WO2009036568A1 - Procédés et compositions pour traiter des tumeurs et des infections virales - Google Patents

Procédés et compositions pour traiter des tumeurs et des infections virales Download PDF

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WO2009036568A1
WO2009036568A1 PCT/CA2008/001653 CA2008001653W WO2009036568A1 WO 2009036568 A1 WO2009036568 A1 WO 2009036568A1 CA 2008001653 W CA2008001653 W CA 2008001653W WO 2009036568 A1 WO2009036568 A1 WO 2009036568A1
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antigen
tumor
mammal
cells
administered
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PCT/CA2008/001653
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English (en)
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Tak W. Mak
Pamela S. Ohashi
Thomas Calzascia
Marc Pellegrini
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University Health Network
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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2046—IL-7
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/46444—Interleukins [IL]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • 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
    • A61P31/12—Antivirals
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae

Definitions

  • the invention relates to methods, pharmaceutical compositions and kits for treating a tumor or a viral infection.
  • An immune response involves the basic idea of self/non-self discrimination, a process that is accomplished by means of recognition mechanisms. These recognition mechanisms are used to eliminate undesirable materials from the body, and are an essential part of the body's defense mechanisms. Antigens are materials the body recognizes as foreign. The immune response is a reaction against the antigen in an effort to eliminate it from the body. The generation of an immune response is helpful in the treatment of many diseases, including cancers and pathogenic infections. Strategies for acquiring or increasing immunity against threatening agents such as cancer cells or pathogens have been pursued for a long time. Cancer immunotherapy involves therapeutic methods that utilize the immune system to reject cancer cells or a tumor.
  • the main goal in cancer immunotherapy is to stimulate the patient's immune system to attack the malignant tumor cells that are responsible for the disease. This can be either through immunization of the patient, in which case the patient's own immune system is trained to recognize tumor cells as targets to be destroyed, or through the administration of therapeutic antibodies as drugs, in which case the patient's immune system is recruited to destroy tumor cells by the therapeutic antibodies.
  • Vaccines for treating cancer and many pathogen infections are currently under development.
  • Tumors and chronically infected cells can escape clearance by the immune system using several mechanisms (Speiser, D. E., et al, J Exp Med 186, 645-653 (1997)). Since the immune system responds to the environmental factors it encounters on the basis of discrimination between self and non-self, many kinds of tumor cells that arise as a result of the onset of cancer are more or less tolerated by the patient's own immune system since the tumor cells are essentially the patient's own cells that are growing, dividing and spreading without proper regulatory control.
  • IL-7 interleukin-7
  • the invention relates to methods of treating a tumor in a mammal, comprising administering to the mammal IL-7 and an antigen of the tumor in an amount sufficient to induce an immunogenic response thereto.
  • the invention features methods of increasing an antigen-mediated immunogenic response to a tumor in a mammal, comprising administering to the mammal IL-7 and an antigen of the tumor.
  • the immunogenic response is an increase in antigen-specific T cell numbers, T H 17 cells and/or natural killer cells in the mammal.
  • the invention features a method of prolonging survival of a mammal having a tumor, comprising administering to the mammal IL-7 and an antigen of the tumor in an amount sufficient to induce an immunogenic response thereto.
  • the invention features a method of decreasing TGF-/3 receptor signaling in a mammal having a tumor, comprising administering to the mammal IL-7 and an antigen of the tumor in an amount sufficient to induce an immunogenic response thereto.
  • the decreased signaling results in increased levels of Smurf-2 and/or decreased levels of Cbl-b in the T cells of the mammal.
  • the antigen is comprised in a vaccine, the antigen of the tumor is expressed on the tumor and/or the antigen is obtained from the tumor.
  • the antigen is administered prior to administration of IL-7; the antigen is administered at the same time as IL-7; or IL-7 is administered more than one time after the antigen is administered.
  • prior to treatment the mammal is diagnosed as having a tumor; the mammal is a human and/or administration of the IL-7 and antigen of the tumor is intratumoral, subcutaneous, intradermal, intravenous or intraperitoneal.
  • the invention features a method of treating a viral infection in a mammal, comprising administering to the mammal IL-7 and a viral antigen in an amount sufficient to induce an immunogenic response thereto.
  • the antigen is comprised in a vaccine; the antigen is administered prior to administration of IL-7; IL-7 is administered more than one time after the antigen is administered; prior to treatment the mammal is diagnosed as having a viral infection; the mammal is a human; and/or administration of the IL-7 and viral antigen is intratumoral, subcutaneous, intradermal, intravenous or intraperitoneal.
  • the invention features the use of IL-7 and an antigen of a tumor for treating a tumor in a mammal.
  • the invention features the use of IL-7 and a viral antigen for treating a viral infection in a mammal.
  • the invention features the use of IL-7 and an antigen of a tumor in the manufacture of a medicament for the treatment of a tumor in a mammal.
  • the invention features the use of IL- 7 and a viral antigen in the manufacture of a medicament for the treatment of a viral infection in a mammal.
  • the invention features a pharmaceutical composition for treating a tumor in a mammal, comprising IL-7, an antigen of a tumor and a pharmaceutically acceptable carrier.
  • the invention features a pharmaceutical composition for treating a viral infection in a mammal, comprising IL-7, a viral antigen and a pharmaceutically acceptable carrier.
  • the invention features a kit comprising IL-7, an antigen of a tumor and a pharmaceutically acceptable carrier.
  • the invention features a kit comprising IL-7, a viral antigen and a pharmaceutically acceptable carrier.
  • the present invention has a number of advantages.
  • the invention provides new methods and pharmaceutical compositions for effectively treating subjects with cancer or a viral infection.
  • FIG. IA is a bar graph showing the survival (in days) of RIP-TAG2 (unvaccinated or vaccinated with LCMV) or RIP (GP x TAG2) (unvaccinated or vaccimnated with LCMV) administered either IL-7 or PBS.
  • Data represents averages and SD obtained from 12 mice of each genotype and in each treatment arm. Differences in survival between RIP (GP x TAG2) vaccinated / IL-7-treated mice and RIP (GP x TAG2) vaccinated / PBS-treated mice was statistically significant (*p ⁇ 0.0001).
  • FIG. IB is a graph showing the random blood glucose levels (mM) over time (days post LCMV infection) in C57BL/6 mice, RIP (GP x TAG2) mice administered IL-7, RIP (GP x TAG2) mice administered PBS or RIP-T AG2 mice. Data represents averages of 4 mice in each group. Error bars indicate SD and are only shown for data points where there is a statistically significant difference between IL-7 and PBS-treated RIP (GP x TAG2) animals (p ⁇ 0.008).
  • FIG. 2 A is a series of images of histological sections of the pancreases of RIP (GP x TAG2) LCMV-vaccinated mice administered IL-7 or PBS after the last dose of IL-7 or PBS was administered or 1 week later. The sections were stained with an anti-CD4 stain or an anti-CD8 stain (the magnification is equivalent for all sections).
  • 2B is a series of bar graphs showing the absolute cell numbers quantitated in spleen, inguinal lymph nodes (ILN) and pancreatic draining lymph nodes (PDLN) of LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice at the completion of 2 weeks of administration of either IL-7 or PBS.
  • G granulocytes
  • M monocytes
  • B B cells.
  • the activation status of CD4 and GP33 tetramer positive CD8 cells was assessed using CD69 (69), CD25 (25) and CD44 (44) markers.
  • CD8 T cells were also stained with GP276 and NP396 tetramers as indicated. Bars and data points represent means from 4-6 mice in each treatment arm and error bars represent SD.
  • Statistically significant differences between IL-7 and PBS treatments are indicated with asterisks (* p ⁇ 0.04, ** p ⁇ 0.005, *** p ⁇ 0.0005).
  • FIG. 2C is a series of graphs showing the numbers of GP33-specific CD8 T cells over time (days after the commencement of administration of IL-7 or PBS) in LCMV-vaccinated tumor- bearing RIP (GP x TAG2) mice administered either IL-7 or PBS. Tetramer staining was used to quantitate the number of antigen-specific CD8 T cells in these mice. Arrows indicate the end of treatment. Data points represent means from 4-6 mice in each treatment arm and error bars represent SD. Asterisks indicate p values (* p ⁇ 0.04, ** p ⁇ 0.005, *** p ⁇ 0.0005).
  • FIG. 1 is a series of graphs showing the numbers of GP33-specific CD8 T cells over time (days after the commencement of administration of IL-7 or PBS) in LCMV-vaccinated tumor- bearing RIP (GP x TAG2) mice administered either IL-7 or PBS. Tetramer staining was used to quantitate the number of antigen-specific CD8 T cells in
  • 2D is a set of bar graphs showing the numbers of antigen-specific T cells in the pancreases of RIP (GP x TAG2) mice vaccinated with LCMV.
  • the absolute number of CD4 GP61- specific T cells was quantitated in the pancreases of the mice after 7 days of IL-7 or PBS treatment (left graph).
  • the number of antigen-specific CD8 T cells in the pancreases of the mice 1 week after IL-7 or PBS treatment was quantitated using tetramer staining (right graph). Bar graphs represent averages of 4-6 mice and error bars represent SEM. Statistically significant differences are indicated with asterisks (* p ⁇ 0.04, ** p ⁇ 0.03, *** p ⁇ 0.01).
  • FIG. 3B is a series of histograms showing the in vivo survival and expansion of adoptively transferred antigen-specific CD8 T cells isolated from C57BL/6 mice. Also in the bottom panels of FIG. 3B is a set of bar graphs showing the absolute number of antigen-specific carboxyfluoroscein diacetate succinimidyl ester (CFSE)-labeled CD8 T cells recovered from these mice. The bar graphs represent averages from 3 mice and error bars represent SD. P values for statistically significant differences are indicated.
  • FIG. 3B is a series of histograms showing the in vivo survival and expansion of adoptively transferred antigen-specific CD8 T cells isolated from C57BL/6 mice. Also in the bottom panels of FIG. 3B is a set of bar graphs showing the absolute number of antigen-specific carboxyfluoroscein diacetate succinimidyl ester (CFSE)-labeled CD8 T cells recovered from these mice. The bar graphs represent averages from 3 mice and error bars represent SD. P values for statistical
  • 3C is a graph showing the ex vivo cytolytic activity (determined by the ability of the cells to kill peptide pulsed target cells and measured as a percent of PG33-specific killing) of pancreatic infiltrating lymphocytes (PILS) from LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice after 7 days of IL-7 treatment at different dilutions of PILS. Data points represent averages of 3 mice and error bars represent SEM. Results are representative of 3 separate experiments with a total of 9 mice in each group. Statistically significant differences between IL-7 and PBS treatments are indicated with asterisks which correspond to the following p values: * p ⁇ 0.03, ** p ⁇ 0.01, *** p ⁇ 0.002.
  • FIG. 3D is a graph showing the ex vivo degranulation kinetic of GP33-specific pancreatic infiltrating CD8 lymphocytes (assessed as the amount of CD107a levels on CD8 + H-2Db/GP33 + cells over time after stimulation of the cells) from LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice after 7 days of IL-7 or PBS treatment.
  • the data represent the average and SD of results obtained from 3 independent mice in the IL-7 group and 3 mice (pooled lymphocytes) in the PBS group.
  • FIG. 3E is a set of histograms showing granzyme-B expression in PILs isolated from LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice after 7 days of PBS or IL-7 administration.
  • the top histogram shows granzyme-B expression in mice receiving IL-7, while the bottom panel shows granzyme-B expression in animals receiving PBS.
  • the results are representative of 3 independent experiments performed on 12 mice.
  • FIG. 3F is a set of dot plots showing the percent of IL-2 and IL- 17 producing CD4 PILs of PBS and IL-7-treated LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice re-stimulated ex vivo, as determined by flow cytometry after 4 hours of re-stimulation. Dot plots were gated on CD4 T cells.
  • FIG. 3 G is a set of bar graphs showing the percent of IL-2 and IL- 17 producing CD4 PILs of PBS and IL-7-treated LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice re-stimulated ex vivo, as determined by flow cytometry after 4 hours of re-stimulation.
  • FIG. 3H is a set of bar graphs showing the absolute cell numbers quantitated in inguinal lymph nodes (ILN), pancreatic draining lymph nodes (PDLN) and pancreas infiltrating lymphocytes (PIL) of LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice after 1 week of treatment with either IL-7 or PBS.
  • the proportion of NKLl + cells that are CD3 " , CD3 + CD8 " CD4 " (CD3 + DN), CD3 + CD8 + CD4 " (CD3 + CD8 + ), and CD3 + CD8 CD4 + (CD3 + CD4 + ) are indicated. Bars and data points represent means from 3 mice in each treatment arm and error bars represent SD. Statistically significant differences between IL-7 and PBS treatments are indicated with asterisks, which correspond to the indicated p values.
  • IL-7 IL-7
  • PBS-treated LCMV-vaccinated tumor-bearing RIP GP x TAG2 mice administered IL-7 or PBS.
  • Data points represent values from individual mice and p values indicate statistically significant differences.
  • FIG. 5B is a series of histograms showing CFSE proliferation profiles of effector CD4 + T cells from BL57/6 mice (pre-treated in vivo with PBS or IL-7 for 3 days) cultured with APC, CD3 mAb and Treg at the indicated ratios (Treg:T effector).
  • the numerical values indicate the absolute number of CFSE-labeled CD4 + T effector cells after 3 days in culture, the proportion of dividing cells is also indicated.
  • FIG. 5C is series of images of histological tumor sections from RIP-TAG2 mice, stained for TGF- ⁇ , PDL-I and PDL-2.
  • FIG. 5D is a set of histograms showing expression of PD-I on CD4 and CD8 PILS obtained from LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice treated for 1 week with IL-7 or PBS.
  • FIG. 5E is a set of histograms showing CFSE proliferation profiles of CD8 + T cells isolated from C57BL/6 mice and cultured with APC, CD3 mAb and TGF- ⁇ at a concentration of 10 ng/niL either in the presence of IL- 7 (500 ng/mL) or control PBS. Numerical values indicate the absolute number of CFSE-labeled CD8 + T cells that have undergone at least one division after 3 days in culture ( ⁇ SD), the proportion of dividing cells is also indicated. Histograms are representative of 6 mice in each group and the experiment was repeated twice. FIG.
  • 6A is an image of a western blot showing levels of phosphorylated Stat5 (p-Stat5), phosphorylated Stat3 (p-Stat3), Stat5, Stat3, Fox03a, p27, survivin and actin in purified CD8 T cells from na ⁇ ve C57BL/6 mice treated for 1 hr or 7 days with PBS or IL-7 and purified CD8 T cells from LCMV-infected C57BL/6 mice treated with PBS or IL-7 between days 3 and 8 post infection.
  • 6B is an image of a western blot showing levels of phosphorylated Smad3 (p-Smad3), phosphorylated Smad2 (pSmad2), Smad2, Smad3, and tubulin in purified CD8 T cells from na ⁇ ve C57BL/6 mice treated for 1 hr or 7 days with PBS or IL-7 and purified CD8 T cells from LCMV- infected C57BL/6 mice treated with PBS or IL-7 between days 3 and 8 post infection.
  • p-Smad3 phosphorylated Smad3
  • pSmad2 phosphorylated Smad2
  • tubulin tubulin
  • FIG. 6C is an image of a western blot showing levels of TGF-/3 RII, Smurf2, Smad2, Smad7 and tubulin in purified CD8 T cells from na ⁇ ve C57BL/6 mice treated for 1 hr or 7 days with PBS or IL-7 and purified CD8 T cells from LCMV-infected C57BL/6 mice treated with PBS or IL-7 between days 3 and 8 post infection.
  • FIG. 6D is an image of a western blot showing levels of Cbl-b and tubulin in purified CD8 T cells from na ⁇ ve C57BL/6 mice treated for 1 hr or 7 days with PBS or IL-7 and purified CD8 T cells from LCMV-infected C57BL/6 mice treated with PBS or IL-7 between days 3 and 8 post infection.
  • FIG. 6E is an image of a western blot showing levels of Cbl-b and tubulin in lysates prepared from human purified CD8 T cells incubated with 500ng/mL IL-7 for 1 hour.
  • FIG. 6F is an image of a western blot showing levels of Fox03a, Cbl-b, TGF-/3 RII and tubulin in purified CD44 hi CD8 T cells or CD44 hi CD4 T cells isolated from LCMV-infected
  • mice C57BL/6 mice 8 days post infection. One hour prior to T cell isolation, the mice received 20 ⁇ g IL- 7 or PBS intravenously.
  • FIG. 7A is a set of histograms showing CD127 (IL-7 receptor- ⁇ ) levels on na ⁇ ve C57BL/6 mice or in vivo LCMV activated CD8 T cells from C57BL/6 mice 8 days after infection. CD127 staining is compared to isotype control antibody staining (Ctrl).
  • FIG. 7B is a series of histograms showing CFSE dilution in CD8 and CD4, CD127 hi and CD127 10 adoptively transferred cells from C57BL/6 mice. Histograms are gated on congenic markers and CD8 or CD4 labeled splenic T cells. Inset histograms show CD 127 levels on the gated population compared to isotype control antibody, levels were consistent with the initial levels immediately prior to transfer. Numbers represent the proportion of dividing cells.
  • FIG. 7C is a bar graph showing the percent of dividing CD8 and CD4 T, CD127 hl and CD127 10 adoptively transferred cells of the experiment represented by FIG. 7B. Averages and SD were calculated from data of 3 independent experiments.
  • FIG. 7D is a bar graph showing survival and proliferation for the cells in the experiment represented by FIG. 7B, with "% recovered cells” comparing the number of cells transferred versus the number recovered from the spleens. Averages and SD were calculated from data of 3 independent experiments.
  • FIG. 7E is a set of histograms showing CD 127 levels compared to isotype control antibody staining (Ctrl) on CD4 and CD8 T cells isolated from PILs of LCMV-infected tumor bearing mice treated for 9 days with IL-7 or PBS.
  • the present invention relates to methods of treating a tumor in a mammal by administering to the mammal interleukin-7 (IL-7) and an antigen of the tumor in an amount sufficient to induce an immunogenic response thereto.
  • IL-7 interleukin-7
  • a tumor is meant any tumor or cancer, excluding haematological and lymphoid tumors, and includes metastatic cancer.
  • the tumor is a malignant (i.e., neoplastic) tumor.
  • malignant tumors include, for example, renal carcinoma, prostate carcinoma, colon carcinoma, pancreas carcinoma, lung carcinoma, breast carcinoma, liver carcinoma, brain carcinoma, stomach carcinoma, kidney carcinoma, esophageal carcinoma, and carcinomas of the gastrointestinal tract or reproductive tract (e.g., cervix, ovaries, endometrium, etc.).
  • the tumor may be, for example, a solid tumor such as primary tumor of ductal epithelial cell origin.
  • IL-7 for use in the invention can be obtained from a number of commercial sources.
  • the IL-7 is human IL-7.
  • IL-7 is available in a recombinant human form from, for example, Cytheris Corp. (Issy les Moulineaux, France).
  • the recombinant human IL-7 may also be a fully glycosylated form and can be obtained, for example, from Cytheris Corp.
  • IL-7 is used in combination with an antigen of a tumor to induce an immunogenic response for the treatment of the tumor.
  • tumor antigen or "antigen of a tumor” is meant a substance expressed by a tumor or a tumor cell.
  • a tumor antigen is recognized by the adaptive immune system that stimulates the body to find and eliminate cancer cells.
  • a tumor antigen includes, for example, cancer cells, parts of cancer cells or pure tumor antigens (i.e., substances isolated from tumor cells).
  • the endogenous tumor itself, a portion of the endogenous tumor or cells from the endogenous tumor may be used as the tumor antigen.
  • tumor antigens are well known in the art, and include antigens that are expressed exclusively by tumor cells or that are expressed on both tumor cells and normal cells.
  • Specific examples of tumor antigens include, but are not limited to products of mutated oncogenes or tumor suppressor genes (e.g., mutant ras and mutant p53 gene products), oncofetal antigens, and abnormal proteins produced by cells infected with an oncovirus such as Epstein Barr Virus or a human papilloma virus.
  • tumor antigens include HER2, NY-ESO-I, MEGA A4, MAGEs, BAGE, GAGE , CDK-I, MUM-I and CASP-8, MAGEs, MART 1, p97 melanoma antigen and CEA (carcinoembryonic antigen).
  • the antigen may be a protein or an antigenic portion of a protein.
  • the antigen of the tumor is expressed on the tumor of the subject who is going to be treated with the tumor antigen and IL-7.
  • the tumor antigen may be prepared or isolated from the tumor of a subject diagnosed with cancer, for example, by way of a biopsy or other surgical procedure.
  • the tumour may be obtained from the subject who is to receive the therapy.
  • the tumor may be obtained from a subject other than the subject who is to receive the therapy, for example, a subject who has the same type of tumor.
  • Tumor antigens can be prepared in a number of ways.
  • the tumor- infiltrating lymphocytes (TILs) can be isolated from the tumor sample obtained from the subject.
  • the TILs can be expanded in vitro and used as the antigen for administration, by re-introduction, to the subject in combination with IL-7 as described herein.
  • the TILs can be isolated and one or more antigens on these cells can be determined. These antigens can then be purified, manufactured or generated and used as the antigen to be delivered to the subject in the therapeutic methods described here.
  • tumor antigens can be determined from the tumor sample itself, without first isolating TILs from the sample, and then purified, manufactured or generated.
  • the tumor antigen can be prepared outside of the body and administered to the subject in need of treatment through, for example, a vaccine.
  • vaccine or “vaccination” or “immunizer” is meant a biological preparation which is used to establish or improve immunity to a particular disease.
  • the vaccine may be a tumor vaccine, in which case, the biological preparation may comprise either tumor cells, tumor antigen, nucleic acid that encodes tumor antigen, cells expressing tumor antigen, or a mixture thereof.
  • a vaccine may employ only one antigen, or more than one antigen.
  • the vaccine stimulates an immune response within the patient, which may result in partial or complete treatment of the disease, for example, a viral infection or a tumor.
  • the antigen may be produced inside the body.
  • IL-7 can be administered once a day, twice a day or once every other day following administration of the tumor antigen.
  • Tumor antigens useful in the methods described herein may be prepared by any suitable method.
  • the antigen may be isolated and purified from a selected tissue using conventional methods.
  • affinity columns employing antibodies or fragments thereof for specific adsorption of the desired antigen can be used to advantage.
  • the nature of the purification method will, of course, depend on the nature of the antigen obtained.
  • antigens that are proteins or peptides
  • protein or peptide antigens may be prepared using recombinant nucleic acid technologies. Procedures for the production of pure antigens from the DNA encoding the desired antigen are well known to those skilled in the art (see, for example, Current Protocols in Molecular Biology, Ausubel et al. (Eds.), John Wily & Sons, Inc. (2007)). Briefly, the preferred DNA is expressed in a suitable recombinant expression vector such as those adapted for E.
  • yeast such as Saccharomyces cerevisiae or Pichia pastoris
  • filamentous fungi such as Aspergillus nidulans.
  • the yeast, fungi or bacteria can be grown in continuous culture producing recombinant protein which may be then be isolated and purified. Alternately, higher organisms may be used for recombinant protein production.
  • the encoding DNA may be expressed in an insect virus expression vector such as recombinant baculovirus and the resulting recombinant baculovirus then used to infect susceptible cultured SF9 cells (Spodotera frugiperda insect cells) to produce the protein product of the DNA.
  • Other expression systems commonly used include those appropriate for production of proteins in mammalian cells, such as CHO cells or even plant cells. The choice of host will determine the nature of the post-translational processing, and is a consideration in devising purification techniques.
  • Recombinant nucleic acid technologies may also be used to produce portions of the desired antigen rather than the entire antigen. For example, it maybe desirable to express the extracellular domain without the intracellular and/or transmembrane domains to facilitate purification of membrane associated antigen. Similarly, it may be desirable to express just the epitopes of choice eliminating unrelated or competing epitopes. All of these may be accomplished through techniques well known to those skilled in the art. Techniques for identifying peptides representing important epitopes of the antigen are well known, and are summarized in Berzofsky, J. A. and Berkower I. J., Fundamental Immunology 2nd edition, Raven Press, (1989) W. E. Paul (Ed.).
  • peptides and proteins can be prepared using standard chemical synthesis methods, preferably the commercially available solid-phase-based techniques. These techniques are well known (see, for example, Merrifield R. B., J Am Chem Soc 85, 2149-2154 (1983)) and automated systems to conduct them can be purchased and employed according to the manufacturer's instructions.
  • the methods of the present invention are used to treat a tumor.
  • a therapy e.g., IL-7 plus an antigen of the tumor, chemotherapy, radiation therapy
  • treatment of a patient's tumor may reduce or inhibit tumor growth, or may induce partial or complete tumor regression.
  • Treatment of a tumor may include increasing the survival rate of the patient as compared to a patient that does not receive the therapy.
  • the therapies described herein are administered in an amount sufficient to induce an immunogenic response.
  • an "sufficient to induce an immunogenic response”, an “effective amount” or an “immunologically effective amount” means that the administration of that amount to a subject, either in a single dose or as part of a series, is effective for inducing an immune reaction and preferably, for treating a tumor or cancer.
  • Vaccination protocols for the administration of IL-7 and the tumor antigen may be designed to induce treat an existing cancer or tumor.
  • Induction of an immune response by administration of a tumor antigen may be achieved by conventional techniques that are well-known to those skilled in the art of vaccine production and delivery.
  • the IL-7 may be administered at the same time as the tumor antigen, or after administration of the tumor antigen.
  • additional doses of IL-7 can be administered one of more times after administration of the tumor antigen. For example, IL-7 can be administered once a day, twice a day or once every other day following administration of the tumor antigen.
  • the mammal for example, a human patient, to be treated according to the methods of the invention is diagnosed prior to treatment as having a tumor.
  • Methods for diagnosing a tumor are known to those skilled in the art.
  • the dosage regimen for treatment of a subject with the tumor antigen and IL-7 is based on a variety of factors, including the type mammal to be treated, the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. Methods for the delivery of the IL-7 and tumor antigen are known to those skilled in the art. A number of delivery routes are available for injection of the therapy, including intratumoral, subcutaneous, intradermal, intravenous and intraperitoneal routes. Other modes of delivery include topical, oral and nasal delivery, using biocompatible particles, such as liposomal formulations.
  • the therapeutic methods described herein can also be used to increase an antigen-mediated immunogenic response to a tumor in a mammal.
  • the immunogenic response can be, for example, an increase in antigen- specific T cell numbers in the mammal; an increase in the number of T H 17 cells in the mammal and/or increase in the number of natural killer cells in the mammal.
  • the increase in the cells types may be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more increase as compared to those samples not receiving IL-7 and a tumor antigen. Methods for assessing the numbers of these cell types are known in the art and are described herein. Flow cytometric techniques using antibodies that specifically bind to particular cells is a standard method for determining the amount of a specific type of a cell in a sample.
  • the therapeutic methods described herein can also be used to prolong the survival of a subject, for example, a mammal such as a human, having a tumor.
  • Malignant tumors can be slow growing or fast growing. If left untreated, virtually all malignant tumors are fatal. Survival of a mammal may be prolonged by weeks, months or years, or by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more as compared to subjects who do not receive the therapy.
  • TGF-/3 receptor signaling can be decreased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to subjects who do not receive the therapy.
  • Decreased TGF- ⁇ receptor signaling can be assessed in a number of ways. An increased level of Smurf-2 in a sample, for example, a T cell sample, is an indication that there is decreased TGF-/3 receptor signaling in the sample.
  • the level of Smurf-2 may be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more as compared to a sample from a subjects who does not receive the therapy.
  • a decreased level of Cbl-b in a sample indicates that there is decreased TGF-/3 receptor signaling in the sample.
  • the level of Cbl-b can be decreased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to a sample from a subjects who does not receive the therapy
  • the levels of Smurf-2 and Cbl-b can be determined using a number of standard techniques, such as western blot analysis.
  • the present invention describes a number of effects caused by administration of IL-7.
  • the present invention also relates to methods of treating a viral infection in a mammal, comprising administering to the mammal IL-7 and a viral antigen in an amount sufficient to induce an immunogenic response thereto.
  • a viral infection is meant an infection caused by the presence of a virus in the body.
  • Viral infections include chronic or persistent viral infections, which are viral infections that are able to infect a host and reproduce within the cells of a host over a prolonged period of time-usually weeks, months or years, before proving fatal.
  • Viruses giving rise to chronic infections that which may be treated in accordance with the present invention include, for example, the human papilloma viruses (HPV), Herpes simplex and other herpes viruses, the viruses of hepatitis B and C as well as other hepatitis viruses, and the measles virus, all of which can produce important clinical diseases. Prolonged infection may ultimately lead to the induction of disease which may be, e. g., in the case of hepatitis C virus liver cancer, fatal to the patient.
  • Other chronic viral infections which may be treated in accordance with the present invention include Epstein Barr virus (EBV), as well as other viruses such as those which may be associated with tumours.
  • viral infections can be treated through the administration of a viral antigen and IL-7.
  • viral antigen is meant a substance recognized by the adaptive immune system that stimulates the body to find and eliminate a pathogen causing a viral infection.
  • the viral antigen may be, for example, a viral protein or a portion of a viral protein.
  • Methods for generating a viral antigen for use in the present invention are know to those skilled in the art and are also discussed herein with respect to tumor antigens.
  • treatment or “amelioration” of a viral infection is meant that a therapy (e.g., a vaccine or an anti-viral drug), administered either alone or in combination with other therapies, alleviates the viral infection in at least some patients to which the therapy is administered.
  • a therapy e.g., a vaccine or an anti-viral drug
  • treatment of a viral infection may result in decreasing or eradicating the viral load in the patient, or may result in a decrease or elimination in one or more symptoms associate with a viral infection.
  • the dosage regimen for treatment of a subject with the viral antigen and IL-7 is based on a variety of factors, including the type of mammal to be treated, the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. Methods for the delivery of the IL-7 and viral antigen are known to those skilled in the art. A number of delivery routes are available for injection of the therapy, including intratumoral, subcutaneous, intradermal, intravenous and intraperitoneal, routes. Other modes of delivery include topical, oral and nasal delivery, using biocompatible particles, such as liposomal formulations.
  • the therapeutic methods of the present invention may be provided to a subject alone as a single therapy, or may be used in combination with other therapies such as chemotherapy or radiotherapy, or drug therapy.
  • Mammals that can be treated according to the methods of the present invention include humans, apes, cats, dogs, mice, gorillas, hamsters and horses. In one embodiment, the mammal is a human.
  • the invention also features pharmaceutical compositions for treating a tumor or a viral infection in a mammal.
  • the pharmaceutical composition for treating a tumor in a mammal comprises IL-7, an antigen of the tumor and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition for treating a viral infection in a mammal comprises IL-7, a viral antigen and a pharmaceutically acceptable carrier.
  • a "pharmaceutically acceptable carrier” means a carrier that is physiologically acceptable to the treated subject while retaining the therapeutic properties of the compound with which it is administered.
  • One exemplary pharmaceutically acceptable carrier is physiological saline.
  • Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's
  • the pharmaceutical compositions can be formulated in general by mixing the IL-7 or the antigen with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions may additionally include one or more of the following reagents: excipients, buffers, stabilisers or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier and other additional reagents will depend on the route of administration, which may be by any convenient route for those of skill in the art, though is preferably by injection, e. g., cutaneous, subcutaneous, intra-dermal, intratumoral or intraperitoneal.
  • the pharmaceutical composition and methods described herein for treating a tumor or a viral infection may include an adjuvant.
  • adjuvants that can be used include, for example, a mineral oil emulsion, an aluminum compound, and a surface active material such as saponin, lysolecithin, retinal, Quil A.RTM., some liposomes, and pluronic polymer formulations. See, for example, Fundamental Immunology, edited by William E. Paul, at p. 1008, Raven Press, New York 1989. Techniques for preparing adjuvant-antigen preparations for injection are well known in the art. See, for example, Terry M. Phillips, Analytical Techniques in Immunochemistry, pp. 307-10, Marcel Dekker, New York, 1992. Combinations of adjuvants can also be used.
  • An adjuvant can be administered prior to, simultaneously with, or following the administration of the tumor or viral antigen and IL-7.
  • the active ingredient is preferably in the form of a parenterally acceptable aqueous solution which has suitable pH, isotonicity and stability.
  • Oral administration may be used, in which case the pharmaceutical composition may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • the pharmaceutical formulations can be packaged into kits for easy delivery of the formulations to a subject in need of therapy.
  • the kit may include one or more containers containing a tumor or viral antigen and one or more containers containing IL-7.
  • the antigen and/or IL-7 may each be already formulated with a pharmaceutically acceptable carrier and/or other excipients, or they may each be contained in individual containers for combination prior to administration.
  • the kit may further comprise other excipients as well as instructions for combining and administering the contents of the kit to the subject in need of therapy.
  • Example 1 IL-7 prolongs survival of vaccinated tumor-bearing mice
  • mice expressing the SV40 large T antigen (TAG) under the control of the rat insulin promoter (RIP), also known as RIP-T AG2 mice, develop pancreatic ⁇ -islet cell tumors. As the tumors grow, they progressively secrete more insulin and blood glucose readings can be used to monitor tumor burden. These mice have been crossed with transgenic animals expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) under the control of RIP to produce RIP (GP x TAG2) mice.
  • TAG SV40 large T antigen
  • RIP rat insulin promoter
  • mice Although tumors in the intercrossed mice express GP they are not cleared by the immune system. If these mice are infected with LCMV, mimicking a live viral anti-tumor vaccine, a limited increase in survival is observed (FIG. IA). This abrogated effect is likely due to the termination of the immune response coincident with the eradication of virus despite the persistence of antigen in tumors and memory T cells in the host. Therefore the virally induced anti-tumor immune response cannot be maintained by the tumor, indicating the need for more complex approaches to augment and sustain anti-tumor immunity in vivo.
  • One approach is to treat LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice with IL-7.
  • C57BL/6 (H-2 b ), CD45.1 congenic C57BL/6 and Thyl.l congenic C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
  • the derivations of RIP-GP, RIP-T AG2, RIP (GP x TAG2), Pl 4 and SMARTA transgenic mice and Cbl-b deficient mice have been described previously (Hanahan, D., Nature 315, 115-122 (1985); Ohashi, P.S., et al, Cell 65, 305-317 (1991); thoughr, supra; Oxenius, A., Bachmann, M.F., Zinkernagel, R.M.
  • mice were kept in a specific pathogen-free environment until infected with LCMV at which time animals were moved to a quarantine room. Mice were maintained at the Ontario Cancer Institute (Toronto, Canada) animal facility according to institutional guidelines. Blood glucose levels were measured from blood obtained from the tail veins of tumor mice using the Accu Check Advantage kit according to the manufacturer's instructions (Roche Diagnostics, Basel, Switzerland). Testing commenced when mice were 6 weeks old and continued weekly until two consecutive readings below 5 mM were obtained at which stage mice were assigned to an experimental protocol.
  • mice of the indicated genotypes were immunized i.v. with LCMV Armstrong strain (3,000 PFU except P14 mice which received 2 x 10 5 PFU).
  • Tumor mice i.e., RIP (GP x TAG2) mice
  • mice Eight days after infection, which coincided with the peak of the CTL response, viral elimination and the onset of the contraction phase, mice were injected i.p. twice daily with 10 ⁇ g o ⁇ E.coli derived recombinant IL- 7 or phosphate-buffered saline (PBS) for 2 weeks.
  • PBS phosphate-buffered saline
  • rgIL-7 fully glycosylated IL-7
  • rgIL-7 (Cytheris) was injected subcutaneously once daily.
  • LCMV infected RIP GP x TAG2 mice receiving IL-7 twice daily for two weeks had prolonged survival rates as compared to RIP (GP x TAG2) mice treated with PBS twice daily alone.
  • the long-term survival of IL-7-treated RIP GP x TAG2 mice was enhanced by more than 100 days compared to control mice (FIGs. IA and 1C). This profound increase in survival was dependent on efficient vaccination and the expression of GP on the tumor, as the effect was not observed in uninfected RIP (GP x TAG2) mice treated with IL-7 nor in IL-7 treated LCMV-infected RJP-T AG2 mice (FIG. IA).
  • Glucose measurements were performed on C57BL/6 mice, RIP (GP x TAG2) mice treated with either IL-7 or with PBS and RIP-T AG2 mice after LCMV infection and during and after treatment with PBS or IL-7.
  • the LCMV infected, IL-7-treated RIP (GP x TAG2) mice tended to have a faster onset and more pronounced level of euglycemia or mild hyperglycemia than PBS- treated, LCMV infected RIP (GP x TAG2) mice, reaching statistical significance at late time points, suggesting an enhanced anti-tumor response (FIG. IB).
  • PBS-treated RIP GP x TAG2 mice had died.
  • Example 2 Anti-tumor immune responses are magnified and sustained in mice treated with IL-7 after LCMV infection
  • LCMV-vaccinated RIP mice administered IL-I or PBS, as described in
  • Example 1 were sacrificed at the completion of IL-7 or PBS treatment or 1 week following the last dose of IL-7 or PBS. Sections of the mice's pancreases were stained for CD4 and CD8 cells. This staining revealed that the tumors in the IL-7-treated mice were heavily infiltrated with both CD4 + and CD8 + T cells compared to PBS-treated mice which consistently had larger tumors with fewer immune cells (FIG. 2A). The lymphocytic infiltrates observed in IL-7-treated mice persisted even 1 week after the cessation of therapy (FIG. 2A).
  • the absolute numbers of lymphocytes, granulocytes and macrophages in the pancreatic draining lymph nodes (PDLN), the non-draining inguinal lymph nodes (ILN), the spleen and the pancreases of the LCMV-vaccinated RIP (GP x TAG2) mice at the completion of 2 weeks of IL-7 or PBS treatment were also quantitated.
  • IL-7-treated tumor mice showed a 3.5 to 10-fold increase in both CD4 + and CD8 + T cell numbers compared to PBS controls (FIG. 2B).
  • B cell numbers were also increased in IL-7-treated mice but there were no differences in the number of granulocytes or macrophages in the two treatment arms.
  • Tetramers were produced by conjugating monomers, H-2D b /GP33 (KAVYNFATM (SEQ ID NO.: I)), H-2D b /GP276 (SGVENPGGYCL (SEQ ID NO.: 2)) and H-2D b /NP396 (FQPQNGQFI (SEQ ID NO.: 3)), with Extravidin-PE (Sigma).
  • Monomers were obtained from the Canadian Network for Vaccines and Immuno therapeutics (Montreal, QC). Cells were stained with tetramers for 1 hr at 4 0 C and were subsequently analyzed by flow cytometry.
  • IL-7 therapy did not skew the representation of GP33- and GP276-specific CD8 T cells compared to PBS controls.
  • a significant but less pronounced increase in GP33- and GP276-specific CD8 + T cells was also seen in the spleen (FIG. 2B) and ILN. This difference may reflect continued and enhanced expansion in nodes draining the tumors.
  • the increase in antigen-specific cells persisted after the cessation of IL-7 treatment but consistent with the importance of this survival factor, the cell numbers slowly declined with time (FIG. 2C).
  • pancreatic infiltrating leukocytes were isolated from tumor-bearing RIP (GP x TAG2) mice that had been infected with LCMV and treated for 7 days with PBS or IL-7.
  • the absolute numbers of CD4 GP61 -specific T cells was quantitated in the pancreases of tumor mice after 7 days of IL-7 or PBS treatment by determining the proportion of cells producing cytokine (TNF- ⁇ , IFN- ⁇ and IL-2) after ex vivo stimulation with GP61 peptide.
  • T cell depleted splenocytes were obtained using IMag cell separation system (BD Pharmingen) and were used to in vitro restimulate antigen-specific cells.
  • the T cell-depleted preparations were plated at a density of 3x10 6 cells/well in a 24 well plate and 1 ⁇ g/ml of GP61 peptide (GLNGPDIYKGVQFKSVEFD (SEQ ID NO.: 4), New England Peptide), or an irrelevant OVA323 control peptide (IS Q A VH AAH AEINE AGR (SEQ ID NO.: 5)) was added to the cultures.
  • pancreatic infiltrating leukocytes 4 ⁇ l ⁇ 5 purified pancreatic infiltrating leukocytes were added to each well in the presence of the intracellular protein transport inhibitor monensin (BD Pharmingen). Cells were harvested after 4 h and labeled with a CD4 mAb, then fixed overnight with 2% paraformaldehyde. After fixation cells were permeabilized with Perm/Wash solution (BD Pharmingen) and then incubated for 30 min with mAb specific for TNF- ⁇ , IFN- ⁇ and IL-2.
  • BD Pharmingen intracellular protein transport inhibitor monensin
  • IL-7-treated RIP GP x TAG2 mice
  • IL-7-treated RIP GP x TAG2 mice
  • FIG. 7A This indicates that the residual levels of IL-7 receptor are sufficient to support T cell survival and proliferation when exogenous IL-7 is administered.
  • IL-7 could be modulating and improving antigen presenting cell (APC) function and thereby enhancing T cell priming. This latter possibility however is less likely as IL-7 is administered well after LCMV infection and hence subsequent to priming of the immune response.
  • APC antigen presenting cell
  • virus activated GP-specific CD8 + or CD4 + T cells were isolated from mice expressing a T cell receptor (TCR) specific for the LCMV-GP33/H-2D b or the LCMV-GP61/I-A b restricted epitopes respectively (known as P14 and SMARTA mice, respectively).
  • TCR T cell receptor
  • P14 or SMARTA TCR transgenic mice were made as previously described (Oxenius et al., supra; Pircher et al., surpr ⁇ ).
  • P14 or SMARTA TCR transgenic mice were first infected with LCMV and at the height of the immunological response, just prior to the contraction phase, splenic T cells were isolated.
  • Single cell suspensions were prepared from the splenic T cells and CD8 + or CD4 + T cells, respectively, were purified by magnetic bead negative depletion according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in media alone or with the addition of 500 ng/ml IL-7 at a density of 5 x 10 5 cells/ml. At specified time points cell survival was assayed using P.I. staining and flow cytometry.
  • Labeled cells were adoptively transferred to C57BL/6 mice that had been infected with LCMV 8 days earlier.
  • the mice were treated with PBS or IL-7, and after 6 days of PBS or IL-7 treatment, the mice's spleens and ILN were harvested.
  • CD45.1 + P14 CD8 + T cells were analyzed by flow cytometric analysis.
  • the histograms presented in FIG. 3B show the proportion of cells that have divided and diluted their CFSE.
  • FIG. 3B when in vivo primed antigen-specific CD8 + CFSE- labeled T cells were adoptively transferred into hosts that had been infected with LCMV 8 days previously, it was determined that 6 days of IL-7 treatment enhanced both survival of cells and expansion as evidenced by the dilution of the CFSE label (FIG. 3B).
  • This experiment was repeated with 3 mice in each treatment group and histograms are representative of all mice. In contrast, the same cells transferred into PBS-treated mice failed to proliferate and did not persist in the host.
  • Example 4 Antigen-specific T cells treated in vivo with IL-7 show enhanced cytolytic activity
  • IL-7 antigen-specific T cells
  • PILs were isolated from LCMV-vaccinated tumor-bearing RIP (GP x TAG2) mice after 7 days of IL-7 treatment.
  • PILS were isolated as follows. Pancreatic tissue was dissected into small pieces and incubated for 30 min in Hanks balanced salt solution (HBSS, Gibco Invitrogen, Carlsbad, CA) with the addition of EDTA (1.3mM) at 37°C.
  • HBSS Hanks balanced salt solution
  • Tissue was recovered by centrifugation and resuspended in RPMI 1640 (Invitrogen) supplemented with 0.7 mg/ml Collagenase D (Roche) and lOU/ml DNase I (Roche). After 1 hr of collagenase/DNase digestion at 37°C, tissue debris was filtered through a 70 ⁇ m cell strainer and centrifuged. The pellet was re-suspended in 5 ml 44% Percoll and underlayed with 5 ml 67.5% Percoll and subsequently centrifuged for 20 min at 1600 rpm.
  • the leukocyte- enriched fraction was collected from the 44%-67.5% Percoll interphase and washed in HBSS supplemented with 5% fetal calf serum (Hyclone, Logan, UT).
  • the cytolytic activity of PILs was determined by their ability to kill peptide pulsed target cells (i.e., GP33-specific cytotoxic activity). Briefly, EL-4 target cells were incubated with 200 ⁇ Ci 51 Cr and 10 "8 M GP33 peptide or non-stimulatory AV peptide (SGPSNTPPEI) (SEQ ID NO: 6) for 1 h at 37°C.
  • the target cells were washed three times and plated at 2000 cells per well with serially diluted PIL effectors, isolated from experimental mice as described, in 96-well round-bottomed plates. Plates were centrifuged at 800 rpm for 1 min and incubated for 4 h at 37 0 C. Supernatant activity was measured using a Wallac Wizard counter (Perkin Elmer). Percentage of specific lysis was calculated as (cpm sample release - cpm spontaneous release)/(cpm maximal release - cpm spontaneous release) x 100.
  • GP33-specific pancreatic infiltrating CD8 lymphocytes isolated from vaccinated IL-7-treated mice showed a more rapid and robust kinetic and capacity to degranulate (FIGs. 3D and 3E).
  • Degranulation was measured as follows. PILs were mixed with GP33-pulsed (10 "8 M) EL- 4 cells at a concentration of 5xl0 5 /ml and 10 6 /ml, respectively and kept on ice. The mix was then supplemented with 1 ⁇ l/ml of monensin solution (100Ox stock from e-bioscience), 1 ⁇ l/ml GolgiPlug (BD Biosciences) and 5 ⁇ g/ml CD107a-FITC Ab.
  • the cells were plated in round- bottomed 96-well plates (200 ⁇ l/well) and rapidly brought up to 37°C to allow degranulation. Samples were then harvested at the indicated times and kept on ice until the last sample was collected. Cells were then washed and stained with CD8 and H-2Db/GP33 tetramers as described above. CD 107a levels were then assessed on CD8 + H-2Db/GP33 + cells by flow cytometry analysis, gating GP33-tetramer + CD8 + cells.
  • GP33-specific pancreatic infiltrating CD8 lymphocytes isolated from vaccinated IL-7-treated mice also showed a log increase in granzyme B levels (as assayed by collecting PILs as described above, labeling them with a CD8 mAb, and fixing, permeabilizing and staining directly ex vivo with mAb specific for granzyme B).
  • Example 5 IL-7 creates an IL-2, TH 17 and NK-rich milieu
  • the enhanced killer activity of antigen-specific T cells from RIP could be a direct effect of IL-7 on CTL or alternatively IL-7 may indirectly create a milieu that is more conducive to efficient effector activity.
  • T H 17 CD4 T cells that secrete IL- 17 (T H 17) (which are known to be critical in promoting inflammation and inducing pathology in several animal models of autoimmunity) were studied.
  • the CD4 T cells in the PIL populations isolated from LCMV -vaccinated tumor-bearing RIP (GP x TAG2) mice after 7 days of IL-7 or PBS treatment were characterized by re- stimulating these cells ex vivo and analyzing their cytokine production.
  • the cells were cultured in the presence of PMA (100 ng/niL), ionomycin (100 ng/mL) and monensin for 4h. The cells were then harvested labeled, fixed, permeabilized, stained with mAb specific for IL- 17 and IL-2 and assessed for IL2 and IL- 17 levels. In addition to an 1 1-fold increase in the proportion of cells that produce IL-2, there was an 18-fold increase in the number of IL- 17 producing CD4 T cells in IL-7-treated mice (FIGs. 3F and 3G). A similar increase was also seen in the number of NK and NKT cells infiltrating the pancreas of IL-7-treated LCMV -vaccinated tumor mice (FIG. 3H). These increases in IL-2 producing cells, NK and T H 17 cells in tumor mice treated with IL-7 versus PBS may create a pathogenic milieu that overcomes tumor- associated inhibitory factors.
  • Example 6 Enhanced cytokine production in IL-7-treated mice after LCMV infection
  • mice were injected intravenously with 20 ⁇ g IL-7 or PBS and sacrificed 1 hour later for serum collection (na ⁇ ve lhr), alternatively na ⁇ ve mice were injected subcutaneously once daily for 7 days with 10 ⁇ g IL-7 or PBS and serum was subsequently collected (na ⁇ ve 7d).
  • LCMV- infected mice received 10 ⁇ g of IL-7 or PBS subcutaneously once daily for 5 days commencing 3 days post infection or 8 days post infection after which serum was collected for cytokine analysis. Blood from the mice was collected post mortem by cardiac puncture and serum cytokine levels were assayed using the SearchLight Array Service (Pierce Biotechnology, Woburn, MA).
  • IL-7 did not result in any significant increase in cytokine levels 1 hr post treatment compared to PBS-treated mice (FIG. 4).
  • Daily subcutaneous injection of IL-7 over 7 days also had little effect on serum cytokine levels.
  • a dramatic increase in numerous cytokines was observed when IL-7 was administered daily subcutaneously for five days starting 3 or 8 days following LCMV infection.
  • Three to ten fold increases in serum IL-6, IL- l ⁇ , IL-I ⁇ , IL-12, IL-18, IFN ⁇ , TNF- ⁇ , Rantes, MIPl ⁇ and MIP l ⁇ were observed (FIG. 4).
  • Example 7 IL- 7 treatment renders T effector cells refractory to inhibitory networks
  • IL-7-treated mice could be due to the suppression of negative regulators of immune responses. It has been suggested that IL-7 may inhibit the ability of Treg to suppress effector cells in vitro.
  • LCMV-vaccinated IL-7-treated RIP GP x TAG2
  • tumor-bearing mice there was an increase in Foxp3 + Treg numbers in ILN, PDLN and in the pancreas. Foxp3 staining was performed using a mouse regulatory T cell staining kit (eBioscience San Diego, CA).
  • IL-7 when the increase in Foxp3 + Treg numbers was compared to the increase in total T cell numbers IL-7 was found to be associated with a reduction in the representation of Treg amongst total T cells in ILN and PDLN. This is consistent with the finding described herein that IL-7 promotes a cytokine milieu that favors T H I 7 differentiation over Tregs.
  • IL-7 may suppress the function of Treg or alternatively it may render antigen-specific effector cells refractory to inhibition.
  • T effector or Treg were isolated from mice treated for 3 days with IL-7 or PBS and an in vitro suppression assay was performed as follows.
  • CD4 + effector cells and CD4 + CD25 + regulatory T cells were isolated from BL57B/6 mice pre-treated with either IL-7 or PBS using a CD4 + CD25 + Regulatory T cell isolation kit (Miltenyi Biotec Bergisch Gladbach, Germany). The manufacturer's protocol was modified by ultra purifying the negatively depleted CD4 + T cell fraction by performing 2 depletion runs using the autoMACS Separator (Miltenyi Biotec Bergisch Gladbach, Germany).
  • CD4 + cells were then positively sorted for CD25 + cells and the CD4 + CD25 " effector cells were collected in the negative fraction.
  • the latter cells were ultra purified by using a CD4 + negative selection kit (Miltenyi Biotec Bergisch Gladbach, Germany).
  • CD4 + CD25 + regulatory T cells were plated in 96 well round-bottom plates starting at 10 5 cells per well (2:1 Treg:Teffector ratio) and cells were then serially diluted two fold.
  • CD4 + effector cells were added to wells along with 1.5 x 10 5 APC and 0.25 ⁇ g/ml CD3 mAb (eBioscience San Diego, CA) in medium alone or medium supplemented with 500 ng/ml IL-7.
  • Proliferation of CD4 + CFSE-labeled and P.I. negative cells was assessed using a FACSCanto (BD, Franklin Lakes, NJ) and Flow Jo software (Tree Star, Ashland, OR) after 3 days.
  • FACSCanto BD, Franklin Lakes, NJ
  • Flow Jo software Te Star, Ashland, OR
  • Treg are not the only inhibitory network that may function in tumors.
  • Factors such as TGF- ⁇ , IL-10, PDL-I and PDL-2 may all synergize in abrogating potential anti-tumor and antiviral responses. It has been proposed that IL-7 may down regulate TGF- ⁇ production and signaling in fibrosarcomas.
  • Tumors in uninfected RIP-T AG2 mice produce TGF- ⁇ and express PDL-I but not PDL-2 (FIG. 5C) and this was not affected by 7 days of IL-7 treatment.
  • Tumor-infiltrating T cells would likely be susceptible to inhibition given their high expression of PD-I, as assessed by treating LCMV-infected tumor bearing RIP (GP x TAG2) mice were for 1 week with IL-7 or PBS, harvesting the PILs, and measuring PD-I expression on CD8 and CD4 T cells (FIG. 5D).
  • C57/BL6 mice were isolated using a CD8+ T cell isolation kit (Miltenyi Biotec), CFSE-labeled and plated with APC and CD3 as described above for CD4 T cells in the presence or absence of 10 ng/ml human TGF- ⁇ 1 (Peprotech) and also in the presence of absence of IL-7 or PBS. Proliferation was assessed by flow-cytometry 3 days later. It was found that TGF- ⁇ -induced inhibition of CD8 T cell proliferation in vitro was reversed by the addition of IL-7 to the culture medium (FIG. 5E).
  • the refractory state of IL-7-treated effector cells to both Treg and TGF- ⁇ effects may contribute to the enhanced capacity of these cells to degranulate and kill targets as Treg have been reported to inhibit the degranulation efficiency of tumor-specific CD8 T cells in a TGF- ⁇ -dependent manner.
  • Example 8 IL-7 impairs in vivo TGF- ⁇ signaling
  • IL-7 was injected intravenously or subcutaneously in na ⁇ ve or LCMV-infected C57BL/6mice and T cells were harvested and sorted at various time points (1 hour, 7 days or 5 days (starting after a 3 day post LCMV infection) (d3-8)).
  • Direct ex vivo western blot analysis was performed to interrogate intracellular signaling pathways, avoiding any potential in vitro artifact.
  • Western blot analyses were performed as follows.
  • the indicated cell populations were directly sorted from spleens using Miltenyi isolation kits and magnetic column depletion (Miltenyi Biotec, Auburn, CA). Cell suspensions were strictly kept on ice and separations were performed at 4°C. Sorted cells were lysed in buffer containing 150 mM NaCL, 2OmM HEPES (pH 7.0), 10% glycerol, 1% Triton X- 100, protease and phosphatase inhibitors. Lysates were boiled for 5 min in SDS loading buffer and resolved using SDS-PAGE and western blot analysis.
  • Stat3, Stat5, Smad2, Smad3, pSmad2, pSmad3, p27 and Smad7 Cell Signaling, Danvers, MA
  • pStat5, FOX03a Millipore/Upstate, Lake Placid, NY
  • Cbl-b Santa Cruz Technology, Santa Cruz, CA
  • biotin-TGF- ⁇ RII Ab R & D Systems, Minneapolis, MN
  • Smurf2 and Survivin Novus, Littleton, CO.
  • Both Stat5 and Stat3 were phosphorylated in CD8 T cells harvested and sorted from na ⁇ ve mice lhr after intravenous injection of 20 ⁇ g of IL-7 (FIG. 6A).
  • phosphorylation could not be detected if na ⁇ ve mice were treated for 7 days with daily subcutaneous injections of 10 ⁇ g of IL-7 nor if LCMV-infected mice were treated for 5 days with daily subcutaneous injections starting 3 days post infection. This most likely reflects the transient nature of the phosphorylation events or the asynchronous activation of cells following prolonged treatment. Similarly, phosphorylation of AKT or ERK1/2 was undetectable at all time points. However, the downstream degradation of FOXO3a and p27 were apparent at all time points and in all conditions following IL-7 treatment (FIG. 6A).
  • Example 9 IL-7 modulates TGF- ⁇ signaling by regulating Smurf2 expression
  • TGF- ⁇ receptor II A downregulation of TGF- ⁇ receptor II was observed in all IL-7 treatment regimens described in Example 8 (FIG. 6C).
  • Smurf2, a C2-WW-HECT domain ubiquitin ligase, and Smad7 have been shown to target Smad2 and or TGF- ⁇ receptor for degradation. Indeed, consistent with the refractory nature of T cells towards TGF- ⁇ inhibition, it was found that Smurf2, but not Smad7, was upregulated in CD8 T cells treated in vivo with IL-7 under all conditions (FIG. 6C). Recently, the relative proportions of TGF- ⁇ and IL-6 have been implicated in skewing cells towards a Treg or pro-inflammatory T H 17 phenotype.
  • TGF- ⁇ synergizes with IL-6 and other cytokines to favor T H 17 differentiation.
  • IL-7 appears to compromise but not completely block TGF- ⁇ signaling and hence in the presence of augmented IL-6 serum levels found in IL-7-treated LCMV-infected mice it would strongly favor the differentiation of T H I 7 cells in vivo.
  • These molecular events may explain thel ⁇ fold increase in the number of IL- 17 producing CD4 T cells in the PIL population of IL-7-treated tumor bearing RIP (GP x TAG2) mice (FIGs. 3F and 3G).
  • Example 10 Cbl-b levels in T cells are negatively regulated by IL-7 in vivo
  • Cbl-b deficient T cells are known to be resistant to TGF- ⁇ and Treg suppression much like IL-7-treated cells.
  • the question of whether IL-7 could be regulating Cbl-b levels in T cells was investigated next be measuring Cbl-b levels in the C57BL/6 mice treated as described in Example 8.
  • Cbl-b levels in CD8 T cells FIG. 6D.
  • Human T cells were purified from Ficoll enriched PBMCs using a human CD8 T cell isolation kit (Miltenyi Biotec, Auburn, CA).
  • the human cells were incubated with 500 ng/mL IL-7 for 1 hour then harvested, lysed and analyzed using western blot techniques (FIG. 6E).
  • the differences in Cbl-b levels and TGF- ⁇ signaling in CD8 T cells isolated from mice treated with IL-7 for prolonged periods may be due to an expansion of phenotypically different cells. No significant differences were observed in activation markers between CD8 T cells isolated from IL-7 or PBS-treated LCMV-infected mice at day 8 post infection when cells were harvested for western blot analysis.
  • FIG. 7A Most activated T cells isolated from C57BL/6 mice on day 8 following LCMV infection express low levels of IL-7 receptor- ⁇ (CD127) (FIG. 7A). The data herein suggest that despite these low IL-7 receptor levels, cells are still able to respond to exogenously administered IL-7. This is further supported by the finding that CD127 10 T cells harvested from LCMV-infected C57BL/6 mice rapidly expand when transferred into LCMV-infected recipients receiving IL-7 compared to PBS (FIGs. 7B to 7D).
  • CD8 and CD4 T cells were sorted from the spleens of LCMV-infected Thy 1.1 or CD45.1 congenic mice 8 days post infection using a FACSAria (BD, Franklin Lakes, NJ). Approximately 0.5-3 x 10 6 of each cell population were CFSE labeled and transferred into Thyl .2 host mice which had been infected with LCMV 8 days previously. Recipient mice were treated with IL-7 or PBS administered subcutaneously every day, after 7 days cells were recovered and analyzed by flow cytometry.
  • CD4 and CD8 T cells amongst the PIL population in tumor bearing RIP (GP x TAG2) mice treated with PBS or IL-7 for 9 days expressed high CD 127 levels (FIG. 7E). This may reflect reacquisition of high CD 127 expression after clearance of LCMV from these tissues or de novo priming of cells in the PDLN that do not downregulate CD127 in the absence of acute viral infection.

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Abstract

L'invention concerne des procédés pour traiter une tumeur chez un mammifère, le procédé comportant l'administration au mammifère d'interleukine-7 (IL-7) et d'un antigène tumoral en une quantité suffisante pour induire une réponse immunogène chez celui-ci, des compositions pharmaceutiques et sur des kits comportant de l'IL-7 et un antigène tumoral. L'invention concerne également des procédés pour traiter une infection virale chez un mammifère, le procédé comportant l'administration au mammifère d'IL-7 et d'un antigène viral en une quantité suffisante pour induire une réponse immunogène chez celui-ci, des compositions pharmaceutiques et sur des kits comprenant de l'IL-7 et un antigène viral.
PCT/CA2008/001653 2007-09-19 2008-09-19 Procédés et compositions pour traiter des tumeurs et des infections virales WO2009036568A1 (fr)

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WO2011051657A1 (fr) * 2009-10-26 2011-05-05 St George's Hospital Medical School Protéine a comme adjuvant de vaccin
WO2016196211A1 (fr) * 2015-05-29 2016-12-08 Armo Biosciences, Inc. Procédés d'utilisation de l'interleukine-10 pour le traitement de maladies et de troubles
WO2017062491A1 (fr) * 2015-10-05 2017-04-13 Jing-Feng Huang Compositions thérapeutiques pour le traitement du syndrome de l'œil sec et de maladies de surface oculaire associées
US9823255B2 (en) 2013-06-17 2017-11-21 Armo Biosciences, Inc. Method for assessing protein identity and stability
US9943568B2 (en) 2013-04-18 2018-04-17 Armo Biosciences, Inc. Methods of using pegylated interleukin-10 for treating cancer
US10010588B2 (en) 2013-08-30 2018-07-03 Armo Biosciences, Inc. Methods of using pegylated interleukin-10 for treating hyperlipidemia
US10143726B2 (en) 2014-10-22 2018-12-04 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders
US10195274B2 (en) 2015-05-28 2019-02-05 Armo Biosciences Inc. Method of modulating a chimeric antigen receptor t cell immune response by administering IL-10
US10293043B2 (en) 2014-06-02 2019-05-21 Armo Biosciences, Inc. Methods of lowering serum cholesterol
US10350270B2 (en) 2014-10-14 2019-07-16 Armo Biosciences, Inc. Interleukin-15 compositions and uses thereof
US10398761B2 (en) 2015-08-25 2019-09-03 Armo Biosciences, Inc. Methods of using combinations of PEG-IL-10 and IL-15 for treating cancers
US10618970B2 (en) 2015-02-03 2020-04-14 Armo Biosciences, Inc. Method of treating cancer with IL-10 and antibodies that induce ADCC
US11413332B2 (en) 2013-11-11 2022-08-16 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011051657A1 (fr) * 2009-10-26 2011-05-05 St George's Hospital Medical School Protéine a comme adjuvant de vaccin
US10357545B2 (en) 2013-04-18 2019-07-23 Armo Biosciences, Inc. Methods of using interleukin-10 for treating solid tumors
US9943568B2 (en) 2013-04-18 2018-04-17 Armo Biosciences, Inc. Methods of using pegylated interleukin-10 for treating cancer
US9823255B2 (en) 2013-06-17 2017-11-21 Armo Biosciences, Inc. Method for assessing protein identity and stability
US10010588B2 (en) 2013-08-30 2018-07-03 Armo Biosciences, Inc. Methods of using pegylated interleukin-10 for treating hyperlipidemia
US11413332B2 (en) 2013-11-11 2022-08-16 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders
US10293043B2 (en) 2014-06-02 2019-05-21 Armo Biosciences, Inc. Methods of lowering serum cholesterol
US10350270B2 (en) 2014-10-14 2019-07-16 Armo Biosciences, Inc. Interleukin-15 compositions and uses thereof
US10143726B2 (en) 2014-10-22 2018-12-04 Armo Biosciences, Inc. Methods of using interleukin-10 for treating diseases and disorders
US10653751B2 (en) 2014-10-22 2020-05-19 Armo Biosciences Inc. Methods of treating cancer metastasis by using interleukin-10
US10618970B2 (en) 2015-02-03 2020-04-14 Armo Biosciences, Inc. Method of treating cancer with IL-10 and antibodies that induce ADCC
US10195274B2 (en) 2015-05-28 2019-02-05 Armo Biosciences Inc. Method of modulating a chimeric antigen receptor t cell immune response by administering IL-10
WO2016196211A1 (fr) * 2015-05-29 2016-12-08 Armo Biosciences, Inc. Procédés d'utilisation de l'interleukine-10 pour le traitement de maladies et de troubles
US10398761B2 (en) 2015-08-25 2019-09-03 Armo Biosciences, Inc. Methods of using combinations of PEG-IL-10 and IL-15 for treating cancers
WO2017062491A1 (fr) * 2015-10-05 2017-04-13 Jing-Feng Huang Compositions thérapeutiques pour le traitement du syndrome de l'œil sec et de maladies de surface oculaire associées

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