, or may be 5 million to 70 million organisms per ml, or 1 million to 60 million organisms per ml, or may be 5 hundred million to 50 hundred million organisms per ml, or may be 10 hundred million to 40 hundred million organisms per ml, or any integer within these ranges. A therapeutically or prophylactically effective amount of an antigen of a pathogenic bacterial species may range from 0.1nM to 0.1nM, 0.05nM to 15 μ M, or any integer from 0.01nM to 10 μ M.

It should be noted that the dosage concentration and range may vary with the severity of the disease state to be alleviated, or may vary with the immune response of the individual. Generally, the goal is to achieve an adequate immune response. For administration by subcutaneous or intradermal injection, the extent of the immune response can be determined, for example, by the size (e.g., from 0.25 inch to 4 inches in diameter) of the delayed local immune skin response at the injection site. The dosage required to achieve an appropriate immune response may vary depending on the individual (and their immune system) and the response desired. Standardized dosages may also be used.

In the case of subcutaneous or intradermal administration, if the goal is to achieve a2 inch local skin response using a bacterial composition, the total amount can range, for example, from 2 million bacteria (e.g., 0.001ml of vaccine at a concentration of 20 million organisms per ml) to greater than 200 million bacteria (e.g., 1ml of vaccine at a concentration of 200 million organisms per ml). The concentration of the individual bacterial species or antigens thereof within the composition may also be considered. For example, if the concentration of a particular pathogenic bacterial species, the cell size of that species, or the antigenic load thereof is higher in a vaccine relative to other pathogenic bacterial species, the local immune skin response of an individual may be attributed to its response to that particular bacterial species. In certain embodiments, the immune system of an individual may be more intense than one bacterial species within a vaccine, for example depending on the past history of infection exposure to a particular species, then the dose or composition may be adjusted accordingly for that individual.

The timing and dosage of treatment can be adjusted over time for a particular individual according to the individual's needs and the professional judgment of the person administering or instructing the administration of the composition (e.g., the timing can be daily, every other day, weekly, monthly). For example, in the case of subcutaneous or intradermal administration, the composition may be administered every other day. An initial dose of about 0.05ml can be administered subcutaneously, followed by an increase of 0.01-0.02ml every other day until a sufficient cutaneous response is administered at the injection site (e.g., a delayed response of visibly redness at the injection site of 1 inch to 2 inches in diameter). Once this sufficient immune response is achieved, the dose may be continued as a maintenance dose. The maintenance dose can be adjusted from time to achieve the desired apparent skin response (inflammation) at the injection site. Administration may be for an administration duration of, for example, at least 2 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years or more.

In some embodiments, the invention may comprise administering the antigen composition to one or more epithelial tissues via a parenteral route. For example, for application to the skin by intradermal or subcutaneous injection; applied to the lung epithelium by inhalation. Thus, in some embodiments, the antigen compositions of the present invention are administered to elicit an immune response in non-intestinal tissue, such as epithelial tissue. In some embodiments, one or more parenteral routes of administration may be combined with one or more other routes of administration, such as intratumoral, intramuscular, or intravenous administration.

In various aspects of the invention, the antigenic composition administered to a patient may be characterized as having an antigenic signature, i.e., a combination of antigens or epitopes sufficient to be specific, such that the antigenic composition is capable of eliciting an immune response, e.g., an adaptive immune response, specific for a particular pathogen.

The amount of active compound (e.g., a bacterial species, a virus, a protozoan or a parasite, or antigens thereof) in the compositions of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, a single pill may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be advantageously formulated in dosage unit form for ease of administration and uniformity of dosage.

In the case of an antigenic preparation (similar to a vaccine), an immunologically effective amount of a compound of the invention can be provided alone or in combination with other compounds, with immunological adjuvants. The compounds may also be linked to a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin, to increase immunogenicity. An antigenic composition ("vaccine") is a composition comprising materials that elicit a desired immune response. The antigen composition may select, activate or expand memory B, T cells, neutrophils, monocytes or macrophages of the immune system, for example, to reduce or eliminate symptoms of IBD. In some embodiments, a specific pathogenic microorganism, virus, viral antigen, bacterium, bacterial antigen, or composition thereof of the invention is capable of eliciting a desired immune response in the absence of any other agent, and thus can be considered an antigenic composition. In some embodiments, the antigenic composition includes a suitable carrier, such as an adjuvant, which is an agent that acts in a non-specific manner to increase the immune response to a particular antigen or population of antigens, enabling a reduction in the quality of the antigen in any given vaccine administration, or a reduction in the frequency of doses required to produce a desired immune response. The bacterial antigenic composition may comprise live or dead bacteria capable of eliciting an immune response against antigenic determinants normally associated with the bacteria. In some embodiments, the antigenic composition may comprise live bacteria with less virulent strains (attenuated) and thereby induce less severe infections. In some embodiments, the antigenic composition may comprise a live, attenuated, or killed virus capable of eliciting an immune response against antigenic determinants normally associated with the virus.

An antigen composition containing a biocidal organism for administration by injection may be prepared as follows. The organisms can be grown in a suitable medium and washed with physiological salt solution. The organisms can then be centrifuged, resuspended in a saline solution, and killed with heat. The suspensions can be standardized by direct microscopic counting, mixed with the required amounts and stored in suitable containers, which can be tested in an approved manner for safety, shelf life and sterility. In addition to organisms and/or antigens thereof, a kill formulation suitable for administration to humans may include a phenolic preservative (e.g., 0.4%) and/or sodium chloride (e.g., about 0.9%). The composition may also contain trace amounts of brain heart infusion (bovine), peptone, yeast extract, agar, sheep blood, dextrose, sodium phosphate, and/or other media components.

In some embodiments, the antigenic composition may be used as an aerosol for inhalation.

Generally, the compositions of the present invention should be used without substantially inducing toxicity. Toxicity of the compounds of the invention can be determined using standard methods, e.g., by testing in cell cultures or experimental animals, and determining the therapeutic index, i.e., the ratio between LD50 (the dose lethal to 50% of the population) and LD100 (the dose lethal to 100% of the population).

In some embodiments, bacteria that are members of the endogenous flora of a particular region of the GIT may be used to formulate the antigenic compositions of the present invention. The table 1 lists a number of bacterial species, together with the biological domain, where each species may form part of the endogenous flora. For example, fastidious bacteria (Abiotrophia spp.) are typically members of the endogenous flora of the oral cavity.

Table 1: common flora of human bacteria (endogenous bacteria human pathogen)

Endogenous microbial flora, such as bacteria, reach tissues by continuous or bacteremic dissemination for pathogenesis. Under favorable conditions, all endogenous organisms can become pathogenic and locally invade and disseminate to adjacent tissues and organs by continuous dissemination. The endogenous bacterial flora of the skin, mouth and colon is understood to be a species that can also be used for the dissemination of bacteremia. Bacteria that are members of a particular endogenous flora domain can thus induce an infection in the tissues or organs to which these bacteria can disseminate. Thus, one aspect of the invention includes the use of an endogenous microbial pathogen to treat symptomatic IBD located in the region of the GIT where endogenous bacteria can disseminate to induce infection. The columns of table 2 list the domains of endogenous flora. The rows of Table 2 list the regions of the GIT where IBD may be located. Thus, one aspect of the invention includes the use of endogenous microbial pathogens to formulate antigenic compositions or to select existing formulations with pathogens for use in the treatment of IBD located in the region of the GIT where the pathogen can spread to induce infection. Thus, in an alternative embodiment, IBD that is symptomatic in the area listed in the first column of table 2 may be treated with an antigenic composition comprising antigenic determinants specific for microbial pathogens that are members of the endogenous flora of one or more of the endogenous flora domains listed in the first row of table 2 and are represented by X's or checkmarks in the appropriate row.

Table 2: tissue/organ pathogenicity of endogenous flora

Tissue/organ site	Oral cavity	Stomach (stomach)	Duodenum/jejunum	Ileum	Colon
						Oral cavity	x
Tonsil	x
						Nasopharynx/sinus	x
Esophagus		x
						Stomach (stomach)		x
Small intestine			x	x
						Colon/rectum			x
Anus					x

According to the combined information of tables 1 and 2, the list of IBDs in the specific region of the GIT shown in column 1 of table 2 can be treated by an antigenic composition comprising antigenic determinants of the corresponding bacterial species of table 1, so that the column headings of table 2 can actually be replaced by the bacterial species of table 1.

In some embodiments, the pathogen used in the present invention may be an exogenous bacterial pathogen. For example, the organisms listed in table 3 may be used as microbial pathogens to formulate antigenic compositions, or antigenic compositions with those pathogens may be selected for use in treating IBD located in the GIT region listed by the relevant organisms of table 3. In some embodiments, antigenic determinants of endogenous and exogenous bacterial species targeted to a particular tissue or organ can be used in combination. For example, antigenic compositions derived from or specific for clostridium difficile may be used to treat IBD located in the colon.

Table 3: exogenous bacterial human pathogens and their sites of infection at the GIT

In some embodiments, the pathogen used in the present invention may be a viral pathogen. Table 4 provides an exemplary listing of viral pathogens along with tissues and organs that are reported to be pathogens for each viral species. Accordingly, one aspect of the present invention provides for the treatment of IBD located in the region of the GIT identified as being adjacent to the viral names in table 4 using an immunogenic composition specific for the given virus.

Table 4: viral human pathogens and sites of infection thereof

The cumulative information of tables 1 to 4 provides a broad identification of pathogens that can be used in the antigenic compositions of the present invention, as well as the identification of the GIT regions in which these organisms are pathogenic, and accordingly the GIT regions in which IBD is located that can be treated by the antigenic preparations of the present invention.

In some embodiments, the pathogen selected for use in the antigenic composition of the present invention may be one that is a common cause of acute infection of the GIT region where the IBD to be treated is located. Table 5 identifies this type of bacterial and viral pathogens along with the regions of the GIT where they normally induce infection. Thus, in selected embodiments, IBD that fall within the GIT region identified in the first column of table 5 may be treated by an antigenic composition comprising antigenic determinants for one or more pathogenic organisms listed in the second column of table 5.

Table 5: common causes of acute infections (bacterial and viral) of selected GIT regions

The particular organisms that normally induce infection in a particular area of the GIT may vary by geographic location. Thus, table 5 is not an exhaustive list of common pathogens for all geographical locations and species groups. It is understood that for a particular region of the GIT of the present invention, the general clinical microbiologist in the art can determine the common pathogenic species in a particular geographic region or population.

Human being is a host for a wide range of Gastrointestinal parasites (including various protozoa and parasites), which for the purposes of the present invention constitutes the GIT pathogen (Schafer, T.W.), Skopic, A. Parasitis of the smalllintestinine.Curr Gastroenterol reporters 2006; 8: 312-20; Jennigan, J. Guerrant, R.L., Pearson, R.D. Parasitic infections of the said small intestinal intestine.Gut.Gut 1994; 35: 93; rienger & S. Slostris. mesenteroides Distinguis.Distilla.8. Schizosticta, Schizosaccharomyces pombe (Microsporum), Microsporum (Microsporum ), Microsporum (Microsporum), Microsporum, phylum, and other, phylum, Microsporum, phylum, Microsporum, and other, Microsporum, etc.) (Salmon, phylum, Microsporum, phylum, Microsporum, phylum, Microphylum, phylum, Microphylum, phylum, Microphylum, phylum, Microphylum, phylum, Microphylum, phylum, Microphylum, phylum, Microphylum, phylum, Microphylum, phylum, Microphylum, phylum.

In selected embodiments, the invention includes a diagnostic step to assess the prior exposure of a patient to an organism. For example, the diagnostic step can include obtaining a history of exposure to the selected pathogen and/or evaluating the patient's immune response to the selected pathogen. For example, serological tests may be performed to detect antibodies to selected pathogens in the patient's serum. For this aspect of the invention, the antigenic determinants of the selected pathogen may be selected for use in the immunogenic composition for the selected patient based on the diagnostic indication that the patient has had one or more prior exposures to the pathogen, for example by the presence of antibodies to the antigenic determinants of the pathogen in the patient's serum.

In other selected embodiments, the invention includes a diagnostic step to evaluate the immunological response of a patient to treatment with the selected immunogenic composition. For example, the diagnostic step may include assessing the patient's immune response to the antigenic determinants of the immunogenic composition, for example using a serological test to determine antibodies to those antigenic determinants. For this aspect of the invention, if the evaluation indicates the presence of an active immunological response to an antigenic determinant of the composition, the treatment with the selected immunogenic composition may be continued, and if the evaluation indicates the absence of a sufficiently active immunological response to an antigenic determinant of the immunogenic composition, the vaccine treatment may be discontinued and an alternative treatment with a different immunogenic composition may be started.

While various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in light of the general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numerical ranges include the numbers defining the range. The word "comprising" is used herein in an open-ended fashion, that is, substantially identical to the word "comprising", and the word "comprising" has a comparable meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an item" includes more than one of such item. Citation of a document herein is not an admission that such document is prior art to the present invention. Any prior document and all publications (including but not limited to patents and patent publications) cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as if fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and figures.

In some embodiments, the invention excludes steps comprising medical or surgical treatment.

The following examples illustrate embodiments of the invention.

Example 1: clinical research

Bacterial compositions

In studies blinded by the participants, five killed bacterial compositions have been used to treat a wide range of cancer types and stages, as shown below:

1.Bayer Corporation MRV^TM"Bayer MRV" (Hollister-Steir Laboratories, Spokane, WA, U.S. A.) contains the following bacterial species:

the vaccine was prepared for the following indications: rhinitis, infectious asthma, chronic sinusitis, nasal polyps, and chronic serous otitis media. The treatment of cancer is not indicated as the intended use of the vaccine. The vaccine also includes the following components: 0.4% phenol, 0.9% NaCl, traces of brain heart infusion (bovine), peptone, yeast extract, sheep blood, dextrose and sodium phosphate.

Stallergenes MRV "Stallergenes MRV" (Laboratories des Stallergenes, S.A., Fresnel, France) comprising the following:

the vaccine was prepared for the same indication as the MRV vaccine, i.e. recurrent respiratory tract infections and listed cancers as contraindications.

As shown below, these MRV vaccines containing many common pulmonary pathogens were surprisingly found to be effective in treating lung cancer.

Polyvaccinum Forte (PVF; Biomed S.A., Krakow, Poland) comprising the following:

the vaccine is prepared for chronic and recurrent inflammatory disease states of the upper and lower respiratory and genitourinary tracts, including nasopharyngitis, recurrent laryngitis, tracheitis, bronchitis, otitis media, chronic and recurrent neuralgia of the trigeminal and occipital nerves, sciatica, brachial neuritis, intercostal neuralgia, chronic vesicoureteritis, vaginitis, adnexitis, and endometritis. The treatment of cancer is not indicated as the intended use of the vaccine.

Notably, although the total concentration of bacteria in PVF is the same as MRV (Bayer and Stallergenes), patients typically exhibit a significant inflammatory immune response to PVF compositions injected subcutaneously at doses lower than those typically required to achieve a similar skin response using MRV compositions, meaning that the immune response is similar to a novel component of the Polyvaccinum Forte vaccine, such as e. As shown below, surprisingly, PVF comprising escherichia coli (a common pathogen of colon, abdomen, kidney, ovary, peritoneum, liver and pancreas) has been found to be effective in treating cancer in colon, peritoneal lymph nodes, kidney, ovary, peritoneum, liver and pancreas.

Staphage Lysate (Delmont Laboratories Inc., Swartmore, PA, USA) comprising the following:

staphylococcus aureus

As shown below, surprisingly, Staphage lysote comprising staphylococcus aureus (a common pathogen of breast and bone) has been found to be effective in treating breast and bone cancers.

Administration of MRV, Staphage Lysate and PVF

The bacterial composition (vaccine) is a suspension that kills bacterial cells, so the suspension is gently shaken before use to ensure uniform distribution before removal of the agent (dose) from the vial, and is administered subcutaneously three times a week on monday, wednesday and friday. The patient is advised to continue treatment for at least 6 months. The required dose of vaccine is determined by the sufficiency of the immune response to the vaccine. Starting with a very small dose (0.05cc), the dose is gradually increased (0.01-0.02 cc each time) until a sufficient immune response is achieved. This delayed local response occurs at the injection site within 2-48 hours after injection and lasts up to 72 hours or more. The goal was to achieve pink/red circular plaques of 1 to 2 inches in diameter at the injection site, indicating adequate immune stimulation. Once this response is achieved, the dose is maintained at the level required to achieve the response. If the response is significantly below 2 inches (e.g., half an inch), the dose is increased, and if it is significantly greater than 2 inches (e.g., 3 inches), the dose is decreased. This local immune response usually occurs within the first 24 hours after injection. The patient is asked to check for the response, and if present, measured or marked. The maintenance dose required to achieve this adequate immune response varies significantly, depending on the individual's immune response, as low as 0.001cc for some people and as much as 2cc for others. The vaccine must be stored in a refrigerator (2 to 8 ℃). Common sites for injection are the upper arm, thigh or abdomen. The exact site of each injection was varied so as not to give it to sites where pink/red color still existed. A known contraindication for vaccines is allergy to any component of the vaccine.

A fifth vaccine, a polymicrobial oral vaccine, is used in an alternative aspect of the invention, which is as follows:

respivax, manufactured by BB-NCIPD Ltd (Bulgaria). The oral vaccine comprises the following lyophilized bactericidal species:

administration of Respivax

Respivax oral vaccines are manufactured for the treatment of chronic respiratory infections and contain many of the most common respiratory pathogens, including many of the most common causes of pulmonary infections. Patients were treated with a dose of one 50mg tablet per day, providing 1.25X 10 per species per dose⁹Equivalent per cell. The patient is prescribed the above doses for a continuous period of at least 6 months.

As shown below, surprisingly, Respivax oral vaccines containing many common pulmonary pathogens were found to be effective in treating lung cancer.

Example 1A: cancer of the lung

This part relates to primary cancer in or metastasis to the lung, which is treated by microbial pathogens in the lung, such as endogenous respiratory bacterial flora.

Patients were eligible for the lung cancer study if they were initially diagnosed with stage 3B or 4 (inoperable) cancer. Lung Cancer Staging is performed using standard methods, such as those described in AJCC: Cancer Staging Handbook (6 th edition) 2002; Springer-Verlag New York: Editors: Fredrick Greene, David Page and Irvin flashing, or in the International Union age Cancer: TNM Classification of MalignantTumors (6 th edition) 2002; Wiley-Liss Geneva Switzerland Editors: L.H.Sobin and dC.H.Witterind. For example, lung cancer can be classified as follows:

clinical and pathological classification of TNM lung

Stage classification of TNM subtypes

Charts with diagnostic codes 162.9 (lung cancer) and 197 (metastatic cancer) were collected manually and electronically. Information is collected on these patients, such as the date of diagnosis, the date of death, and the stage of cancer. The patient's chart is examined to determine the date of diagnosis and the stage of the cancer. Patients were excluded from the analysis for the following reasons: 1) a stage of error; 2) data loss; 3) there is no icon; or 4) the chart does not arrive on time for data analysis. 20 patients were excluded from the study because their charts did not arrive, or there was insufficient information, 6 of which were MRV users. The study group included a total of 108 patients: 50 employed MRV vaccines, and 58 not employed MRV vaccines.

The survival comparisons of patients with MRV and patients without MRV who were initially diagnosed with stage 3B and 4 lung cancer and SEER standard survival data (fig. 1) with patients initially diagnosed with stage 3B and 4 lung cancer were as follows:

survival (as above) ratio (fig. 2) for only those patients including MRV with at least two months was as follows:

median survival and 1-year survival, 3-year survival and 5-year survival were substantially better in the group treated with MRV (containing bacteria that normally induce lung infections), demonstrating the effectiveness of this vaccine for lung cancer treatment. Patients treated with the MRV vaccine for at least 2 months had higher survival rates and the effectiveness of the vaccine for lung cancer treatment was also demonstrated.

Alternative analyses are performed based on data from patient populations where MRV compositions are not feasible, thereby addressing the sensory potential for bias resulting from diseased patients being more likely to select a new treatment (using MRV) and healthy patients being less likely to receive use of the antigenic composition of the present invention. Comparison of the survival of MRV patients for whom MRV compositions are feasible (indicated as "lung 1") with the survival of non-MRV patients for whom MRV compositions are not feasible (indicated as "lung 2") precludes some of these selection deviations, providing a clearer and more accurate illustration of MRV treatment, as shown in figure 3.

In some embodiments, a particular significant clinical benefit has been obtained from antigenic bacterial compositions used by injection at a repetitive frequency (i.e., three times a week) for an extended period of time, e.g., at least 2 months, 3 months, 4 months, 5 months, 6 months or 12 months, or 2 years, 3 years, 4 years or 5 years (in the case of advanced cancers such as inoperable lung cancer, longer periods may be more beneficial). This type of treatment can be performed to provide long lasting, prolonged immune stimulation. The survival advantage of MRV treatment is even more clearly shown in figure 4 when the above analysis is limited to patients treated with MRV for at least 2 months.

As shown in fig. 4, one-year survival of patients with stage 3B or 4 lung cancer treated with MRV for at least two months was 70%, relative to a one-year survival of only 48% for the non-MRV lung 2 group and 23% for the SEER database group. The 3-year survival rate for the MRV group was more than 4-fold compared to both non-MRV patients and SEER enrollment. None of the non-MRV groups in the lung 2 study survived 5 years, whereas 15% of patients treated with MRV for a minimum period of 2 months survived 5 years after diagnosis. The above results are very encouraging and surprising in the case of disease, such as inoperable lung cancer, which is considered to be absolute and has a typical 5-year survival rate of only 3% (SEER enrollment).

Survival curves were very significant when analysis of patient data was limited to patients treated with MRV for at least 6 months, as shown in figure 5. More than 60% of patients live in year 3, more than 10 times the survival of non-MRV groups and SEER enrolment. 36% of patients treated with MRV for at least 6 months (5 out of 14 patients) survived 5 years after diagnosis compared to only 3% for SEER data and 0% for the non-MRV group. These significant results are very promising and surprising in the case where cancer diagnosis is considered to be absolute. Thus, in some embodiments, a cancer, such as an advanced cancer (e.g., inoperable lung cancer), can be treated for a duration of administration of at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, 2 years, 3 years, 4 years, 5 years, or indefinitely.

Limiting the analysis to those patients treated with MRV for a minimum period of time (e.g., 6 months) causes a bias in favor of the MRV group, since MRV patients who survive less than this period of time are excluded from the group, including those who die before they can complete 6 months of treatment. Detailed statistical analysis of this deviation by compensation excluded short-term survivors of the non-MRV and SEER groups showed that this deviation plays a very minor role in the very significant survival advantage of patients treated with MRV for at least 6 months.

In stage 3B lung cancer, the cancer is confined to the lung, and a targeted anti-cancer therapeutic response may thus be stimulated by vaccines comprising pulmonary pathogens, such as MRV, according to aspects of the present invention. In stage 4 lung cancer, the cancer has metastasized to distant organs that are not subject to targeted stimulation by pulmonary pathogens according to the methods of the present invention. Thus, according to some embodiments, patients with stage 3B lung cancer may be selected for treatment with an MRV vaccine, as all cancers are confined to the lungs and thus can be targeted by an MRV vaccine. Comparison of survival curves shows the benefit of MRV treatment even more clearly when analysis of patient data is limited to patients with stage 3B lung cancer. As shown in fig. 11, one-year survival for patients with stage 3B lung cancer treated with MRV was 76% relative to one-year survival of only 53% for the non-MRV lung 2 group and 23% for the SEER database group. The 3-year survival rate for the MRV group was 3-fold for non-MRV patients and 6-fold higher than for SEER registrations. None of the non-MRV groups survived for 5 years, while 14% of the patients at stage 3B treated with MRV survived 5 years after diagnosis. The above results are very encouraging and surprising in the case of disease, such as inoperable stage 3B lung cancer, which is considered to be absolute and has a typical 5-year survival rate of only 5% (SEER enrollment).

Because some patients have not had their initial visit for many months or even one or two years after diagnosis, their inclusion in the survival curve skews the curve towards longer survival. To determine if the deviation affects the difference in survival curves, survival was analyzed from the date of initial visit, which excluded the deviation, as shown in fig. 12. Comparison of the survival curves for stage 3B lung cancer patients in fig. 12 shows even greater survival benefit of MRV treatment than that shown in fig. 11, indicating that the benefit of MRV treatment is partially masked in fig. 11. As shown in fig. 12, the 1-year survival rate (date from initial visit) for stage 3B lung cancer patients treated with MRV was 57% compared to the 1-year survival of only 21% for stage 3B patients not treated with MRV. Although stage 3B patients not treated with MRV survived 3 years, the 3-year survival rate for stage 3B patients treated with MRV was 33% and the 5-year survival rate was 14%, a significant and unexpected result.

The benefit of early treatment with MRV is clearly shown when the analysis is limited to the initial visit of 3B lung cancer patients diagnosed within 3 months. As shown in fig. 13, although all stage 3B lung cancer patients who were initially visited within 3 months of diagnosis died within 1 year of diagnosis, 70% of the 3B lung cancer patients treated with MRV within 3 months of diagnosis survived 1 year, 40% survived 3 years, and 20% survived 5 years, a very significant survival benefit for early stage MRV treatment.

One aspect of the invention includes treating or metastasis to the lung of primary lung cancer with an antigenic composition comprising antigenic determinants of a microbial pathogen known as a pulmonary pathogen, such as an exogenous pulmonary pathogen or a pathogen that is a member of the endogenous flora of the respiratory system. For example, antigenic determinants of the endogenous bacterial respiratory flora species (see table 5) that most frequently induce infections in the lung can be used to treat primary and metastatic cancers located in the lung: streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae. Likewise, common viral pulmonary pathogens from table 5 may be selected for use in some embodiments. Alternatively, based on the pathogenicity information provided in table 2, a more exhaustive list of endogenous pulmonary pathogens may be selected from table 1. In other alternative embodiments, the viral pulmonary pathogens listed in table 4 may be used. And in other alternative embodiments, exogenous bacterial pulmonary pathogens from table 3 may be used to formulate the antigenic compositions of the present invention, i.e., selected from: achromobacter, actinomadura, Alcaligenes, Microsporean, Bacillus anthracis, other Bacillus, Pasteurella, Bartonella transversa, Burger bacteria ulcerosa, Bordetella hopcalis, Bordetella parapertussis, Bordetella pertussis, Bordetella burgdorferi, Bordetella hudonovani, Brucella hutchuensis, Burkholderia arborescens, Burkholderia farina, Burkholderia pseudorhizi, Campylobacter fetus, Cytophaga canis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia pneumoniae, Violacerobacter, Chlamydia psittaci, Corynebacterium pseudotuberculosis, Corynebackii, Francisella tularensis, Gordonia, Legionella, Leptospira community, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium tuberculosis, Other mycobacteria, nocardia, orientia tsutsugamushi, oxalaria, pseudomonas aeruginosa, other pseudomonas, rhodococcus, rickettsia kansei, rickettsia pustuloside, rickettsia rickettsii, typhus rickettsia.

For example, because MRV compositions contain many of the most common pulmonary pathogens, these vaccines can be used to treat primary lung cancer or lung metastases, as shown by the cumulative data presented herein, and these vaccines can be used in many case reports. In light of the foregoing results, one aspect of the present invention comprises treating or metastasis to the lung of primary lung cancer with an antigenic composition comprising antigenic determinants of a microbial pathogen known to be pathogenic in the lung, such as an exogenous pulmonary pathogen or a pathogen that is a member of the endogenous flora of the respiratory system. In selected embodiments, antigenic determinants of common pulmonary pathogens may be used to treat primary and metastatic cancers located in the lung, for example, antigenic determinants from one or more of the following bacterial species or viral types: streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, influenza virus, adenovirus, respiratory syncytial virus, and parainfluenza virus. In other selected embodiments, the antigenic determinants of streptococcus pneumoniae (the most common cause of bacterial lung infection) may be used alone or in combination with other pathogens most common to the lung to treat cancer in the lung.

Primary lung cancer may also be derived from bronchial tissue, and thus, in some embodiments, antigenic compositions comprising antigenic determinants of microbial pathogens known to induce bronchial infections may be used to treat patients with cancers localized to bronchial tissue, including but not limited to the following common causes of bronchial infections: mycoplasma pneumoniae, chlamydophila pneumoniae, bordetella pertussis, streptococcus pneumoniae, haemophilus influenzae, influenza virus, adenovirus, rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus or coxsackie virus. Lung cancer (or lung metastases) located in lung and bronchial tissue may be treated with an antigenic composition comprising antigenic determinants of microbial pathogens known to induce lung and bronchial infections (e.g., streptococcus pneumoniae, haemophilus influenzae, and mycoplasma pneumoniae are all common lung and bronchial pathogens), or alternatively with an antigenic composition comprising antigenic determinants of microbial pathogens known to induce lung infections and antigenic determinants of microbial pathogens known to induce bronchial infections.

Example 1B: breast cancer with metastasis to bone or lung

The most common cause of breast and bone infections is staphylococcus aureus. Thus, in one aspect of the invention, an antigenic composition comprising an antigenic determinant of staphylococcus aureus may be used to treat breast cancer with metastasis to bone. The significant cases of patient r (ptr) treated with the staphylococcus aureus vaccine shown in the following case report indicate the efficacy of this approach for treating breast cancer with bone metastasis. As shown in fig. 6, in some of the cumulative 52 patients, survival of breast cancer patients with metastasis to bone and/or lung with MRV containing staphylococcus aureus (n ═ 19) was better than that of patients not treated with MRV vaccine (n ═ 33):

in accordance with the foregoing results, one aspect of the present invention includes treating primary cancer or metastasis to the breast with an antigenic composition comprising antigenic determinants of a microorganism known to induce breast infection, and treating primary cancer or metastasis to the bone with an antigenic composition comprising antigenic determinants of a bacterial species or a virus known to induce bone infection. In selected embodiments, a vaccine comprising an antigenic determinant of staphylococcus aureus (the most common cause of breast and bone infections) can be used alone or in combination with the most common pathogens of other breast to treat cancer of the breast, or alone or in combination with the most common pathogens of other bone to treat cancer of the bone.

Example 1C: metastasis to bone

In patients with prostate cancer, one of the most common sites of metastasis is bone. In one aspect of the invention, MRV compositions comprising antigenic determinants of staphylococcus aureus, the most common cause of bone infection, may be used to treat metastasis to bone, for example in patients with or already with primary prostate cancer. Figure 7 is a graph comparing survival of some cumulative metastatic prostate cancer patients who have undergone surgery or radiation to destroy their prostate (and thus, the primary tumor) and have detectable cancer restricted to bone metastasis. As shown, patients treated with MRV (n ═ 4) survived substantially better than patients not treated with MRV (n ═ 7).

In light of the foregoing results, one aspect of the invention comprises treating primary bone cancer or metastasis to bone with an antigenic composition comprising an antigenic determinant of a microbial pathogen known to induce bone infection, such as an exogenous bone pathogen or a pathogen that is a member of the endogenous flora of the skin, mouth or colon. For example, in selected embodiments, antigenic determinants from one or more of the following microbial species from the list of common osteopathic pathogens may be used to treat primary and metastatic cancers located in bone: staphylococcus aureus, coagulase-negative staphylococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other streptococci, Escherichia coli, Pseudomonas, Enterobacter, Bacillus proteus, Serratia, parvovirus B19, rubella virus, and hepatitis B virus. In other selected embodiments, Staphylococcus aureus, the most common cause of bone infection, may be used alone or in combination with other most common bone pathogens to treat cancer of the bone.

Example 1D: cancer located in the colon

Treatment with the PVF composition has been shown to improve survival in colon cancer patients (see fig. 8), as shown by comparison of the four following groups of colon cancer patients.

A stage 4 colon cancer patient treated with MRV.

Stage 4 colon cancer patient, who was not treated with vaccine.

Stage 4 colon cancer patients treated with PVF vaccine.

Stage 4 colon cancer patients from SEER (detection, epidemiology and end results) database.

This example shows that patients with colon cancer treated with PVF, which comprises escherichia coli, the most common cause of colonic bacterial infection, have substantially improved survival.

Patients were eligible for the first two groups of the study if they exhibited stage 4 colon cancer. Patients were excluded from this analysis for the following reasons:

incorrect diagnosis

Incorrect period

Loss of essential data (e.g., death date)

Without icon

For data analysis, the graphs did not arrive at us on time.

The patient group included a total of 136 stage 4 colon cancer patients: 15 were used with PVF vaccine, 56 with MRV vaccine, and 65 without vaccine. The results are shown in fig. 8 as follows:

median survival for patients with stage 4 colon cancer treated with PVF (which contains escherichia coli, one of the most common colonic pathogens) was more than twice that of patients treated with MRV (which does not contain colonic pathogens) or patients not treated with vaccine and four times that of SEER enrollment. All 15 patients treated with PVF were still alive 10 months after diagnosis compared to only 71% of the MRV group, 69% of the non-vaccine group and only 45% of the SEER registrations. The 30-month survival of the PVF group was twice that of the MRV and vaccine-free groups and almost four times that of SEER enrollment.

The rank-sum test (wilcoxon test) indicated statistically significant survival differences between patients treated with the PVF vaccine and the MRV group (p 0.0246) and the vaccine-free group (p 0.0433). This significantly takes into account the minimum size of the PVF group (n-15), indicating substantial therapeutic effect. As these results demonstrate, PVF compositions comprising the most common cause of escherichia coli, i.e. colonic bacterial infection, are effective treatments for colon cancer.

Those patients who provided the immunological treatment of the invention within 3 months of diagnosis have been analyzed for survival (i.e., excluding those patients who were long-term survivors prior to providing treatment). The results of this analysis are shown in fig. 9. As shown, the "MRV" and "vaccine-free" survival curves of fig. 9 shifted substantially to the left (indicating that selection bias towards "long-term" survivors can shift these curves manually to the right in fig. 8), while significantly, the PVF curves in fig. 9 were actually more to the right than the curves of fig. 8, indicating that the benefit of early treatment with PVF (i.e., within 3 months of diagnosis) was far superior to the bias of any long-term survivors excluded in fig. 9. This analysis provides compelling evidence that the benefits of PVF treatment of stage 4 colon cancer can be greater than that shown in figure 8, and that the benefit is greater the earlier treatment with the composition of the invention is initiated after diagnosis.

In light of the foregoing results, one aspect of the present invention relates to the treatment of colon cancer with an antigenic composition comprising an antigenic determinant of a microbial pathogen known as a colonic pathogen, such as a pathogen that is a member of the endogenous flora of the colon or an exogenous colonic pathogen. For example, antigenic determinants of the following microbial species may be used to treat primary and metastatic cancers located in the colon: escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteritidis, Yersinia enterocolitica, Shigella flexneri; adenovirus, astrovirus, calicivirus, norwalk virus, rotavirus, or cytomegalovirus. For example, cancer localized to the colon may be treated with a PVF composition comprising a colonic bacillus or an alternative formulation comprising only antigenic determinants of a colonic pathogen. In other selected embodiments, the antigenic determinants of E.coli, the most common bacterial cause of colonic infection, may be used alone or in combination with other most common pathogens of the colon to treat cancer of the colon.

Example 1E: use of Respivax (an oral vaccine) for treating lung cancer

The oral Respivax vaccine was administered as described above with a dose of one 50mg tablet per day, providing 1.25X 10 per species per dose⁹Equivalent per cell. The patient is advised to continue the above dose for at least 6 months.

As shown in fig. 10, the survival of stage 3B lung cancer patients treated with oral Respivax antigen was substantially better than patients not treated with the antigen composition. Median survival for patients treated with Respivax was 37 months compared to median survival for only 20 months without treatment with the antigen composition vaccine. 40% of patients treated with Respivax survive 5 years after diagnosis, while none of untreated patients survive more than 2 years.

In light of the foregoing results, one aspect of the present invention comprises treating primary cancer of the lung or metastasis to the lung with oral administration of an antigenic composition comprising antigenic determinants of microbial pathogens that normally induce lung infections.

Example 2: case report

These case reports indicate patients that make up the patient population reflected in the cumulative study described above and illustrate other aspects of the invention. In particular, individual case reports for patients a-N show surprising results in some patients treated with anti-inflammatory therapy, while case reports for patients O-AA show a general patient population that includes many effective vaccine treatment examples in the absence of anti-inflammatory therapy.

MRV of lung cancer with and without anti-inflammatory drugs

Patient a (pta): in 9 months of 0, PtA found right upper chest pain with wheezing. These symptoms persist and in 1 month of year 1, she has a chest X-ray examination, which shows that a 7cm X8 cm sized packet is found in the right lung apex. Fine needle aspiration is positive for non-small cell lung cancer. On day 27 of month 1 of year 1, MRI showed that the subclavian artery was affected, which made it unacceptable for surgical resection, and thus PtA was diagnosed as inoperable advanced lung cancer in stage 3B. She underwent short-term palliative radiation and reduced chemotherapy. She was told to have advanced cancer with a life expectancy of 3 to 6 months.

On 29 days 4 months of year 1, PtA began treatment with MRV vaccine 3 times per week. On the same day she also started treatment with non-steroidal anti-inflammatory drugs (NSAIDs): indometacin 50mg was taken 4 times daily and ingested as an antioxidant supplement and vitamin D. After 18 months, by 10 months of year 2, the tumor size was significantly reduced, with a diameter of 3cm, and by 19 days of year 5, at 19 days of year 5, only residual scar remained after 4 years of treatment with the combined ingestion of MRV vaccine, indomethacin, antioxidant vitamins and vitamin D. PtA continued to be treated with MRV vaccine and adjuvant anti-inflammatory drug combination therapy for more than 4 years until 5 months of 5 years, when there was no evidence of residual cancer, although she was diagnosed with advanced, inoperable lung cancer more than 4 years ago. Over 14 years since the diagnosis of advanced lung cancer, PtA is known to feel well with no evidence of residual cancer.

In light of the foregoing results, one aspect of the present invention comprises the use of an antigenic composition comprising antigenic determinants of microbial pathogens that normally induce lung infections to treat lung cancer.

In light of the foregoing results, another aspect of the invention includes repeated administration of the immunogenic composition relatively frequently over a relatively long period of time.

The use of anti-inflammatory agents such as antioxidants, vitamin D and indomethacin in combination with targeted MRV therapy is associated with substantially improved survival over that of otherwise similar cases where the adjunctive anti-inflammatory agent modality in combination with the compositions of the present invention is not used. For example, in an otherwise similar case (where no anti-inflammatory drug is administered), patient B was diagnosed with non-small cell lung cancer stage 3B, refractory to surgery, and died within 3 months of diagnosis. These cases provide evidence of synergy between the antigenic compositions of the present invention and anti-inflammatory drug therapy.

In light of the foregoing results, one aspect of the present invention includes the use of an antigenic composition comprising antigenic determinants of a microbial pathogen that is pathogenic to a target organ or tissue, and adjunctive anti-inflammatory therapy to achieve a synergistic effect for the treatment of cancer.

MRV of lung cancer with and without anti-inflammatory drugs

Patient c (ptc): in the spring of year 0, PtC begins to develop pain in its upper right chest region. Pain persisted and on day 5 of 10 months of year 0 he underwent chest X-ray examination showing that a 12cm X11 cm sized packet actually occupied the entire right upper lung lobe. The fine needle aspiration is positive for the poorly differentiated non-small cell lung cancer. Thoracoscopy was performed 12 months and 7 days of 0, and tumors were found to invade the chest wall and superior vena cava, so PtC tumors were inoperable (i.e., stage 3B). PtC was subjected to short-term palliative radiation and reduced chemotherapy. He was told to have advanced cancer with a life expectancy of 3 to 6 months. By day 27 of 1 month of 1 year, rapidly growing tumors have grown to 14cm by 11.5 cm.

On day 9 of month 2 of year 1, PtC began treatment with indomethacin 50mg 4 times daily, antioxidant vitamins and vitamin D. After 3 weeks, 3 months and 1 day of 1 year, PtC started to be treated 3 times a week with MRV vaccine. By 6 months of year 1, PtC felt well and ran 3 to 4 times a week, 8km each. Chest X-ray examination showed a reduction in tumor size to 11cm in diameter on

day

6, 4 of year 1. PtC is always feeling well, which makes its life rich and vigorous, and works all over again and continues all over physical activity. PtC continued treatment with a combination therapy of MRV vaccine and adjuvant anti-inflammatory drugs (indomethacin, antioxidants and vitamin D) for more than 16 months until 7 months and 24 days of the 2 nd year, at which time treatment with indomethacin was discontinued (due to impaired renal function, which is known to be a potential side effect of chronic use of indomethacin). After 6 months, 12 months of 2 years, 22 months after targeted vaccine therapy, MRV treatment was discontinued (since MRV was ineffective beyond this date). PtC felt well until 6 months of 6, when he was diagnosed with bilateral lung cancer recurrence, which resulted in his death on 26 days 5 months of 7 years, i.e., 6.5 years after he was diagnosed with advanced lung cancer and was told to survive 3 to 6 months.

In this case, in the face of a diagnosis that would typically die within 1 year, the use of a supplemental anti-inflammatory agent comprising an antioxidant, vitamin D and indomethacin in combination with targeted MRV therapy for more than 16 months is associated with a substantial improvement in survival that is greater than that of other similar cases in which the supplemental anti-inflammatory agent modality is not used in combination with the composition of the present invention, and inoperable lung cancer dies within 8 months of diagnosis. These cases provide evidence of synergy between the antigenic compositions of the present invention and anti-inflammatory drug therapy.

PVF for colon cancer with metastasis to the liver and lung without the use of anti-inflammatory drugs

Patient e (pte): PtE surgical resection of colon cancer was performed on day 17 of

year

0, 6 months, followed by chemotherapy. On

day

8, 15 of year 0 he was diagnosed with stage 4 cancer with metastasis to the liver and lungs, with a very poor prognosis. On day 10/20 of year 0, PtE began antioxidant and vitamin D regimen treatment, and on day 10 of month 12 of year 0, he began treatment with the PVF composition 3 times per week, during which he continued to combine antioxidant and vitamin D. In 9 months of year 1, he began using Celebrex^TM100mg was treated 2 times daily. Despite the very poor initial prognosis, PtE remained and felt good at the time of last contact more than 3 years after diagnosis with advanced metastatic colon cancer.

In light of the foregoing results, one aspect of the present invention includes the use of an antigenic composition comprising antigenic determinants of microbial pathogens known to be pathogenic to the colon, liver and lung for the treatment of cancer of the colon, liver and lung.

Of the 15 patients diagnosed with stage 4 colon cancer and treated with PVF, the patient with the shortest survival, patient F, was not treated with an anti-inflammatory drug, unlike PtE. These cases provide an anti-inflammatory modality (i.e., Celebrex) in combination with targeted PVF therapy^TMAntioxidants and vitamin D) have a synergistic effect that helps PtE prolong survival, longer than in similar cases where these adjunctive anti-inflammatory modalities are not used in combination with the compositions of the invention.

With anti-inflammatory drugsPVF for use with colon cancer with metastasis to the lung

Patient g (ptg): PtG developed rectal bleeding in 5 months of year 0 and was diagnosed as colon cancer. He underwent surgery, chemotherapy and radiation, but developed metastases to the lungs on

day

8, 16 of year 1 (stage 4 cancer), a poor prognosis for this late stage diagnosis. He began the antioxidant vitamin and vitamin D regimen at 6 months of year 0 and began taking NSAID celebrex 100mg 2 times daily at 23 days 9 months of year 1. On month 3 of year 3, he started PVF vaccine treatment 3 times a week, and he continued the treatment until month 4 of year 4, when he developed brain metastases, which resulted in his death on day 2 of year 6 of year 4, i.e. almost 3 years after diagnosis of stage 4 colon cancer. PtG survived substantially longer than expected for stage 4 diagnosis of colon cancer with metastases to the lungs. In this regard, the present invention provides a means of using anti-inflammatory drugs in combination with an immunogenic composition such as PVF to achieve synergy.

Patient h (pth): PtH was diagnosed as colon cancer metastasizing to the liver and lungs on day 2, 13 of year 0. On day 11 of 1 month of 1 year, he was prescribed an antioxidant and vitamin D regimen. However, in month 3 of year 1, he started the investigation of chemotherapy and at that time the supplements should be discontinued on request of the research coordinator. He was not treated with any NSAID. On day 12 of 5 months of 1 year, he started the treatment with PVF, and he took this treatment 3 times a week until he died after 2.5 months. When compared to other similar cases involving the use of anti-inflammatory drugs, the cases indicated: if the adjunctive anti-inflammatory modality is not administered concurrently with the targeted antigen activity therapy, there is a lack of synergy that may accompany the use of adjunctive anti-inflammatory modalities.

In summary, the use of adjunctive anti-inflammatory drugs including antioxidants, vitamin D and celecoxib in combination with targeted antigen activation therapy for stage 4 colon cancer patients treated with targeted PVF vaccine therapy was associated with substantially improved survival that was much longer than that of two cases in which these adjunctive anti-inflammatory drug regimens were not used in combination with the vaccine, providing evidence that synergy was demonstrated.

For pancreatic cancer with metastasis to lung, liver and abdominal lymph nodes with and without anti-inflammatory drugs PVF

Patient i (pti): at 8 months of year 1, PtI was diagnosed as pancreatic cancer, when he had undergone surgical resection of his pancreas (i.e., whapple surgery). However, in month 7 of year 2 he developed bilateral metastasis to the lung and in month 2 of year 4 cancer recurrence in the pancreatic region with abdominal and hepatic metastasis. This is a late diagnosis with a very poor prognosis. PtI schedule starting on 27 days 9 months of 2 years antioxidant vitamins, vitamin D, high dose turmeric (curcumin), fish oil (9 mg daily), resveratrol and green tea (36 cup equivalent daily), all of which are anti-inflammatory, all of which are taken continuously. In 3 months of the 3 rd year, he started treatment with celebrex 100mg 2 times daily, and he took more than 20 months. At 5 months of year 4, PtI began to be treated with PVF 3 times a week, from which he continued to use regularly for more than 3 years. PtI survives more than 5 years after diagnosis of advanced metastatic pancreatic cancer, a significantly prolonged survival in cases where the diagnosis has a very poor prognosis. This case provides evidence that the high dose of multiple anti-inflammatory modalities (i.e., celebrex, antioxidants, vitamin D, turmeric, fish oil, resveratrol, green tea) taken in combination with the PVF composition formed a synergistic effect that contributed to the dramatic survival of PtI for 5 years after the development of metastatic pancreatic cancer (a diagnosis of death typically within 6 months).

In light of the foregoing results, one aspect of the present invention includes the use of an antigenic composition comprising antigenic determinants of microbial pathogens known to induce infections of the pancreas, abdominal lymph nodes, liver and lungs for the treatment of cancers of the pancreas, abdominal lymph nodes, liver and lungs.

Patient j (ptj) was essentially the same diagnosis as PtI (i.e., pancreatic cancer with metastasis to the abdominal lymph nodes, lungs, and liver). PtJ did not receive any other anti-inflammatory drugs along with the PVF vaccine except for antioxidant and vitamin D, which died within 4 months of diagnosis, while PtI, taken in combination with the PVF vaccine in high doses, many other anti-inflammatory drug modalities (i.e., Celbor, turmeric, fish oil, resveratrol, and green tea), survived 5 years after diagnosis. These cases provide evidence of synergy of high doses of multiple anti-inflammatory modalities and targeted vaccine therapies.

MRV for breast cancer with metastasis to bone

Patient k (ptk): in month 3 of year 0, PtK developed persistent pain in the neck and back. On day 7 and 28 of year 0, she was diagnosed with stage 4 breast cancer that metastasized to the spinous processes of the cervical spine, which is an intractable diagnosis. She was surgically removed two breast tumors (axillary lymph nodes positive) and had palliative radiation of metastases in her spinal cord. On day 18 of month 1 of year 1, PtK treatment with antioxidants and vitamin D was started and NSAID indomethacin 50mg was administered 4 times daily. After 3 days, on day 21 of 1 month of 1 year, she began treatment with an MRV composition containing staphylococcus aureus, the most common pathogen of breast and bone. Although there is no documentation of the exact length of time that treatment with this combination MRV/indomethacin/antioxidant/vitamin D is continued, the patient is given enough vaccine (20ml) at the usual dose and frequency (i.e. 3 times per week) for nearly 2 years, and PtK states that she completed the recommended course of treatment at home. Interestingly, PtK remained viable and at least associated with us 13 years after stage 4 breast cancer, which was diagnosed as metastasizing to bone.

In light of the foregoing results, one aspect of the present invention comprises the use of an antigenic composition comprising antigenic determinants of microbial pathogens known as pathogens of breast and bone infections to treat breast and bone cancers.

Unlike patient K, patient l (ptl) was diagnosed with metastatic breast cancer to bone on day 10, 11 of year 0. She was not given NSAIDs or other anti-inflammatory drugs. On day 27 of month 2 of year 1, PtL began treatment with MRV. She died after 9 months, i.e., 11 months and 4 days of 1 year, only 1 year after the diagnosis of stage 4 breast cancer with metastasis to bone. PtK and PtL demonstrate the potential for synergistic treatment with anti-inflammatory drugs and the antigenic compositions of the present invention.

MRV for breast cancer with metastasis to bone and without anti-inflammatory drugs

Patient m (ptm): PtM was diagnosed as metastatic stage 4 breast cancer on

days

6, 15 of year 0. For sustained pain relief she started taking 250mg of the NSAID naproxen 2 times daily and she started taking antioxidants and vitamin D10 months in the 3 rd year. After 3 months she started treatment with MRV vaccine (which contains staphylococcus aureus, the most common pathogen of the mammary glands and bones) in combination with these anti-inflammatory medications (i.e. naproxen, antioxidants and vitamin D) on

day

1, 15 of year 4. PtM survives for more than 9 years after the first diagnosis of stage 4 breast cancer with metastasis to bone, an unusually long survival period given the often very poor prognosis associated with this diagnosis.

In light of the foregoing results, one aspect of the present invention comprises the use of an antigenic composition comprising antigenic determinants of microbial pathogens known to be common causes of breast and bone infections to treat breast and bone cancers.

Unlike PtM, patient n (ptn): PtN was diagnosed as having stage 4 cancer metastasizing to bone on

day

4, 8 of year 0. She started taking antioxidants and vitamin D on 24 days 4 months of year 0. However, before starting the MRV treatment she was given warfarin as a blood diluent, limiting the supplementation of vitamin E and vitamin C, both important antioxidants leading to possible complications if used in combination with warfarin. In addition, NSAIDs cannot be administered in this situation because they prohibit their use with warfarin. PtN began treatment with MRV on day 2 at 6 months of year 1. She died in 8 months of the 2 nd year after 14 months. In this case, it is likely that the synergy of using targeted vaccine therapy without an auxiliary anti-inflammatory drug (i.e., NSAID, vitamin E and therapeutic dose of vitamin C) limits its possible benefits.

In summary, in the 4 th breast cancer case with metastasis to bone treated with the targeted MRV therapy detailed above, the use of adjunctive anti-inflammatory drugs in combination with MRV was associated with substantially improved survival, which was much longer than that of 2 cases in which the adjunctive anti-inflammatory drug regimen was not used in combination with the vaccine, providing evidence of synergy.

MRV for metastasis to the lung

Patient o (PtO) was diagnosed as kidney cancer at 6 months of year 0 with bilateral lung and bone (left femur) metastases, which is generally considered a refractory advanced diagnosis and poor prognosis, he started MRV treatment at 10 days of 8 months of year 0 and continued regular treatment (3 times per week) for 16 months (after which MRV was no longer used), he started experimental drug treatment at 7 months of 9 months of year 0, peginterferon α -2 a. his left femur was "pinned" due to metastases risk, but due to surgical complications, required amputation below the mid-thigh of the left leg for 9 months of year 2, his cancerous right kidney was resected for 10 months of year 2, PET scans did not reveal intrapulmonary cancer, and no evidence of further bone metastases, PtO survived for more than 9 years after diagnosis of bilateral lung metastases, which is a striking result.

In light of the foregoing results, one aspect of the present invention comprises the use of an antigenic composition comprising an antigenic determinant of a microbial pathogen known as a pneumopathogen to treat metastasis to the lung.

MRV for metastasis to bone and lung

Patient p (ptp) was diagnosed as renal cancer at 7 months of 0 and right nephrectomy was performed. In month 12 of year 4 he developed bone (bilateral femur) and lung (bilateral) metastases. PtP reduced conventional treatment and treatment with MRV started 4 months of year 5, he continued the treatment regularly, 3 times a week for 18 months. The health status of PtP improves and he resumes normal daily activities. X-ray and imaging of the chest and femur did not show progression, and the disease stabilized in the lungs and femur in 18 months of PtP MRV treatment.

In light of the foregoing results, one aspect of the present invention includes the use of an antigenic composition comprising antigenic determinants of microbial pathogens that are common causes of lung and bone infections to treat metastasis to lung and bone.

MRV for metastasis to the lung

Patient q (ptq) was diagnosed with colon cancer and possibly metastases to the lungs in month 6 of year 0. At that time, the primary colon tumor was completely resected, leaving only a few lung metastases. PtQ treatment with MRV was started on day 12, 11 of year 0, and she continued the treatment 3 times a week for 4 months. On day 19 of 4 months of 1 year, after 6 months of chemotherapy, she underwent surgery to remove only the remaining visible lung lesions, which were identified as metastatic lesions. Even with chemotherapy after surgical resection of visible metastases, the diagnosis of colon cancer with lung metastases is poor prognosis. Despite her poor initial prognosis, PtQ remained an excellent health state and no evidence of cancer more than 10 years after she was initially diagnosed with lung metastasis and treated with MRV.

Staphylococcus aureus for breast cancer with metastasis to bone

Patient r (ptr): PtR was diagnosed as breast cancer and transferred to her sternum, femoral and cervical spinous processes at 5 months of year 0, which is an incurable cancer with poor prognosis. She was treated with radiation and tamoxifen. In month 5 of year 4, she developed additional metastatic areas in her lumbar spinous process and she began treatment with megestrol. At 11 months of 4, she began treatment with a vaccine containing only staphylococcus aureus (Staphage lysotate vaccine), which is the most common cause of both breast and bone infections, and therefore its vaccine was the agent of choice for the treatment of breast and bone cancer. She continued regular treatment with the vaccine for 5 years. Despite metastatic breast cancer with multiple bone metastases, PtR survived for more than 17 years, which is a striking survival in the case of incurable metastatic breast cancer and is conclusive evidence of the promise of targeted vaccine therapies for the treatment of breast cancer.

In light of the foregoing results, one aspect of the present invention comprises the use of an antigenic composition comprising antigenic determinants of microbial pathogens known to be the most common causes of breast and bone infections to treat breast and bone cancers.

This embodiment shows that a formulation comprising only antigenic determinants of pathogenic organisms most common to tissues may be of particular advantage, as also shown in the following mouse model data. Consistent with this view, we have found enhanced efficacy of Respivax in treating cancers located in the lung, unlike MRV, in response to the fact that Respivax formulations are somewhat more desirable because they contain higher concentrations of pathogenic species that are most common in inducing lung infections (i.e., Respivax has a 67% bacterial cell count consisting of the species most common in inducing lung infections, whereas MRV vaccines consist of only 30% of the species most common in inducing lung infections).

In light of the foregoing results, one aspect of the present invention includes formulating the antigenic composition so that antigenic determinants of microbial pathogens known to be common causes of infection are prescribed to predominate in the formulation proportions, and the most common causes of infection receive the greatest advantage. For example, the proportion of antigenic determinants from pathogens known to be common causes of infection may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%.

Thus, in some embodiments, the present invention provides antigenic compositions in which a defined proportion of antigenic determinants selected according to the present invention is used, relative to any other antigenic determinants in the composition. For example, an antigenic composition may contain greater than X% of antigenic determinants from a pathogenic (or common pathogenic or most common pathogenic) species, where X may be, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100 (or any integer between 10 and 100). For example, at least X% of the antigenic determinants in the antigenic composition can be specific for a microbial pathogen that is pathogenic (or commonly pathogenic or most commonly pathogenic) within a particular organ or tissue of the patient in which the cancer is situated. With other measures of the total number of microbial pathogens in the antigen composition, at least X% can be selected as the microbial pathogen that is pathogenic (or commonly pathogenic or most commonly pathogenic) within a particular organ or tissue of the patient in which the cancer is located. In some embodiments, the antigenic composition can accordingly consist essentially of antigenic determinants of one or more microbial pathogens that are each pathogenic within the specific organ or tissue of the patient in which the cancer is situated. In selected embodiments, the antigenic composition may consist essentially or entirely of antigenic determinants of microbial pathogens that are commonly pathogenic in the particular organ or tissue of the patient in which the cancer is situated. In other selected embodiments, the antigenic antigen composition may consist essentially of, or consist entirely of, antigenic determinants of microbial pathogens (or pathogens) that are the most commonly pathogenic within the particular organ or tissue of the patient in which the cancer is situated.

For various aspects of the invention, organisms are characterized by the frequency with which they are pathogenic. In this regard, the invention also relates to the frequency with which the endogenous flora is pathogenic. For clarity, in this regard, a characterization of pathogenicity frequency, such as a designation of "generally pathogenic," generally relates to the proportion of infection in a particular organ or tissue that is generally attributable to a particular organism but not to the frequency with which the tissue colonizes with microorganisms that convert to pathogenic infections. In north america, a major human infection is understood to be induced by endogenous organisms even though these organisms are usually present as part of the endogenous flora that does not induce the infection. For example, although streptococcus pneumoniae is a common cause of pulmonary infection (i.e. pneumonia) in humans (and is therefore designated as "commonly pathogenic" in the lungs), it is precisely streptococcus pneumoniae that is normally present as part of the endogenous flora of the respiratory tract that does not induce infection and is therefore not generally pathogenic in nature for endogenous colonization.

MRV for multiple myeloma

Patient s (pts) was diagnosed with multiple myeloma in the fall of year 0 (stage 3A), with multiple lesions in bone scans, including the skull, humerus, and pelvis. He was treated with standard chemotherapy (melphalan and prednisone) for 6 months. However, in the 12 rd year of 3, he had a pathological fracture in his right femur due to his disease, which required wire nail fixation and partial irradiation. On day 28 of 4 months of 4 years, PtS began treatment with MRV, which contained Staphylococcus aureus, a common cause of sepsis, which continued for more than 13 years until 17 years when the vaccine was no longer used. Remarkably, PtS survived almost 25 years after diagnosis of multiple myeloma, which is indeed an unexpected outcome considering his "late" diagnosis.

In light of the foregoing results, one aspect of the present invention comprises treating hematologic cancer by administering an antigenic composition comprising antigenic determinants known to be microbial pathogens that cause leukemia.

In light of the foregoing results, as well as shown in other patient case reports detailed herein, other aspects of the invention relate to the administration of immunogenic compositions repeatedly, relatively frequently, over relatively long periods of time, as described elsewhere herein.

PVF for colon cancer with liver and abdominal lymph node metastasis

Patient t (ptt) was diagnosed as colon cancer at 9 months of year 0 and was treated with resection of its primary tumor (and subsequent chemotherapy). After 10 months, she developed liver metastases that were surgically excised in 7 months of year 1. PtT remained in good condition until 6 months of 7, when she was diagnosed with recurrent disease-an inoperable abdominal lymph node mass immediately adjacent to the aorta and spinous processes that blocked her left urinary canal, requiring insertion of a nephrostomy tube. In month 10 of year 7, PtT was considered advanced and treated with palliative radiation. She started treatment with PVF on day 17 of 11 months of the 7 th year, from which she continued the treatment every 1 day. PtT survives almost 4 years after being diagnosed as advanced recurrent metastatic colon cancer.

In light of the foregoing results, one aspect of the present invention includes the use of an antigenic composition comprising antigenic determinants of a microbial pathogen known to induce abdominal lymph node infection for the treatment of cancer in abdominal lymph nodes.

MRV for metastasis to skin and perineum

Patient u (ptu) was diagnosed as colon cancer at 11 months of year 0 and was treated with resection of the primary tumor. He was diagnosed with stage 4 cancer in month 7 of year 2 due to metastasis to perineum (i.e., perianal/genital soft tissue region) and skin. He further surgically removed as much of the cancer in the perineum as possible (cancer spreading to the edges of past surgery) and received subsequent radiation and chemotherapy. The only sites known to have residual cancer are in the skin and perineum. PtU beginning on day 25 of month 5 of year 3, MRV, which contains Staphylococcus aureus, a common cause of cutaneous and perineal infections, he continued the treatment 3 times a week for 5 months. Despite his poor initial prognosis, PtU was in an excellent health state for more than 10 years after diagnosis of stage 4 cancer with metastasis to perineum and skin.

In light of the foregoing results, one aspect of the present invention comprises treating cancer of the skin and perineum by administering an antigenic composition comprising antigenic determinants of microbial pathogens known to be common causes of skin and perineal infections.

PVF for peritoneal transfer

Patient v (ptv) was diagnosed with breast cancer at 5 months of year 0, when she underwent curative breast surgery and adjuvant chemotherapy. In month 1 of 12, she developed abdominal pain and ascites and was diagnosed with peritoneal metastasis, a poor prognosis. On day 5 of 8 months of 12 years, PtV began treatment with PVF, which contained escherichia coli, a common cause of peritoneal infection, which she continued regularly for 1 year. Her tumor markers and ascites decreased and at 8 months of 13 years, i.e. 1 year after PVF treatment, she had undergone abdominal surgery for an unrelated medical condition, when the operator did not find any evidence of prior peritoneal cancer. PtV interrupts the use of vaccines. PtV survived last association 3 years and 9 months after being diagnosed as advanced peritoneal metastasis.

In light of the foregoing results, one aspect of the present invention includes treating peritoneal metastasis by administering an antigenic composition comprising antigenic determinants of a microbial pathogen known to induce peritoneal infection.

PVF for ovarian and pelvic cancers

Patient w (ptw) was diagnosed with stage 3B poorly differentiated ovarian cancer in fall of year 0. She was operated at 11 months of year 0, with left ovaries removed, but the cancer could not be completely removed, and therefore her risk of recurrence was very high. She underwent full-course post-operative chemotherapy. However, her tumor markers began to rise in year 2 and were diagnosed as recurrence of the right ovarian region in month 1 of year 3. She was surgically removed the right ovarian mass at 2 months of 3 years, but the cancer was still not accurately removed completely and she was subsequently treated with chemotherapy. However, she had another recurrence in the pelvic region and retroperitoneal lymphadenopathy in month 12 of year 3. She started on day 5 of month 1 of year 4 with the PVF vaccine, which contained escherichia coli with ovarian and pelvic infection predisposing factors, and she continued the treatment for 6 months. She had elevated to 2600 and had the tumor marker decreased to the 300 range. PtW survived and felt very well at the time of last contact, 9 months after being diagnosed with recurrent ovarian cancer. It should be noted that her tumor markers decreased after PVF treatment.

In light of the foregoing results, one aspect of the invention includes treating ovarian and pelvic cancers by administering an antigenic composition comprising antigenic determinants of microbial pathogens known to induce ovarian and pelvic region infections.

MRV for follicular non-Hodgkin lymphoma

Patient y (pty): diagnosed as stage 4A follicular non-hodgkin's lymphoma, with extensive overt lymphadenopathy (i.e., enlarged lymph glands). He refused all conventional treatments. PtY was initially treated with MRV compositions that contain multiple pathogens that commonly induce lymph node infections in the head and neck, axilla, mediastinum and inguinal regions. In addition, he began treatment with the intake of multivitamins/supplements, a healthy diet and other immune enhancing therapies. He continues to use the vaccine regularly for more than 3 years, when his lymph gland size starts to decrease greatly and he feels good. The resolution of lymphadenopathy continues and imaging shows that a previously large amount of lymphadenopathy has almost completely resolved. PtY felt well and no perceptible lymphadenopathy: clear and obvious healing. 5 years after he was initially diagnosed with stage 4A follicular non-hodgkin's lymphoma, PtY had no signs of recurrence and had passed active and healthy life. His stage 4A follicular non-hodgkin's lymphoma was completely alleviated by treatment with the MRV vaccine.

In light of the foregoing results, one aspect of the present invention comprises treating lymphoma by administering an antigenic composition comprising antigenic determinants of a microbial pathogen known to be a common cause of lymph node infection in the area of the lymphoma.

PVF for colon cancer with metastasis to liver and kidney

Patient z (ptz) was diagnosed with metastatic spread of previously treated colon cancer, with metastases to the liver and possibly other metastases to bilateral kidneys. The liver metastases were excised. The prognosis for colon cancer in this stage (i.e., stage 4) is poor and the benefits of other conventional therapies (i.e., chemotherapy) are limited. PtZ chemotherapy was initially rejected. At 3 months after diagnosis of metastatic colon cancer, PtZ began treatment with Polyvaccinum Forte (PVF), which contains E.coli, a common cause of colon, liver and kidney infections. Additionally, PtZ began to be treated with a multivitamin/supplement regimen and a healthy diet. He continues to use the vaccine and vitamin and supplement regimen on a regular basis and starts chemotherapy. Although her overall course of disease progresses slowly, recurrence of lung and liver metastases occurs, and 28 months after he initially diagnosed with metastatic disease, he is stable in weight and he is well-minded. PtZ felt good after 3 years (36 months) after diagnosis of stage 4 colon cancer, except for nausea and slight weight loss associated with chemotherapy.

In light of the foregoing results, one aspect of the present invention comprises treating cancer of the colon, liver and kidney by administering an antigenic composition comprising antigenic determinants of microbial pathogens known to be pathogenic in the colon, liver and kidney.

PVF for colon cancer with liver, hepatic portal lymph nodes and lungs

Patient aa (ptaa) was diagnosed with metastatic colon cancer with liver, hepatic portal lymph nodes and lung metastases. The prognosis for colon cancer in this stage (i.e., stage 4) is very poor (i.e., "advanced" cancer), and the benefits of conventional therapy (i.e., chemotherapy) are limited. PtAA started chemotherapy, but terminated treatment due to side effects after about 5 months of his diagnosis, when he started treatment with a polyvalent Forte (containing bacterial species that induce infections in colon, liver, abdominal lymph nodes and lung), 1 time every 2 days, and was on a multivitamin/supplement regimen and a healthy diet. Later CT scans of PtAA confirmed: although the size of both liver metastases grew moderately (3.4cm to 4.5cm and 1.2cm to 3.0cm), the size of the necrotic hepatic portal lymph nodes did not change from his diagnosis and the size of the pulmonary metastases did not change. Although the prognosis is very poor, PtAA continues to feel very good in the last 1 year after diagnosis of advanced cancer.

In light of the foregoing results, one aspect of the present invention comprises treating cancer of the colon, liver, abdominal lymph nodes and lungs by administering an antigenic composition comprising antigenic determinants of microbial pathogens known to be pathogenic in the colon, liver, abdominal lymph nodes and lungs.

Example 3: microbial pathogens

In an alternative aspect, the invention utilizes a microbial antigen, such as a bacterial or viral antigen, to formulate an antigenic composition, wherein the microbial species is selected based on the tissue or organ in which the microbe is known to induce an infection. The bacterial host flora is the most common pathogen, which causes the vast majority of infectious episodes in most animals, including humans. For example, host flora is infected by primary contact, or contact and invasion following mucosal injury, e.g. by blood vessels, trauma, chemical injury, or damage caused by primary infection.

For microbial pathogens, toxicity and infectious capacity are a combination of the ability of the microorganism to adhere, produce enzymes, survive in immune products (complement, antibodies), and survive in the microbicidal activity of macrophages and neutrophils. Some bacteria, including endogenous bacteria, may be sufficiently toxic to induce a single microbial infection, while others are synergistically effective against multiple microbial infections. In general, it is often not possible to ascertain the specific role that individual microorganisms play in a mixed-infection environment. In some cases, the invention utilizes, in some embodiments, the microbial species associated with acute infections, as acute infections may provide more appropriate immune stimulation.

In some embodiments, bacteria that are members of the endogenous flora of a particular region may be used to formulate the antigenic compositions of the present invention. The panel of table 6 lists a number of bacterial species, along with the biological region, where each species may form part of the endogenous flora. For example, fastidious bacteria are typical members of the endogenous flora of the respiratory tract and oral cavity.

Table 6: common flora of human bacteria (endogenous bacteria human pathogen)

Endogenous microbial flora such as bacteria have been spread to tissues for pathogenesis either by continuous dissemination or by bacteremia. Under favorable conditions, all endogenous organisms can become pathogenic and locally invade and disseminate to adjacent tissues and organs by continuous dissemination. The endogenous bacterial flora of the skin, mouth and colon is understood to be a species that can also be used for the dissemination of bacteremia. Bacteria that are members of a particular endogenous flora domain can thus induce an infection in the tissues or organs to which these bacteria can disseminate. Accordingly, one aspect of the invention includes the use of an endogenous microbial pathogen to treat cancer in a tissue or organ in which the endogenous bacteria can disseminate to induce infection. The column of table 7 lists 9 regions of endogenous flora: skin, respiratory system, genitalia, GU system, mouth, stomach, duodenum/jejunum, ileum and colon. The rows of table 7 list the organs or tissues in which the cancer may be located. Thus, one aspect of the invention includes the use of endogenous microbial pathogens to formulate antigenic compositions or to select existing formulations with pathogens for use in the treatment of cancer located in tissues or organs where the pathogens can spread to induce infection. Thus, in an alternative embodiment, a tumor located in a tissue or organ listed in the first column of table 7 may be treated with an antigenic composition comprising antigenic determinants specific for microbial pathogens that are members of the endogenous flora of one or more of the endogenous flora domains listed in the first row of table 7 and are represented by X's or checkmarks in the appropriate row. For example, a tumor located within the prostate may be treated with an antigenic composition comprising antigenic determinants specific for microbial pathogens or pathogens derived from the GU system and/or reproductive system. In table 6, the number of bacterial species endogenous to the endogenous flora domain listed in table 7 and the corresponding endogenous flora domain are listed. Accordingly, one aspect of the present invention includes the use of an antigenic composition comprising the antigenic determinants of the bacterial species listed in table 6 for the treatment of cancer located in the tissues listed in table 7, wherein the region of endogenous flora associated with the tumor site in table 7 matches the region of endogenous flora associated with the bacterial species in table 6.

Table 7: tissue/organ pathogenicity of endogenous flora

Bacteria reach tissues/organs by serial dissemination (x) or by bacteremia dissemination (□).

According to the combined information of tables 6 and 7, the cancer located in the tissue or organ shown in column 1 of table 7 can be treated by an antigenic composition comprising the antigenic determinants of the respective bacterial species of table 6, so that the column headings of table 7 can be actually replaced by the bacterial species of table 6.

In some embodiments, the microbial pathogen used in the present invention may be an exogenous bacterial pathogen. For example, the organisms listed in table 8 may be used as microbial pathogens to formulate antigenic compositions, or antigenic compositions with those pathogens may be selected for use in treating cancer located in tissues or organs listed by the relevant organisms of table 8. In some embodiments, antigenic determinants of endogenous and exogenous bacterial species targeted to a particular tissue or organ can be used in combination. For example, antigenic compositions derived from or specific for clostridium difficile may be used to treat cancers localized in the colon.

Table 8: exogenous bacterial human pathogens and sites of infection therewith

In some embodiments, the microbial pathogen used in the present invention may be a viral pathogen. Table 9 provides an exemplary listing of viral pathogens along with tissues and organs that are reported to be pathogens for each viral species. Thus, one aspect of the invention includes the use of an immunogenic composition specific for a given virus to treat cancer in a defined organ or tissue located next to the viral name of figure 9. For example, antigenic compositions derived from or specific for vaccine viruses may be used to treat cancers located in the skin, blood tissues, lymph nodes, brain, spinal cord, eyes, or heart.

Table 9: viral human pathogens and sites of infection thereof

The cumulative information of tables 6 to 9 provides a broad determination of microbial pathogens that can be used in the antigenic compositions of the invention, along with a determination of the tissues or organs in which these organisms are pathogenic, and accordingly the tissues or organs in which the cancer is located that can be treated by the antigenic preparation.

In some embodiments, the microbial pathogens selected for use in the antigenic compositions of the present invention can be pathogens that are common causes of acute infection of the tissue or organ in which the cancer to be treated is located Table 10 identifies bacterial and viral pathogens of this type along with tissues and organs in which they normally induce infection accordingly, in selected embodiments, cancers that belong to tissues identified in the first column of Table 10 can be treated with antigenic compositions that contain antigenic determinants for one or more pathogenic organisms listed in the second column of Table 10.

Table 10: common causes of acute infections (bacterial and viral) at various tissue/organ sites

In selected embodiments, specific microbial pathogens may be suitable for treatment of a particular cancer, examples of which are listed in table 10. These are exemplary embodiments and are not exhaustive of the alternative formulations used in accordance with the present invention.

The particular organism that normally induces an infection in a particular tissue or organ may vary by geographic location. For example, mycobacterium tuberculosis is a more common cause of pulmonary infection in some geographic areas and populations than in others, and thus, while mycobacterium tuberculosis is not a common pulmonary pathogen in some geographic and species groups, it may be a common pulmonary pathogen in others. Thus, table 10 is not exhaustive of common pathogens for all geographical locations and species groups. It is understood that for a particular tissue or organ site of the invention, the general clinical microbiologist in the art can determine the common pathogenic species in a particular geographic region or population. For veterinary use, there are of course specific pathogens that are common in selected tissues of the selected species, and this may also vary geographically.

In selected embodiments, the invention includes a diagnostic step to evaluate prior exposure of a patient to a microbial pathogen. For example, the diagnostic step can include obtaining a history of exposure to the selected pathogen and/or evaluating the patient's immune response to the selected pathogen. For example, serological tests can be performed to detect antibodies to selected pathogens in patient sera. For this aspect of the invention, the antigenic determinants of the selected microbial pathogen may be selected for use in the immunogenic composition for the selected patient based on the diagnostic indication that the patient has had one or more prior exposures to the pathogen, for example by the presence of antibodies to the antigenic determinants of the pathogen in the patient's serum.

As discussed for patient R, in selected embodiments, the microbial pathogen selected for use in the antigenic composition of the invention may be the most common pathogen that induces an acute infection in the tissue or organ in which the cancer to be treated is located, which may provide particular benefits as evidenced by the case of patient R. For example, for the treatment of bone cancer, staphylococcus aureus may be the selected species; for the treatment of cancer in lung tissue, streptococcus pneumoniae will be selected; for the treatment of breast cancer, staphylococcus aureus will be selected; for the treatment of cancer of the kidney or bladder, e.coli will be the species of choice; for the treatment of colon cancer, E.coli will be the species of choice. It is to be understood that clinical microbiologists in the art are able to determine the most common pathogenic species, i.e., bacteria or viruses, for each particular tissue or organ according to the present invention. In selected embodiments, only the antigenic determinants of the most common pathogens of a particular tissue or organ can be used to treat cancer in that tissue or organ. In an alternative embodiment, the antigenic determinants of the most common pathogens of a particular tissue or organ can be used in combination with antigenic determinants of other pathogens known to be pathogenic within that particular tissue or organ, preferably the other pathogens are selected from the more common pathogens.

In some embodiments, the present invention provides antigenic compositions in which a selected threshold ratio of antigenic determinants according to the present invention is used, relative to any other antigenic determinants in the composition. For example, an antigenic composition may contain greater than X% of antigenic determinants from a pathogenic (or common pathogenic or most common pathogenic) species, where X may be, for example, 10, 30, 40, 50, 60, 70, 80, 90, 95, or 100 (or any integer between 10 and 100). For example, at least X% of the antigenic determinants in the antigenic composition can be specific for a microbial pathogen that is pathogenic (or commonly pathogenic or most commonly pathogenic) within a particular organ or tissue of the patient in which the cancer is situated. With other measures of the total number of microbial pathogens in the antigen composition, at least X% can be selected as the microbial pathogen that is pathogenic (or commonly pathogenic or most commonly pathogenic) within a particular organ or tissue of the patient in which the cancer is located. In some embodiments, the antigenic composition may accordingly consist essentially of antigenic determinants of one or more microbial pathogens that are each pathogenic (or generally pathogenic or most generally pathogenic) within the particular organ or tissue of the patient in which the cancer is situated. The following data shows the surprising efficacy of these selected formulations:

(1) the use of MRV, which contains many common respiratory pathogens as well as staphylococcus aureus, the most common pathogen for both breast and bone, was found to be helpful in treating breast cancer with metastasis to bone (see figure 6). However, the survival benefit (survival of patients treated with MRV compared to survival of patients not treated with MRV) was moderate (i.e., median survival of patients treated with vaccine compared to median survival of 26 months of patients not treated with vaccine, median survival of 31 months of patients treated with vaccine). On the other hand, one patient (patient R) treated with a vaccine specifically targeting breast and bone cancer (i.e. containing only staphylococcus aureus, the most common cause of both breast and bone infections) had a significant survival benefit, surviving for more than 17 years. The inclusion of other bacterial species in the MRV that do not induce (or far from commonly induce) bone infections and often induce infections elsewhere (i.e. respiratory tract) appears to substantially diminish the benefit of the vaccine for cancer treatment of the breast and bone.

(2) Using Respivax^TMThe survival of the treated stage 3B lung cancer patients (i.e., median survival at 38 months and 5-year survival at 40%) was substantially greater than the survival of the stage 3B lung cancer patients treated with MRV (i.e., median survival at 18 months and 5-year survival at 14%). Respivax^TMIn fact, contain higher concentrations of species than MRV that normally cause lung infections. Respivax^TM67% of the bacterial cell count is made up of species that normally induce lung infection, while 30% of the MRV bacterial cell count is made up of species that normally induce lung infection. Thus, a composition containing a greater proportion of the most common bacteria inducing lung infections, i.e. Respivax^TMLung cancer appears to be more effective than MRV treatment.

(3) Stage 4 colon cancer patients treated with MRV (which does not contain any colonic pathogens) survived worse than patients not treated with the vaccine. This suggests that treatment with antigenic determinants not from microorganisms that are pathogenic in the organ or tissue in which the cancer is located is not only ineffective, but may also worsen.

(4) The murine studies are shown below, and in particular, the cancer cell model data relates to treatment with only the klebsiella pneumoniae antigenic determinant, as compared to treatment with klebsiella pneumoniae along with other antigens.

Thus, the use of targeted antigenic compositions derived from microbial pathogens that are pathogenic to a particular organ or tissue herein provides evidence for a gradual increase in pathogenicity from pathogenicity to general pathogenicity to the most general pathogenicity for the treatment of cancer within that particular organ or tissue.

In some embodiments, the invention includes the use of bacterial or viral vaccines (e.g., polio vaccine, haemophilus influenzae vaccine, meningococcal vaccine, pneumococcal vaccine, influenza vaccine, hepatitis b vaccine, hepatitis a vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, measles vaccine, mumps vaccine, rubella vaccine, varicella vaccine, BCG vaccine, cholera vaccine, japanese encephalitis vaccine, rabies vaccine, typhoid vaccine, yellow fever vaccine, smallpox vaccine, etc.) approved for other purposes for use as a cancer treatment by selecting a vaccine containing a pathogen (or antigenic component of an antigen) that is pathogenic within a particular organ or tissue of a patient in whom the cancer is located, upon review of tables 6-10. For example, a streptococcus pneumoniae vaccine, whole cell vaccine or vaccine consisting of one or more antigenic components of streptococcus pneumoniae (e.g., pneumococcal polysaccharide-23-valent), can be used to treat cancer at any of the following sites where streptococcus pneumoniae is listed as a common pathogen in table 10: pulmonal lymph nodes, hematological cancers, bone, meninges, spinal cord, eye/orbit, sinus, thyroid, bronchus, lung, pleura, or peritoneum. As another example, a hepatitis b virus vaccine can be used to treat cancer at any of the following sites, where hepatitis b virus is listed as a pathogen in table 9: liver, pancreatic or hematological cancers.

In some embodiments, selected compositions and methods are specifically excluded from the scope of the present invention. For example, the use of the following microbial pathogens in the treatment of the following cancers is excluded from some embodiments so that the claimed invention may be extended to specific embodiments (in addition to one or more of the following):

a) for example, the treatment of gastric and colon cancer by injection with BCG (mycobacterium bovis);

b) for example, treatment of lung cancer by mycobacterium w by injection;

c) for example, non-small cell lung cancer by injection with mycobacterium vaccae;

d) for example, treatment of melanin streams by injection with corynebacterium parvum;

e) for example, treatment of gastric cancer by injection with streptococcus pyogenes;

f) treatment of lung cancer or acute myeloid leukemia, e.g., by injection with nocardia rubra;

g) for example, treatment of cervical cancer by injection with lactobacillus casei;

h) for example, treatment of lymphoma and lung cancer with pseudomonas aeruginosa by injection;

i) treatment of melanoma, for example, by injection with vaccinia virus;

j) treatment of melanoma with rabies virus, e.g., by injection;

k) a composition consisting of a combination of antigens of the following species is used for the treatment of primary (or, alternatively, metastatic) cancer located in the lung of an animal (or, alternatively, a human), for example by oral administration: streptococcus pneumoniae; neisseria catarrhalis; streptococcus pyogenes; haemophilus influenzae; staphylococcus aureus bacteria; klebsiella pneumoniae.

l) compositions consisting of the combined antigens of the following species are used for the treatment of primary (or, alternatively, metastatic) cancer located in the lung of an animal (or, alternatively, a human), for example by oral administration: streptococcus pneumoniae; neisseria catarrhalis; streptococcus pyogenes; haemophilus influenzae; staphylococcus aureus bacteria; klebsiella pneumoniae; klebsiella odorifera (Klebsiella ozaenae); and (4) grass green streptococcus.

Example 4: murine study

In the following murine studies, the following common materials were used: PBS (Gibco), and the mice were 7-week-old female C57 BL/6.

Example 4A: cancer of the lung

This section relates to a Lewis lung cancer mouse model. The bacterial vaccines used in this experiment were as follows: streptococcus pneumoniae [ cells and broths ]]Vaccine (lot # J049-1[ 2X 10 ]⁹](ii) a Klebsiella pneumoniae [ cells and broths ]]Vaccine (lot # J046-1[ 2X 10 ]⁹](ii) a Staphylococcus aureus [ cells and Broth ]]Vaccine (lot # J041-2[ 10X 10 ]⁹](ii) a Coli (colonic isolate) [ cells and Broth]Vaccine (lot # J047-1[ 6X 10 ]⁹](ii) a Salmonella enterica [ cells and broth]Vaccine (lot # J31[ 15X 10 ]⁹](ii) a Klebsiella pneumoniae [ cell only ]]Vaccine (lot # J048-1[ 2X 10 ]⁹](ii) a And medium only (Klebsiella pneumoniae medium) (lot # J046-1). Mice were treated according to the experimental groups defined in table 11.

Table 11: experimental groups for Lung cancer mouse model

Specifically, groups of mice were pretreated subcutaneously with bacterial vaccines on days-10, -8, -6, -4, and-2. On day 0, mice were challenged intravenously with Lewis lung carcinoma cells (Cedarlane lot #508714) at a dose of 10e 5. Thereafter, mice were treated subcutaneously with vaccine injections every other day for the duration of the experiment as defined in table 11. Control group was treated with medium only. Animal body weights were measured every 4 days and recorded. The experiment was terminated when the mice began to develop disease. Thereafter, all mice were humanely sacrificed and the lungs were surgically removed and weighed. Lungs were placed in vials with Buoin fluid and the number of lung nodules in each group was counted. These results are shown in table 12. A typical example of these lungs is depicted in fig. 14.

Table 12: number of distinct lung tumors per group of mice

The lung weight of mice injected with tumor cells was then compared to the lung weight of mice injected with PBS alone to determine the tumor burden and hence the efficacy of the vaccine treatment. These results are shown in table 13.

Table 13: mean lung weight (mg) and tumor weight suppression (compared to control) in mice immunized with a killed bacterial vaccine in an intravenous Lewis lung cancer model

Example 4B: skin cancer

This section relates to mouse models of skin cancer. The bacterial vaccines used in this experiment were as follows: streptococcus pneumoniae [ cells and broths ]]Vaccine (lot # J049-1[ 2X 10 ]⁹](ii) a Klebsiella pneumoniae [ cells and broths ]]Vaccine (lot # J046)-1[2×10⁹](ii) a Staphylococcus aureus [ cells and Broth ]]Vaccine (lot # J041-2[ 10X 10 ]⁹](ii) a Coli (colonic isolate) [ cells and Broth]Vaccine (lot # J047-1[ 6X 10 ]⁹](ii) a Salmonella enterica [ cells and broth]Vaccine (lot # J31[ 15X 10 ]⁹](ii) a Staphylococcus aureus [ cell only ]]Vaccine (lot # J041-2[ 10X 10 ]⁹](ii) a And medium only (Staphylococcus aureus medium) (lot # J041-1). Mice were treated according to the experimental groups defined in table 14.

Table 14: experimental groups for mouse model of skin cancer

Specifically, groups of mice were pretreated subcutaneously with bacterial vaccines on days-10, -8, -6, -4, and-2. Mice were challenged intravenously with 2 × 10e6 doses of B16 melanoma cells (lot # 3995448; ATCC CRL-6323) on day 0. Thereafter, mice were treated subcutaneously with vaccine injections every other day for the duration of the experiment as defined in table 14. Control group was treated with medium only. Animal body weights were measured every 4 days and recorded. Once the tumor was palpable, tumor diameter was measured every other day using calipers. The experiment was terminated when mice began to develop disease or tumors of either group reached 20mm in diameter. Thereafter, all mice were sacrificed humanely. The mean volume of tumors present in the groups of mice described herein is shown in figure 15.

Example 4C: cancer of colon

This section relates to a mouse model of colon cancer. The bacterial vaccines used in this experiment were as follows: streptococcus pneumoniae [ cells and broths ]]Vaccine (lot # J049-1[ 2X 10 ]⁹](ii) a Klebsiella pneumoniae [ cells and broths ]]Vaccine (lot # J046-1[ 2X 10 ]⁹](ii) a Staphylococcus aureus [ cells and Broth ]]Vaccine (lot # J041-2[ 10X 10 ]⁹](ii) a Coli (colonic isolate) [ cells and Broth]Vaccine (lot # J047-1[ 6X 1 ]0⁹](ii) a Coli (prostate isolate) [ cells and Broth]Vaccine (lot # J040-2[ 6X 10 ]⁹](ii) a Salmonella enterica [ cells and broth]Vaccine (lot # J31[ 15X 10 ]⁹](ii) a And medium only (E.coli medium) (lot # J040-1). Mice were treated according to the experimental groups defined in table 15.

Table 15: experimental grouping of Colon cancer mouse models

Specifically, groups of mice were pretreated subcutaneously with bacterial vaccines on days-10, -8, -6, -4, and-2. Mice were challenged intraperitoneally with MC-38collo adenocarcinoma cells (administered by Dr. Jeff Schlom Lab, NCI) at a dose of 2 × 10e5 on day 0. Thereafter, mice were treated subcutaneously with vaccine injections every other day for the duration of the experiment as defined in table 15. Control group was treated with medium only. Mice were observed for the following clinical factors: weight, cold touch, diarrhea, tachypnea, eye closure, decreased activity, piloerection and convulsions. When mice began to show clinical signs of morbidity, mice were humanely sacrificed and the day was defined as the day of death. The survival data for this experiment is depicted in fig. 16.

In addition, healthy versus sick/dead mice included in this example were counted on day 29 of the experiment as defined in table 16.

Table 16: health score for morbidity/mortality group^＊＊

Health is 10 points; only tumors were scored 5; 2 points of tumor + ascites; and death is 0 point.

TABLE 17 survival summary of each mouse group from colon cancer experiments

MST median survival time

ILS-increase in Life time compared to control group

Comparison with placebo-treated control group

Overall comparative survival:

logarithmic rank (Mantel Cox) p ═ 0.001

Breslow (generalized rank sum test) p ═ 0.003

Tarone-Ware p＝0.002

Example 4 summary

Skin model:

there were significant therapeutic advantages for the group treated with staphylococcus aureus (cells only). This study demonstrates the effectiveness of killed staphylococcus aureus for skin cancer treatment in mouse models, consistent with the fact that staphylococcus aureus is the most common cause of skin infection in mice. For slowing or inhibiting tumor growth, the data are consistent with using the immunogenic compositions of the invention. A staphylococcus aureus cell only vaccine, in which only the cells are collected by centrifugation of the cells and broth to remove the medium and exotoxin, and then reconstituted with physiological saline, is more effective than a staphylococcus aureus cell and broth vaccine, which contains both the medium and exotoxin. This may be because staphylococcus aureus exotoxins inhibit immune functions, such as leukocidin, which can kill white blood cells. Thus, the invention includes embodiments in which the antigenic determinants to be used in the immunogenic composition are isolated from the immunomodulatory compounds produced by the microbial pathogen of interest.

Lung model:

the data presented in this example indicate that there is substantial tumor suppression using an immunogenic composition from klebsiella pneumoniae, consistent with the fact that klebsiella pneumoniae is a common cause of lung infection in mice. In mice (but not usually humans), salmonella enterica can induce pneumonia, which is consistent with the beneficial effects of this vaccine in mouse lung models. Unlike humans, in which streptococcus pneumoniae is a common pulmonary pathogen, streptococcus pneumoniae is relatively rare among pulmonary pathogens in mice (although streptococcus pneumoniae can induce pneumonia in mice). Coli and s.aureus are able to induce lung infections in mice rarely, consistent with their mild benefits shown herein. This example demonstrates that the killed klebsiella pneumoniae vaccine is significantly effective for the treatment of lung cancer in mice, particularly illustrating embodiments in which the immunogenic composition comprises an antigen that is only the most common pathogenic organism (see group 2, group 6).

Colon model:

salmonella enterica is the most common cause of gastrointestinal and intraperitoneal infections in mice, consistent with the beneficial effects shown herein in the treatment of gastrointestinal and intraperitoneal cancer in mice.

Of the immunogenic compositions used in this example (i.e., salmonella enterica, escherichia coli, staphylococcus aureus, klebsiella pneumoniae, streptococcus pneumoniae), salmonella enterica is the most common mouse g.i./intraperitoneal pathogen. Escherichia coli is the second most common g.i./intraperitoneal pathogen in mice. Staphylococcus aureus and klebsiella pneumoniae can be found as part of the colonic flora and can induce g.i./intraperitoneal infections, although far less common. Streptococcus pneumoniae does not induce g.i./intraperitoneal infection. Accordingly, salmonella enterica vaccines showed substantial benefit in this mouse colon tumor model, escherichia coli vaccines showed moderate benefit, staphylococcus aureus and klebsiella pneumoniae vaccines showed mild benefit, and streptococcus pneumoniae showed no benefit. In humans, salmonella species induce g.i. infections, and thus, salmonella enterica and other salmonella vaccines would be expected to be helpful in treating colon cancer in humans.

Example 5: animal model

Example 5A: exemplary effects of Heat-inactivated Klebsiella pneumoniae antigen compositions on monocyte/macrophage and dendritic cell populations in mice

The following methods and materials were used in this example:

mice for these studies, 7-8 week old C57BL/6 female mice were scheduled from Harlan Labs (Livermore, Calif.).

Antibodies and reagents the following antibodies were used in this example: anti-I-A/I-E FITC (MHC class II; M5/114.15.2); anti-Gr-1 PE (RB6-8C 5); anti-CD 11b PerCP-Cy5 (M1/70); anti-CD 11c APC (N418); anti-CD 4FITC (GK 1.5); anti-NK1.1PE (PK 136); anti-CD 8a eFluor780 (53-6.7); anti-CD 44APC (IM 7). All antibodies were obtained from eBioscience (San Diego, CA). Liberase TM and DNAse I were obtained from Roche. All media were from HyClone (Fisher).

Treatment with antigen composition klebsiella pneumoniae (KO12[5.0OD600 units ]) with thermal kill of phenol was diluted 1/10 in PBS containing 0.4% phenol and injected 100 μ Ι subcutaneously into 4 mice on

days

0, 2, 4 and 6. Control mice (n-5) were injected with PBS on

days

0, 2, 4 and 6.

Bronchoalveolar lavage mice were sacrificed on day 7 and bronchoalveolar lavage (BAL) was performed by exposing the trachea and then inserting a 22G catheter connected to a 1ml syringe. 1ml of PBS was injected into the lungs and excised and placed in a 1.5ml microcentrifuge tube. Lungs were washed sequentially 3 times more with 1ml PBS and the liquids were pooled. The first wash from each mouse was centrifuged at 400xg and the supernatant frozen for cytokine analysis. The final 3ml of lavage fluid was centrifuged and the cells were combined with the cell pellet from the first lavage. Cells were counted and stained with antibodies specific for MHC class II, Ly6G/C, CD11b and CD11 c. After staining, cells were washed and analyzed on a FACS Calibur flow cytometer.

Lung digestion after BAL was performed, lungs were placed in 5ml RPMI containing 417.5. mu.g/ml Liberase TL (Roche) and 200. mu.g/ml DNAse I (Roche). Then, the lungs were digested at 37 ℃ for 30 minutes. After digestion, the lungs were passed through a 70um cell filter to produce a separate cell suspension. The cells were then centrifuged, washed, resuspended in FACS buffer (PBS with 2% FCS and 5mM EDTA) and counted. After counting, cells were stained and analyzed by FACS using the same antigen as BAL cells.

Peritoneal lavage following BAL, 1ml of PBS was injected into the peritoneum of the mice using a 1ml syringe attached to a 25G needle. The abdomen was rubbed for 1 minute and 0.5ml of PBS was recovered from the peritoneum using a 1ml pipette. The lavage solution was placed into a 1.5ml centrifuge tube, centrifuged at 400Xg for 5 minutes, and resuspended in FACS buffer prior to staining and FACS analysis.

Spleen and lymph node analysis spleens and draining lymph nodes were removed after BAL and peritoneal lavage and placed in PBS. Spleen was destroyed by trituration through a 70 μm cell filter (Fisher) and lymph nodes were destroyed using the rubber end of the embolus from a 1ml syringe. After disruption, individual cell suspensions from spleen and lymph nodes were centrifuged, washed once with FACS buffer, and resuspended in FACS buffer prior to counting, staining and FACS analysis.

FACS analysis cells were stained on ice for 20 minutes in 96-well plates using 50 μ Ι of antibody diluted in FACS buffer. After 20 minutes, 100 μ l of FACs buffer was added to the wells and the plates were centrifuged at 400Xg for 5 minutes. Subsequently, the medium was removed and the cells were washed 1 more times with FACS buffer. After the final wash, cells were resuspended in 200 μ l FACS buffer and data were acquired using a FACS Calibur flow cytometer (BD). A minimum of 20,000 field events were collected for all samples except BAL, with a minimum of 5,000 events collected for BAL.

The following results were obtained in this example.

Normal mice without tumors were treated with the klebsiella pneumoniae antigen composition on

days

0, 2, 4 and 6. On day 7, mice were sacrificed and bronchoalveolar lavage fluid, lung tissue, peritoneal lavage fluid, lymph nodes and spleen were analyzed for changes in monocytes and macrophages. The increased number of acute inflammatory blood monocytes/macrophages was defined by the high expression of CD11b and Gr-1 (same marker as Ly6 c) and F4/80 for lymph node drainage, the injection site of the klebsiella pneumoniae antigen composition, was observed (see, fig. 17A). These acute inflammatory monocytes/macrophages also express very high levels of MHC class II molecules, indicating exposure to cellular antigens. Importantly, treatment of mice with the klebsiella pneumoniae antigen composition for 1 week resulted in an increase in the frequency of acute inflammatory monocytes in the bronchoalveolar lavage fluid and in the lungs (i.e., the target organ) of the treated mice, but not in the spleen or peritoneum, suggesting that treatment can induce specific return of monocytes to the lungs without affecting other organs (see fig. 17B). Monocytes were able to differentiate into Dendritic Cells (DCs) in the lung and consistent with our observation of a significant increase in monocyte recruitment, a significant increase in the frequency of cells representing the marker of mature DCs was also observed (see: fig. 17C).

As shown in figure 17, treatment with the klebsiella pneumoniae antigen composition for 7 days resulted in a significant increase in acute inflammatory monocytes and dendritic cells in the mouse lungs (compared to treatment with placebo-PBS). As shown in fig. 17, mice were treated with klebsiella pneumoniae antigen composition or PBS on

days

0, 2, 4, and 6. On day 7, mice were sacrificed and the total number of A) and B) inflammatory monocytes (CD11B + Gr-1+ cells) and C) dendritic cells (CD11C + MHC II + type cells) was determined by flow cytometry in the lung and spleen. Error bars indicated at A) represent the average of 4-5 mice per group.

Example 5B illustrates the effect of heat-inactivated Klebsiella pneumoniae antigen compositions and heat-inactivated Escherichia coli antigen compositions on monocyte/macrophage, dendritic cell and effector cell populations of mice

The following methods and materials were used in this example:

for these studies, 7-8 week old C57BL/6 female mice were scheduled from Harlan Labs (Livermore, Calif.).

The following antibodies were used: anti-I-A/I-E FITC (MHC class II; M5/114.15.2); anti-Gr-1 PE (RB6-8C 5); anti-CD 11b PerCP-Cy5 (M1/70); anti-CD 11c APC (N418); anti-CD 4FITC (GK 1.5); anti-NK1.1PE (PK 136); anti-CD 8a eFluor780 (53-6.7); anti-CD 44APC (IM 7). All antibodies were obtained from eBioscience (san diego, CA). Liberase TM and DNAse I were obtained from Roche. All media were from HyClone (Fisher).

Klebsiella pneumoniae (K.pneumoniae; lot KO 12; 5.0OD600 units) with thermal kill of phenol was diluted 1/10 in PBS containing 0.4% phenol and injected 100 μ l subcutaneously into 5 mice on

days

0, 2, 4 and 6. Heat killed E.coli (lot; 5.0OD600 units) was diluted 1/10 in 0.4% phenol and injected 100. mu.l subcutaneously into 5 mice on

days

0, 2, 4 and 6. Control mice (n-5) were injected with PBS on

days

0, 2, 4 and 6.

Mice were sacrificed on day 7 and bronchoalveolar lavage (BAL) was performed by exposing the trachea and then inserting a 22G catheter connected to a 1ml syringe. 1ml of PBS was injected into the lungs and excised and placed in a 1.5ml microcentrifuge tube. Lungs were washed sequentially 3 times more with 1ml PBS and the liquids were pooled. The first wash from each mouse was centrifuged at 400 × g and the supernatant was frozen for cytokine analysis. The final 3ml of lavage fluid was centrifuged and the cells were combined with the cell pellet from the first lavage. Cells were counted and stained with antibodies specific for MHC class II, Ly6G/C, CD11b and CD. After staining, cells were washed and analyzed on a FACS Calibur flow cytometer.

After BAL, lungs were placed in 5ml RPMI containing 417.5. mu.g/ml Liberase TL (Roche) and 200. mu.g/ml DNAse I (Roche). Then, the lungs were digested at 37 ℃ for 30 minutes. After digestion, the lungs were passed through a 70 μm cell filter to produce a separate cell suspension. The cells were then centrifuged, washed, resuspended in FACS buffer (PBS with 2% FCS and 5mM EDTA) and counted. After counting, cells were stained and analyzed by FACS using the same antigen as BAL cells.

After BAL, 1ml of PBS was injected into the peritoneum of the mice using a 1ml syringe connected to a 25G needle. The abdomen was rubbed for 1 minute and 0.5ml of PBS was recovered from the peritoneum using a 1ml pipette. The lavage solution was placed into a 1.5ml centrifuge tube, centrifuged at 400Xg for 5 minutes, and resuspended in FACS buffer prior to staining and FACS analysis.

Spleen and draining lymph nodes were removed after BAL and peritoneal lavage and placed in PBS. Spleen was destroyed by trituration through a 70 μm cell filter (Fisher) and lymph nodes were destroyed using the rubber end of the embolus from a 1ml syringe. After disruption, individual cell suspensions from spleen and lymph nodes were centrifuged, washed once with FACS buffer, and resuspended in FACS buffer prior to counting, staining and FACS analysis.

The following results were obtained in this example:

as shown in fig. 18, mice were treated with the klebsiella pneumoniae antigen composition, the escherichia coli antigen composition, or PBS on

days

0, 2, 4, and 6. On day 7, mice were sacrificed and the total number of inflammatory monocytes (CD11b + Gr-1+ cells) and dendritic cells (CD11c + MHC II + type cells) was determined by flow cytometry in peritoneal lavage fluid, lungs, lymphoid tuberculosis spleen. Error bars in figure 18 represent standard deviations from 5 mice. P-value <.05 using Student's t-test

Figure 18 shows that treatment with the klebsiella pneumoniae antigen composition, but not the escherichia coli antigen composition, significantly increased the number of monocytes and DCs in the mouse lungs. In contrast to the lung, Klebsiella pneumoniae did not lead to an increase in monocytes in the peritoneum of mice, which is caused by E.coli. Importantly, there was only a slight increase in the number of inflammatory monocytes and no increase in DC in the spleen of mice treated with klebsiella pneumoniae or escherichia coli, indicating that the therapeutic effect is not general, but actually site-specific for a specific organ. In addition to considering therapeutic effects on inflammatory monocytes and DCs in the mouse lung, we also considered changes in other cytokines, such as the cytotoxin CD8T cells, CD4T helper cells, and Natural Killer (NK), all of which may play a role in antitumor immunity.

Figure 19 shows that the klebsiella pneumoniae antigen composition, but not the PBS or escherichia coli antigen composition, resulted in a significant increase in the frequency and total number of NK cells, CD4, and CD8 cells in the lungs of treated mice. This example is the first illustration of our common knowledge that subcutaneous injection of a biocidal species, which normally induces lung infection, promotes leukocyte accumulation in the lung without any inflammation at this site. Furthermore, we also exemplify the specificity of the effect for the targeted site, and it is also specific for the bacterial component of the treatment used.

As shown in fig. 19, mice were treated with the klebsiella pneumoniae antigen composition, the escherichia coli antigen composition, or PBS on

days

0, 2, 4, and 6. On day 7, mice were sacrificed and the total number of CD4T cells, CD8T cells, and Natural Killer (NK) cells was determined by flow cytometry. Error bars represent the standard deviation of the values obtained from 5 mice per group. P-value <.05 using Student's t-test

Example 5C illustrates the effect of heat and phenol inactivated klebsiella pneumoniae (k. pneumoconiae) antigen compositions on the anti-tumor response of mice, and the status of inflammatory monocytes and dendritic cells following treatment of tumor-bearing mice

The following methods and materials were used in this example:

lewis lung cancer cell lines derived from a C57BL/6 background were obtained from ATCC (Manassas, Va.). Cells were maintained in Dulbecco's Modified Eagles medium (ATCC, Manassas, Va.) containing 10% FCS. Cells were allowed to grow at 5% CO₂The wet 37 ℃ incubator. Prior to tumor inoculation, cells were detached from the culture plates using 0.25% trypsin and 0.53mM EDTA. Cells were washed in PBS and washed at 8x10⁶Individual cells/ml were resuspended and mice were injected intravenously with 200. mu.l (4X 10)⁵Individual cells).

The following antigen compositions were used in this study: a klebsiella pneumoniae antigen composition with heat inactivation of phenol (lot KO12) and a klebsiella pneumoniae antigen composition with heat inactivation of phenol (lot KO 25). The heat-inactivated Klebsiella pneumoniae and phenol-inactivated Klebsiella pneumoniae were concentrated to 5.0OD600 units. Beginning 2 days after tumor injection, every other day 0.1ml of klebsiella pneumoniae diluted 1/10 in PBS with 0.4% phenol was injected subcutaneously.

Analysis of inflammatory monocytes, DCs, T cells and NK cells. All analyses were performed according to the method used in example 5B above.

Table 18: experimental grouping and dosing protocol for example 5C

¹Dulbecco's phosphate buffered saline with phenol²With thermal destruction of phenol³Phenol kill with phenol

The following results were obtained in this example:

this example is set up to determine whether the presence of a tumor affects the recruitment of cells to the lung and whether an increase in the effect of the treatment over time leads to a further increase in cell recruitment with ongoing treatment, thereby possibly determining a better therapeutic effect with prolonged treatment. Furthermore, we wanted to determine that the phenol inactivated Klebsiella antigen composition is more effective than the heat inactivated Klebsiella pneumoniae antigen composition.

Figure 20 clearly shows that by day 9 (i.e. 4 treatments with the klebsiella pneumoniae antigen composition), there was a significant increase in the number of acute inflammatory monocytes, DCs, T cells and NK cells in the lungs of mice treated with the klebsiella pneumoniae antigen composition inactivated by heat or phenol, many times more than in the lungs of mice treated with placebo (saline-PBS), and furthermore, a significant targeted intracellular immune response in the lungs elicited by the klebsiella pneumoniae antigen composition treatment. On day 9, it is recommended that phenol deactivation be more effective than thermal deactivation, but this trend reverses on day 16. Importantly, however, with reference to the number of cells in the lungs on days 9 and 16, it is evident that there is an accumulative effect of bacterial treatment on the recruitment of cells to the lungs. For example, in the group treated with the phenol inactivated klebsiella pneumoniae antigen composition, about 100,000 acute inflammatory monocytes were present in the lungs of mice on day 9, and by day 16, the value increased substantially to 400,000, indicating a substantial increase in response with continued treatment. The same increased therapeutic response that continued with treatment occurred in mice treated with heat-inactivated bacteria. Importantly, this cumulative therapeutic effect was observed for all other cell types analyzed as well. In this study, there were no demonstrable statistically significant differences in immune cell recruitment with heat-or phenol-inactivated klebsiella pneumoniae antigen compositions.

As shown in FIG. 20, the drug was used at 4X10 on day 0⁵lewis lung carcinoma cells were injected intravenously into mice. Subsequently, from day 2 onwards, mice were treated every other day with a klebsiella pneumoniae antigen composition produced by heat inactivation or phenol inactivation, or with PBS. Mice were sacrificed on days 9 and 16 and the total number of (a) inflammatory monocytes (CD11B + Gr-1+) and DCs (CD11c + Iab +) or (B) CD4T cells, CD8T cells and Natural Killer (NK) cells was determined by flow cytometry. The bars represent: PBS treatment group, mice treated with heat inactivated klebsiella pneumoniae antigen composition, and mice treated with phenol inactivated klebsiella pneumoniae antigen composition. Error bars represent the standard deviation of the values obtained from 5 mice per group. P-value using Student's t-test<.05

The results of this study demonstrate an increased immune response in the targeted tissue with continued treatment.

Example 5D shows the effects of heat, radiation and phenol inactivation on the Klebsiella pneumoniae antigenic composition, including cytokine recruitment to the mouse lungs, and the effect of phenol as a preservative having any effect

The following methods and materials were used in this example:

In this study, a klebsiella pneumoniae antigen composition with thermal kill of phenol (KO12), a klebsiella pneumoniae antigen composition without thermal kill of phenol (KO25), a klebsiella pneumoniae antigen composition without radiation of phenol (KO24), and a klebsiella pneumoniae antigen composition without phenol kill (KO25) were used. All bacterial preparations were at a concentration of 5.0OD units in saline. For the 1/10 dilution, 1ml of the bacterial formulation was added to 9ml of DPBS and mixed immediately, then mixed again before injection. For the 1/100 dilution, 0.1ml of the bacterial formulation was added to 9.9ml of DPBS and mixed immediately, then mixed again before injection. For the dilution of the heat-killed Klebsiella pneumoniae antigen composition with phenol, a DPBS solution containing 0.4% phenol (w/v) was used, and the dilution was performed as above. To prepare a 0.4% solution of phenol in DPBS, a 5% solution of phenol was first prepared by adding 0.5g of solid phenol (Sigma Aldrich, st. louis, MO) to 10ml of DPBS (Hyclone, Logan, UT). The solution was filtered through a 0.22um filter (Millipore, Billerica, Mass.) and stored at 4 ℃. Immediately before use, 1ml of a 5% phenol solution was diluted in 12.5ml of DPBS and used to prepare the bacterial preparation.

Treatment with antigen composition 5 mice per group were treated subcutaneously on

days

0, 2, 4 and 6 with either 0.1ml of a heat killed klebsiella pneumoniae antigen composition diluted 1/10 in PBS or with 0.4% phenol, 0.1ml of a irradiated klebsiella pneumoniae antigen composition diluted 1/10 in PBS, or 1/10 diluted with PBS or with 0.4% phenol. On day 7, mice were sacrificed and cytokine recruitment to the lungs was analyzed as in example 5B.

The following results were obtained in this example:

in this example, we used cytokine recruitment to the lung as a surrogate for potency to compare the potency of klebsiella pneumoniae antigen compositions inactivated by various methods. Figure 21 shows that the addition of phenol (0.4%) as a preservative increases efficacy for the klebsiella pneumoniae antigen composition of thermal and phenol kill as measured by intracellular recruitment. In some embodiments, small amounts of phenol (i.e., 0.4%, as a preservative) can stabilize various components of the bacterial cell wall, such as components critical in antigen profiling recognition and activation of optimal targeting responses. In comparing 3 formulations containing phenol as a preservative (i.e., heat-killed, phenol-killed, and radiation-killed), the irradiated klebsiella pneumoniae antigen composition resulted in maximal recruitment of acute inflammatory monocytes, DC, NK cells, and T cells to the lungs, followed by the phenol-killed klebsiella pneumoniae antigen composition, and the heat-killed klebsiella pneumoniae antigen composition resulted in minimal recruitment of cells.

As shown in figure 21, mice were treated with klebsiella pneumoniae antigen compositions on

days

0, 2, 4, and 6, wherein the antigen compositions were irradiated with heat inactivated (HKWP) or without (HKnp) phenol preservative, with phenol preservative and with phenol inactivated (PKWP) or without phenol preservative and with phenol inactivated (PKnp), or with phenol preservative (IRWP). Mice were sacrificed on day 7 and the total number of (a) inflammatory monocytes (CD11B + Gr-1+) and DCs (CD11c + Iab +) or (B) CD4T cells, CD8T cells and Natural Killer (NK) cells was determined by flow cytometry. Error bars represent the standard deviation of the values from 5 mice per group. P-value <.05 compared to mice treated with IRWP using Student's t-test

Example 6: clinical studies involving advanced epithelial cancers

Overview of treatment

The following patients with different advanced cancers have undergone treatment with heat-inactivated targeted bacterial antigen compositions. A full informed written consent (Fully in-formed written content) was received for each patient and in each case. Treatment consisted of repeated (3/week) subcutaneous injections of precise amounts of vaccine in the abdominal region. The dose was gradually increased in each patient in order to achieve an adequate skin response (3-5cm redness lasting ca. 24h). For each patient, the Case Report Form (CRF) records the skin response and possible clinical effects and/or side effects associated with the treatment. Typical and concomitant treatments and patient responses to therapy are briefly described.

Patient # 1:

a 53 year old male patient with advanced melanoma ICD10: C43, 11/2005 was first diagnosed with lesions under the right toenail one year later, i.e. histology 12/2006: advanced malignant melanoma; the patient refuses the big toe amputation; 5/2008, lymphatic vessel transfer to the right leg, with a 100% increase in leg circumference swelling at the first appearance of targeted bacterial antigen composition treatment (9/2008): 80% on the Ka-score, no pretreatment.

Treatment for 6 months 09/2008-04/2009:

12 x intraperitoneal ozone (O3) insufflation;

42 × local high frequency superheat (13.56 Mhz);

18 times moderate general overheating 38.5 ℃;

s.c. 6 month treatment with staphylococcus aureus antigen composition;

orthomolecular medicine: high dose vitamin C infusion (0.5g/kg/BW), vitamin D3; 2.000 iu/day, 200 mg/day of artemisinin succinate, 100 mg/day of celecoxib, and low dose naltrexone; agaricus campestris (Cordyceps, Ganoderma, Lentinus Edodes), selenium 200 uc/day, curcumin 3.000 mg/day, and proteolytic enzyme (Wobemumgos)

Evaluation details:

PET 7/2008 SUV is 4.81 in the right big toe, in the knee: 5.01

PET 12/2008 SUV is 3.80 in the right big toe, in the knee: 4.02

We started the treatment at the end of 9/2008

At that time (9/2008), there had been clinically significant inguinal lymph nodes, which were not observed on 7/2008 PET.

05/2010: good clinical status, PET determined complete remission with a 100% karman score.

Patient # 2:

a 48 year old female patient with advanced bilateral breast cancer ICD10: C50.9, a first diagnosis of 4/2008; histologically invasive ductal adenocarcinoma of the breast, ER/PR location, unknown Her2, T1/T2, N1 (sentinel lymph node axilla), M0G 3; multiple treatments with sodium bicarbonate injections and repeated surgery on both mammary glands; patients reject the proposed bilateral mastectomy and do not have "healthy (in sano)" resection (e.g., the margin of breast tumor resection will not have tumor cells). The patient still refuses chemotherapy and/or hormonal therapy. The kappa score was 90% with no foreknowledge.

8 months of treatment 03/2009-11/2009:

3 × autologous dendritic cell therapy combined with:

3 times long-term moderate general overheating, 40 degree in 8 hours

57 × local hyperthermia to both mammary glands (13.56 Mhz);

s.c. 6 month treatment with staphylococcus aureus antigen composition;

orthomolecular medicine: high dose vitamin C infusion (0.5g/kg/BW), low dose naltrexone, medicinal mushroom (Cordyceps, Ganoderma, Lentinus Edodes), Curcuma rhizome 3.000 mg/day, zinc

Evaluation details:

3/2009 treatment is not started.

05/2010: bilateral breast MRI showed no detectable deformity, good clinical status, Ka's score 100%

Patient # 3:

a 73 year old female patient with advanced NSCLC cancer, FIGO IIc, ICD10: C34, the first diagnosis being 6/2009; histological clear cell adenocarcinoma of the lung, T4, N1, M0G 3; she passed through the newly assisted CHT; recurrence after 3 cycles of neoadjuvant CHT indicated no T2 tumor; however, she underwent left lung resection surgery, i.e., R0 resection, and mediastinal lymphadenectomy; the Ka-score was 90%.

7 months of treatment 08/2009-03/2010:

08/2009 chemotherapy with plectrum/cisplatin was initiated up to 10/2009 in combination with:

4X Long-term moderate general overheating, 40 ℃ within 8 hours (1-2/2009)

Chest 20X local high frequency superheat (13.56Mhz) (8-10/2009)

10/2009 left Pneumectomy R0 resection (unequivocally against other secondary CHT)

Treatment with Klebsiella pneumoniae antigen composition s.c. for 6 months (08/2009-02/2010)

Orthomolecular medicine Thymopeptide i.m., Indometacin, cimetidine, high dose vitamin C infusion (0.5g/kg/BW), ALA/N regimen (low dose naltrexone and α lipoic acid), medical mushroom (Ganoderma), Curcuma longa 3.000 mg/day, Zinc, melatonin, ionized oxygen inhalation

Evaluation details:

08/2009 treatment is not started.

05/2010: CT chest and tumor markers CEA, NSE and CYFRA showed complete remission, good medical condition, with a 100% on the karman scale.

Patient # 4:

a 50 year old female patient with advanced breast cancer ICD10: C50 with disseminated liver and lung metastases, first diagnosed at 8/1990; histological non-differentiated cirrhosis type breast adenocarcinoma, pT1c, N1, M0G 3; she underwent multiple chemotherapies within 20 years; 11/2004 first wound recurrence, 12/2004 left mastectomy, 6 × CHT Epitax and chest wall radiation; 9/2005 first diagnosis of disseminated liver and lung metastases: furthermore, 8 × CHT with Epitax is up to 3/2006. Reappearance indicates that at this point in time the progressive disease she began treatment with the targeted bacterial antigen composition. The Ka-score was 90%.

Treatment for 4 years 03/2006-03/2010:

03/2006 begin treatment with a multiple vaccine forte vaccine in combination with:

3 × autologous dendritic cell therapy in combination with (6-8/2006):

3X Long-term moderate Total superheat (LD-WBH), 40 ℃ within 8 hours (1-2/2009)

Chest and liver 25X local high frequency overheating (13.56Mhz) (3-6/2006)

Treatment with Klebsiella pneumoniae antigen composition for 8 months (11/2008-7/2009)

10/2009, TM CEA and CA15/3 began to rise again

02-03/20092 x autologous dendritic cell therapy without LD-WBH

04/2010 Heat ablation of liver metastasis (unchanged lung metastasis)

Orthomolecular medicine: thymus polypeptide i.m., indomethacin, cimetidine, high dose vitamin C infusion (0.5g/kg/BW), turmeric 3.000 mg/day, zinc, proteolytic enzyme

Evaluation details:

we started the treatment at the end of 03/2006

05/2010: CT Thorax indicates stable disease within 4 years, progressive disease of liver metastasis, good clinical status, 100% on the charpy score.

Patient # 5:

a 66 year old male patient with advanced prostate cancer ICD10: C61 with disseminated bone and lymphatic metastases, first diagnosis 01/1997; histologically undifferentiated prostate adenocarcinoma, pT3, N1, M1G 3; he passes multiple hormones and CHT within 13 years; the patient was in a good clinical state, self-diagnosis began with metastatic prostate cancer for 13 years; the Ka-score was 90%.

11 years of treatment 11/1999-05/2010:

13 year-old antiandrogen, buserelin, norreyde, Consolid, Trenantone, elipidity, and β -sitosterol

06/2006-12/2007 begin mixed bacterial vaccine therapy with multiple vaccine forte vaccine

Regular chemotherapy with Taxotere 140mg every 3-4 weeks starting from 03/2008

50 × moderate systemic fever, 39 ℃ within 3 hours (1999-2009)

18 months of treatment with a staphylococcus aureus antigen composition (11/2008-05/2010, uninterrupted)

05-06/20092 x autologous dendritic cell therapy without moderate WBH 39 °, 3h

Orthomolecular medicine: wheatgrass, cimetidine, zitaetidine, high dose vitamin C infusion (0.5g/kg/BW), turmeric 3.000 mg/day, boswellia serrata (India), 400mg 4X 4/day, zinc, proteolytic enzyme

Evaluation details:

we began treatment at the end of 1999

05/2010: bone scanning indicates stable disease conditions; currently, PSA is 89ng/ml, good medical status, 90% Karschner score.

Patient # 6:

a 52 year old female patient with advanced primary peritoneal cancer ICD10: C48.2, with disseminated peritoneal cancer, first diagnosed at 06/2003; histologically undifferentiated peritoneal adenocarcinoma pT3, N1, M1G 3; she underwent debulking OP by bilateral ovariectomy and hysterectomy, and adjuvant chemotherapy with paclitaxel/carboplatin: progressive disease, and change of paclitaxel/carboplatin according to SD up to 8/2008; progressive disease with disseminated peritoneal LK metastasis and treatment with carboplatin third line CHT and starting with targeted bacterial antigen compositions; the patient is in good medical condition; the Ka-score was 100%.

Treatment for 4 months 05-09/2009

5 × carboplatin chemotherapy (line 3), then:

5X Long-term moderate Total superheat (LD-WBH), 40 ℃ within 8 hours (05-09/2009)

20X local hyperthermia (13.56Mhz) (05-09/2009)

Treatment with E.coli (colon) antigen composition for 2 months (05-07/2009)

Orthomolecular medicine: high dose vitamin C infusion (0.5g/kg/BW), high dose artichoke and silymarin extracts (liver)

Evaluation details:

we started the treatment at the end of 5/2009

04/2010: CT abdominal and tumor markers indicate complete remission, good clinical status, a Ka-score of 100%

Patient # 7:

a 50 year old female patient with inoperable pancreatic cancer ICD10: C25.9, and cancer infiltrating the major blood vessels; ultrasound images and CT evidence indicate invasion of the superior mesenteric vein, a first diagnosis at 02/2009; histologically undifferentiated pancreatic adenocarcinoma pT3, N1, M1G3, and with peritoneal carcinoma; she was exposed to topical irradiation and low dose capecitabine as a radiosensitizer; when she started treatment in our clinic, the cancer was not yet unresectable; the Ka-score was 100%.

2 months of treatment 06-07/2009: (Escherichia coli SSI for 11 months)

1 × autologous NDV-activated dendritic cell therapy combined with:

1X Long-term moderate general overheating, 40 ℃ in 8 hours (7-10/2009)

4 × moderate general overheating 38.5 °;

abdominal 15X local high frequency overheating (13.56Mhz) (06-07/2009)

11 months of treatment with E.coli (colon) antigen composition (06/2009 to date and onwards)

Orthomolecular medicine: thymus polypeptide i.m; medical mushrooms (ganoderma, cordyceps, shiitake mushroom); high dose vitamin C infusion (0.5g/kg/BW), high dose proteolytic enzyme therapy (wbenczym phylogenzym), cimetidine.

Evaluation details:

we started the treatment at the end of 6/2009

05/2010: complete remission, NED; PET 02/2010 indicated no glucose uptake; tumor marker normal CA19/9: 4; good clinical status, Ka's score 100%

Example 7: dose studies (QB28 study)

To examine the effect of the dose, mice were treated with different doses of the klebsiella pneumoniae antigen composition or PBS control. All mice (C57BL/6) began treatment with the Klebsiella pneumoniae antigen composition or PBS on days-10, -8, -6, -4, and-2 using the following injection sites (first injection: abdominal right groin; second injection: abdominal right axilla; third injection: abdominal left axilla; 4 th injection: abdominal left groin, etc.). All mice then received 3x10 by intravenous injection into the lateral tail vein of the mice⁵Tumor inoculation dose for each Lewis lung cancer cell. The doses of antigen composition or PBS were as follows: i) PBS 0.1 ml; ii) klebsiella pneumoniae, 0.1mL OD600 ═ 1.67; iii) Klebsiella pneumoniae, 0.1mL OD600 ═ 0.5. Mice received the klebsiella pneumoniae antigen composition or PBS and were treated on

days

2, 4, 6 and 8. The experiment was terminated on day 10; all mice were sacrificed, their lungs surgically removed, washed in water, weighed, then placed in Bouins solution for fixation and then counted for 24 hours. As shown in fig. 22, each data point represents the number of tumor nodules from one mouse. A picture of a typical lung from these experiments is shown in figure 23. Figure 23 shows that lungs from mice treated with PBS control exhibit a large number of nodules. By comparison, figure 23 shows that the lungs from mice treated with the klebsiella pneumoniae antigen composition exhibit fewer nodules, comparable to the ratio shown in figure 22.

QB30 in other studies of the effect of doses of the klebsiella pneumoniae antigen composition, it has also been shown that if the dose of the klebsiella pneumoniae antigen composition is too low, it is not effective for the treatment of lung cancer. In these studies, the results are shown in FIG. 24, where all mice (C57BL/6) began treatment with the Klebsiella pneumoniae antigen composition or PBS on days-10, -8, -6, -4, and-2, using the following injection sites (first injection: abdominal right groin; second injection: abdominal right axilla, third injection: abdominal left axilla; 4 th injection: abdominal left groin, etc.). All mice then received 3x10 by intravenous injection into the lateral tail vein of the mice⁵Tumor inoculation dose of individual Lewis lung cancer cells. The doses of antigen composition or PBS were as follows: i) PBS 0.1 ml; ii) klebsiella pneumoniae, 0.1mL OD600 ═ 0.5; iii) Klebsiella pneumoniae, 0.1mL OD600 ═ 0.05. Mice continued to receive either the klebsiella pneumoniae antigen composition or PBS for treatment on

days

2, 4, 6, 8, 10, 12, 14 and 16. The experiment was terminated on day 18; all mice were sacrificed, their lungs surgically removed, washed in water, weighed, then placed in Bouins solution for fixation and then counted for 24 hours. As shown in fig. 24, each data point is represented byNumber of tumor nodules from one mouse.

The results from QB28 and QB30 indicate that extremely high doses of the klebsiella pneumoniae antigen composition are ineffective, perhaps due to over-stimulation of the host immune system. The results from this study also indicate that low doses are ineffective, perhaps due to inadequate stimulation of the host immune system.

Example 8: cisplatin potency study

To examine the effect between cisplatin and chemotherapy and the dose of the klebsiella pneumoniae antigen composition, a QB38 study was completed. Briefly, all mice received 3 × 10 intravenous injections into the lateral tail vein on day 0 of the study⁵Tumor inoculation dose of individual Lewis lung cancer cells. After this inoculation, all mice were pooled in one cage and then randomly assigned to their respective cages to control bias data. Mice in the cisplatin group were injected intraperitoneally with 10mg/kg of the drug on the +2 morning of the study. Control mice were injected with control (PBS). Thereafter, in the afternoon of +2 days of the study, mice received either the klebsiella pneumoniae antigen composition or PBS and these injections were continued on

days

4, 6, 8, 10 and 12. Study termination on day 14; thereafter, all mice were sacrificed, their lungs surgically removed, washed in water, weighed, then placed in Bouins solution for fixation and then counted for 24 hours. As shown in figure 25, each data point represents the number of tumor nodules from one mouse based on whether the mouse was treated with PBS, a klebsiella pneumoniae antigen composition in combination with cisplatin, or cisplatin alone. The results summarized in figure 25 show that mice treated with the klebsiella pneumoniae antigen composition and cisplatin present fewer nodules than those treated with cisplatin alone, the klebsiella pneumoniae antigen composition alone, or the PBS control.

Other experiments were set up to determine whether the klebsiella pneumoniae antigen composition therapy could be used to synergize with platinum-based chemotherapy to extend the survival of mice bearing Lewis lung tumors in the lungs. 4 groups of 5C 57BL/6 female mice all received 10 i.v. injections on day 0⁵And Lewis lung cancer cells. Groups 3 and 4 received a single dose of 10mg/kg cisplatin intraperitoneally on day 2. Starting on day 4, mice received klebsiella pneumoniae or Placebo (PBS) every 2 days until death. Fig. 26 shows survival of all mice and p-values of various survival curves calculated using the log rank test are shown. Mice treated with cisplatin alone increased survival of mice with lung tumors and median survival increased from 16 days for the PBS group to 23 days for the PBS + cisplatin group. Treatment with klebsiella pneumoniae alone also increased survival of mice compared to PBS (survival average of klebsiella pneumoniae of 19 days versus PBS of 16 days). Most importantly, cisplatin chemotherapy together with klebsiella pneumoniae therapy had the greatest therapeutic benefit (median survival of 32 days), indicating a synergistic effect between platinum-based chemotherapy and antigenic composition therapy in a mouse model of lung cancer. The p-values of the data generated in this QB45 study are shown in table 19 below.

TABLE 19 survival data (QB45)

As shown in this example, the antigenic composition can be used on the same day as cisplatin or after a period of cisplatin administration. As used herein, the term "antigenic composition" refers to a composition comprising antigens of one or more microbial species. As used herein, the term "microbial species" may relate to a viral pathogen or a bacterial pathogen or a fungal pathogen, as detailed herein.

Other studies were set to explore whether there was a synergistic effect between platinum-based chemotherapy and the klebsiella pneumoniae antigenic composition for pneumonia treatment in mice. All mice received 10 i.v. injections on day 0⁵And Lewis lung cancer. Starting on day 0, mice received the antigen composition or PBS subcutaneously every 2 days. On day 12, some mice were treated intraperitoneally (10mg/kg) with cisplatin. Subsequently (day 13), 3 mice from each group were bled and blood cells were stained with anti-CD 11b antibody and analyzed by FACS. FIG. 27 is a schematic view ofThe curves indicate the frequency of CD11b + myeloid cells in blood at day 13. Each dot represents a single mouse. The results of this study indicate that antigenic composition therapy, when administered with cisplatin, increases the frequency of myeloid cells (monocytes, macrophages, granulocytes, dendritic cells) in the blood. This may enhance the immunity of the animal by providing greater amounts of these innate myeloid cells that are capable of responding to tumors or pathogens. Furthermore, because myelosuppression is a clinically important side effect of chemotherapy and the result is a reduction of myeloid cells in the blood, which can lead to a delay/discontinuation of chemotherapy or susceptibility to infection, the use of antigenic composition therapy to enhance myeloid cells during chemotherapy can help reduce the risk of this important and clinically relevant side effect of chemotherapy.

Example 9: macrophage study (MOA13 study)

To examine the effect of macrophages on embodiments of the invention, a MOA13 study was performed. Briefly, mice were inoculated with 3 × 10⁵Individual Lewis lung carcinoma cells or HBSS injected as vehicle control. For mice vaccinated with Lewis lung cancer cells, they were pooled in a single cage, where they were subsequently transferred randomly to their respective cages. Thereafter, on

days

2, 4, 6 and 8, mice were treated with the klebsiella pneumoniae antigen composition or PBS. Thereafter, all mice were sacrificed on day 9. To obtain information about bone marrow and NK cell frequency, 20 mice (5 from each group) had their lungs surgically removed and the lungs were digested with releasese and dnase. After digestion and cell formulation, a separate cell suspension was obtained. Thereafter, a portion of the cells were transferred to a 96-well round bottom plate to be stained with bone marrow-specific antibodies (CD11b +, NK1.1+) and NK-specific antibodies (NK1.1+, CD11b +). For previous cell types, a valve plate (gate) was used to select only CD11b + and NK 1.1-cell populations. Thereafter, cell data acquisition was performed using BD FACSCalibu and analysis was performed using FlowJo. Statistical analysis and graphical representation were performed using Excel and GraphPad Prism. The results are shown in fig. 28. Briefly, using a composition of Klebsiella pneumoniae antigensTreatment resulted in an increase in myeloid cells (probably monocytes and macrophages) and CD11b + NK cells in the lungs of mice so treated.

For information related to cytokine production in lung tissue, the following experiment was performed. Mice were inoculated with 3X10⁵Individual Lewis lung carcinoma cells or HBSS injected as vehicle control. For mice vaccinated with Lewis lung cancer cells, they were pooled in a single cage, where they were subsequently transferred randomly to their respective cages. Thereafter, on

days

2, 4, 6 and 8, mice were treated with the klebsiella pneumoniae antigen composition or PBS. Thereafter, mice were sacrificed on day 9. To obtain information on cytokines in lung tissue, bronchoalveolar lavage was performed on the lungs. Immediately thereafter, the lungs were surgically removed after lavage and placed in pre-weighed vials containing PBS and protease inhibitors. Thereafter, lung tissue was homogenized, centrifuged, and the supernatant was coated on an ELISA kit (eBioscience); ELISA analysis was performed according to manufacturer's instructions. Statistical analysis and graphical representation were performed using Excel and GraphPad Prism. Data are expressed as pg cytokines per mg of original lung tissue. Each data point in fig. 29 is from a single mouse value. As shown in FIG. 29, treatment with the Klebsiella pneumoniae antigenic composition resulted in an increase in the production of anti-tumor (IL-12, MCP-1, GMCSF, IL-6) cytokines in lung tissue.

For information related to cytokine production in bronchoalveolar lavage (BAL) fluid, the following experiments were performed. Mice were inoculated with 3X10⁵Individual Lewis lung carcinoma cells or HBSS injected as vehicle control. For mice vaccinated with Lewis lung cancer cells, they were pooled in a single cage, where they were subsequently transferred randomly to their respective cages. Thereafter, mice were treated with klebsiella pneumoniae or PBS on

days

2, 4, 6 and 8 of the experiment. Thereafter, all mice were sacrificed on day 9. To obtain information about cytokine production in the lungs, bronchoalveolar lavage was performed on the lungs of mice. The solution from the lavage was placed into a vial and stored at-80 ℃ until the time when the ELISA assay was performed. Thereafter, ELI is performed according to manufacturer's guidelinesAnd (6) SA analysis. Statistical analysis and graphical representation were performed using Excel and GraphPad Prism. Data are expressed as pg/ml of lavage fluid, as shown in FIG. 30. Each data point represents a value obtained from one mouse. As shown in figure 30, treatment with the klebsiella pneumoniae antigen composition had no effect on cytokine production in BAL fluid.

For examination of the M1/M2 macrophage phenotype in the model described herein, NOS2 and Arg1 levels were monitored in the lung. The experiment was carried out as follows: briefly, mice were inoculated with 3x10⁵Individual Lewis lung carcinoma cells or HBSS injected as vehicle control. For mice vaccinated with Lewis lung cancer cells, they were pooled in a single cage, where they were subsequently transferred randomly to their respective cages. Thereafter, mice were treated with klebsiella pneumoniae or PBS on

days

2, 4, 6 and 8 of the experiment. Thereafter, all mice were sacrificed on day 9. To obtain information about the expression of NOS2 and Arg1 genes, 20 mice (5 from each group) had their lungs surgically removed. Thereafter, a small portion of these lungs was cut using sterile techniques and RNAlater was placed to stabilize the RNA material for further gene analysis. Total RNA was extracted from lung tissue using a kit from Qiagen and used according to the manufacturer's protocol. The cDNA kit is used to convert a quantity of RNA into cDNA (Qiagen), which is then converted by instructions provided by the manufacturer. qPCR was performed using a primer set to the specific methods Nos2 and Arg1(Nos 2: forward-CGCTTTGCCACG GACGAGA; reverse-AGGAAGGCAGCGGGCACAT; Arg 1: forward-GGTCCACCCTGACCTATGTG; reverse-GCAAGCCAATGTACACGATG). Statistical analysis was performed using Excel and GraphPad Prism. As shown in figure 31, treatment with the klebsiella pneumoniae antigen composition resulted in an increase in the Nos2/Arg1 ratio in the lung, which was associated with an increased anti-tumor response.

For the study of M1/M2 responses in the in vivo model described herein, CD206 (mannitol receptor) expression was monitored. Briefly, mice were inoculated with 3 × 10⁵Individual Lewis lung carcinoma cells or HBSS injected as vehicle control. For mice vaccinated with Lewis lung carcinoma cells, they were pooled in a single cage where they were subsequently randomizedTransferred to their respective cages. Thereafter, mice were treated with klebsiella pneumoniae or PBS on

days

2, 4, 6 and 8 of the experiment. Thereafter, all mice were sacrificed on day 9. To obtain information about CD206 expression, 20 mice (5 from each group) had their lungs surgically removed and digested with the releasing enzyme TL and dnase. After digestion and cell formulation, a separate cell suspension was obtained. A portion of the cells were transferred to a 96-well round bottom plate for staining using CD 206-specific antibodies (clone MR5D3) obtained from Cedarlane Labs (Burlington, ON). Since CD206 is located both intracellularly and extracellularly, cells were first fixed with paraformaldehyde and stained with antibodies in a permeabilization solution. Analysis was performed using FlowJo. Statistical analysis and graphical representation were performed using Excel and GraphPad Prism. The results are shown in fig. 32. As shown in figure 32, treatment with klebsiella pneumoniae resulted in a reduction in CD206 expression on pulmonary macrophages in the presence (PBS + LL2 and klebsiella pneumoniae 1/10X + LL2) or absence (PBS and klebsiella pneumoniae 1/10X) of lung tumors. The klebsiella pneumoniae antigen composition reduces the frequency of CD206 expression from about 20% (PBS + LL2 group) to about 10% (klebsiella pneumoniae 1/10X + LL2 group) in the presence of lung cancer. A similar decrease in CD206 expression was observed in klebsiella pneumoniae treated animals without any lung cancer (PBS and klebsiella pneumoniae 1/10X).

For the study of the M1/M2 phenotype in the in vivo model described herein, F4/80+ macrophage expression was monitored. Briefly, mice were inoculated with 3 × 10⁵Individual Lewis lung carcinoma cells or HBSS injected as vehicle control. For mice vaccinated with Lewis lung cancer cells, they were pooled in a single cage, where they were subsequently transferred randomly to their respective cages. Thereafter, mice were treated with klebsiella pneumoniae or PBS on

days

2, 4, 6 and 8 of the experiment. Thereafter, all mice were sacrificed on day 9. To obtain information relating to the expression of F4/80+ by macrophages, 20 mice (5 from each group) had their lungs surgically removed and digested with the releasing enzyme TL and DNase. After digestion and cell formulation, to obtainTo a separate cell suspension. A portion of the cells were transferred to a 96-well round bottom plate for staining with the F4/80 monoclonal antibody. The acquisition of cellular data was performed using BD FACSCalibur. Analysis was performed using FlowJo. Statistical analysis and graphical representation were performed using Excel and GraphPad Prism. As shown in FIG. 33, treatment with the Klebsiella pneumoniae antigen composition resulted in a reduction of F4/80+ macrophages in the lungs. This decrease is thought to be associated with a decrease in M2-like macrophages.

Example 10: site-specific study (MOA14 study)

For the study of the M1/M2 phenotype of the in vivo model described herein for use with the antigenic compositions described herein, the following experiments were performed. Briefly, on

days

0, 2, 4 and 6, 5 mice per group were treated with PBS, an escherichia coli colon antigen composition or a klebsiella pneumoniae antigen composition. On day 7 of the experiment, mice were sacrificed and bronchoalveolar lavage was performed. Subsequently, the lungs and proximal colon are removed and enzymatically digested. After digestion, the collected cells were washed and stained with antibodies specific for: I-A/I-E FITC (MHC class II; M5/114.15.2); anti-Gr-1 PE (RB6-8C5 _; anti-CD 11bPerCP-Cy5 (M1/70); anti-CD 11C APC (N418); all antibodies were obtained from eBioscience (San Diego, CA); lung cells were counted to determine the total number of cells (colon is counted, as we did not remove an equal amount of colon between samples); after staining for 20 minutes, cells were washed and analyzed with FACS. each data point correspondingly shown in fig. 34 represents the frequency of CD11b + Gr-1+ cells screened in real time for each mouse.

In addition and as shown in fig. 35, when monocytes in the lungs were detected based on the experimental methods detailed herein, it was found that while the e.coli and klebsiella pneumoniae antigen compositions increased the frequency of monocytes in the lungs of mice, the klebsiella pneumoniae antigen compositions were more effective when counted against total. Referring to FIG. 35, the left-most graph shows the frequency of CD11b + Gr-1+ (inflammatory monocytes) cells in the lungs; the right-most graph shows the total amount of CD11b + Gr-1+ cells in the lung.

To detect the phenotype of macrophages present in a tumor, M1-like and M2-like macrophages were detected using the in vivo model described herein for use with the antigenic compositions described herein. As shown in fig. 36, the graph shows the frequency of M1-like (see left panel of fig. 36) TAM (tissue-associated macrophages) or M2-like (see right panel of fig. 36) in subQ 4T1 tumors at day 8 post tumor transplantation. As detailed herein, M1-like macrophages are defined as higher CD11b +/Gr-1-/mhc ii type; m2-like macrophages are defined as lower CD11b +/Gr-1-/MHC class II.

Example 11: indometacin potency and anti-inflammatory drug research

To examine the efficacy of antigenic composition therapy in combination with indomethacin. Briefly, experiments were designed in which 10 or 11 mice in each of the 4 groups with the treatment started on day 4 after tumor inoculation. The mice received 50,000 4T1 mammary tumor cells subcutaneously on day 0. Thereafter, 4 groups were processed as follows: 1) once daily indomethacin (in drinking water) + every other day PBS was administered subcutaneously; 2) s. aureus antigen composition was administered subcutaneously every day (in drinking water) + every two days; 3) PBS was given subcutaneously once daily (in drinking water) + everytwo days; and 4) once daily control vehicle (in drinking water) + every two days s.c. administration of the staphylococcus aureus antigen composition. For fig. 37, the left-most graph of the graph represents tumor volume for each group at day 15 of the experiment. The right-most graph shows the frequency and composition of CD11b + cells in the tumor at day 11 of the experiment. The frequency of CD11b + cells was significantly increased in both indomethacin-treated groups compared to the control. These results indicate the efficacy of the combination of a staphylococcus aureus antigen composition with an anti-inflammatory drug, such as indomethacin. In addition and as shown in fig. 38 herein, indomethacin treatment resulted in an increase in CD11b + cells at the time point of day 22.

To further examine the efficacy of antigenic composition therapy in combination with indomethacin, studies were designed. Briefly, four groups of 10-11 Balb/c mice received 10,000 4T1 cancer cells subcutaneously on day 0. Thereafter, the treatment was as follows: the first group received indomethacin (14 μ g/ml in water) from day 4) + PBS on

days

4, 6, 8, etc.; the second group received indomethacin (14 μ g/ml in water) and staphylococcus aureus antigen composition [0.1ml of 0.5OD600nm stock ] + starting on day 4+ PBS on

days

4, 6, 8, etc.; the third group received PBS starting on

days

4, 6, 8, etc.; and the fourth group received a staphylococcus aureus antigen composition [0.1ml of 0.5OD600nm stock ] on

days

4, 6, 8, etc. Tumor volume measurements were taken on

days

15, 19 and 22, and the data are shown in figure 39 for four (4) groups of mice. The data shown in figure 39 indicate that there is synergy prior to treatment with the antigen composition and the anti-inflammatory drug (e.g., indomethacin) in the subcutaneous cancer model.

On day 11 of the experiment detailed above, the tumors were excised and digested. Thereafter, digested tumors were stained with anti-CD 11b and analyzed with FACS (n-3 per group of mice). The results are depicted in figure 40 herein and demonstrate an increase in the frequency of CD11b + cells in tumors of indomethacin-treated mice 11 days post-inoculation.

On day 22 of the experiment detailed above, the tumors were excised and digested. Thereafter, digested tumors were stained with anti-CD 11b and analyzed with FACS (n-7 for each group of mice). The results are depicted in figure 41 herein and demonstrate an increased frequency of CD11b + cells in tumors of indomethacin-treated mice 22 days post-inoculation. Furthermore, figure 42 shows that indomethacin treatment induced an increase in CD11b + CD94+ myeloid cells in the tumor, as found 22 days post-inoculation.

To further examine whether antigenic composition treatment is associated with anti-inflammatory response, quantitative PCR was performed on the 22 nd day of the experiment using whole tumor samples by known methods. Targeting gene products Fizz1 and Ym 1. Fizz1 and Ym1 have been reported to be associated with M2 macrophages (see, e.g., Wong et al (2010) Eur. J. Immunol.40(8): 2296-307). The results are depicted in fig. 43 herein. As shown in figure 43, the relative expression of Fizz1 and Ym1 was increased in tumors in which treatment with indomethacin and the antigenic composition was present (e.g., as compared to treatment with indomethacin alone).

On day 22 of the experiment, relative expression levels of Arg1 and Fizz1 were detected in the tumor and spleen of mice. As shown in figure 44, Arg1 and Fizz1 expression was increased in the spleen of mice treated with indomethacin and antigen composition. On day 22 of the experiment, the relative expression levels of Nos2 and Ym1 were detected in the tumor and spleen of mice. Relative expression levels are depicted in figure 45 herein for four (4) groups of mice.

To further examine the anti-inflammatory response associated with the treatment with the antigenic compositions, experiments were designed as follows. Briefly, on day 0, mice were given Lewis lung tumors [ PBS and klebsiella pneumoniae antigen composition ] or no tumors [ PBS (no tumor) or klebsiella pneumoniae antigen composition (no tumor) ]. Mice received the Klebsiella pneumoniae antigen composition [0.1ml of 0.5OD500nm stock ] or PBS on

days

2, 4, 6, and 8. Mice were sacrificed on day 9 and their lungs were removed and homogenized. IFN γ was measured in lung homogenates using ELISA. The results are shown in figure 46 herein. Treatment with the klebsiella pneumoniae antigen composition reduces IFN γ production in the lungs of mice with or without lung tumors.

To further examine the anti-inflammatory response associated with the treatment with the antigenic compositions, the following experiments were designed. Briefly, bone marrow macrophages are cultured overnight in culture medium or medium supplemented with LPS or in medium supplemented with a klebsiella pneumoniae antigen composition. The media were tested for IL-12 and IL-10. The results are shown in FIG. 47 herein, and indicate that IL-12 was not identified. As shown in FIG. 47, bone marrow macrophages were cultured overnight with IL-10-produced Klebsiella pneumoniae antigen compositions. IL-10 is known to be associated with anti-inflammatory responses (see, e.g., Bazzoni et al (2010) Eur. J. Immunol.40(9): 2360-8).

To further examine the anti-inflammatory response associated with the treatment with the antigenic compositions, the following experiments were designed. Briefly, the 4T1 tumor model was used to determine the effect of Staphylococcus Aureus (SA) antigen composition therapy in synergy with the anti-inflammatory drug indomethacin. In this study, four (4) groups of 10 Balb/c female mice received 50,000 4T1 breast cancer cells subcutaneously on day 0. The first group received PBS subcutaneously every two days beginning on day 4. The second group received SA [0.1ml of 0.5OD600nm ] subcutaneously every two days starting on day 4. The third and fourth groups received 14ug/ml indomethacin (indo) and subcutaneously PBS and SA in their drinking water every two days starting on day 4, respectively. On day 22, mice were sacrificed and tumors from 7-8 mice per group were harvested and placed in RNA depositors. Subsequently, we analyzed the tumor for IL-10 expression by real-time PCR. The results in fig. 48 show that mice treated with indomethacin and PBS had a significant increase in tumor IL-10 expression. Treatment with indomethacin and SA resulted in even greater amounts of IL-10, indicating that SA increased the anti-inflammatory effects of indomethacin in this tumor model. Since IL-10 is widely known to be an anti-inflammatory cytokine, these results suggest that SA acts synergistically with indomethacin in an anti-inflammatory manner. It is important to note that tumors were largest in the PBS group, followed by the SA combination Indo-PBS group. Tumors in the Indo-SA group were minimal at day 22. The anti-inflammatory effects detailed herein can be used to address various inflammatory diseases (see table 20 herein).

TABLE 20 list of chronic inflammatory diseases

Example 12: inflammatory bowel disease study

This embodiment provides for the use of an antigen formulation comprising killed escherichia coli for a clinically effective treatment of patients with crohn's disease over a three month course of treatment. During the course of treatment, the patient becomes asymptomatic and discontinues use of the anti-inflammatory drug.

The patient initially provided a pain report in the large intestine area with prednisone and Imuran simultaneously^TMAnd (6) treating.

Treatment was initiated with subcutaneous inoculation of a killing agent derived from whole e.coli of an e.coli strain collected from a patient with e.coli colonic infection. The dosing regimen included subcutaneous administration every two days, starting with a 0.05ml dose, increasing the volume gradually until a light pink/red skin response of 2 inches in diameter was achieved within 24 hours after injection at the injection site. The dose ultimately required to achieve the skin response is 0.09-0.11ml in the patient and this dose continues every two days as a maintenance dose.

One week after starting treatment with the antigen preparation that completely killed the e.coli, the patient reported that the pain had disappeared. Within about 2 months, the patient stopped treatment with prednisone and continued to have 150mg of Imuran per day^TMThe dosage of (a). Subsequently, the patient also discontinued using Imuran^TM。

2 months after starting treatment with the E.coli composition, patients self-administered 0.09-0.11ml of E.coli formulation every other day. The patient self-adjusted the dose in order to elicit a local inflammatory response, as evidenced by a pink spot of about 2 inches in diameter at the site of administration for about 2 days.

Example 13: fungus research

An assay using the pulmonary mouse system, detailed herein, was performed that determined that mice that had received water contaminated with a particular fungal species (penicillium marneffei), known to induce respiratory infections in mice and humans, exhibited reduced tumor burden as evidenced and discussed by the bacterial pulmonary pathogen fraction.

Many fungal species are known to be associated with various organs or tissues as detailed in table 21 below, where water infected by the fungal species also appears. Thus, and based on the experimental principles detailed herein, the specificity demonstrated herein in connection with bacterial and viral pathogens should likewise extend to fungal pathogens.

TABLE 21 fungal species and Association with organs/tissues

Other embodiments

While various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in light of the general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numerical ranges include the numbers defining the range. In the specification, the word "comprising" is used in an open-ended fashion, that is, substantially equivalent to the word "including, but not limited to," and that the word "comprises" has a comparable meaning. Citation of a document herein shall not be construed as an admission that such document is prior art to the present invention. All publications (e.g., each individual publication) incorporated by reference herein are expressly and individually indicated to be incorporated by reference herein in their entirety. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and figures.

Claims

1. Use of an anti-inflammatory composition comprising antigenic determinants of one or more microbial species that are pathogenic in bone in the manufacture of a medicament for treating a disease characterized by inflammation in bone in a subject.

2. The use according to claim 1, wherein the microbial species is selected from parvovirus B19, rubella virus, hepatitis B virus, staphylococcus aureus, coagulase-negative staphylococcus, streptococcus pyogenes, streptococcus pneumoniae, streptococcus agalactiae, other streptococci, escherichia coli, pseudomonas, enterobacter, proteus or serratia.

3. The use of claim 2, wherein the microbial species is streptococcus pyogenes, streptococcus pneumoniae or streptococcus agalactiae.

4. The use of claim 2, wherein the microbial species is staphylococcus aureus.

5. The use according to claim 2, wherein the disease is temporal arthritis (giant cell arthritis), juvenile arthritis, psoriatic arthritis, responsive arthritis (Reiter's syndrome), rheumatoid arthritis, arthritis associated with origin and stop inflammation, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, osteoarthritis or paget's disease of the bone.

6. The use of claim 2, wherein the anti-inflammatory composition is for administration of consecutive doses at dosing intervals of one hour to one month over an administration duration of at least one week.

7. The use of claim 6, wherein the anti-inflammatory composition is for eliciting a local inflammatory immune response at the site of administration.

8. The use of claim 7, wherein the anti-inflammatory composition is for intradermal or subcutaneous use.

9. The use of claim 8, wherein the local inflammatory immune response at the site of administration occurs within 1-48 hours.

10. The use of claim 9, wherein the duration of administration is at least two weeks.

11. The use of claim 10, wherein the anti-inflammatory composition is for daily or every other day use.

12. The use of claim 10, wherein the anti-inflammatory composition is a total pathogen kill composition.

13. Use of an anti-inflammatory composition comprising antigenic determinants of one or more microbial species that are pathogenic in bone in the manufacture of a medicament for treating a disease characterized by inflammation in bone in a subject; wherein the microbial species is Staphylococcus aureus and the disease is rheumatoid arthritis.

14. The use of claim 13, wherein the anti-inflammatory composition is a whole cell killing composition.

15. The use of claim 13, wherein the anti-inflammatory composition is for administration of consecutive doses at an administration interval of one hour to one month over an administration duration of at least two weeks.

16. The use of claim 15, wherein the anti-inflammatory composition is for intradermal or subcutaneous use.

17. The use of claim 16, wherein the anti-inflammatory composition is for daily or every other day use.