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WO2013030670A2 - Activation antigénique de fusobacterium ciblé à un tissu de la réponse immunitaire pour traiter des cancers de l'intestin - Google Patents

Activation antigénique de fusobacterium ciblé à un tissu de la réponse immunitaire pour traiter des cancers de l'intestin Download PDF

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Publication number
WO2013030670A2
WO2013030670A2 PCT/IB2012/002065 IB2012002065W WO2013030670A2 WO 2013030670 A2 WO2013030670 A2 WO 2013030670A2 IB 2012002065 W IB2012002065 W IB 2012002065W WO 2013030670 A2 WO2013030670 A2 WO 2013030670A2
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fusobacterium
macrophages
cells
cancer
protein
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WO2013030670A3 (fr
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Harold David Gunn
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Qu Biologics Inc
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Qu Biologics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/114—Fusobacterium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • Helicobacter pylori alone are responsible for an estimated 15% of the global cancer burden, based on strength of the association and prevalence of infection (Parkin, "The global health burden of infection-associated cancers in the year 2002," Int. J. Cancer (2006) 1 1 8:3030-3044). Metagenomics methods using new sequencing technologies provide a useful approach for detecting tumor-associated microorganisms that may be overlooked by culture or histology based methods.
  • Colorectal carcinoma is the second leading cause of cancer deaths, responsible for approximately 655,000 deaths per year worldwide (World Health Organization fact sheet # 297, 2009). It is also one of the first and best genetically characterized cancers and specific somatic mutations on oncogenes and tumor suppressor genes have been found to be associated with progression from adenomatous lesions (polyps) to invasive carcinoma (Vogelstein, et al, "Genetic alterations during colorectal-tumor development,” N. Engl. J. Med. (1 988) 31 9: 525-532).
  • aspects of the invention include, among other inventions as summarized below, methods of treating intestinal, e.g., lower intestinal such as colon and/or rectal, cancers by administering a composition that is antigenically specific for a Fusobacterium, such as Fusobacterium nucleatum. Accordingly, a method for treating a human subject for an intestinal cancer, such as a cancer of the lower intestine, e.g., colon and/or rectum, is provided.
  • the method involves administering to the subject a medicament having an effective amount of a Fusobacterium antigenic composition comprising a whole killed bacterial cell composition, wherein the Fusobacterium, e.g., Fusobacterium nucleatum, is pathogenic in the specific organ or tissue of the subject within which the cancer is situated.
  • the medicament may be administered to the subject in an amount and for a time that is effective to modulate an immune response.
  • the modulation of the immune response may involve a shift in the activation state of macrophages.
  • the modulation of the immune response may involve shifting from a M2-like macrophage response to a M1 -like macrophage response.
  • the modulation of the immune response may involve a shift from M1 - like macrophages to M2-like macrophages, as those terms are defined herein.
  • the method may further involve measuring a characteristic of the immune response.
  • comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells.
  • the following cells inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I+ cells, CD4+ T cells, CD8+ T cells, or NK cells.
  • macrophages may include any one or more of the following: M1 -like
  • comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages.
  • the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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.
  • cytokines may be produced as a result of a shift in an activation state of the macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages.
  • the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ 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 differential gene expression may be produced as a result of a shift in an activation state of the macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages.
  • the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • a diagnostic step may be used to identify the target intestinal cancer, prior to producing the Fusobacterium antigenic composition targeted to the site of the cancer.
  • the antigenic composition may be sufficiently specific that it would be capable of eliciting an immune response in the mammal specific to the microbial pathogen.
  • the antigenic composition may be a bacterial composition, for example derived from a Fusobacterial species.
  • the microbial pathogen may be killed. In alternative embodiments, the microbial pathogen may be live or attenuated.
  • Immunogenic compositions of the invention may also be formulated or administered with anti-inflammatory modalities, such as an NSAID.
  • the site of administration may be at a site distant from the site of the cancer, for example in an organ or tissue that is not intestinal within which the cancer is situated, for example the skin or subcutaneous tissue.
  • the antigenic composition may for example be formulated for
  • the dosing or formulation of the antigenic composition may be adjusted in order to produce a localized immune reaction visible in the skin at the site of administration, for example an area of inflammation from 2mm to 1 00mm in diameter appearing, for example, 2 - 48 hours after administration and lasting, for example, 2 - 72 hours or longer.
  • the antigenic composition may be formulated for repeated
  • subcutaneous or intradermal administration for example at alternating successive sites.
  • the invention involves methods of treating a mammal for an intestinal cancer, e.g., a cancer of the lower intestine, such as the colon and/or rectum.
  • the treatment may anticipate the development of the cancer in the target tissue, for example if the site of a primary tumor suggests the likelihood of metastasis to a particular tissue or organ, then the patient may be prophylactically treated to prevent or ameliorate metastasis to that tissue or organ.
  • the method may include administering to the subject an effective amount of an antigenic composition comprising antigenic determinants that together are specific for at least one Fusobacterium microbial pathogen.
  • the antigenic composition may be administered, for example by subcutaneous or intradermal injection at an administration site, in successive doses given at a dosage interval, for example of between one hour and one month, over a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.
  • Each injection dose may for example be metered so that it is effective to cause visible localized inflammation at the administration site, appearing, for example, 1 - 48 hours after injection.
  • methods for treating cancers of the intestine (or a portion thereof, e.g., the lower intestine or a portion thereof, such as the colon and/or rectum) in a subject by administering one or more antigens of one or more Fusobacterium microbial pathogens, such as Fusobacterium nucleatum, that are pathogenic in the specific organ or tissue.
  • Fusobacterium microbial pathogens such as Fusobacterium nucleatum
  • the Fusobacterium antigen may be administered by administering a whole microbial species.
  • the method may, for example, include administering at least two or more microbial species, or administering at least three or more microbial species, and the microbes may be bacteria or viruses, where at least one of the microbial species in such embodiments is a Fusobacterium.
  • the method may further include administering a supplement or an adjuvant.
  • An aspect of the invention involves administering antigenic
  • compositions so as to elicit an immune response in said subject.
  • the Fusobacterium microbial pathogen in the antigenic composition may be killed, and thus rendered non-infectious.
  • the antigenic composition is administered at a site distant from the cancer site, and in selected embodiments of this kind, methods of the invention may be carried out so that they do not produce infection at the cancer site.
  • treatment may be carried out so as to provide a variety of outcomes.
  • treatment may: provoke an immune reaction that is effective to inhibit or ameliorate the growth or proliferation of a cancer; inhibit the growth or proliferation of cancer cells or tumors; cause remission of a cancer; improve quality of life; reduce the risk of recurrence of a cancer; inhibit metastasis of a cancer; or, improve patient survival rates in a patient population.
  • extending the life expectancy of a patient, or patient population means to increase the number of patients who survive for a given period of time following a particular diagnosis.
  • treatment may be of patients who have not responded to other treatments, such as patients for whom a chemotherapy or surgery has not been an effective treatment.
  • Treatment in alternative embodiments may for example be before or after onset of cancer.
  • prophylactic treatment may be undertaken, for example of patients diagnosed as being at risk of a particular cancer.
  • a patient having a genetic or lifestyle predisposition to cancer of a certain tissue or organ may be treated with an immunogenic composition comprising antigenic determinants of a pathogen that is pathogenic in that organ or tissue.
  • the prophylactic treatment of metastasis may be undertaken, so that patients having a primary cancer with a propensity to metastasize to a particular tissue or organ may be treated with an immunogenic composition comprising antigenic determinants of a pathogen that is pathogenic in that organ or tissue.
  • Figure 1 Relative abundance of microbial genomes in tumor and control specimens. Numbers of read pairs that matched known microbial sequences were normalized according to sequencing depth for both tumor and matched normal samples. The abundance of bacterial read pairs ranged from zero to a maximum of 46,795 represented by a transition from white to red on a log scale. Fusobacterium nucleatum sequences were present in the tumor samples at levels 2-fold or greater than in normal samples in 9 out of the 1 1 subjects. The mean over abundance across all subjects was 86-fold. Figure 2. Relative Abundance of Fusobacterium in Tumor versus Normal Colorectal Carcinoma Biopsies.
  • cross_match -minmatch 29 -minscore 59 -masklevel 1 01
  • FIG. 4 In the Immunofluorescence micrograph, CC53 shows a very long, flexible cell morphology. Green is actin (Caco-2 cells), orange are invasive and internalized bacteria, purple are bacteria external to the cell. Figure S1. Number of sequencing read pairs that match known microbial genomes are shown for the 25 most abundant genomes.
  • Figure S2 Distribution of hits from colorectal carcinoma RNA-Seq data to the annotated F.nucleatum subsp. nucleatum ATCC 25586 genome. The total number of read pair hits was 80, 1 18.
  • Fusobacterium in their tumor biopsies versus matched normal tissue were compared to those patients with less than 5X relative amounts of Fusobacterium.
  • a significantly higher number of patients from the high Fusobacterium group (A) had more tumor spreading in their lymph nodes as measured by their surgical TNM scores than the low Fusobacterium group (B) (one-tailed Fisher's exact test p-value 0.0035).
  • the invention relates to the surprising discovery that administration, for example at a site distant from an intestinal cancer, of a Fusobacterium microbial pathogen, such as a killed Fusobacterium microbial pathogen, that are pathogenic in the intestine or a portion thereof, e.g., the lower intestine or portion thereof, e.g., the colon and/or rectum, is effective in treating cancer situated in that target tissue or organ.
  • a Fusobacterium microbial pathogen such as a killed Fusobacterium microbial pathogen
  • the invention provides Fusobacterium antigenic compositions derived from Fusobacterium, including whole killed bacterial species, or components thereof, for the treatment of cancer, and methods for using the same.
  • Fusobacterium microbial pathogen such as Fusobacterium nucleatum.
  • antigenic determinants are sufficiently characteristic of the pathogen that they could be used to raise an immune response, such as an adaptive immune response, against the pathogen in the patient, if the antigenic determinants were to be administered in an appropriate manner to have that effect. It will be recognized that the antigenic determinants need not be so specific that they are characteristic of only one particular strain or species of pathogen, since even a specific immune response against a particular pathogen may be cross reactive with other closely related organisms that are also naturally pathogenic in the tissue or organ in which the cancer is situated and that the antigenic composition is formulated or selected to target.
  • the compositions of pathogenic microbes may be used for treating primary cancer sites and/or sites of metastasis.
  • the microbial compositions may be used for the treatment of a cancer at a particular site, regardless of whether the cancer is a primary cancer or a metastasis.
  • the composition may be directed to the treatment of each cancer site, or may be a combined composition for both the primary cancer and the metastatic site(s).
  • three different compositions including one or more species that are known to be Fusobacterium pathogens, one or more species that are known to be lung pathogens and one or more species that are known to be bone pathogens, or a combined composition thereof may be used.
  • the compositions may be administered in different locations at the same time or at different times.
  • both a microbial composition including one or more
  • Fusobacterium bacterial species and a microbial composition including one or more bacterial species (or viruses) which commonly cause bone infection may be used.
  • the antigenic compositions may be used for treating or preventing cancers at primary sites or for treating or preventing metastasis.
  • a Fusobacterium antigenic composition may be used to appropriately stimulate the immune system to defend against the development of cancer within the colon.
  • carcinomas which are the predominant cancers and are cancers of epithelial cells or cells covering the external or internal surfaces of organs, glands, or other body structures (for e.g., skin, uterus, lung, breast, prostate, stomach, bowel), and which tend to metastasize; sarcomas, which are derived from connective or supportive tissue (for e.g., bone, cartilage, tendons, ligaments, fat, muscle); and hematologic tumors, which are derived from bone marrow and lymphatic tissue.
  • Carcinomas may be adenocarcinomas (which generally develop in organs or glands capable of secretion, such as breast, lung, colon, prostate or bladder) or may be squamous cell carcinomas (which originate in the squamous epithelium and generally develop in most areas of the body). Sarcomas may be adenocarcinomas (which generally develop in organs or glands capable of secretion, such as breast, lung, colon, prostate or bladder) or may be squamous cell carcinomas (which originate in the squamous epithelium and generally develop in most areas of the body). Sarcomas may be adenocarcinomas (which generally develop in organs or glands capable of secretion, such as breast, lung, colon, prostate or bladder) or may be squamous cell carcinomas (which originate in the squamous epithelium and generally develop in most areas of the body). Sarcomas may be adenocarcinomas (which generally develop in organs or glands capable of secreti
  • osteosarcomas or osteogenic sarcomas (bone), chondrosarcomas (cartilage), leiomyosarcomas (smooth muscle), rhabdomyosarcomas (skeletal muscle), mesothelial sarcomas or mesotheliomas (membranous lining of body cavities), fibrosarcomas (fibrous tissue), angiosarcomas or hemangioendotheliomas (blood vessels), liposarcomas (adipose tissue), gliomas or astrocytomas
  • Hematologic tumors may be myelomas, which originate in the plasma cells of bone marrow; leukemias which may be "liquid cancers" and are cancers of the bone marrow and may be myelogenous or granulocytic leukemia (myeloid and granulocytic white blood cells), lymphatic, lymphocytic, or lymphoblastic leukemias (lymphoid and lymphocytic blood cells) or polycythemia vera or erythremia (various blood cell products, but with red cells predominating); or lymphomas, which may be solid tumors and which develop in the glands or nodes of the lymphatic system, and which may be Hodgkin or Non-Hodgkin lymphomas.
  • mixed type cancers such as a
  • lung cancers are generally small cell lung cancers or non-small cell lung cancers, which may be squamous cell carcinoma, adenocarcinoma, or large cell carcinoma; skin cancers are generally basal cell cancers, squamous cell cancers, or melanomas. Lymphomas may arise in the lymph nodes associated with the head, neck and chest, as well as in the abdominal lymph nodes or in the axillary or inguinal lymph nodes. Identification and classification of types and stages of cancers may be performed by using for example information provided by the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute, which is an
  • the SEER Program currently collects and publishes cancer incidence and survival data from 14 population- based cancer registries and three supplemental registries covering
  • the program routinely collects data on patient demographics, primary tumor site, morphology, stage at diagnosis, first course of treatment, and follow-up for vital status, and is the only comprehensive source of population-based information in the United States that includes stage of cancer at the time of diagnosis and survival rates within each stage. Information on more than 3 million in situ and invasive cancer cases is included in the SEER database, and approximately 170,000 new cases are added each year within the SEER coverage areas.
  • the incidence and survival data of the SEER Program may be used to access standard survival for a particular cancer site and stage.
  • specific criteria may be selected from the database, including date of diagnosis and exact stage (for example, in the case of the lung cancer example herein, the years were selected to match the time-frame of the retrospective review, and stage 3B and 4 lung cancer were selected; and in the case of the colon cancer example herein, the years were also selected to match the timeframe of the retrospective review, and the stage 4 colon cancer was selected).
  • Cancers may also be named based on the organ in which they originate i.e., the "primary site,” for example, cancer of the breast, brain, lung, liver, skin, prostate, testicle, bladder, colon and rectum, cervix, uterus, etc. This naming persists even if the cancer metastasizes to another part of the body that is different from the primary site.
  • treatment is directed to the site of the cancer, not type of cancer, so that a cancer of any type that is situated in the lung, for example, would be treated on the basis of this
  • a “cancer” or “neoplasm” is any unwanted growth of cells serving no physiological function.
  • a cancer cell has been released from its normal cell division control, i.e., a cell whose growth is not regulated by the ordinary biochemical and physical influences in the cellular environment.
  • cancer is a general term for diseases characterized by abnormal uncontrolled cell growth.
  • a cancer cell proliferates to form clonal cells that are malignant.
  • the lump or cell mass, "neoplasm” or “tumor” is generally capable of invading and destroying surrounding normal tissues.
  • malignancy is meant as an abnormal growth of any cell type or tissue that has a deleterious effect in that organism having the abnormal growth.
  • malignancy or “cancer” includes cell growths that are technically benign but which carry the risk of becoming malignant. Cancer cells may spread from their original site to other parts of the body through the lymphatic system or blood stream in a process known as "metastasis.” Many cancers are refractory to treatment and prove fatal. Examples of cancers or neoplasms include, without limitation, transformed and immortalized cells, tumors, carcinomas, in various organs and tissues as described herein or known to those of skill in the art.
  • a “cell” is the basic structural and functional unit of a living organism. In higher organisms, e.g., animals, cells having similar structure and function generally aggregate into “tissues” that perform particular functions. Thus, a tissue includes a collection of similar cells and surrounding intercellular substances, e.g., epithelial tissue, connective tissue, muscle, nerve.
  • An “organ” is a fully differentiated structural and functional unit in a higher organism that may be composed of different types of tissues and is specialized for some particular function, e.g., kidney, heart, brain, liver, etc. Accordingly, by “specific organ, tissue, or cell” is meant herein to include any particular organ, and to include the cells and tissues found in that organ.
  • “Pathogenic” agents are agents, such as microbes, such as bacteria or viruses, which are known to cause infection in a host in nature, and in this sense, "pathogenic” is used in the context of the present invention to mean “naturally pathogenic”. Although a wide variety of microbes may be capable of causing infection under artificial conditions, such as artificial innoculations of a microbe into a tissue, the range of microbes that naturally cause infection is necessarily limited, and well established by medical practice.
  • An “infection” is the state or condition in which the body or a part of it is invaded by a pathogenic agent (e.g., a microbe, such as a bacterium) which, under favorable conditions, multiplies and produces effects that are injurious (Taber's Cyclopedic Medical Dictionary, 14th Ed., C.L Thomas, Ed., F.A. Davis Company, PA, USA).
  • a pathogenic agent e.g., a microbe, such as a bacterium
  • An infection may not always be apparent clinically and may result in only localized cellular injury. Infections may remain subclinical, and temporary if the body's defensive mechanisms are effective. Infections may spread locally to become clinically apparent as an acute, a subacute, or a chronic clinical infection or disease state.
  • a local infection may also become systemic when the pathogenic agent gains access to the lymphatic or vascular system (On-Line Medical Dictionary, http://cancerweb.ncl.ac.uk/omd/). Infection is usually accompanied by inflammation, but inflammation may occur without infection.
  • Inflammation is the characteristic tissue reaction to injury (marked by swelling, redness, heat, and pain), and includes the successive changes that occur in living tissue when it is injured. Infection and inflammation are different conditions, although one may arise from the other (Taber's Cyclopedic Medical Dictionary, supra). Accordingly, inflammation may occur without infection and infection may occur without inflammation (although infection by pathogenic bacteria or viruses typically results in inflammation). Inflammation is
  • redness characterized by the following symptoms: redness (rubor), heat (calor), swelling (tumor), pain (dolor). Localized visible inflammation on the skin may be apparent from a combination of these symptoms, particularly redness at a site of administration.
  • a "subject" is an animal, for e.g., a mammal, to whom the specific pathogenic bacteria, bacterial antigens, viruses, viral antigens or compositions thereof of the invention may be administered.
  • a subject may be a patient, e.g., a human, suffering from a cancer, or suspected of having a cancer, or at risk for developing a cancer.
  • a subject may also be an experimental animal, e.g., an animal model of a cancer, as is described in Example 5.
  • the terms "subject” and “patient” may be used interchangeably, and may include a human, a non-human mammal, a non- human primate, a rat, mouse, dog, etc.
  • a healthy subject may be a human who is not suffering from a cancer or suspected of having a cancer, or who is not suffering from a chronic disorder or condition.
  • a "healthy subject” may also be a subject who is not immunocompromised. By immunocompromised is meant any condition in which the immune system functions in an abnormal or incomplete manner. Immunocompromization may be due to disease, certain medications, or conditions present at birth. Immunocompromised subjects may be found more frequently among 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 both M1 -like macrophages and M2-like macrophages as described herein), B cells, T cells (including helper T cells, natural killer cells, cytotoxic T cells, T cells), such as induction or activation by the antigen(s) in a composition or vaccine, following administration of the composition or vaccine.
  • An immune response to a composition or vaccine thus generally includes the development in the host animal of a cellular and/or antibody-mediated response to the composition or vaccine of interest. In some embodiments, the immune response is such that it will also result in slowing or stopping the progression of a cancer in the animal.
  • An immune response includes both cellular immune responses and humoral immune responses as understood by those persons skilled in the art.
  • bacteria which generally exist in symbiotic or commensal relationships with the host animal.
  • bacteria many species of normally harmless bacteria are found in healthy animals, and are usually localized to the surface of specific organs and tissues. Often, these bacteria aid in the normal functioning of the body.
  • symbiotic Escherichia coli bacteria may be found in the intestine, where they promote immunity and reduce the risk of infection with more virulent pathogens.
  • Bacteria that are generally harmless can cause infection in healthy subjects, with results ranging from mild to severe infection to death. Whether or not a bacterium is pathogenic (i.e., causes infection) depends to some extent on factors such as the route of entry and access to specific host cells, tissues, or organs; the intrinsic virulence of the bacterium; the amount of the bacteria present at the site of potential infection; or the health of the host animal. Thus, bacteria that are normally harmless can become pathogenic given favorable conditions for infection, and even the most virulent bacterium requires specific circumstances to cause infection. Accordingly, microbial species that are members of the normal flora can be pathogens when they move beyond their normal ecological role in the endogenous flora.
  • endogenous species can cause infection outside of their ecological niche in regions of anatomical proximity, for example by contiguous spread. When this occurs, these normally harmless endogenous bacteria are considered pathogenic.
  • Specific bacterial species and viruses are known to cause infections in specific cells, tissues, or organs in otherwise healthy subjects.
  • bacterial species are classified operationally as collections of similar strains (which generally refers to groups of presumed common ancestry with identifiable physiological but usually not morphological distinctions, and which may be identified using serological techniques against bacterial surface antigens).
  • each bacterial species e.g., Streptococcus pneumoniae
  • serotypes 1 , 3, 4, 7, 8, and 1 2 are most frequently responsible for pneumococcal disease in humans.
  • compositions of the invention include antigens of Fusobacterium pathogenic microbial species that are pathogenic in the intestine or a portion thereof, e.g., the large intestine or portion thereof, such as the colon or rectum.
  • the compositions may include whole cells of bacterial species, or may include extracts or preparations of the pathogenic bacterial species of the invention, such as cell wall or cell membrane extracts, or whole cells, or exotoxins, or whole cells and exotoxins.
  • compositions may also include one or more isolated antigens from one or more of the Fusobacterium pathogenic bacterial species of the invention; in some embodiments, such compositions may be useful in situations where it may be necessary to precisely administer a specific dose of a particular antigen, or may be useful if administering a whole bacterial species or components thereof (e.g., toxins) may be harmful.
  • Pathogenic bacterial species may be available commercially (from, for example, ATCC (Manassas, VA, USA), or may be clinical isolates from subjects having a bacterial infection of a tissue or organ (e.g., pneumonia).
  • compositions of the invention can be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans.
  • compounds for example, nucleic acid molecules, small molecules, peptides, or peptide analogues
  • liposome for example, an adjuvant, or any pharmaceutically acceptable carrier
  • pharmaceutically acceptable carrier or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier can be suitable for any appropriate form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal,
  • intramuscular, sublingual, inhalational, intratumoral or oral administration is intramuscular, sublingual, inhalational, intratumoral or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound (i.e., the specific bacteria, bacterial antigens, or compositions thereof of the
  • compositions of the invention use thereof in the pharmaceutical compositions of the invention is contemplated.
  • Supplementary active compounds can also be incorporated into the compositions.
  • treatment with bacterial antigens according to the invention may be combined with more traditional and existing therapies for cancer, such as chemotherapy, radiation therapy, surgery, etc., or with any other therapy intended to stimulate the immune system, reduce inflammation or otherwise benefit the subject, such as nutrients, vitamins and supplements.
  • vitamin A, vitamin D, vitamin E, vitamin C, vitamin B complex, selenium, zinc, co- enzyme Q1 0, beta carotene, fish oil, curcumin, green tea, bromelain, resveratrol, ground flaxseed, garlic, lycopene, milk thistle, melatonin, other antioxidants, cimetidine, indomethacin, or COX-2 Inhibitors e.g., CelebrexTM [celecoxib] or VioxxTM [rofecoxib]
  • COX-2 Inhibitors e.g., CelebrexTM [celecoxib] or VioxxTM [rofecoxib]
  • CelebrexTM [celecoxib] or VioxxTM [rofecoxib] may be also be administered to the subject.
  • Any appropriate route 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 oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • excipients for example, lactose
  • aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate
  • the pathogenic bacterial species are administered to an individual in an amount effective to stop or slow progression or metastasis of the cancer, or to increase survival of the subject (relative to, for example, prognoses derived from the SEER database) depending on the disorder.
  • an “effective amount” of a pathogenic microbial species or antigen thereof according to the invention includes a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of the cancer cells or tumors, prevention of carcinogenic processes, slowing the growth of the tumor, or an increase in survival time beyond that which is expected using for example the SEER database.
  • a therapeutically effective amount of a pathogenic microbial (bacterial or viral) species or antigen(s) thereof may vary according to 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.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount may also be one in which any toxic or detrimental effects of the pathogenic bacterial species or virus or antigen thereof are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention of cancer, prevention of metastasis, slowing the growth of the tumor, reduction or elimination of the cancer cells, tissues, organs, or tumors, or an increase in survival time beyond that which is expected using for example the SEER database.
  • a prophylactic dose is used in subjects prior to or at an earlier stage of cancer, so that a prophylactically effective amount may be less than a therapeutically effective amount.
  • an exemplary range for therapeutically or prophylactically effective amounts of one or more pathogenic bacterial species may be about 1 million to 100,000 million organisms per ml, or may be 100 million to 7000 million organisms per ml, or may be 500 million to 6000 million organisms per ml, or may be 1 000 million to 5000 million organisms per ml, or may be 2000 million to 4000 million organisms per ml, or any integer within these ranges.
  • the total concentration of bacteria per ml may range from 1 million to 100,000 million organisms per ml, or may be 50 million to 7000 million organisms per ml, or may be 1 00 million to 6000 million organisms per ml, or may be 500 million to 5000 million organisms per ml, or may be 1 000 million to 4000 million organisms per ml, or any integer within these ranges.
  • the range for therapeutically or prophylactically effective amounts of antigens of a pathogenic bacterial species may be any integer from 0.1 nM- 0.1 M, 0.1 nM-0.05M, 0.05 ⁇ -15 ⁇ or 0.01 ⁇ -10 ⁇ .
  • the concentration of antigenic compositions utilized herein may be determined by using the OD600.
  • a dosage of an antigenic composition equating with 5.0 OD600 may be utilized herein. The foregoing is provided as an example and is non-limiting in terms of methods and procedures by which a dosage can be determined.
  • dosage concentrations and ranges may vary with the severity of the condition to be alleviated, or may vary with the subject's immune response. In general, the goal is to achieve an adequate immune response.
  • the extent of an immune response may be determined, for example, by size of delayed local immune skin reaction at the site of injection (e.g., from 0.25 inch to 4 inch diameter).
  • the dose 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.
  • the total bacterial composition dose may, for example, range from 2 million bacteria (e.g., 0.001 ml of a vaccine with a concentration of 2,000 million organisms per ml) to more than 20,000 million bacteria (e.g., 1 ml of a vaccine with a concentration of 20,000 million organisms per ml).
  • concentrations of individual bacterial species or antigens thereof within a composition may also be considered.
  • the local immune skin reaction of an individual may be likely due to its response to this specific bacterial species.
  • the immune system of an individual may respond more strongly to one bacterial species within a composition than another, depending for example on past history of exposure to infection by a particular species, so the dosage or composition may be adjusted accordingly for that individual.
  • an immune response will not be monitored by way of a skin reaction.
  • the effective treatment of such animals with antigenic compositions may not result in corresponding skin reactions.
  • a person skilled in the art will understand that there are alternate ways in which an immune response can be monitored besides relying on the presence or absence of a skin reaction.
  • the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • the compositions may be administered every second day.
  • An initial dose of approximately 0.05 ml may be administered subcutaneously, followed by increases from 0.01 -0.02 ml every second day until an adequate skin reaction is achieved at the injection site (for example, a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site). Once this adequate immune reaction is achieved, this dosing is continued as a maintenance dose.
  • the maintenance dose may be adjusted from time to time to achieve the desired visible skin reaction (inflammation) at the injection site. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.
  • Oral dosages may for example range from 10 million to 1 ,000,000 million organisms per dose, comprising antigenic determinants of one or more species. Oral dosages may be given, for example, from 4 times per day, daily or weekly. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.
  • the invention may include antigenic compositions administered sublingually or by inhalation, or administered to one or more epithelial tissues (i.e., skin by intradermal or subcutaneous injection; lung epithelium by inhalation; gastrointestinal mucosa by oral ingestion; mouth mucosa by sublingual administration) simultaneously or sequentially.
  • epithelial tissues i.e., skin by intradermal or subcutaneous injection; lung epithelium by inhalation; gastrointestinal mucosa by oral ingestion; mouth mucosa by sublingual administration
  • the antigenic compositions of the invention are administered so as to provoke an immune response in an epithelial tissue.
  • one or more epithelial routes of administration may be combined with one or more additional routes of administration, such as intratumoral, intramuscular or intravenous administration.
  • the antigenic compositions that are administered to a patient may be characterized as having an antigenic signature, i.e., a combination of antigens or epitopes that are sufficiently specific that the antigenic composition is capable of eliciting an immune response that is specific to a particular pathogen, such as an adaptive immune response.
  • an antigenic signature i.e., a combination of antigens or epitopes that are sufficiently specific that the antigenic composition is capable of eliciting an immune response that is specific to a particular pathogen, such as an adaptive immune response.
  • the non-adaptive or non-specific activation of the immune response that is mediated by these specific antigenic compositions is effective to treat cancers situated in the tissues in which the particular pathogen is pathogenic.
  • routes of administration and dosage ranges set forth herein are exemplary only and do not limit the route of administration and dosage ranges that may be selected by medical practitioners.
  • the amount of active compound (e.g., pathogenic bacterial species or viruses or antigens thereof) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be
  • parenteral 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. It may be advantageous to formulate parenteral
  • compositions in dosage unit form for ease of administration and uniformity of dosage.
  • an immunogenically effective amount of a compound of the invention can be provided, alone or in combination with other compounds, with an immunological adjuvant.
  • the compound may also be linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance
  • An antigenic composition is a composition that includes materials that elicit a desired immune response.
  • An antigenic composition may select, activate or expand, without limitation: memory B, T cells, neutrophils, monocytes or macrophages of the immune system to, for example, reduce or eliminate the growth or proliferation of cancerous cells or tissue.
  • the specific pathogenic microbe, virus, viral antigens, bacteria, bacterial antigens, or compositions thereof of the invention are capable of eliciting the desired immune response in the absence of any other agent, and may therefore be considered to be an antigenic composition.
  • an 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 specific antigen, or to a group of antigens, enabling the reduction of the quantity of antigen in any given vaccine dose, or the reduction of the frequency of dosage required to generate the desired immune response.
  • a bacterial antigenic composition may include live or dead bacteria capable of inducing an immune response against antigenic determinants normally associated with the bacteria.
  • an antigenic composition may include live bacteria that are of less virulent strains
  • the antigenic composition may include live, attenuated or dead viruses capable of inducing an immune response against antigenic determinants normally associated with the virus.
  • An antigenic composition comprising killed bacteria for administration by injection may be made as follows.
  • the bacteria may be grown in suitable media, and washed with physiological salt solution.
  • the bacteria may then be centrifuged, resuspended in saline solution, and killed with either heat or phenol.
  • the suspensions may be standardized by direct microscopic count, mixed in required amounts, and stored in appropriate containers, which may be tested for safety, shelf life, and sterility in an approved manner.
  • a killed bacterial vaccine suitable for administration to humans may include 0.4% phenol preservative and/or 0.9% sodium chloride.
  • the bacterial vaccine may also include trace amounts of brain heart infusion (beef), peptones, yeast extract, agar, sheep blood, dextrose, sodium phosphate and/or other media components.
  • the bacterial vaccine may be used in tablet or capsule form or drops for oral ingestion, as an aerosol for inhalation, or as drops, aerosol or tablet form for sublingual administration.
  • the concentrations of specific bacterial species in compositions for subcutaneous or intradermal injection may be about 1 million to 100,000 million organisms per ml, or may be 100 million to 7000 million organisms per ml, or may be 500 million to 6000 million organisms per ml, or may be 1 000 million to 5000 million organisms per ml, or may be 2000 million to 4000 million organisms per ml, or any integer within these ranges.
  • the total concentration of bacteria per ml may range from 1 million to 100,000 million organisms per ml, or may be 50 million to 7000 million organisms per ml, or may be 100 million to 6000 million organisms per ml, or may be 500 million to 5000 million organisms per ml, or may be 1 000 million to 4000 million organisms per ml, or any integer within these ranges.
  • the pathogenic bacterial species and antigens thereof of the invention should be used without causing substantial toxicity.
  • Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population).
  • the invention involves the use of an anti-inflammatory in conjunction with vaccinations.
  • a wide variety of antiinflammatory treatments may be employed, including effective amounts of non- steroidal anti-inflammatory drugs (NSAIDs), including but not limited to:
  • diclofenac potassium diclofenac sodium, etodolac, indomethicin, ketorolac tromethamine, sulindac, tometin sodium, celecoxib, meloxicam, valdecoxib, floctafenine, mefenamic acid, nabumetone, meloxicam, piroxicam, tenoxicam, fenoprofen calcium, flubiprofen, ibuprofen, ketoprofen, naproxen, naproxen sodium, oxaprozin, tiaprofenic acid, acetylsalicylic acid, diflunisal, choline magnesium trisalicylate, choline salicylate, triethanolamine salicylate, COX1 inhibitors, COX2 inhibitors (e.g., VioxxTM, and CelebrexTM).
  • COX1 inhibitors e.g., VioxxTM, and CelebrexTM
  • COX2 inhibitors e
  • herbs and natural health products may also be used to provide anti-inflammatory treatment, including but not limited to: green tea, fish oil, vitamin D, antioxidant vitamins and minerals ⁇ e.g., B carotene, vitamin A, vitamin C, vitamin D, vitamin E, co-enzyme Q10, selenium, etc.), resveratrol, turmeric, bromelain, boswellia, feverfew, quercetin, ginger, rosemary, oregano, cayenne, clove, nutmeg, willowbark.
  • antioxidant vitamins and minerals ⁇ e.g., B carotene, vitamin A, vitamin C, vitamin D, vitamin E, co-enzyme Q10, selenium, etc.
  • resveratrol turmeric, bromelain, boswellia, feverfew, quercetin, ginger, rosemary, oregano, cayenne, clove, nutmeg, willowbark.
  • Alternative anti-inflammatory modalities may also include lifestyle modifications, such as: exercise, weight loss, smoking cessation, stress reduction, seeking social support, treatment of depression, stress management, abdominal breath work and dietary change (such as adopting a mediterranean diet, a low glycemic diet, eating non-charred foods, including foods having omega-3 fatty acids).
  • lifestyle modifications such as: exercise, weight loss, smoking cessation, stress reduction, seeking social support, treatment of depression, stress management, abdominal breath work and dietary change (such as adopting a mediterranean diet, a low glycemic diet, eating non-charred foods, including foods having omega-3 fatty acids).
  • a method of comparing immune responses involves administering to an animal having an intestine, a
  • medicament having an antigenic composition having antigenic determinants selected or formulated so that together the antigenic determinants are specific for at least one Fusobacterium that is pathogenic in the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum), extracting a quantifiable immune sample from the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum), measuring a characteristic of the immune response in the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum), in the quantifiable immune sample following the administration of the medicament, and, comparing the characteristic of the immune response in the quantifiable immune sample to a corresponding characteristic of the immune response in a reference intestinal immune sample, e.g., where the reference immune sample may be
  • the formulations of the invention thereby facilitate activation of an immune response to a cancer in the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum).
  • the immune response may be characterized as an immune response that includes a shift in an activation state of macrophages.
  • the shift in macrophages may include a shift from M2-like macrophages to M1 -like macrophages.
  • the compositions disclosed may, for example, include killed or attenuated microbial pathogens, such as whole killed bacterial cells, and may be administered at sites distant from the cancer, for example the skin or subcutaneous tissue.
  • microbial species of endogenous flora that are known to cause infection in the relevant organ or tissue may be used in the formulation of the antigenic compositions.
  • exogenous microbial pathogens that are known to cause infection in the relevant organ or tissue may be used in the formulation of the antigenic compositions.
  • the administration of the immunogenic compositions may be repeated relatively frequently over a relatively long period of time.
  • dosages may be adjusted so that injections reproduce a consistent, visible, delayed inflammatory immune reaction at the successive site or sites of administration.
  • Comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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.
  • comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages or a population of macrophages.
  • the macrophages or the population of macrophages may shift from being M2-like macrophages or a population of M2- like macrophages to being M1 -like macrophages or a population of M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages or a population of M1 -like macrophages to being M2-like macrophages or a population of M2-like macrophages.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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 cytokines are produced as a result of a shift in an activation state of the macrophages.
  • the macrophages shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages shift from being M1 -like macrophages to being M2- like macrophages.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells:
  • the macrophages may include any one or more of the following: M1 -like
  • macrophages or M2-like macrophages.
  • the differential gene expression is produced as a result of a shift in an activation state of the macrophages.
  • macrophages may shift from being M2-like
  • macrophages to being M1 -like macrophages. Further and optionally, the macrophages shift from being M1 -like macrophages to being M2-like
  • the medicament may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week.
  • the medicament may be administered intradermal ⁇ or subcutaneously.
  • the medicament may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week.
  • the medicament may be administered intradermal ⁇ or subcutaneously.
  • the medicament may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week.
  • the medicament may be administered intradermal ⁇ or subcutaneously.
  • the medicament may be administered intradermal ⁇ or subcutaneously.
  • the medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site.
  • the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours.
  • the animal may be a mammal.
  • the animal may be a human or a mouse.
  • a method of selecting a therapeutic preparation suitable for treating an individual for an intestinal cancer such as a cancer of the lower intestine, e.g., colon and/or rectum.
  • the method involves providing an animal having a cancer of the intestine (, such as a cancer of the lower intestine, e.g., colon and/or rectum), providing a test preparation having one or more antigenic determinants of a Fusobacterium which is pathogenic in the corresponding specific organ or tissue in a healthy individual, measuring a characteristic of the immune response in a reference immune sample obtained from the organ or tissue of the animal, administering the test preparation to the animal, measuring a characteristic of the immune response in a quantifiable immune sample obtained from a corresponding organ or tissue of the animal, comparing the characteristic of the immune response in the in the reference and quantifiable immune samples, and treating an enhanced characteristic of the immune response in the quantifiable immune sample compared to the reference immune sample as an indication of the suitability of the test preparation as
  • comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells.
  • the macrophages may include any one or more of the following: M1 -like
  • comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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 cytokines are produced as a result of a shift in an activate state of the macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ 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 differential gene expression may be produced as a result of a shift in an activation state of the macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • a method of selectively targeting an immune response to a cancerous intestine (or portion thereof, such as the lower intestine, e.g., colon and/or rectum), in a human subject involves administering to the subject a medicament having an effective amount of a Fusobacterium (e.g., Fusobacterium nucleatum, antigenic composition, wherein the Fusobacterium may be pathogenic in the specific organ in which the cancer is situated and the antigenic composition comprises antigenic determinants that together are specific for the microbial pathogen.
  • the antigenic composition may include a whole killed bacterial cell composition.
  • the medicament may be administered to the subject in an amount and for a time that is effective to up-regulate an immune response in the cancerous organ or tissue of the subject.
  • the method may further involve measuring a characteristic of the immune response.
  • a method of monitoring efficacy of a treatment regime in an individual being treated for an intestinal cancer such as a cancer of the lower intestine, e.g., colon and/or rectum.
  • the method involves measuring a characteristic of an immune response in a post-treatment immune sample obtained from the specific organ or tissue after the individual has been subject to the treatment regime for a period of time, wherein the presence of a characteristic of the immune response which is greater in magnitude than would be expected had the individual not been subject to the treatment regime, is indicative of the efficacy of the treatment regime; and the treatment regime involves administering a preparation comprising one or more antigenic determinants of a microbial pathogen which is pathogenic in the corresponding specific organ or tissue in a healthy subject.
  • measuring the characteristic of the immune response may involve determining an indication of the number of inflammatory monocytes in a sample of the organ or tissue.
  • measuring the characteristic of the immune response may involve 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.
  • measuring the characteristic of the immune response may involve determining an indication of the number of CD1 1 b+ Gr-1 + cells in a sample of the organ or tissue or determining an indication of the number of dendritic cells in a sample of the organ or tissue. Further and optionally, measuring the characteristic of the immune response may involve determining an indication of the number of CD1 1 c+ MHC class II+ cells in a sample of the organ or tissue or determining an indication of the number of CD4+ T cells in a sample of the organ or tissue or determining an indication of the number of CD8+ T cells in a sample of the organ or tissue.
  • measuring the magnitude of the immune response may involve determining an indication of the number of NK cells in a sample of the organ or tissue. Further and optionally, comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells.
  • the macrophages may include any one or more of the following: M1 -like
  • comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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 cytokines may be produced as a result of a shift in an activation state of the macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ 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 differential gene expression may be produced as a result of a shift in an activation state of the macrophages.
  • the macrophages may shift from being M2-like macrophages to being M1 -like macrophages.
  • the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.
  • the invention provides methods for formulating an immunogenic composition for treating an intestinal cancer, such as a cancer of the lower intestine, e.g., colon and/or rectum, in a mammal, such as human patient.
  • the method may include selecting at least one Fusobacterium that is naturally pathogenic in the intestine or target portion thereof the mammal within which the cancer is situated.
  • An antigenic composition may be produced that includes antigenic determinants that together are specific for or
  • the medicament may be any medicament.
  • the medicament may be administered intradermal ⁇ or subcutaneously.
  • the medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site.
  • the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours.
  • a visible localized inflammatory immune response may not always be present in all circumstances despite an immune response being initiated.
  • the profile (and relative change in characterization) of immune cells from a subject undergoing an immune reaction can be compared with those from a subject that is not undergoing an immune reaction.
  • the animal may be a mammal.
  • the animal may be a human or a mouse.
  • the foregoing examples are provided as examples only and are not meant to be limiting.
  • Fusobacterium nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa.
  • BCCA-TTR BC Cancer AgencyTumor Tissue Repository
  • BCCA REB BC Cancer AgencyTumor Tissue Repository
  • RNA quality and concentration was assessed using Agilent Bioanalyzer 2000 RNA Nanochips. Ribosomal RNAs were depleted from 1 mg of total RNA using the manufacturer's protocol for the RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen). Depletion was assessed using Agilent Bioanalyzer 2000 RNA Nanochips.
  • Each paired-end library was PCR amplified for 15 cycles using the standard lllumina PE1 PCR primer plus one of 12 modified PE2 primers, each including a unique six base insertion as an index sequence.
  • Libraries prepared using indexed primers were then combined in pools of 1 1 each (one tumor pool, one control pool) gel purified, and then sequenced on the lllumina GAIIx platform. One lane of 75 bp paired end sequence was obtained for each of the two pools.
  • Paired-end sequence reads from indexed tumor and adjacent normal sample libraries were processed as described. (Moore, et al., 201 1 , supra) Briefly, corresponding human RNA-seq libraries were aligned with bwa (version 0.5.4 [sample -o 1000, default options] (Li, et al. "Fast and accurate long-read alignment with Burrows-Wheeler transform," Bioinformatics (2010) 26:589-595), sequentially against human rRNA, cDNA and genome reference sequences (Flicek, et al., "Ensemble 201 1 ,” Nuc. Acids Res. (201 1 ) 39: D800-D806).
  • read pair count was reported for each Genbank accession in our lAdb, sorted in decreasing order by the sum of unambiguous pairs and PERL scripts were developed to mine these data.
  • a custom TaqMan primer/probe set was designed to amplify
  • Fusobacterium nucleatum DNA that matched the contiguous sequence from the WTSS experiment.
  • the cycle threshold (Ct) values for Fusobacterium were normalized to the amount of human biopsy gDNA in each reaction by using a primer/probe set for the reference gene, prostaglandin transporter (PGT), as previously described (Wilson, et al. "DNA copy-number analysis in bipolar disorder and schizophrenia reveals aberrations in genes involved in glutamate signaling," Hum. Mol. Genet. (2006)15:743-749).
  • PGT prostaglandin transporter
  • the fold difference (2 ct ) in Fusobacterium abundance in tumor versus normal tissue was calculated by subtracting ACtt umor from ACt normal where ACt is the difference in threshold cycle number for the test and reference assay.
  • Isolated biopsy DNA was quantified by PicoGreen Assay (Invitrogen) on a
  • CAACCATTACTTTAACTCTACCATGTTCA 3' (SEQ ID NO:01 ); Fusobacteria reverse primer, 5' G TTG ACTTTAC AG A AG G AG ATT ATG T AAA AATC 3' (SEQ ID NO:02); Fusobacteria FAM
  • Frozen tumor sections were thawed and immediately placed into 500 ml of pre-reduced phosphate buffered saline, and the tissue agitated and gently broken up using a pipette fitted with a sterile, wide-bore, plugged tip. 100 ml aliquots of this suspension were directly spread onto pre-reduced fastidious anaerobe agar (FAA) plates supplemented with 5% defibrinated sheep blood (DSB), and incubated for 1 0 days in a humidified anaerobe chamber (Ruskinn Bug Box). Plates were inspected every 2 days for growth, and all colonies were picked and streak-purified on further pre-reduced FAA+5% DSB plates.
  • FAA fastidious anaerobe agar
  • DSB defibrinated sheep blood
  • a product size of 495bp confirmed that the isolate belonged to the Fusobacterium genus, and a further PCR to partially amplify 16S rRNA gene was carried out using the same DNA template using primers and conditions as defined by Ben-Dov et al. (Ben-Dov, et al. "Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity," Appl. Environ. Microbiol. (2006) 72:6902-6906); this product was sent for Sanger sequence analysis to MWG Operon, and obtained traces confirmed F.nucleatum as the species.
  • Fusobacterium genomic DNA was sonicated and size fractions between 175 to 200 bp and 400 to 450 bp were isolated following PAGE.
  • WGSS Paired- end lllumina libraries were prepared from each size fraction as described previously with the following modifications: the final PCR amplification was increased to 15 cycles and contained the standard lllumina PE1 PCR primer and an indexed PE2 primer as detailed above for RNA-Seq library construction (Shah, et al., 2009, supra; Morin, et al., 201 0 supra). A total of 92.0M paired 1 00 nt reads were obtained from a single lane of the lllumina HiSeq instrument.
  • Fusobacterium tumor isolate contigs were in turn aligned onto the reordered Fusobacterium sp. 3_1_36A2 HMP genome assembly and ordered/oriented according to that genome sequence, using the same cross_match parameters. Threeway cross_match alignments between the ordered Fusobacterium genomes were performed and plotted using hive plots (www.hiveplot.com).
  • Caco-2 cell invasion assays with CC53 were carried out in triplicate using a differential staining immunofluorescence procedure as previously described (Strauss, et al. "Invasive potential of gut mucosa-derived Fusobacterium nucleatum positively correlates with IBD status of the host," Inflammatory Bowel Disease (201 1 ) in press). Briefly, bacterial cultures were grown to late log phase according to pre-determined growth-curve data, and normalized for cell number using McFarland standards. Caco-2 cells were grown to 80% confluence on glass coverslips in 24-well plates and infected at a multiplicity of infection of 100:1 (bacterial cells:intestinal cells).
  • Infected cells were maintained at 37°C, 5% CO2 for 4 hours following infection, after which time cells were washed with PBS to remove non-adherent bacteria, and then fixed with 2.5% paraformaldehyde, and blocked in 10% (v/v) normal goat serum.
  • Prepared polyclonal antibodies were diluted to 1 /500, applied to coverslips, and incubated for 1 hr at 37°C. Coverslips were then incubated with donkey anti-rabbit (EAV AS1 ) or anti-rat (EAV_AS2) Alexa 350 (1 /1 00) (Molecular Probes), permeabilized by the addition of 0.1 % TritonX100, and then reincubated with prepared polyclonal antibodies, as above.
  • Reads were filtered for base quality and low complexity, then aligned pairwise to human rRNA and cDNA (Flicek, et al. , 201 1 , supra), and genome (hg18) reference sequences using bwa (Li, et al., 2010, supra), as previously described (Moore, et al., 201 1 , supra). Aligned reads were removed from the data set, leaving 34.9M pairs (Table S1 , Appendix A). These residual read pairs were then used to search a custom database containing accessions for all Refseq bacterial and
  • F. nucleatum was the organism with the highest number of hits overall (21 % of all alignments) and nine of the eleven subjects showed at least two-fold higher read counts in tumor relative to corresponding control tissue (Fig 1 ). The mean over-abundance in tumor was 86-fold. The majority of the hits were to highly abundant F.nucleatum ribosomal transcripts but other non- ribosomal F.nucleatum gene products were also detected (Fig S2).
  • the initial metagenomics screen described above involved interrogation of expressed genes, however, once we established F.nucleatum as a candidate pathogen we switched to analysis of gDNA because a larger amount of high quality DNA than RNA was obtainable from the frozen tissue sections.
  • F.nucleatum as a candidate pathogen we switched to analysis of gDNA because a larger amount of high quality DNA than RNA was obtainable from the frozen tissue sections.
  • We conducted qPCR on gDNA isolated from an additional 88 colorectal carcinomas and matched normal specimens and confirmed an over representation of F.nucleatum in tumor versus matched normal specimens (p 2.5E-6, two-tailed ratio t-test) (Fig 2).
  • Caco-2 cells were grown on glass coverslips, infected with CC53 culture (at a multiplicity of infection of 100:1 ), and then differentially stained with anti- Fusobacterium antibodies conjugated to different fluorophores before and after Caco-2 cell permeabilization. We confirmed the invasiveness of CC53 in this model system (Fig 4).
  • Fusobacterium nucleatum is an invasive (Swidsinski, et al., Acute appendicitis is characterized by local invasion with Fusobacterium
  • F. nucleatum is a well-known pathogen, but it has not been associated previously with cancer of the Gl tract or any other cancer site.
  • Our observation of an over-representation of F.nucleatum in colorectal tumor specimens was largely unexpected, given that F.nucleatum is generally regarded as an oral pathogen, and it is not an abundant constituent of the normal gut microbiota (Weiss, et al., "Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose-binding adhesin," Oral Microbiol. Immunol. (2000) 15:371 -377).
  • F.nucleatum is indicated to be involved in tumorigenesis, e.g., through pro-inflammatory mechanisms, particularly with colon cancer.
  • three of the 99 tumor sections were comprised entirely of the adenomatous component of adenocarcinoma specimens.
  • two out of these three had very high Fusobacterium content and, in fact, one of these gave the highest tumor normal ratio of all samples.
  • Fusobacterium infection is related to the early stages of tumor progression and is therefore an appropriate target for vaccination and/or antimicrobial therapy.
  • an antigenic composition comprised of antigens of Fusobacterium nucleatum could be used to treat cancer of the colon/rectum.
  • Table S Host sequence subtraction RNA-seq data from eleven colorectal carcinoma and matched normal specimens.

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Abstract

Des aspects de l'invention comprennent des méthodes de traitement de cancers de l'intestin, par exemple de l'intestin inférieur, tel que du côlon et/ou du rectum, par l'administration d'une composition qui est spécifique du point de vue antigénique pour un Fusobacterium, tel que Fusobacterium nucleatum.
PCT/IB2012/002065 2011-08-29 2012-08-24 Activation antigénique de fusobacterium ciblé à un tissu de la réponse immunitaire pour traiter des cancers de l'intestin Ceased WO2013030670A2 (fr)

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US11226340B2 (en) 2016-04-26 2022-01-18 Qu Biologics Inc. Therapeutically triggering an innate immune response in a target tissue
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US11226340B2 (en) 2016-04-26 2022-01-18 Qu Biologics Inc. Therapeutically triggering an innate immune response in a target tissue
WO2024103187A1 (fr) * 2022-11-18 2024-05-23 Provincial Health Services Authority Anticorps dérivés de fap2 et vaccins contre fusobacterium

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