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WO2008051220A1 - Interaction d'il-27 et d'il-2 dans le traitement de tumeurs - Google Patents

Interaction d'il-27 et d'il-2 dans le traitement de tumeurs Download PDF

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WO2008051220A1
WO2008051220A1 PCT/US2006/041534 US2006041534W WO2008051220A1 WO 2008051220 A1 WO2008051220 A1 WO 2008051220A1 US 2006041534 W US2006041534 W US 2006041534W WO 2008051220 A1 WO2008051220 A1 WO 2008051220A1
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administration
tumor
cells
tbj
administered
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Rosalba Salcedo
Jon M. Wigginton
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US Department of Health and Human Services
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    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • 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]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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 A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 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 A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • the present disclosure is generally related to methods of treating cancer, for example methods for treating neuroblastoma, melanoma, lymphoma, leukemia, and renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma.
  • Neuroblastoma is the most common extracranial solid tumor in children, with highly variable biologic features and clinical outcome.
  • the prognosis of patients with high-risk neuroblastoma is poor overall, and has fueled an intense effort to develop new therapeutic approaches, particularly for those with widespread metastatic disease.
  • imrnunotherapeutic agents including cytokines in preclinical tumor models and/or early phase clinical studies in children with neuroblastoma.
  • IL-2 interleukin-2
  • IL- 12 interleukin- 12
  • the clinical efficacy of single cytokines or immunocytokines in treating neuroblastoma has been modest, and is associated with significant deleterious side effects that limit the usefulness of these therapies.
  • an effective anti-cancer therapeutic for the treatment of neuroblastoma as well as other types of cancer, such as melanoma, lymphoma, leukemia, and renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma.
  • one embodiment of the disclosure is a method of treating tumors such as neuroblastoma, melanoma, leukemia, lymphoma, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma or lung carcinoma in a subject.
  • the method includes administering to the subject a therapeutically effective amount of IL-2 and a therapeutically effective amount of IL- 27, thereby treating the tumor.
  • the IL-2 and IL-27 are administered in a manner that the two interleukins interact to provide an improved tumor response.
  • the two interleukins can, for example, be administered concurrently, substantially concurrently, or in any other manner that allows their improved therapeutic effect to be achieved.
  • IL-2 is intermittently administered while IL-27 is continuously administered (for example by constitutive expression, such as local expression at a tumor site).
  • a method for treating metastatic neuroblastoma in a subject which method includes administering to the subject a therapeutically effective amount of IL-2 and a therapeutically effective amount of IL-27.
  • IL-2 is administered systemically and the IL-27 is administered locally, and this combination treats the metastatic neuroblastoma.
  • FIG l is a pair of graphs showing that IL-27 and IL-2 synergistically enhance proliferation and IFN- ⁇ production by murine splenocytes.
  • Normal murine splenocytes were cultured in the presence or absence of IL-27 (50ng/ml) +/- IL-2 (50 IU/ml) or medium alone for 48 hours.
  • Proliferative responses in the respective groups were assessed by 3 H-thymidine (1 ⁇ Ci/well) incorporation as described in Example 1. Values shown represent the mean value +/- standard error of the mean (SEM) for triplicate samples (FIG IA).
  • FIG 2 is a survival curve showing that combined delivery of IL-27 and IL-2 mediates complete tumor regression and long term survival in mice bearing disseminated TBJ neuroblastoma metastases.
  • Cohorts of 10 A/J mice per group were injected intravenously with 1 x 10 5 TBJ FLAG or TBJ IL-27 neuroblastoma tumor cells on day 0. Mice were then treated with either IL-2 or medium alone on days 5-9, 12-16, 19-23, and 26-30 post-tumor implantation as described in Example 1 , and were monitored for survival.
  • FIG 3 is a series of digital images and a graph showing that combined delivery of IL-27 and IL-2 mediates complete regression of disseminated TBJ neuroblastoma metastases in the liver.
  • FIG 4 is a pair of graphs showing that combined delivery of IL-27 and IL-2 mediates complete regression of induced neuroblastoma metastases in the bone marrow.
  • Cohorts of 10 albino Jackson (A/J) mice/group were injected intravenously with 1x10 5 TBJ IL-27 or TBJ FLAG (control) neuroblastoma tumor cells on day 0 as described in Example 1.
  • Mice were then treated with either IL-2 or vehicle alone on days 5-9, 12-16, 19-23, and 26-30 post tumor cell implantation. Mice were harvested individually as they became sick, bone marrow was extracted, and tumor burden in each marrow specimen was assessed by colony assay in the presence of G418 (1 mg/ml) as described in Example 1.
  • *p
  • mice 10 mice/group were injected with 1x10 5 TBJ FLAG or TBJ IL-27 cells on day 0, followed by therapy with IL-2 or vehicle on days 5-9, 12- 16 post tumor cell injection.
  • Nineteen days post-tumor injection mice were euthanized and bone marrow cells were isolated as described in Example 1.
  • Single cell suspensions containing both marrow cells and metastatic neuroblastoma tumor cells were prepared and injected subcutaneously (1 x 10 6 cells/injection) into naive mice to assess the tumorigenicity of contaminating neuroblastoma metastases in the bone marrow. Tumor size was monitored twice a week.
  • FIG 5 is a survival curve showing the role of T cells versus natural killer cells (NK) in the anti-tumor activity of IL-27/IL-2 therapy.
  • Cohorts of 10 wild type A/J mice were injected intravenously with either TBJ-IL-27 or TBJ-FLAG tumor cells (1 x 10 5 cells/animal) on day 0.
  • Mice were concurrently depleted of NK cells, CD4 + T cells or CD8 + T cells as described in Example 1.
  • Mice were treated with either IL-2 or medium alone on days 5-9, 12-16, 19-23, and 26-30 post-tumor implantation as described in Example 1, and were monitored for survival. Mice surviving at the last follow-up point were tumor-free.
  • FIG 6 is a pair of graphs showing that IL-27 inhibits IL-2-induced increases in the proportion of CD4+CD25+FoxP3+ T cells within tumor-infiltrating lymphocytes (TIL) from TBJ neuroblastoma tumors.
  • TIL tumor-infiltrating lymphocytes
  • FIG 7 is a graph showing that combined exposure to IL-27 and IL-2 synergistically enhances immune responsiveness: IFN- ⁇ production. Spleens were resected under sterile conditions from mice bearing disseminated TBJ IL-27 or TBJ FLAG (control) tumors at day 15 post-tumor implantation.
  • FIG 8 is a pair of graphs showing that combined exposure to IL-27 and IL-2 synergistically enhances tumor-specific immune responsiveness: generation of neuroblastoma-specific cytotoxic lymphocyte (CTL) activity.
  • CTL neuroblastoma-specific cytotoxic lymphocyte
  • Spleens were resected under sterile conditions from mice bearing disseminated TBJ IL-27 or TBJ FLAG (control) tumors at day 15 post-tumor implantation.
  • Single cell suspensions were prepared and splenocytes were re-stimulated with irradiated TBJ IL-27 or TBJ- FLAG tumor cells +/- IL-2 for six days as described in Example 1.
  • the cytolytic activity directed against TBJ FLAG or irrelevant syngeneic SA-I tumor cells was assayed by ' ' 'indium release assay.
  • FIG 9 is a series of three histograms showing the efficacy of various doses and administration schedules of IL-2.
  • FIG 9A A/J mice were injected with 1 x 10 5 TBJ cells intravenously on day -5.
  • rhIL-2 Intraperitoneal treatment started on day 0 as follows: Gl : Vehicle control; G2 (split pulse): 200,000 ILVqAM, qPM day 0, 3, 7, 10, 14, and 17; G3 (chronic): 100,000 IU/day 0-4, 7-11, and 14-18; G4: (chronic): 200,000 IU/day 0-4, 7-11, and 14-18; G5 (intermittent): 200,000 IU/day 0, 2, 4, 7, 9, 11, 16, andl8.
  • the survival proportions were as follows: G2: 10%, G3: 30%, G4: 22%, Gp5: 22%.
  • FIG 9B 1 x 10 5 TBJ parental, TBJ-FLAG or TBJ-IL-27 were injected intravenously on day - 5. Mice were treated with IL-2 (100,000 IU on days 0-4, 7-11, and 14-18) or vehicle alone. Although IL-2 prolonged survival of the TBJ-IL-27 group, the effect was not very profound. Because chronic injection of IL-2 at 100,000 IU/injection was somewhat effective, this was increased to 200,000 IU/injection for FIG 9C. Thus, 1 x 10 5 TBJ parental, TBJ-FLAG, or TBJ-IL-27 were injected IV on day -5.
  • mice were treated with IL-2 (200,000 IU on days 0-4, 7-11, 14-18, and 21-25) or vehicle ⁇ alone.
  • SEQ ID NO: 1 is a synthetic linker
  • one embodiment of the disclosure is a method of treating a tumor that responds to immunotherapy, and the IL-2 and IL-27 interact in their effects to stimulate an immune response and treat the tumor.
  • the tumor is a neuroblastoma, melanoma, leukemia, lymphoma, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, or lung carcinoma.
  • the method includes administering to the subject a therapeutically effective combined amount of IL-2 and IL-27, thereby treating the tumor.
  • administration of the IL-2 is by systemic administration, for instance by intravenous, intra-muscular, subcutaneous, intra-arterial, or intra-peritoneal administration.
  • administration of the IL-2 is by local administration, for instance by local injection, including injection of a cell that expresses the IL-2 (for example by constitutive expression of the IL-2 at the tumor site), adoptive transfer of T- W
  • lymphocytes expressing IL-2 anaerobic bacterial administration, administration via alpha(v)beta(3)-targeted nanoparticles, or liposomal delivery of IL-2.
  • Administration of the IL-27 also can be by systemic administration, for instance by intravenous, intra-muscular, subcutaneous, intra-arterial, or intra- peritoneal administration, or by local administration, for instance by local injection, including injection of a cell that expresses the IL-27 (for example by constitutive expression of the IL-27 at the tumor site), adoptive transfer of T-lymphocytes expressing IL-27, anaerobic bacterial administration, administration via alpha(v)beta(3)-targeted nanoparticles, or liposomal delivery of IL-27.
  • administration of the IL-2 is by systemic administration, and administration of the IL-27 is by local administration.
  • the IL-27 is constitutively expressed in the tumor and the IL-2 is intermittently administered to the subject, and in still more particular examples the IL-2 is intermittently administered by systemic administration no more than once per day.
  • the effective amount of IL-2 is from about 30,000 IU/kg to about 600,000 IU/kg, whereas in other embodiments, the effective amount of IL-27 is from about 30 ng/kg to about 3,000 ng/kg.
  • the IL-2 and the IL-27 are administered substantially concurrently, whereas in other examples the IL-27 is administered before the IL-2.
  • Substantially concurrent administration can include administration within a few minutes to a few hours of one another, for example within an hour or a day of one another.
  • an advantage of some embodiments of the method is that the IL-2 and IL 27 can be administered in a manner that reduces side effects from the drugs.
  • the dose of IL-2 can be reduced to at least partly avoid IL-2 toxicity (such as capillary leak syndrome) while the combined effects of the IL-2 and IL-27 enhances the anti-tumor efficacy of the treatment.
  • Co-administration of the IL-2 and IL-27 potentiates the effectiveness of the anti-tumor response over the response that would be seen with either agent alone.
  • the timing of the administration of IL-2 can vary, for instance in certain embodiments the IL-2 is administered daily, or in a repeating cycle of daily administration for about three to seven days, followed by no administration for about two to nine days.
  • the timing of the administration of IL-27 is similarly variable, and in some embodiments the IL-27 is administered daily, whereas in other embodiments the IL-27 is administered in a repeating cycle of daily administration for about three to seven days, followed by no administration for about two to seven days. For both IL-2 and IL-27, the cycle of administration is repeated in some embodiments from about two to about ten times.
  • the subject has metastatic neuroblastoma.
  • the subject is human, wherein in other embodiments the subject is a veterinary subject.
  • the method is a method of treating metastatic neuroblastoma in a subject that includes administering to the subject a therapeutically effective amount of IL-2 and a therapeutically effective amount of IL- 27.
  • the IL-2 is administered systemically and the IL-27 is administered locally, thereby treating the metastatic neuroblastoma.
  • compositions for use in stimulating an anti-tumor response include an amount of IL-2 and IL-27 effective to stimulate an immune response and treat the tumor. //. Abbreviations AJJ albino Jackson
  • TBJ a cell clone from C-1300 neuroblastoma cells.
  • Adoptive transfer by T lymphocytes involves the passive transfer of antitumor-reactive cells into a host in order to mediate tumor regression.
  • the transfer of immune lymphoid cells can eradicate widely disseminated tumors and establish long-term systemic immunity, for example when T lymphocytes overexpressing a gene of interest are administered locally. See, for example, Duval et al, (2006) Clinical Cancer Research Vol. 12, 1229-1236; and Sussman et al, (2004) Annals of Surgical Oncology, VoI 1, Issue 4 296-306 for a more thorough discussion of the techniques involved.
  • Alpha(v)beta(3)-targeted nanoparticles can be used to selectively deliver a gene to tumor vasculature.
  • An alpha(v)beta(3)-targeted nanoparticle is a cross between two molecules: a "catalytic" antibody, and a small drug molecule, which are linked by a linker molecule.
  • a "catalytic" antibody also called immunoglobulins
  • antibodies are proteins produced by immune cells that are designed to recognize a wide range of foreign pathogens. After a pathogen enters the bloodstream, antibodies target antigens (proteins, carbohydrate molecules, and other pieces of the pathogen) specific to that pathogen. These antibodies then alert the immune system to the presence of the pathogen and attract lethal "effector" immune cells to the site of infection.
  • the small-molecule portion targets cancer cells.
  • the compounds target alpha(v)beta(3) integrin, which are expressed by endothelial cells during angiogenesis.
  • Many cancer cells like breast, ovarian and prostate cancer, also express integrins on their surface, providing for a potential double-strike against the tumor itself as well as its key blood supply.
  • Anaerobic bacterial administration such as by toxin-free Clostridium novyi, occurs when spores of the anaerobic bacterium Clostridium n ⁇ vyz-NT are systemically injected into animals, and they germinate exclusively within the hypoxic regions of cancers.
  • the germinated bacteria destroy adjacent tumor cells but spare a rim of well oxygenated tumor cells that subsequently expand.
  • the mechanism underlying this effect is immune-mediated, because cured subjects reject a subsequent challenge of the same tumor.
  • the induced immune response when combined with the bacteriolytic effects of C. novyi- ⁇ NT, eradicates even large, established tumors. For a more thorough discussion of this technique see Agrawal et al, (2004) Proc Natl Acad Sci USA. 101(42):15172-7.
  • Animal living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • the term mammal includes both human and non- human mammals.
  • the term “subject” includes both human and veterinary subjects. Therefore, the general term “subject” is understood to include all animals, including, but not limited to, humans, or veterinary subjects, such as other primates, dogs, cats, horses, and cows.
  • Anti-cancer agent an anti-neoplastic agent.
  • Anti-cancer agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and bbr3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example, capecitabine),
  • Anti-cancer agents often are used in combination with one another, and the IL-2 and IL-27-based therapies disclosed herein can be administered in combination with one or more other anti-cancer agents.
  • Breast carcinoma a cancer of breast tissue. Worldwide, it is the most common form of cancer in females, affecting, at some time in their lives, approximately one out of nine to thirteen women who reach age ninety in the Western world. It is the second most fatal cancer in women (after lung cancer), and the number of cases has significantly increased since the 1970s. Because the breast is composed of identical tissues in males and females, breast cancer can also occur in males, although cases of male breast cancer account for less than one percent of the total.
  • Cancer or tumor a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion or by implantation into distant sites by metastasis. Metastasis is defined as the stage in which cancer cells are transported through the bloodstream or lymphatic system. Cancer may affect people at all ages, but risk tends to increase with age, due to the fact that DNA damage becomes more apparent in aging DNA. It is one of the principal causes of death in developed countries.
  • cancer There are many types of cancer. Severity of symptoms depends on the site and character of the malignancy and whether there is metastasis. A definitive diagnosis usually requires the histologic examination of tissue by a pathologist. Once diagnosed, cancer is usually treated with a combination of surgery, chemotherapy and radiotherapy.
  • the unregulated growth that characterizes cancer is caused by damage to DNA, resulting in mutations to genes that encode for proteins controlling cell division. Many mutation events may be required to transform a normal cell into a malignant cell. These mutations can be caused by chemical carcinogens, by close exposure to radioactive materials, or by certain viruses that can insert their DNA into the human genome. Mutations occur spontaneously, and may be passed down from one generation to the next as a result of mutations within germ lines. Many forms of cancer are associated with exposure to environmental factors such as tobacco smoke, radiation, alcohol and certain viruses. While some of these risk factors can be avoided or reduced, there is no known way to entirely avoid the disease. Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor.
  • carcinomas are malignant tumors derived from epithelial cells. This group represents the most common cancers, including the common forms of breast, prostate, lung and colon cancer.
  • Lymphomas and leukemias include malignant tumors derived from blood and bone marrow cells.
  • Sarcomas are malignant tumors derived from connective tissue or mesenchymal cells.
  • Mesotheliomas are tumors derived from the mesothelial cells lining the peritoneum and the pleura.
  • Gliomas are tumors derived from glia, the most common type of brain cell.
  • Germinomas are tumors derived from germ cells, normally found in the testicle and ovary.
  • Choriocarcinomas are malignant tumors derived from the placenta.
  • cancers for which combination IL-2 and IL-27 therapy is useful include neuroblastomas, renal cell carcinomas, melanomas, leukemias, colon carcinomas, breast carcinomas, ovarian carcinomas, prostate carcinomas, lung carcinomas, and lymphomas, including non-Hodgkin's lymphomas.
  • Colon carcinoma includes carcinomas of the colon, rectum and appendix.
  • Colon cancer is the third most common form of cancer and the second leading cause of death among cancers in the Western world. Many colorectal cancers are thought to arise from adenomatous polyps in the colon. These growths are usually benign, but some develop into cancer over time. The majority of colon cancers are found in the sigmoid colon and at the rectosigmoid junction. These cancers are usually small, annular and ulcerated. The next common site is the caecum, where the tumors tend to be bulky and papilloform. The majority of the time, the diagnosis of localized colon cancer is through colonoscopy. Therapy is usually through surgery, which in many cases is followed by chemotherapy.
  • DNA deoxyribonucleic acid: a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid).
  • the repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule.
  • Encode a polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • Functional fragments and variants of a polypeptide included are those fragments and variants that maintain at least one function of the parent polypeptide. It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more of the polypeptide's functions. First, the genetic code is well known to be degenerate, and thus different codons encode the same amino acids. Second, even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein (see Stryer, Biochemistry 4th Ed., (c) W. Freeman & Co., New York, NY, 1995). Third, part of a polypeptide chain can be deleted without impairing or eliminating all of its functions.
  • sequence variants in a protein may retain the full function of an entire protein.
  • insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1998).
  • Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vzvo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids.
  • Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, sumoylation, labeling, for example, with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art.
  • a variety of methods for labeling polypeptides and labels useful for such purposes are well known in the art, and include radioactive isotopes such as 32 P, ligands that bind to or are bound by labeled specific binding partners (for example, antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands. Functional fragments and variants can be of varying length.
  • a fragment may consist of 10 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more amino acid residues.
  • Immune response A response of a cell of the immune system, such as a B cell or a T cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").
  • a "parameter of an immune response” is any particular measurable aspect of an immune response, including, but not limited to, cytokine secretion (IL-6, IL-IO, IFN- ⁇ , etc.), immunoglobulin production, dendritic cell maturation, and proliferation of a cell of the immune system.
  • a "substantial" increase in a parameter of the immune response is a significant increase in this parameter as compared to a control.
  • Specific, non-limiting examples of a substantial increase are at least about a 50% increase, at least about a 75% increase, at least about a 90% increase, at least about a 100% increase, at least about a 200% increase, at least about a 300% increase, and at least about a 500% increase.
  • One of skill in the art can readily identify a significant increase using known statistical methods.
  • Immunotherapy an array of strategies of treatment based upon the concept of modulating the immune system to achieve a prophylactic and/or therapeutic goal. Cancer immunotherapy attempts to stimulate the immune system to reject and destroy tumors.
  • BCG immunotherapy for early stage (non-invasive) bladder cancer utilizes instillation of attenuated live bacteria into the bladder, and is effective in preventing recurrence in up to 2/3 of cases.
  • Topical immunotherapy utilizes an immune .enhancement cream (imiquimod) which is an interferon producer causing the patients own killer T cells to destroy warts, actinic keratoses, basal cell cancer, squamous cell cancer, cutaneous lymphoma, and superficial malignant melanoma.
  • imiquimod immune .enhancement cream
  • Injection immunotherapy uses mumps, Candida or trichophytin antigen injections to treat warts (HPV induced tumors).
  • Dendritic cell based immunotherapy utilizes dendritic cells to activate a cytotoxic response towards an antigen.
  • a tumor that responds to immunotherapy is one that responds to treatment with one or more interleukins, for instance IL-2, IL- 12, or IL-27.
  • Specific, non-limiting examples of tumors that respond to immunotherapy include neuroblastoma, melanoma, lymphoma, leukemia, and renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma.
  • IL-27 and IL-2 interact favorably to enhance immune responsiveness. For instance, the combination of IL-2 and IL-27 synergistically enhances proliferative responses by splenocytes and IFN- ⁇ production. Additionally, the combination of IL-2 and IL-27 produces a more robust anti-tumor effect than either interleukin produces alone. This permits IL-2 to be administered at a lower dose, yet still retain its anti-tumor efficacy, for example against neuroblastoma, lymphoma, leukemia, melanoma, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma..
  • IL-2 Interleukin-2
  • IL-2 Interleukin-2
  • IL-2 a T cell growth factor that binds to a specific tripartite receptor on T cells.
  • IL-2 Interleukin-2
  • Administering IL-2 in high doses is comparable to inducing a controlled state of septic shock.
  • IL-2 low blood pressure, low systemic vascular resistance, high cardiac output, grade 3/4 hematologic toxicity, hepatic toxicity, renal toxicity, and pulmonary edema have all been documented. Toxicity is nearly always reversible.
  • the typical regimen for administering IL-2 alone is from about 600,000 to about 720,000 IU/kg, an average of 50 million units of IL-2 per dose given three times a day as a bolus over 15 minutes. The maximum most patients can tolerate is 14 doses. A rest period of 5 to 9 days between cycles is recommended, and patients must be treated in a step-down situation or ICU.
  • the toxicity associated with IL-2 therapy is a major consideration, and these patients require intensive management.
  • Isolated an "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Labeled a biomolecule attached covalently or noncovalently to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et ah, Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989 and Ausubel et ah, Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley- Intersciences, 1998.
  • ATP can be labeled in any one of its three phosphate groups with radioisotopes such as 32 P or 33 P, or in its sugar moiety with a radioisotope such as 35 S.
  • Leukemia a cancer of the blood or bone marrow characterized by an abnormal proliferation of blood cells, usually white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms. Leukemia is clinically and pathologically split into its acute and chronic forms.
  • Acute leukemia is characterized by the rapid growth of immature blood cells. This crowding makes the bone marrow unable to produce healthy blood cells. Acute forms of leukemia can occur in children and young adults, and acute leukemia is a more common cause of death for children in the US than any other type of malignant disease. Immediate treatment is required due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. If left untreated, the patient will die within months or even weeks.
  • Chronic leukemia is distinguished by the excessive build up of relatively mature, but still abnormal, blood cells. Typically taking months to years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group. Whereas acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy.
  • lymphoid cells lymphoid cells
  • myeloid cells eosinophils, neutrophils, and basophils
  • myeloid or myelogenous leukemia myeloid or myelogenous leukemia. Combining these two classifications provides a total of four main categories: Acute lymphocytic leukemia (also known as Acute Lymphoblastic Leukemia, or ALL) is the most common type of leukemia in young children. This disease also affects adults, especially those age 65 and older.
  • ALL Acute lymphocytic leukemia
  • Acute myelogenous leukemia also known as Acute Myeloid Leukemia, or AML
  • AML Acute Myeloid Leukemia
  • CLL Chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • Liposomal administration lipidic and liposome-mediated gene delivery has been used successfully for transfection with various genes (for reviews, see Templeton and Lasic, MoI. Biotechnol 11 :175-180, 1999; Lee and Huang, Crit. Rev. Ther. Drug Carrier Syst. 14:173-206; and Cooper, Semin. Oncol. 23:172-187, 1996).
  • cationic liposomes are used for their ability to transfect monocytic leukemia cells, and have been shown to be a viable alternative to using viral vectors (de Lima et al, MoI. Membr. Biol. 16:103-109, 1999).
  • Such cationic liposomes also can be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al, Cancer Gene Ther. 3:250-256, 1996).
  • monoclonal antibodies or other appropriate targeting ligands Kao et al, Cancer Gene Ther. 3:250-256, 1996.
  • intravenous administration to tumors using liposomal delivery generally does not require a specific targeting mechanism, since the leakage of the tumor vasculature results in localized administration to the tumor.
  • Lymphoma a variety of cancer that originates in lymphocytes or, more rarely, of histiocytes. Collectively, these cell types form the reticuloendothelial system and circulate in the vessels of the lymphatic system. Just as there are many types of lymphocytes, so there are many types of lymphoma. Lymphomas are part of the broad group of diseases called hematological neoplasms.
  • lymphomas were classified as Hodgkin's lymphoma and non- Hodgkin's lymphoma (all other types of lymphoma). According to the U.S. National Institutes of Health, lymphomas account for about five percent of all cases of cancer in the United States, and Hodgkin's disease in particular accounts for less than one percent of all cases of cancer in the United States. Because the lymphatic system is part of the body's immune system, patients with weakened immune system, such as from HIV infection or from certain drugs or medication, also have a higher incidence of lymphoma.
  • Mature B cell neoplasms include: chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell neoplasms, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lympho
  • MALT lymphoma extranodal marginal zone B cell lymphoma
  • follicular lymphoma mantle cell lymphoma
  • Mature T cell and natural killer (NK) cell neoplasms include: T cell prolymphocyte leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, and anaplastic large cell lymphoma.
  • Hodgkin's lymphomas include: nodular lymphocyte-predominant Hodgkin lymphoma, classical Hodgkin lymphoma, nodular sclerosis, mixed cellularity, lymphocyte-rich, and lymphocyte depleted.
  • Mammal This term includes both human and non-human mammals. Similarly, the term subject includes both human and veterinary subjects.
  • Melanoma a malignant tumor of melanocytes and, less frequently, of retinal pigment epithelial cells (uveal melanoma). While it represents one of the rarer forms of skin cancer, melanoma underlies the majority of skin cancer-related deaths. Despite many years of intensive laboratory and clinical research, the sole effective cure is surgical resection of the primary tumor before it achieves a thickness of greater than 1 mm.
  • Melanoma of the skin accounts for 160,000 new cases worldwide each year, and is more frequent in Caucasian men. It is particularly common in Caucasian populations living in sunny climates About 48,000 deaths worldwide due to malignant melanoma are registered annually.
  • melanoma The diagnosis of melanoma requires experience, as early stages may look identical to harmless moles or not have any color at all. Moles that are irregular in color or shape are suspicious of a malignant melanoma or a premalignant lesion.
  • the treatment includes surgical removal of the tumor; adjuvant treatment; chemo- and immunotherapy, or radiation therapy.
  • the most common types of melanoma include: superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna. Any of these types may produce melanin (and be dark in color) or not (and be amelanotic - not dark). Similarly any subtype may show desmoplasia (dense fibrous reaction with neurotropism) which is a marker of aggressive behaviour and a tendency to local recurrence.
  • Metastasis the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Tumors are classified as either benign or malignant. Malignant tumors can spread by invasion and metastasis while benign tumors cannot (and only grow locally). By definition, the term "cancer" applies only to malignant tumors. Still, some tumors with benign histology can behave as malignant tumors, for example in brain tumors, where treatment has to be as aggressive as with malignant disease.
  • Metastatic tumors are very common in the late stages of cancer.
  • the spread of metastases may occur via the blood or the lymphatics or through both routes.
  • the most common places for the metastases to occur are the adrenals, liver, brain and the bones.
  • prostate cancer usually metastasizes to the bones.
  • colon cancer has a tendency to metastasize to the liver.
  • Stomach cancer often metastasizes to the ovary in women, where it forms a Krukenberg tumor.
  • cancer cells spread to form a new tumor it is called a secondary, or metastatic tumor, and its cells are like those in the original tumor.
  • the secondary tumor is made up of abnormal breast cells (not abnormal lung cells).
  • the disease in the lung is metastatic breast cancer (not lung cancer).
  • Cancer cells also may spread to lymph nodes (regional lymph nodes) near the primary tumor. This is called nodal involvement, positive nodes, or regional disease. Localized spread to regional lymph nodes near the primary tumor is not normally counted as metastasis, although this is a sign of worse prognosis.
  • Neuroblastoma the most common extracranial solid cancer in infancy and childhood.
  • neuroendocrine tumor arising from any neural crest element of the sympathetic nervous system.
  • Other tumors also have similar origins, and show a wide pattern of differentiation ranging from benign ganglioneuroma to partially differentiated ganglioneuroblastoma, to highly malignant neuroblastoma.
  • Antibody to neuron-specific enolase can differentiate neuroblastoma from lymphoma, Ewing's sarcoma, and rhabdomyosarcoma.
  • a rosette pattern in a highly cellular tumor is an additional characteristic of neuroblastoma.
  • cytokines in preclinical tumor models and/or early phase clinical studies in children with neuroblastoma.
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • Nucleotide a monomer that includes a base linked to a sugar, such as a pyrimidine, purine, or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid.
  • a nucleotide is one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • Ovarian carcinoma a malignant ovarian neoplasm. Ovarian cancer is classified according to the histology of the tumor. Lesions differ significantly in clinical features, management, and prognosis.
  • Surface epithelial-stromal tumors are the most common and prototypic ovarian cancers. They are thought to originate from the ovarian surface lining, and include serous cystadenocarcinoma, and mucinous cystadenocarcinoma. Sex cord-stromal tumors include lesions that are hormonally active such as the estrogen-producing granulosa cell tumor and the virilizing arrhenoblastoma. Germ cell tumors originate from dysplastic germ material and tend to occur in young women and girls.
  • Lesions include the dysgerminoma, a form of the choriocarcinoma, and the malignant form of the teratoma. Other lesions include metastasis to the ovary, for instance from breast cancer. Krukenberg cancer is ovarian cancer originating from gastrointestinal cancer.
  • parenteral administered outside of the intestine, e.g., not via the alimentary tract.
  • parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, intra-articularly, or subcutaneously, and various surface applications including intranasal, inhalational, intradermal, and topical application, for instance.
  • Pharmaceutical agent a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell. Incubating includes exposing a target to an agent for a sufficient period of time for the agent to interact with a cell.
  • Contacting includes incubating an agent in solid or in liquid form with a cell.
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable earners useful in this disclosure are conventional. Martin, Remington 's Pharmaceutical Sciences, published by Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of, for instance IL-2 or IL-27. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred.
  • polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • polypeptide fragment refers to a portion of a polypeptide that exhibits at least one useful epitope.
  • functional fragments of a polypeptide refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants of the insulin, are thus included as being of use.
  • Preventing or treating a disease refers to inhibiting the full development of a disease, for example in a person who is at risk for a disease such as neuroblastoma or melanoma.
  • An example of a subject at risk for melanoma is someone who is fair-skinned or has a family history of skin cancer.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • Prostate carcinoma a neoplasm of the prostate. Rates of prostate cancer vary widely across the world. It is least common in South and East Asia, more common in Europe, and most common in the United States. Prostate cancer develops most frequently in men over fifty. It is the second most common type of cancer in men in the United States, where it is responsible for more male deaths than any other cancer except lung cancer. Prostate cancer can be treated with surgery, radiation therapy, hormone therapy, occasionally chemotherapy, or some combination of these. The age and underlying health of the man as well as the extent of spread, appearance under the microscope, and response of the cancer to initial treatment are important in determining the outcome of the disease.
  • Protein a biological molecule expressed by a gene and comprised of amino acids.
  • a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate).
  • Renal cell carcinoma the most common form of kidney cancer arising from the renal tubule, and it is the most common type of kidney cancer in adults. Initial therapy usually involves surgery. Renal cell carcinoma is notoriously resistant to radiation therapy and chemotherapy, although some cases respond to immunotherapy. The prognosis of a subject with renal cell carcinoma varies depending on the size of the tumor, whether it is confined to the kidney or not, and the presence or absence of metastatic spread.
  • the Furhman grading which measures the aggressiveness of the tumor, may also affect survival.
  • the five-year survival rate is around 90-95% for tumors less than 4 cm.
  • survival is still relatively good at 80-85%.
  • the survivability reduces to about 60%. If it has metastasized to the lymph nodes, the 5- year survival is around 5 % to 15 %. If it has spread metastatically to other organs, the 5-year survival at less than 5 %.
  • Sequence identity the similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity.
  • Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • Homologs or orthologs of an IL-2 or IL-27 protein, and the corresponding cDNA sequence will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species that are more closely related (for example, human and chimpanzee sequences), compared to species more distantly related (for example, human and C. elegans sequences). Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman J MoI. Biol.
  • NCBI National Center for Biotechnology Information
  • the Search Tool can be accessed at the NCBI website, together with a description of how to determine sequence identity using this program.
  • nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
  • TBJ a cell clone from C-1300 neuroblastoma cells. While the parent C- 1300 is highly antigenic, locally growing, and non-metastasizing, its clonal counterpart, TBJ, is minimally antigenic and demonstrates not only aggressive local growth but systemic metastases as well.
  • Therapeutically effective amount a quantity of a specified compound (such as IL-2 or IL-27 or a combination if IL-2 and IL-27) required to achieve a desired effect in a subject being treated.
  • a tumor such as neuroblastoma, melanoma, renal cell carcinoma, leukemia, lymphoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, or lung carcinoma in a subject, or a dose sufficient to prevent advancement, or to cause regression of a disease (such as the tumor), or which is capable of relieving symptoms caused by a disease, such as pain, inflammation, neurological symptoms, or fatigue.
  • a therapeutically effective amount of IL-2 and IL-27 can include doses of IL-2 and/or IL-27 that are less than are required for a therapeutic anti-tumor effect they are not given in methods of combined administration.
  • T-Lymphocyte a T cell belonging to group of white blood cells known as lymphocytes.
  • T lymphocytes play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and NK cells by the presence of a special receptor on their cell surface that is called the T cell receptor (TCR).
  • TCR T cell receptor
  • T cells Several different subsets of T cells have been described, each with a distinct function. Cytotoxic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells, since they express the CD 8 glycoprotein at their surface.
  • Helper T cells are part of the adaptive immune system. Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or "help" the immune response. These cells (also called CD4+ T cells) are a target of HIV infection; the virus infects the cell by using the CD4 protein to gain entry. The loss of Th cells as a result of HIV infection leads to the symptoms of AIDS.
  • Regulatory T cells formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T-cell mediated immunity towards the end of an immune reaction. These cells can be distinguished from other T-cells by the presence of an intracellular molecule called FOXP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.
  • Natural Killer T cells are a special kind of lymphocyte that bridges the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigen presented by MHC molecules, NKT cells recognize glycolipid antigen presented by a molecule called CDId. Once activated, these cells can perform functions ascribed to both Th and Tc cells (e.g., cytokine production and release of cytolytic/cell killing molecules).
  • Combined delivery of IL-27 and IL-2 produces a synergistic antitumor effect on metastatic neuroblastoma
  • Neuroblastoma is the most common extracranial solid tumor in children, with highly variable biologic features and clinical outcome.
  • age and stage play a particularly important role in predicting prognosis and clinical outcome (Maris (2005) Curr Opin Pediatr 17: 7- 13; Weinstein et al, (2003) Oncologist 8: 278-292).
  • the outcome of patients with high-risk neuroblastoma is poor overall, and has fueled an intense effort to develop new therapeutic approaches for high-risk patients, including those with widespread metastatic disease.
  • interleukin-2 (IL-2) (Lode et al, (1998) Blood 91 : 1706-1715; Redlinger et al, (2003) JPediatr Surg 38: 199-204), tumor-targeted immunocytokines (Hank et al, (1994) JImmunother 15: 29-37) and interleukin-12 (IL-12) (Siapati et al, (2003) Br J Cancer 88: 1641-1648; Davidoff et al, (1999) Journal of Pediatric Surgery 34: 902-906) have demonstrated some efficacy in pre- clinical neuroblastoma models as well as in the clinical setting (Sondel & Hank
  • IL-27 mediates potent antitumor effects against subcutaneous and orthotopic intradrenal TBJ murine neuroblastoma tumors, resulting in complete durable tumor regression in up to 90% of mice (Salcedo et al, (2004) J Immunol 173: 7170-7182).
  • IL-27 can mediate its potent antitumor effects via mechanisms that are dependent on the induction of endogenous IFN- ⁇ production and the activity of CD4 + and/or CD8 + T cell populations in vivo (Hisada et al , (2004) Cancer Res 64: 1152-1156; Salcedo et al, (2004) J Immunol 173: 7170- 7182; Chiyo et al, (2005) Int J Cancer 115: 437-442). In mice bearing TB J neuroblastoma tumors, IL-27 upregulates MHC class I expression on tumor cells, and enhances the generation of both tumor specific immune responsiveness and immunologic memory responses in mice cured of their original tumors by IL-27.
  • IL-27 mediates overall tumor regression in this model via mechanisms that depend on CD8+ but not CD4+ T cells or NK cells (Salcedo et al, (2004) J Immunol 173: 7170-7182).
  • the antitumor efficacy of IL-27 alone is more modest in mice bearing disseminated TBJ neuroblastoma metastases.
  • IL-27 mediates complete durable tumor regression in only 40% of mice bearing disseminated TBJ IL-27 tumors.
  • IL-2/IL-27 therapy also is useful for treating other diseases that respond to IL-2 therapy, for instance melanoma, Hodgkin's and non-Hodgkin's lymphoma, leukemia, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma.
  • melanoma Hodgkin's and non-Hodgkin's lymphoma
  • leukemia renal cell carcinoma
  • colon carcinoma breast carcinoma
  • ovarian carcinoma prostate carcinoma
  • lung carcinoma lung carcinoma
  • mice Male A/J mice were obtained from the Animal Production Area (Charles River Laboratories, Frederick, MD). Mice were maintained in a dedicated specific pathogen-free environment and generally used between 8 and 10 weeks of age.
  • the TBJ neuroblastoma cell line syngeneic to A/J mice was utilized where indicated herein, and was provided by Dr. Morritz Ziegler (Children's Hospital, Boston, MA).
  • TBJ tumor cells were engineered to over-express murine IL-27 using the p-FLAG- CMV-I vector (Sigma, Saint Louis, MO) containing a fusion sequence encoding the mature coding sequences for murine EBB, followed by the synthetic linker
  • GSGSGGSGGSGSGKL SEQ ID NO: 1
  • the mature coding sequence of mouse p28 as previously described (Roo et ah, (2002) Immunity. 16: 779-790).
  • TBJ neuroblastoma cells were stably transfected with either p-IL27/FLAG-CMV-l (TBJ IL-27) or the empty p-FLAG-CMV-1 vector alone (TBJ-FLAG) using the calcium phosphate method, and cells were subsequently selected in G418 (800 ⁇ g/ml) (Gibco, Invitrogen Corp. Carlsbad, CA).
  • mice syngeneic male A/J mice were injected intravenously with IxIO 5 TBJ IL-27 or control TBJ FLAG tumor cells /animal on day 0.
  • IL-2 (200,000 IU/injection) in 0.2ml Han's buffered saline solution (HBSS) containing 0.1% homologous mouse serum or vehicle alone was administered intraperitoneally on days 5-9, 12-16, 19-23 and 26-30 post-tumor implantation.
  • HBSS Han's buffered saline solution
  • mice bearing metastatic TBJ IL-27 or TBJ FLAG tumors were treated similarly with IL-2 or vehicle alone on days 5-9 and 12-15 post tumor implantation. Cohorts of mice were euthanized on day 16 post-tumor implantation. Livers were then removed for inspection and imaging/quantitation of the metastatic disease burden.
  • mice bearing metastatic TBJ IL- 27 or TBJ FLAG tumors were treated with IL-2 or vehicle alone on days 5-9, 12-16, 19-23 and 26-30 post tumor implantation. Mice were euthanized as they became pre-moribund, and all of the remaining surviving mice were euthanized at day 37 post-tumor implantation. At the time of euthanization, tibias and femurs were removed, and bone marrow was isolated and cultured as described below to assess the frequency of neuroblastoma metastasis via colony assay.
  • mice bearing metastatic TBJ IL-27 or TBJ FLAG tumors were treated with IL-2 or vehicle alone on days 5-9 and 12-16 and mice were euthanized on day 19 post-tumor implantation.
  • Tibias and femurs were resected and bone marrow was isolated as described herein.
  • na ⁇ ve mice were then injected subcutaneously with 1x10 7 nucleated bone marrow cells/mouse. The subsequent growth of tumors in these mice was then monitored twice per week as an indicator of tumor burden in the respective bone marrow specimens.
  • mice were re-challenged subcutaneously with TBJ parental tumor cells (1x10 6 cells/animal) where indicated and then monitored for survival.
  • mice were injected intravenously with 1x10 5 TBJ-FLAG or TBJ IL-27 tumor cells/animal on day 0.
  • mice were injected intraperitoneally with rat anti-mouse CD4 + (GKl .5, diluted 1 :2) or mouse anti-mouse CD8 + (Ly2.2, diluted 1:20) antibodies on day -1 and days 2, 5, 7, 9, 12, 14, 16, 19 and 21 post- tumor implantation.
  • anti-asialoGMl diluted 1 :20, Wako Pure Chemical Industries, Ltd., Osaka, Japan
  • IL-2 or vehicle alone was administered on days 5-9, 12-16, 19-23 and 26-30 post tumor cell implantation. Mice were monitored for survival twice weekly.
  • mice were injected orthotopically in the adrenal gland with 1 x 10 5 TBJ-FLAG or TBJ-IL-27 tumor cells as described elsewhere in detail (Salcedo et ah, (2004) J Immunol 173: 7170-7182), and were then treated with IL-2 (200,000 IU/injection) or vehicle alone intraperitoneally on days 10-12 post tumor cell implantation. Mice were then euthanized and tumors were resected for isolation of TIL as described herein.
  • mice bearing metastatic TBJ neuroblastoma tumors The impact of combined delivery of IL-27 and IL-2 on the generation of immunologic reactivity was assessed in mice bearing metastatic TBJ neuroblastoma tumors.
  • spleens were resected from mice bearing disseminated TBJ IL-27 or TBJ FLAG tumors at day 15 post-tumor cell injection.
  • Spleen cells (Ix 10 6 cells/ml in complete medium) were cultured in the presence of the corresponding irradiated (4,000 rads) TBJ FLAG or TBJ IL-27 tumor cells (2.5x 10 5 cells/ml) with or without IL-2 (50 IU/ml).
  • the cultures were then incubated for three days at 37 0 C, and supernatants were harvested for determination of IFN- ⁇ concentrations by ELISA as described herein.
  • mice bearing TBJ neuroblastoma tumors were resected from mice bearing disseminated TBJ IL-27 or TBJ FLAG tumors at day 15 post-tumor injection.
  • Single cell suspensions were prepared as described above and total splenocytes (Ix 10 7 cells/ml) were incubated in the presence of irradiated (6,000 rads) TBJ FLAG or TBJ IL-27 tumor cells (1 x 10 6 /ml) +/- IL-2 (10 IU/ml) in complete medium.
  • mice bearing induced metastatic TBJ IL-27 or TBJ-FLAG tumors were treated with IL-2 or vehicle alone as described herein.
  • Mice were euthanized on day 16 post tumor implantation, and livers were resected and placed in HBSS for imaging of the disease burden using a slit fiber optic illuminated light table (Lightools Research, San Diego, CA). Images were taken using a Nikon Eclipse E400 Microscope fitted to a Nikon digital camera DXM1200 (Image Systems Inc., Columbia, MD).
  • mice bearing IV induced TBJ IL-27 or TBJ FLAG tumors were treated with IL-2 or vehicle alone as described herein.
  • Femurs and tibias were resected from mice in the respective treatment groups, and bone marrow cells were flushed out with 5 ml of RPMI. Bone marrow cells were then spun down at 1,500 rpm for 5 minutes at 4 0 C, and cells were resuspended at 5x10 6 cells/ml in RPMI containing 5% FCS and 2 mM glutamine.
  • mice bearing these tumors were treated with IL-2 or vehicle alone as described above. Mice were then euthanized , and tumors were carefully dissected and digested with 200 units/ml of collagenase (Invitrogen, Carlsbad, CA) and 100 ⁇ g/ml of DNAse I (Boehringer Mannheim,
  • Single cell suspensions (1 x 10 7 cells/sample) were initially subjected to Fc- receptor block for 15 minutes with rat anti-mouse 24G2 antibody (BD Pharmingen, San Diego, CA ) to limit non-specific antibody binding, and were then diluted 1 :500 in PBS containing 3% FCS, 10 % A/J mouse serum and 0.02 % sodium azide.
  • rat anti-mouse 24G2 antibody BD Pharmingen, San Diego, CA
  • Cells were stained with the following antibodies: PECy7 conjugated rat monoclonal antibody to murine CD4 (L3T4, diluted 1 :500), PE conjugated rat monoclonal antibody to murine CD25 (PC61, diluted 1 :200) or the corresponding isotype controls (BD Pharmingen, San Diego, CA) and incubated for 30 minutes on ice in the dark. Thereafter, cells were washed twice and intracellular staining was performed using a commercially available rat anti-mouse Foxp3 FACS staining set (eBiosciences, San Diego CA).
  • Bioscience-permeabilization solution was added and cells were incubated for 30 minutes on ice in the dark. Cells were then washed twice with PBS (2ml) and samples were then analyzed using an LSR II instrument (Becton-Dickinson Immunocytometry System, San Jose, CA) equipped with a 25 mW coherent Radius laser, a 20 mW coherent Sappire laser and a 17 mW air cooled JDS uniphase HeNe laser. For analysis purposes, the percentage of regulatory T cells (CD4 + CD25 +
  • CD4 + T cell gate lymph nodes cells from na ⁇ ve mice were used as a control to set the markers for CD25 + Fox ⁇ 3 + and CD25 " Foxp3 " populations.
  • mice were monitored for overall survival and/or tumor growth as assessed by abdominal size twice weekly. Survival studies were analyzed by the log-rank test and Kaplan-Meier curves were plotted for survival comparisons. The relative proportion of mice achieving complete durable tumor regression and long-term survival were compared by the Fisher's exact test (FET). Similarly, the proportions of mice rejecting a tumor rechallenge and achieving long-term survival were compared using FET. Interpretations regarding survival duration and complete response rates were in complete interpretative agreement. The proportion of mice developing subcutaneous tumors after injection of bone marrow cells containing metastatic neuroblastoma tumor deposits were compared using the FET.
  • IFN- ⁇ production and CTL responses by murine splenocytes mean values were determined for the respective conditions (duplicate for IFN- ⁇ , triplicate for proliferation and CTL responses), and were compared using the oneway and two-way analysis of variance (ANOVA) procedure.
  • ANOVA analysis of variance
  • pairwise comparisons were done by means of the Tukey HSD procedure. In all cases, the statistical procedures were performed on log transformed values of the dependent variables.
  • pairwise comparisons were performed using the nonparametric Mann- Whitney Wilcoxon test. All p values were two-tailed and were considered significant aXp ⁇ 0.05.
  • IL-27 and IL-2 synergistically enhance proliferative responses and IFN- ⁇ production by murine splenocytes in vitro
  • the proliferation of murine splenocytes treated with the combination of IL-27 and IL-2 was 3.3 to 5.9-fold greater than that observed with splenocytes treated with either single cytokine or medium alone (medium alone: 3694 ⁇ 980.5 cpm, IL-2: 8,936 ⁇ 450.6 cpm, IL-27: 5,035 ⁇ 243.5 cpm, IL-2 + IL-27: 29,633 ⁇ 1969 cpm; p ⁇ 0.001, IL-2 + IL-27 vs. either IL-2 or IL-27 alone or medium alone) (FIG IA).
  • IL-27 and IL-2 mediates synergistic antitumor activity in mice bearing disseminated TBJ neuroblastoma tumors
  • This Example demonstrates that combined delivery of IL-2 and IL-27 mediates a more potent antitumor activity than either single agent alone.
  • a disseminated model of TBJ neuroblastoma metastasis was used that is characterized by the formation of metastasis predominantly in liver, lung and bone marrow.
  • IL-27 alone can mediate complete tumor regression and long-term survival in approximately 40% of mice bearing induced neuroblastoma metastases (Salcedo et ah, (2004) J Immunol 113: 7170-7182).
  • Combined delivery of IL-27 and IL-2 mediates the regression of hepatic TBJ neuroblastoma metastases
  • This Example demonstrates that combined delivery of IL-27 and IL-2 mediates potent antitumor effects against TBJ neuroblastoma tumors in the liver. After intravenous administration, TBJ tumor cells form metastases in the liver, lung and bone marrow. The impact of combined delivery of IL-27 and IL-2 on the development of disease burden in the liver and bone marrow was examined.
  • IL-27 and IL-2 can mediate potent antitumor effects against TBJ neuroblastoma tumors in the liver.
  • mice bearing disseminated TBJ neuroblastoma metastases were harvested individually as they became sick or after long-term survival. Bone marrow cells were isolated as described and the burden of neuroblastoma metastases within the bone marrow compartment was assessed by assay of the formation of tumor colonies grown in methylcellulose in the presence of G418 (1 mg/ml) to provide selective pressure permitting the growth of metastatic tumor cells but not normal bone marrow cells.
  • colonies formed from the bone marrow of eight of nine mice bearing TBJ IL-27, nine of nine mice bearing TBJ FLAG tumors treated with systemic administration of IL-2, and eight of eight control mice bearing TBJ FLAG tumors, while tumor colonies formed from only three often mice bearing TBJ IL-27 tumors and treated with IL-2 (p 0.0198, TBJ IL27 + IL2 vs. TBJ IL-27 alone) (FIG 4A).
  • mice bearing widespread TBJ FLAG or TBJ IL-27 tumors treated with IL-2 or vehicle alone Bone marrow specimens were isolated from mice in the respective groups, and 1x10 7 nucleated bone marrow cells were injected subcutaneously into na ⁇ ve littermate control mice. Tumors formed after injection of bone marrow cells in six often mice (60%) bearing TBJ IL- 27 tumors, seven often mice (70%) bearing TBJ FLAG tumors, and nine often mice (90%) bearing TBJ-FLAG tumors treated with IL-2.
  • p 0.011, TBJ IL-27 + IL2 vs. TBJ IL-27 alone
  • mice bearing disseminated TBJ-IL-27 tumors were treated with IL-2 and concurrently depleted of either NK cells or CD4 + versus CD8 + T cells as described herein, and then monitored for survival.
  • CD8 + T cell depleted vs. either CD4 + T, or NK cell depleted; FIG 5).
  • IL-27 inhibits IL-2 induced expansion of CD4+CD25+Foxp3+ regulatory T cells within the microenvironment of TBJ tumors
  • CD4 + CD25 + T cells CD4 + CD25 + T cells
  • administration of IL-2 increases the frequency of circulating CD4 + CD25 + Foxp3+ regulatory T cells in cancer patients (Ahmadzadeh & Rosenberg (2006) Blood 107: 2409-2414).
  • IL-27 could modulate IL-2 induced alterations in regulatory T cell populations. Studies were performed using orthotopic TBJ Flag or TBJ-IL-27 tumors treated with IL-2 or vehicle alone.
  • p 0.045 (FIG 6). Similar results were obtained using metastatic tumors. Thus, this potentially deleterious effect of IL-2 regarding the increase of tumor infiltrating T regulatory cells was counteracted by IL-27.
  • Splenic effector cells from mice bearing disseminated TBJ FLAG or TBJ IL-27 tumors were harvested fifteen days post tumor implantation and were re-stimulated with the corresponding irradiated TBJ FLAG or TBJ IL-27 tumor cells respectively in the presence (10 IU/ml) or absence of IL-2 for three days. Culture supernatants were then harvested and the production of IFN- ⁇ was assayed by ELISA.
  • EXAMPLE 9 Combined delivery of IL-27 and IL-2 synergistically enhances tumor-specific immunologic reactivity: induction of CTL responses.
  • This Example demonstrates that IL-27 in combination with IL-2 can synergistically enhance the generation of specific cytotoxic activity against TBJ neuroblastoma tumors, and that IL-27 can potentiate the generation of CTL reactivity via mechanisms that act both at the initial sensitization phase as well as the effector phase of CTL generation.
  • Spleens from mice bearing disseminated TBJ FLAG or TBJ IL-27 tumors were resected under sterile conditions at day 15 post-tumor cell implantation, and single cell suspensions of murine splenocytes were expanded in vitro utilizing re-stimulation with either irradiated TBJ FLAG or TBJ TL-TJ cells in the presence or absence of IL-2 (10 IU/ml).
  • IL-2 (10 IU/ml).
  • effector cells were harvested, and the generation of CTL activity against indium labeled TBJ FLAG tumor cells or irrelevant syngeneic SA-I tumor target cells was assessed.
  • Combined IL-2 and IL-27 have a potent immunostimulatory and anti-tumor effect
  • Examples 2-9 demonstrate that combined administration of IL-2 and IL-27 have a potent immunostimulatory and anti-tumor effect.
  • This antitumor effect has been particularly demonstrated against neuroblastoma, but is believed to also have an effect against other tumors susceptible to such immunotherapy, and in particular to have an effect on tumors that are responsive to treatment with IL-27, such as melanoma, lymphoma, leukemia, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma.
  • IL- 12, IL-23 and IL- 27 include a group of heterodimeric IL- 12 related cytokines (IL- 12, IL-23 and IL- 27) ( Roo et al, (2002) Immunity. 16: 779-790; Stern et al, (1990)
  • these immunoregulatory cytokines including IL- 12 (Brunda et al, (1993) J.Exp.Med. 178: 1223-1230; Brunda et ⁇ /., (1996) Cancer Chemother. Pharmacol. 38 Suppl: S16-S21), IL-23 (Lo et al, (2003) J Immunol 111 : 600-607; Wang et al, (2003) M J Cancer 105: 820-824; Chiyo et al, (2004) Anticancer Res 24: 3763-3767), IL-27 (Hisada et al, (2004) Cancer Res 64: 1152- 1156; Salcedo et al, (2004) J Immunol 173: 7170-7182; Chiyo et al, (2005) IntJ Cancer 115: 437-442) and EL-18 (Micallef et al, (1997) Cancer Immunol.
  • Immunother. 43: 361-367 can mediate marked antitumor efficacy in preclinical tumor models. Further, IL-2 can synergistically enhance the antitumor activity of DL- 12 (Pappo et al, (1995) J.Surg.Res. 58: 218-226; Zou et al, (1995) Int.Immunol 7: 1135-1145; Wigginton et al, (1996) J.Natl.Cancer Inst. 88: 38-43; Wigginton et al, (1996) AnnMY.Acad.Sci.
  • IL-18 Redlinger et al, (2003) JPediatr Surg 38: 301-307; Wigginton et al, (2002) J Immunol 169: 4467-4474; Son et al, (2003) J Immunother 26: 234-240
  • IL-27 alone can mediate complete regression and long term survival in up to 90% of mice bearing subcutaneous and orthotopic intra-adrenal primary neuroblastoma tumors (Salcedo et al, (2004) J Immunol 173: 7170-7182). Nonetheless, IL-27 alone mediates complete tumor regression and long-term survival in only 40% of mice bearing disseminated neuroblastoma tumors.
  • IL-2 can enhance the anti- metastatic effects of IL-27 in vivo, with up to 90% of mice bearing disseminated TBJ neuroblastoma metastases achieving complete tumor regression and long term survival. Also described herein are the effects of combined delivery of IL-27 and IL- 2 on disease burden within common sites of neuroblastoma metastasis, including both the liver and bone marrow. Interestingly, both IL-27 alone and combined delivery of IL-27 and IL-2 mediate significant regression of metastatic neuroblastoma tumors in the liver, although the reductions in tumor burden are greatest in the IL-27/IL-2 combination group.
  • IL-27/IL-2 to mediate potent antitumor effects against metastatic neuroblastoma tumors may be particularly useful therapeutically give the high frequency of bone marrow disease in patients with high-risk neuroblastoma (Burchill et al, (1995) Eur J Cancer 31 A: 553-556; Hartmann et al, (1999) Bone Marrow Transplant 23: 789- 795; Cotterill et al, (2000) Eur J Cancer 36: 901-908), and the poor prognosis of these patients.
  • IL-27 alone appears to both directly and indirectly upregulate MHC class I expression on tumor cells in vitro and in vivo, and to enhance the generation of tumor-specific immunologic reactivity and immunologic memory in mice cured of their original TBJ neuroblastoma tumors by IL-27 (Salcedo et al, (2004) J Immunol 173: 7170- 7182). As disclosed herein, administration of IL-2 in combination with IL-27 also induces an effective T cell memory response even in mice bearing advanced metastatic disease. Mice cured of their original disseminated neuroblastoma tumors by combined delivery of IL-27 and IL-2 reject a subsequent re-challenge with TBJ parental tumor cells.
  • IL-27 and IL-2 are clearly dependent on CD8+ T cells, but not CD4+ T cells or NK cells. Further, the combination of IL-27 and IL-2 synergistically enhances the generation of immunologic reactivity as evidenced by the production of IFN- ⁇ in response to ex vivo re-stimulation with irradiated tumor cells.
  • IL-27 can act directly on na ⁇ ve CD8+ T cells to enhance granzyme B expression and the generation CTL reactivity directed against C26 colon carcinoma (Morishima et al, (2005) J Immunol 175: 1686-1693).
  • IL-27 enhanced the generation of antitumor CTL responses via mechanisms that occur at not only the initial sensitization or priming of effector cells, but also at the effector phase when primed effector cells are re-stimulated with tumor cells ex vivo: an unexpected finding suggesting that the immunoregulatory activity of IL-27 may not be tightly restricted to na ⁇ ve T cell populations in vivo.
  • combined exposure to IL-27 and IL-2 synergistically enhances the generation of CTL responses specific for TBJ but not irrelevant syngeneic SA-I tumor cells, and the overall antitumor effects were mediated via CD8 + T cells but not CD4+ T cells or NK cells.
  • IL-2 systemic IL-2 can increase the frequency of circulating CD4 + CD25 + Foxp3+ regulatory T cells in patients with metastatic melanoma or renal cell carcinoma, leading to the suggestion that IL-2- induced expansion of regulatory T cells could compromise the efficacy of IL-2 therapy in these patients (Ahmadzadeh & Rosenberg (2006) Blood 107: 2409-2414). Consistent with these observations, as demonstrated herein, IL-2 administration leads to enhancement of the proportion of regulatory T cells within TIL populations in TBJ murine neuroblastoma tumors. Moreover, IL-2 induced increases in the proportion of CD4+CD25+FoxP3+ regulatory T cells with TIL appeared to be strongly attenuated by concurrent delivery of IL-27.
  • the disclosed methods therefore include administering an effective dose of IL-27 (for example by constitutive expression at a target site such as a tumor) to inhibit CD4+CD25+Foxp3+ regulatory T cell induction.
  • IL-27 mediates potent anti-tumor effects, even in mice bearing disseminated murine neuroblastoma, and the effects of IL-27/IL-2 is particularly pronounced against metastatic tumor in the bone marrow, a common site of disease involvement in children with high-risk neuroblastoma.
  • this disclosure provides insight into the potent immunoregulatory activity mediated by IL-27/IL-2, in that the combination synergistically enhances the generation of tumor-specific CTL responses.
  • IL-27 potentiates these responses both during the initial sensitization of effector cells as well as during the subsequent efferent limb of the response when effector cells are re-stimulated with tumor.
  • EXAMPLE 11 IL-2 and IL-27 Sequence Variants This Example describes variant IL-2 and IL-27 sequences that can be used in lieu of or in conjunction with known IL-2 and IL-27 sequences. A number of specific IL-2 and IL-27 amino acid sequences are known.
  • IL- 2 can refer to any IL-2 amino acid sequence, such as: gi30794290, gi7110653, gil 14052044, gi28178861, gil504137, gil504135, gil504133, gil504127, gi512412, gil6758692, gil992, gi47523136, gi57526778, gi6688671, gil811, gi387384, gi349514, gi49169785, gi386818, and gi438518, all of which are specific, non-limiting examples.
  • IL-27 can refer to any IL-27 amino acid sequence, such as: gi28416913, gi21704110, gil 14661841, and gil 11600677, all of which are specific, non-limiting examples.
  • Variant IL-2 and IL-27 proteins include proteins that differ in amino acid sequence from known IL-2 and IL-27 sequences, but that share at least 60% amino acid sequence identity with a known IL-2 or IL-27 protein.
  • Other variants will share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity.
  • IL-2 or IL-27 nucleotide sequence can be used to produce such variants.
  • the simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties. These conservative substitutions are likely to have minimal impact on the activity of the resultant protein.
  • Table 1 shows amino acids that may be substituted for an original amino acid in a protein, and which are regarded as conservative substitutions.
  • More substantial changes in enzymatic function or other protein features may be obtained by selecting amino acid substitutions that are less conservative than those listed in Table 1. Such changes include changing residues that differ more significantly in their effect on maintaining polypeptide backbone structure (for example, sheet or helical conformation) near the substitution, charge or hydrophobicity of the molecule at the target site, or bulk of a specific side chain.
  • substitutions are generally expected to produce the greatest changes in protein properties: (a) a hydrophilic residue (for example, seryl or threonyl) is substituted for (or by) a hydrophobic residue (for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain (for example, lysyl, arginyl, or histadyl) is substituted for (or by) an electronegative residue (for example, glutamyl or aspartyl); or (d) a residue having a bulky side chain (for example, phenylalanine) is substituted for (or by) one lacking a side chain (for example, glycine).
  • a hydrophilic residue for example, seryl or threonyl
  • a hydrophobic residue for example, leucyl,
  • Variant IL-2 and IL-27-encoding sequences may be produced by standard DNA mutagenesis techniques, for example, Ml 3 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ch. 15. By the use of such techniques, variants may be created that differ in minor ways from known IL-2 and IL-27 sequences. DNA molecules and nucleotide sequences that are derivatives of known IL-2 and IL-27 sequences, and which differ from those disclosed by the deletion, addition, or substitution of nucleotides while still encoding a protein that has at least 60% sequence identity with known IL-2 and IL-27 sequences are comprehended by this disclosure.
  • nucleic acid molecules that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% nucleotide sequence identity with known IL-2 and IL-27 sequences.
  • such variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced.
  • the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to known IL-2 and IL-27 protein sequences.
  • variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences.
  • this disclosure also encompasses nucleic acid sequences that encode a known IL-2 or IL-27 protein, but which vary from the known nucleic acid sequences by virtue of the degeneracy of the genetic code.
  • IL-2 sequence variants can be found in US Patent No:
  • EXAMPLE 12 Dosage ranges and administration schedules This Example provides several exemplary administration schedules and dosage ranges for IL-2 in mice and in humans.
  • A/J mice were injected with 1 x 10 5 TBJ cells intravenously on day -5.
  • Different schedules of rhIL-2 (intraperitoneal) treatment started on day 0 as follows: Gl : Vehicle control; G2 (split pulse): 200,000 ILVqAM, qPM day 0, 3, 7, 10, 14, and 17; G3 (chronic): 100,000 IU/day 0-4, 7-11, and 14-18; G4: (chronic): 200,000 IU/day 0-4, 7-11, and 14-18; G5 (intermittent): 200,000 IU/day 0, 2, 4, 7, 9, 11, 16, andl8 (FIG 9A).
  • the survival proportions were as follows: G2: 10%, G3: 30%, G4: 22%, Gp5: 22%.
  • 1 x 10 5 TBJ parental, TBJ-FLAG or TBJ-IL-27 were injected intravenously on day -5.
  • Mice were treated with IL-2 (100,000 IU on days 0-4, 7-11, and 14-18) or vehicle alone (FIG 9B).
  • IL-2 prolonged survival of the TBJ-IL-27 group, the effect was not very profound. Because chronic injection of IL-2 at 100,000 IU/injection was somewhat effective, this dose was increased to 200,000 IU/injection (FIG 9C).
  • TBJ-IL-27 1 x 10 5 TBJ parental, TBJ-FLAG, or TBJ-IL-27 were injected IV on day -5.
  • Mice were treated with IL-2 (200,000 IU on days 0-4, 7-11, 14-18, and 21-25) or vehicle alone.
  • the human dosage range for IL-2 is from about 3,000 IU/kg to about 2,000,000 IU/kg, or from about 30,000 IU/kg to about 200,000 IU/kg.
  • the human dosage range for IL-27 is from about 3 ng/kg to about 30,000 ng/kg, or from about 30 ng/kg to about 3,000 ng/kg.
  • This Example describes various methods that can be used to administer the IL-2 and IL- 27 to a subject.
  • IL-2 and IL-27 are administered by different routes of administration.
  • IL-2 is administered systemically and IL-27 is administered locally, whereas in another embodiment, IL-2 is administered locally an IL-27 is administered systemically.
  • both IL-2 and IL-27 are administered locally, whereas in other embodiments, both IL-2 and IL-27 are administered systemically.
  • systemic administration can be by injection, for instance intravenous, intra-arterial, subcutaneous, intramuscular, or intra-peritoneal injection.
  • Systemic administration also can include transdermal or inhalational administration.
  • one method of administration to the lungs of an individual is by inhalation through the use of a nebulizer or inhaler.
  • the IL-2 or IL-27 is formulated in an aerosol or particulate and drawn into the lungs using a standard nebulizer well known to those skilled in the art.
  • Transdermal administration can be accomplished, for example, by application of a topical cream or ointment or by using a transdermal patch.
  • An effective amount of IL-2 or IL-27 can be administered in a single dose, or in multiple doses, for example daily or every eight hours, during a course of treatment.
  • a therapeutically effective amount of IL-2 or IL-27 is administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time.
  • pulse doses a bolus administration of IL-2 or IL-27 is provided, followed by a time period wherein the drug is not administered to the subject, followed by a second (and optionally subsequent) bolus administration.
  • pulse doses of IL-2 or IL-27 are administered during the course of a day, during the course of a week, or during the course of a month.
  • the IL-2 or IL-27 is administered to the subject on a schedule that includes several daily doses of the IL-2 or IL-27, followed by a withdrawal period, for example to reduce toxicity.
  • the IL- 2 or IL-27 is administered daily for two, three, four, five, six, seven, or more days in a row, followed by a period in which the drug is not administered for one, two, three, four, five, six, seven, or more days.
  • This cycle is repeated until the desired therapeutic effect is achieved, for example tumor regression or remission. In certain examples, the cycle is repeated from about two to about ten times, or even more.
  • the IL-2 or IL-27 is administered locally. In certain embodiments, this is accomplished by local injection into the body part that is affected by the cancer, for example by injecting or infusing the IL-2 or IL-27 directly into the tumor. In other embodiments, local administration is accomplished by implanting a sustained-release device such as a pump or a micropump, or sustained- release implant, such as a bead or gel that contains the IL-2 or IL-27 and slowly releases the drug into the desired area over time.
  • a sustained-release device such as a pump or a micropump
  • sustained- release implant such as a bead or gel that contains the IL-2 or IL-27 and slowly releases the drug into the desired area over time.
  • a retrovirus is used to deliver the IL-2 or the IL-27 to the desired area.
  • Retroviruses have been considered a preferred vector for gene therapy, with a high efficiency of infection and stable integration and expression (see, e.g., Orkin et al, Prog. Med. Genet. 7:130-142, 1988).
  • the full-length IL-2 or IL-27 gene or cDNA is cloned into a retroviral vector and driven from either its endogenous promoter or from the retroviral LTR (long terminal repeat).
  • viral transfection systems may also be utilized for this type of approach, including adenovirus, adeno-associated virus (AAV) (McLaughlin et al, J. Virol. 62:1963- 1973, 1988), Vaccinia virus (Moss et al, Annu. Rev. Immunol. 5:305-324, 1987),
  • AAV adeno-associated virus
  • Vaccinia virus Moss et al, Annu. Rev. Immunol. 5:305-324, 1987
  • Bovine Papilloma virus (Rasmussen et al, Methods Enzymol. 139:642-654, 1987) or members of the herpesvirus group such as Epstein-Barr virus (Margolskee et al, MoI Cell. Biol. 8:2837 2847, 1988).
  • RNA-DNA hybrid oligonucleotides as described by Cole-Strauss, et al ⁇ Science 273:1386-1389, 1996. This technique allows for site-specific integration of cloned sequences, thereby permitting accurately targeted gene replacement.
  • lipidic and liposome-mediated gene delivery has been used successfully for transfection with various genes (for reviews, see Templeton and Lasic, MoI Biotechnol. 11 :175-180, 1999; Lee and Huang, CHt. Rev. Ther. Drug Carrier Sy st. 14:173-206; and Cooper, Semin. Oncol. 23:172-187, 1996).
  • cationic liposomes are used for their ability to transfect monocytic leukemia cells, and have been shown to be a viable alternative to using viral vectors (de Lima et al, MoI Membr.
  • Such cationic liposomes also can be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al, Cancer Gene Ther. 3:250-256, 1996).
  • monoclonal antibodies or other appropriate targeting ligands Kao et al, Cancer Gene Ther. 3:250-256, 1996.
  • intravenous administration to tumors using liposomal delivery generally does not require a specific targeting mechanism, since the leakage of the tumor vasculature results in localized administration to the tumor.
  • IL-2 and/or IL-27 is delivered via alpha(v)beta(3) integrin nanoparticles.
  • Alpha(v)beta(3)-targeted nanoparticles can be used to selectively deliver a gene to tumor vasculature.
  • An alpha(v)beta(3)-targeted nanoparticle is a cross between two molecules: a "catalytic" antibody, and a small drug molecule, which are linked by a linker molecule.
  • antibodies are proteins produced by immune cells that are designed to recognize a wide range of foreign pathogens. After a pathogen enters the bloodstream, antibodies target antigens (proteins, carbohydrate molecules, and other pieces of the pathogen) specific to that pathogen. These antibodies then alert the immune system to the presence of the pathogen and attract lethal "effector" immune cells to the site of infection.
  • the small-molecule portion targets cancer cells.
  • the compounds target alpha(v)beta(3) integrin, which are expressed by endothelial cells during angiogenesis.
  • Many cancer cells like breast, ovarian and prostate cancer, also express integrins on their surface, providing for a potential double-strike against the tumor itself as well as its key blood supply. » IL-27 delivered by tumor targeted nanoparticles potentiates the generation of cytotoxic T lymphocytes while inhibiting T regulatory cells.
  • peptide binding nucleolin which is expressed by a broad spectrum of tumors as well as in endothelial cells in angiogenice vessels, is another potential targeting molecule to be used in IL-27 nanoparticle delivery approach.
  • IL-27 can directly mediate inhibition of angiogenesis (Shimizu (2006) J Immunol 176:7317-24).
  • Another useful tumor targeting molecule of particular value in treating neuroblastoma is antiganglioside GD2 antibody, which is highly expressed by human neuroblastomas.
  • Nanoparticles can deliver either IL-27 protein or IL-27 DNA.
  • IL-2 and/or IL-27 is administered via adoptive transfer of T lymphocytes overexpressing IL-2 or IL-27.
  • adoptive transfer of T lymphocytes overexpressing IL-2 or IL-27 involves the passive transfer of antitumor-reactive cells into a host in order to mediate tumor regression. The transfer of immune lymphoid cells can eradicate widely disseminated tumors and establish long-term systemic immunity.
  • IL-2- or IL-27-overexpressing T lymphocytes are administered locally or systemically in dose levels ranging from about 10 7 to 10 10 cells/day.
  • IL-27 and/or IL-2 is expressed by a lentiviral vector pG13, and T lymphocytes expressing one or both of the interleukins from tumor-bearing mice or human subjects home into the tumor and mediate tumor regression.
  • T lymphocytes expressing one or both of the interleukins from tumor-bearing mice or human subjects home into the tumor and mediate tumor regression.
  • Clostridium n ⁇ vyz-NT expressing IL-2 and/or IL-27 are systemically injected into animals, they germinate exclusively within the hypoxic regions of cancers.
  • the germinated bacteria destroy adjacent tumor cells but spare a rim of well oxygenated tumor cells that subsequently expand.
  • the mechanism underlying this effect is immune-mediated, because cured subjects reject a subsequent challenge of the same tumor.
  • the induced immune response when combined with the bacteriolytic effects of C. woyyz-NT, eradicates even large, established tumors.
  • the genetically modified anaerobic bacteria are administered intravenously to subjects.
  • an effective dose ranges from about 3,000 IU/kg body weight to about 2,000,000 IU/kg body weight in some specific, non-limiting examples, or in more specific examples from about 30,000 IU/kg body weight to about 600,000 IU/kg body weight, or in yet more specific examples from about 50,000 IU/kg to about 200,000 IU/kg of body weight, based on efficacy.
  • IL-2 is administered in a dose from about 30,000 IU/kg to about 200,000 IU/kg every eight hours, for a daily dose of from about 240,000 IU/Kg to about 600,000 IU/Kg daily.
  • an effective dose ranges from about 3 ng/kg body weight to about 30,000 ng/kg body weight in some specific, non-limiting examples, or in more specific examples from about 30 ng/kg body weight to about 3,000 ⁇ g/kg body weight, or in yet more specific examples from about 100 ng/kg to about 2,000 ng/kg of body weight, based on efficacy.
  • IL-2 is administered by systemic intermittent pulse administration and IL-27 is administered locally at the tumor site.
  • the IL-2 is administered intravenously every day or several times per day (for example every eight hours) for several days (for example from about two to about nine days), followed by a period in which the IL-2 is not administered. This period can range from about two days to about nine days, and limits the toxicity associated with IL-2.
  • the period of IL-administration followed by IL-2 withdrawal is repeated to form an administration cycle.
  • the IL-2 is administered from one to three times per day for two, three, four, five, six, or seven days, followed by two, three, four, five, six, or seven days when IL-2 is not administered.
  • the IL-2 is administered daily for five, six, seven, eight, or nine days, followed by a period of five, six, seven, eight, or nine days in which no IL-2 is administered.
  • the length of the cycles of IL-2 administration and IL-2 withdrawal are adjusted to suit the needs of the individual and the particular disease being treated, and may begin with a higher initial dose, which is then followed by a lower dose in subsequent cycles. For instance, an initial dose of IL-2 may be set to maximize the initial immune response to the IL-2, and then a lower dose may be selected for additional IL-2 administration cycles based on the response of the subject to the initial dose.
  • the number of cycles of administration is adjusted for the particular subject being treated and the disease severity.
  • a shorter course of treatment comprises only one or two cycles.
  • a longer course of administration comprises as many as ten or more cycles. More cycles may be needed to treat a more severe disease or a subject who is resistant to IL-2 toxicity, whereas fewer cycles may be needed to treat a less severe disease or a subject who is prone to IL-2 toxicity.
  • an effective dose ranges from about 3,000 IU/kg body weight to about 2,000,000 IU/kg body weight in some specific, non-limiting examples, or in more specific examples from about 30,000 IU/kg body weight to about 600,000 IU/kg body weight, or in yet more specific examples from about 50,000 IU/kg to about 200,000 IU/kg of body weight, based on efficacy.
  • IL-2 is administered in a dose from about 30,000 IU/kg to about 200,000 IU/kg every eight hours, for a daily dose of from about
  • IU/Kg 240,000 IU/Kg to about 600,000 IU/Kg daily.
  • the upper dose ranges are limited by IL-2 toxicity, which may occur at different doses in different subjects.
  • the lower dose ranges are determined by IL-2 efficacy, which is enhanced by the substantially concurrent administration of IL-27, and which is monitored by tumor regression or remission. Additional dosage regimens can be found in, for example, US Patent Application No: 2003/0235556, although in general the doses required for effective IL-2 therapy are lower when IL-2 is combined with IL-27.
  • IL-27 is administered locally while the IL-2 is administered systemically.
  • Local administration can be accomplished in a number of ways, as described in Example 13, for instance by direct injection or infusion of the protein at the tumor site, by gene therapy using viral vectors, via RNA-DNA hybrid oligonucleotides, via non-infectious methods of delivery, such as lipidic and liposome-mediated gene delivery, via alpha(v)beta(3) integrin nanoparticles, via adoptive transfer of T lymphocytes overexpressing IL-27, or by using anaerobic bacteria, such as toxin-free Clostridium novyi.
  • IL-27 it is desirable to begin administration of IL-27 before the IL-2 administration commences because this increases the efficacy of IL-2 and permits the use of lower IL-2 doses, which in turn lowers IL-2 toxicity.
  • IL-27 administration can be started concurrently with IL-2 administration.
  • Other examples of local delivery can be found in US Patent No: 6,310, 045.
  • the combined IL-2/IL-27 therapy can be administered in conjunction with treatment with other anti-cancer agents.
  • compositions that comprise at least one IL-2 or IL-27 protein or fragment thereof as an active ingredient will normally be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen.
  • the pharmaceutically acceptable carriers and excipients useful in this disclosure are conventional.
  • parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the dosage form of the pharmaceutical composition will be determined by the mode of administration chosen.
  • topical and oral formulations can be employed.
  • Topical preparations can include eye drops, ointments, sprays and the like.
  • Oral formulations may be liquid (for example, syrups, solutions or suspensions), or solid (for example, powders, pills, tablets, or capsules).
  • solid compositions conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
  • the pharmaceutical compositions that comprise IL-2 and/or IL-27 preferably will be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • One possible unit dosage contains approximately 300,000 ng of protein.
  • the amount of active compound administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in an amount effective to achieve the desired effect in the subject being treated.

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Abstract

La présente invention concerne, d'une manière générale, des procédés de traitement du cancer, par exemple des procédés de traitement du neuroblastome, du mélanome, du lymphome, de la leucémie, de l'hypernéphrome, du carcinome du colon, du carcinome de la poitrine, du carcinome des ovaires, du carcinome de la prostate et du carcinome du poumon. Le procédé met à profit la découverte surprenante selon laquelle IL-2 et IL-27 possèdent un effet coopératif anti-tumoral lorsqu'ils sont administrés conjointement à un sujet.
PCT/US2006/041534 2006-10-24 2006-10-24 Interaction d'il-27 et d'il-2 dans le traitement de tumeurs Ceased WO2008051220A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12006354B2 (en) 2017-05-24 2024-06-11 Novartis Ag Antibody-IL2 engrafted proteins and methods of use in the treatment of cancer

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WO2005014642A2 (fr) * 2003-07-21 2005-02-17 Transgene S.A. Nouvelles cytokines multifonctionnelles

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WO2005014642A2 (fr) * 2003-07-21 2005-02-17 Transgene S.A. Nouvelles cytokines multifonctionnelles

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SALCEDO R ET AL: "IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: Role for CD8+ T cells", JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 173, no. 12, 15 December 2004 (2004-12-15), pages 7170 - 7182, XP002345766, ISSN: 0022-1767 *
SALCEDO ROSALBA ET AL: "Immunologic and therapeutic synergy of IL-27 and IL-2: Complete regression of neuroblastoma metastases in the liver and bone marrow", JOURNAL OF IMMUNOTHERAPY, vol. 28, no. 6, November 2005 (2005-11-01), & 20TH ANNUAL SCIENTIFIC MEETING OF THE INTERNATIONAL-SOCIETY-FOR-BIOLO GICAL-THERAPY-OF-CANCER; ALEXANDRIA, VA, USA; NOVEMBER 10 -13, 2005, pages 625, XP009091518, ISSN: 1524-9557 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12006354B2 (en) 2017-05-24 2024-06-11 Novartis Ag Antibody-IL2 engrafted proteins and methods of use in the treatment of cancer

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