WO2008051220A1 - Interaction of il-27 and il-2 for treatment of tumors - Google Patents

Interaction of il-27 and il-2 for treatment of tumors 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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PCT/US2006/041534
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French (fr)
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Rosalba Salcedo
Jon M. Wigginton
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The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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Priority to PCT/US2006/041534 priority Critical patent/WO2008051220A1/en
Publication of WO2008051220A1 publication Critical patent/WO2008051220A1/en

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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4635—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • 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 A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

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.

Abstract

The present disclosure is generally related to methods of treating cancer, for example methods for treating neuroblastoma, melanoma, lymphoma, leukemia, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma. The method makes use of the surprising discovery that IL-2 and IL-27 have a synergistic anti-tumor effect when co-administered to a subject.

Description

INTERACTION OF IL-27 AND IL-2 FOR TREATMENT OF TUMORS
FIELD OF THE DISCLOSURE 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.
BACKGROUND
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. Several studies have evaluated imrnunotherapeutic agents including cytokines in preclinical tumor models and/or early phase clinical studies in children with neuroblastoma. Of these agents, interleukin-2 (IL-2) and interleukin- 12 (IL- 12) have demonstrated some efficacy in pre-clinical neuroblastoma models and in the clinical setting. However, 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.
Given the foregoing, it would be desirable to have 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.
SUMMARY OF THE DISCLOSURE
Disclosed herein is the surprising discovery that the interaction (for example by substantial co-administration) of IL-2 and IL-27 produces a synergistic anti-tumor effect. Thus, 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. In one embodiment, IL-2 is intermittently administered while IL-27 is continuously administered (for example by constitutive expression, such as local expression at a tumor site). Also disclosed is 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. In a particular embodiment, IL-2 is administered systemically and the IL-27 is administered locally, and this combination treats the metastatic neuroblastoma. The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES FIG l is a pair of graphs showing that IL-27 and IL-2 synergistically enhance proliferation and IFN-γ 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 3H-thymidine (1 μCi/well) incorporation as described in Example 1. Values shown represent the mean value +/- standard error of the mean (SEM) for triplicate samples (FIG IA). To investigate the impact of combined IL-27 and IL-2 therapy on the production of IFN-γ, normal spleen cells were cultured in the presence of IL-27 +/- IL-2 or medium alone for 72 hours as described in Example 1. Supernatants were then harvested and the concentration of IFN-γ in the respective culture supernatants was assayed using ELISA. The values shown represent the mean SEM for duplicate samples (FIG IB). *p < 0.001; IL-2 + IL-27 vs. either IL-2 or IL-27 or medium alone.
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 105 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. Cohorts of mice (10 mice/group) bearing established disseminated TBJ IL-27 or TBJ FLAG (control) tumors were treated with IL-2 (200,000 IU/injection) or vehicle alone on days 5-9 and 12-15 post tumor implantation as described in Example 1. Mice were then euthanized at day 16 post tumor implantation and livers were resected for imaging of metastatic disease burden in the respective treatment groups. Light images of the respective organs were captured at IX magnification as described in Example 1. Digital images of 5 livers per group are shown (FIG 3A). The number of macrometastatic lesions at day 16 post-tumor injection was also scored (FIG 3B). *p = 0.035; TBJ IL-27 + IL-2 vs. TBJ IL-27 + vehicle.
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 1x105 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. The data shown represents the proportion of mice developing colonies in each of the respective treatment groups, *p = 0.0198; TBJ IL-27 + IL-2 vs. TBJ IL-27 + vehicle (FIG 4A). In complementary - A -
studies, cohorts of mice (10 mice/group) were injected with 1x105 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 106 cells/injection) into naive mice to assess the tumorigenicity of contaminating neuroblastoma metastases in the bone marrow. Tumor size was monitored twice a week. The proportion of mice that developed a tumor at any time post injection of bone marrow cells from the respective groups is shown in FIG 4B. **p = 0.011, TBJ IL-27 + IL-2 vs. TBJ IL-27 + vehicle.
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 105 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. Mice bearing primary orthotopic TBJ Flag or TBJ-IL-27 tumors treated with vehicle control or IL-2 were euthanized as described Example 1. Tumors were dissected and TIL were isolated and stained with PECy7-conjugated rat anti mouse CD4, APC-conjugated rat anti mouse CD8a, PE-conjugated rat anti-mouse CD25, and FITC-conjugated anti-mouse Foxp3 as described in Example 1. The percentage of CD4+ CD25+ Foxp3+ T cells within the total CD4+ T cell population in the tumors under different treatments was determined. The data shows the mean and SEM of three experiments (FIG 6B), *p = 0.045, TBJ Flag + IL-2 vs. TBJ IL-27 + IL-2. FIG 7 is a graph showing that combined exposure to IL-27 and IL-2 synergistically enhances immune responsiveness: IFN-γ 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. Single cell suspensions were prepared and splenocytes were then re-stimulated with corresponding irradiated TBJ FLAG or TBJ IL-27 tumors cells +/- IL-2 (10 IU/ml) for 72 hours as described in Example 1. IFN-γ concentration in the respective culture supernatants were then assayed by ELISA. Values shown represent the mean +/- SEM of triplicate samples. 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. 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. Combined exposure to IL-27 and IL-2 synergistically enhanced the generation of CTL reactivity (FIG 8A). Additionally, IL-27 mediates effects during both the priming and effector phases of the generation of CTL reactivity and this effect was further enhanced by IL-2 (FIG 8B). This CTL reactivity is tumor specific, since enhancement of reactivity against syngeneic irrelevant SA-I tumor cells was not observed. Values shown represent the mean percentage of specific lysis for each of the respective groups assessed in triplicate at the indicated effector :target (E:T) ratios. FIG 9 is a series of three histograms showing the efficacy of various doses and administration schedules of IL-2. For FIG 9A, A/J mice were injected with 1 x 105 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. The survival proportions were as follows: G2: 10%, G3: 30%, G4: 22%, Gp5: 22%. For FIG 9B5 1 x 105 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 105 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. IL-2 treatment significantly enhanced the survival of the TBJ-IL-27 group, (p =0.2661, TBJ-IL-27 vs. TBJ-IL27 + IL-2).
SEQUENCES SEQ ID NO: 1 is a synthetic linker
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS /. Overview of several embodiments
Disclosed herein is the surprising discovery that co-administration of IL-2 and IL-27 to a subject produces a synergistic anti-tumor effect. Thus, 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. In some embodiments, 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. In some embodiments of the method, administration of the IL-2 is by systemic administration, for instance by intravenous, intra-muscular, subcutaneous, intra-arterial, or intra-peritoneal administration. In other embodiments of the method, 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
- 7 -
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. In particular examples, administration of the IL-2 is by systemic administration, and administration of the IL-27 is by local administration. In even more particular examples, 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.
In some embodiments, 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. In certain examples, 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. For example, 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.
In particular examples of the method, the subject has metastatic neuroblastoma. In certain embodiments the subject is human, wherein in other embodiments the subject is a veterinary subject.
In certain very particular examples, 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. In this particular example of the method, the IL-2 is administered systemically and the IL-27 is administered locally, thereby treating the metastatic neuroblastoma.
Other embodiments are compositions for use in stimulating an anti-tumor response. These compositions include an amount of IL-2 and IL-27 effective to stimulate an immune response and treat the tumor. //. Abbreviations AJJ albino Jackson
ALL acute lymphocytic leukemia
AML acute myelogenous leukemia
ANOVA analysis of variance
CLL chronic lymphocytic leukemia CML chronic myelogenous leukemia
CTL cytotoxic T-lymphocyte
ELISA enzyme-linked immunosorbent assay
FCS fetal calf serum
FET Fisher's exact test IFN-γ interferon-γ
IL-2 interleukin-2
IL-27 interleukin-27
NK natural killer cells
PBS phosphate-buffered saline SEM standard error of the mean
TBJ a cell clone from C-1300 neuroblastoma cells.
TIL tumor-infiltrating lymphocytes III. Explanation of Terms
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of the various embodiments of the disclosure, the following explanation of terms is provided:
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. 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. While many small-molecule drugs are cleared from the blood by the kidneys in a matter of minutes or hours, the large, soluble antibody molecules are designed to remain in the bloodstream for long periods of time. While the antibody portion of the hybrids keeps them circulating, the small-molecule portion targets cancer cells. In the present disclosure, 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.
For a detailed description of targeted delivery via alpha(v)beta(3)-targeted nanoparticles, see for example, Reynolds et al, (2003) Trends MoI Med. 9(l):2-4; and Winter et al, (2006) Arterioscler Thromb Vase Biol. 26(9):2103-9).
Anaerobic bacterial administration: such as by toxin-free Clostridium novyi, occurs when spores of the anaerobic bacterium Clostridium nøvyz-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. Similarly, 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), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfϊmer sodium, and verteporfin; cytokines, such as IL-2 and IL-27; and other agents, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, pentostatin, masoprocol, mitotane, pegaspargase, and tretinoin.
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.
There are a number of different types of breast cancer, including ductal carcinoma in situ, lobular carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, and paget's disease. 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.
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. For example, 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. Several specific, non-limiting examples of 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. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed. Unless otherwise specified, 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. For example, sequence variants in a protein, such as a 5' or 3' variant, may retain the full function of an entire protein. Fourth, 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 32P, 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. For example, 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-γ, etc.), immunoglobulin production, dendritic cell maturation, and proliferation of a cell of the immune system. One of skill in the art can readily determine an increase in any one of these parameters, using known laboratory assays. In one specific non-limiting example, to assess cell proliferation, incorporation of 3H-thymidine can be assessed. 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. For instance, 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. 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. As used herein, 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. Interaction of IL-2 and IL-27: 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-γ 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.. The lower dose of IL-2 is desirable for limiting IL-2 toxicity, which limits the efficacy of IL-2 therapy. Interleukin-2 (IL-2): a T cell growth factor that binds to a specific tripartite receptor on T cells. In dose escalation studies, patients treated with high doses of IL-2 showed clinical responses, although severe toxicity was seen. Response rates were as high as 24% at the highest IL-2 dose in patients with renal cell carcinoma, however the toxicities of treatment were limiting. These toxicities stem from what is known as a capillary leak syndrome. Administering IL-2 in high doses is comparable to inducing a controlled state of septic shock. 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. Dopamine is administered prophylactically and therapeutically for low-urine output, neosynephrine is given for hypotension, acetaminophen for fever and chills, cimetidine for gastric acidity, and oxacillin for neutrophil dysfunction that leads to sepsis. Concomitant antibiotic therapy is essential, as the rate of sepsis without antibiotic therapy is as high as 27%. 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. For example, ATP can be labeled in any one of its three phosphate groups with radioisotopes such as 32P or 33P, or in its sugar moiety with a radioisotope such as 35S.
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.
The diseases are further classified according to the type of abnormal cell found most in the blood. When leukemia affects lymphoid cells (lymphocytes and plasma cells), it is called lymphocytic leukemia. When myeloid cells (eosinophils, neutrophils, and basophils) are affected, the disease is called 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. Acute myelogenous leukemia (also known as Acute Myeloid Leukemia, or AML) occurs more commonly in adults than in children. This type of leukemia was previously called acute nonlymphocytic leukemia. Chronic lymphocytic leukemia (CLL) most often affects adults over the age of 55. It sometimes occurs in younger adults, but it almost never affects children. Chronic myelogenous leukemia (CML) occurs mainly in adults. A very small number of children also develop this disease. The most common forms in adults are AML and CLL, whereas in children ALL is more prevalent.
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). For instance, 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). However, 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.
Traditionally, 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.
Today, lymphomas are classified in three large groups: the B cell tumors, the T cell and natural killer cell tumors, and Hodgkin's lymphoma, as well as other minor groups. 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 lymphoma, Burkitt lymphoma/leukemia, and lymphomatoid granulomatosis. 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.
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. There is also a propensity for certain tumors to seed in particular organs. For example, prostate cancer usually metastasizes to the bones. Similarly, 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.
When 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. This means, for example, that if breast cancer spreads (metastasizes) to the lung, 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. It is a 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. Also a rosette pattern in a highly cellular tumor is an additional characteristic of neuroblastoma.
When the lesion is localized, it is frequently curable. However, long-term survival for children with advanced disease is poor despite aggressive multimodality therapy. Several studies have evaluated imrnunotherapeutic agents including cytokines in preclinical tumor models and/or early phase clinical studies in children with neuroblastoma. Of these agents, interleukin-2 (IL-2) and interleukin-12 (IL- 12) have demonstrated some efficacy in pre-clinical neuroblastoma models and in the clinical setting. However, 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.
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. Generally, 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. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, 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. The term 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.
The term polypeptide fragment refers to a portion of a polypeptide that exhibits at least one useful epitope. The phrase "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: "Preventing" 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.
Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, 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. For larger tumors confined to the kidney without venous invasion, survival is still relatively good at 80-85%. For tumors that extend through the renal capsule and out of the local fascial investments, 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. 147(1):195-197, 1981; Needleman and WunschJ MoI Biol. 48: 443-453, 1970; Pearson and Lipman Proc. Natl. Acad. Sci. USA 85: 2444- 2448, 1988; Higgins and Sharp Gene, 73: 237-244, 1988; Higgins and Sharp CABIOS 5: 151-153, 1989; Corpet et α/. Nuc. Acids Res. 16, 10881-10890, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-165, 1992; and Pearson et al. Meth. MoI. Bio. 24, 307-331, 1994. Furthermore, Altschul et al (J. MoI. Biol. 215:403-410, 1990) present a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.
MoI. Biol. 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. 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. For instance, this can be the amount necessary to treat 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). The abbreviation "T", in T cell, stands for thymus since it is the principal organ for their development.
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).
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
IV. Description of several specific embodiments
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. Among the clinicopathologic features that contribute to existing risk stratification criteria for patients with neuroblastoma, 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). Notably, 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.
Among the approaches under investigation, several studies have evaluated immunotherapeutic agents including cytokines (Siapati et al, (2003) Br J Cancer 88: 1641-1648; Redlinger et al, (2003) J Pediatr Surg 3S: 301-307; Shimizu et al, - z.7 —
(2001) JPediatr Surg 36: 1285-1292) or imniunocytokines (Lode et al, (1998) Blood 91: 1706-1715; Sondel & Hank (1997) Cancer J Sci Am 3 Suppl 1: S121-127; Frost et al, (1997) Cancer 80: 317-333; Hank et al, (1994) JImmunother 15: 29- 37) in preclinical tumor models and/or early phase clinical studies in children with neuroblastoma. Of these agents, 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
(1997) Cancer J Sci Am 3 Suppl 1 : S121-127; Bauer et al, (1995) Cancer 75: 2959- 2965; Bonig et al, (2000) Bone Marrow Transplant 26: 91-96; Toren et al, (2000) Transplantation 70: 1100-1104; Favrot et al, (1989) Cancer Treat Rev 16 Suppl A: 129-142). However, the clinical antitumor efficacy of single cytokines or immunocytokines has been modest (Sondel & Hank (1997) Cancer J Sci Am 3 Suppl 1 : S121-127; Frost et al, (1997) Cancer 80: 317-333; Bauer et al, (1995) Cancer 75: 2959-2965; Bonig et al, (2000) Bone Marrow Transplant 26: 91-96; Toren et al, (2000) Transplantation 70: 1100-1104; Favrot et al, (1989) Cancer Treat Rev 16 Suppl A: 129-142), and is associated with significant deleterious side effects (Roper et al , (1992) American Journal of Pediatric Hematology/Oncology 14: 305- 311).
Based on its potent immunoregulatory activity, recent studies have investigated the antitumor efficacy of IL-27 in preclinical tumor models, including murine models of colon carcinoma and neuroblastoma (Hisada et al , (2004) Cancer Res 64: 1152-1156; Salcedo et al, (2004) J Immunol 173: 7170-7182; Chiyo et al, (2005) MJ Cancer 115: 437-442). 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-γ 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. In turn, 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). However, despite the broad effects of IL-27 against subcutaneous and orthotopic primary neuroblastoma tumors, the antitumor efficacy of IL-27 alone is more modest in mice bearing disseminated TBJ neuroblastoma metastases. In this model, IL-27 mediates complete durable tumor regression in only 40% of mice bearing disseminated TBJ IL-27 tumors.
As described herein, in light of the difficulty in treating patients with metastatic neuroblastoma, the effect of combined delivery of IL-27 and IL-2 was probed in mice bearing disseminated TBJ neuroblastoma tumors. Surprisingly, the combined delivery of IL-27 and IL-2 mediates synergistic antitumor efficacy in mice bearing TBJ neuroblastoma metastases in the liver and bone marrow, and it does so in conjunction with potent effects on the generation of tumor-specific immune responsiveness. In addition to its potent effects on metastatic neuroblastoma, the combination
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. Without further elaboration, it is believed that one skilled in the art can, using this description, utilize the present discoveries to their fullest extent. The following examples are illustrative only, and not limiting of the disclosure in any way whatsoever.
EXAMPLE 1 Materials and methods
This example describes the materials and methods used in the subsequent Examples.
Reagents, Tumor Cells and 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) and the mature coding sequence of mouse p28 as previously described (Pflanz et ah, (2002) Immunity. 16: 779-790). Briefly, 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 μg/ml) (Gibco, Invitrogen Corp. Carlsbad, CA).
Individual clones were isolated by limiting dilution cloning, and were screened subsequently for IL-27-FLAG or FLAG gene expression using RT-PCR. The level of IL-27 protein was also confirmed by FLAG immunoprecipitation followed by Western blot as described previously (Salcedo et al, (2004) J Immunol 173: 7170-7182). The SA-I murine sarcoma cell line (syngeneic to A/J mice) was provided by Dr Suzanne Ostrand-Rosenberg (University of Maryland, Baltimore County, Baltimore, MD). Single chain IL-27 protein generated as previously described (Pflanz et al, (2002) Immunity. 16: 779-790) was used for in vitro studies. Commercially-available recombinant human IL-2 (Chiron, Emeryville, CA) was used for both in vitro and in vivo studies. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985).
Immunoregulatory effects of IL-27 and IL-2 in vitro
The effects of combined treatment with IL-27 and IL-2 on proliferative responses and IFN-γ production by murine splenocytes was assessed in vitro. Single cell suspensions were prepared using spleens from normal A/J mice as described herein. Cells were suspended in complete medium at 2x106 cells/ml in Roswell Park Memorial Institute medium (RPMI) containing 5% FCS, 2 mM glutamine, ImM pyruvate, 5 x 10"5 M 2-mercaptoethanol, 2mM non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 1OmM HEPES. For proliferation assays, 5x 104 cells/well were cultured similarly in round-bottom 96-well plates in the presence or absence of IL-2 (50 IU/ml) and/or IL-27 (50 ng/ml) for 72 hours. 3H-thymidine (1 μCi/well) was added 18 hours prior to harvest, and 3H-thymidine incorporation was determined using a beta counter and standard techniques. For IFN-γ production, Ix 106 spleen cells/ml were incubated in complete medium with IL-27 (50 ng/ml) +/- IL-2 (50 IU/ml) or medium alone for 48 hours. Culture supernatants were then harvested and IFN-γ levels were assessed by ELISA (R&D Systems, Minneapolis, MN). In vivo tumor models and treatment regimens
Where indicated, syngeneic male A/J mice were injected intravenously with IxIO5 TBJ IL-27 or control TBJ FLAG tumor cells /animal on day 0. For survival studies, 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. To assess the impact of combined delivery of IL-27 and IL-2 more specifically on liver metastasis, 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.
To quantitatively assess the impact of combined delivery of IL-27 and IL-2 on neuroblastoma metastases in the bone marrow, 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.
In complementary studies, the tumorigenicity of neuroblastoma metastases in the bone marrow of treated mice was also evaluated. In these studies, 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. Using bone marrow specimens from the respective treatment groups, naϊve mice were then injected subcutaneously with 1x107 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. To evaluate whether specific immunologic memory responses were generated in mice cured of their original tumors by combined delivery of IL-2 and IL-27 as described above, mice were re-challenged subcutaneously with TBJ parental tumor cells (1x106 cells/animal) where indicated and then monitored for survival. To investigate the specific role of T and/or NK cell subsets in the antitumor activity of the IL-27/IL-2 combination, mice were injected intravenously with 1x105 TBJ-FLAG or TBJ IL-27 tumor cells/animal on day 0. To deplete CD4+ versus CD8 T cell subsets respectively in vivo, 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. To deplete NK cells in vivo, anti-asialoGMl (diluted 1 :20, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was administered intraperitoneally on day -1 and days 2, 7, 12, 16, 21 and 26 post-tumor cell implantation. 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.
To investigate the effect of combined delivery of IL-2 and IL27 on the composition of tumor infiltrating lymphocytes (TIL) within TBJ tumors, mice were injected orthotopically in the adrenal gland with 1 x 105 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.
Generation of immunologic responsiveness: IFN- γ production and generation of tumor-specific CTL reactivity
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. Initially, spleens were resected from mice bearing disseminated TBJ IL-27 or TBJ FLAG tumors at day 15 post-tumor cell injection. Spleen cells (Ix 106 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 105 cells/ml) with or without IL-2 (50 IU/ml). The cultures were then incubated for three days at 370C, and supernatants were harvested for determination of IFN-γ concentrations by ELISA as described herein.
To demonstrate the impact of combined delivery of IL-27 and IL-2 on the induction of tumor-specific CTL reactivity in mice bearing TBJ neuroblastoma tumors, spleens 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 107cells/ml) were incubated in the presence of irradiated (6,000 rads) TBJ FLAG or TBJ IL-27 tumor cells (1 x 106/ml) +/- IL-2 (10 IU/ml) in complete medium. Cultures were incubated for six days, and the cytolytic activity of these splenic effector cells was assessed using a standard 11 'indium-release assay. Briefly, 1 x 10 TBJ parental or irrelevant syngeneic SA-I tumor cells were labeled with 10 μCi (C9H6NO)3 "1In (Amersham Health Medi- Physics, Inc., Arlington Heights, IL) for 15 minutes at room temperature. Target cells were washed, resuspended at 1 x 105 cells/ml. Variable numbers of splenic effector cells in complete medium were plated in triplicate in 96-well plate (Costar, Cambridge, MA) and then 1 x 104 labeled target cells/well added to achieve the desired E:T ratio, and plates were then incubated for 18 h at 37 0C. At the conclusion of the incubation, culture supernatants were harvested and counted individually in a gamma counter (Wallac, Turku, Finland). The percentage of specific lysis was calculated as follows: % of specific lysis = (ER-SR) x 100/(MR - SR), where ER = experimental release; SR = spontaneous release; MR= maximum release. Minimum release was determined by measuring target cell supernatant alone. Maximum release was determined by exposing the target cells to 1% SDS.
Imaging of liver metastases
To image the organ-specific impact of combined delivery of IL-27 and IL-2 on metastatic neuroblastoma tumors in the liver, 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).
Bone Marrow Colony Assay
To assess the impact of combined delivery of IL-27 and IL-2 on neuroblastoma metastases in the bone marrow, 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 0C, and cells were resuspended at 5x106 cells/ml in RPMI containing 5% FCS and 2 mM glutamine. One hundred microliters of bone marrow cell suspension (5xlθ5cells) were added to 1.3 ml of methylcellulose media containing 40% base Methylcellulose M3134 ( Metho-Cult, Stem Cell Technologies Inc, Vancouver, BC) and 60% RPMI (20% FCS, 4 mM L- glutamine) in a 35-mm petri dish. Plates were incubated for 12-14 days in humidified atmosphere at 37 0C with 5% CO2. Colony formation from normal bone marrow cells was inhibited by addition of G418 (1 mg/ml), which selectively allowed the formation of colonies of tumor cells carrying the neomycin resistance gene as a component of the IL-27 and FLAG vector control plasmid constructs. Colonies were enumerated on a Nikon SMZ800 stereomicroscope (Nikon, Melville,
NY).
Flow cytometric analysis of tumor infiltrating lymphocytes
To demonstrate the composition of infiltrating lymphocytes within TBJ FLAG or TBJ IL-27 tumors, 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 μg/ml of DNAse I (Boehringer Mannheim,
Mannheim,Germany), at 37 0C for two hours. After digestion, cell aggregates were removed using a 40 μm nylon cell strainer (BD Biosciences, Bedford, MA).
Single cell suspensions (1 x 107 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. 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). Briefly, cell pellets were vortexed and 1 ml of e- Bioscience-Fix/Perm solution was added, followed by incubation on ice for 18 hours in the dark. Cells were then washed once with PBS (ImI) and twice with e-
Bioscience-permeabilization solution (ImI). Thereafter, non-specific binding was blocked again for 15 minutes using rat anti-mouse 24G2 antibody diluted 1 :500 in e- Bioscience-permeabilization solution containing 3% FCS and 10 % homologous A/J mouse serum. Finally, FITC conjugated rat anti mouse Foxp3 (FJK-16s) diluted 1 :100 in e-
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+
Foxp3+) within each tumor was analyzed within the CD4+ T cell gate. Additionally, lymph nodes cells from naϊve mice were used as a control to set the markers for CD25+ Foxρ3+ and CD25"Foxp3" populations.
Statistical methods
Where indicated, 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. For proliferative responses, IFN-γ production and CTL responses by murine splenocytes, mean values were determined for the respective conditions (duplicate for IFN-γ, triplicate for proliferation and CTL responses), and were compared using the oneway and two-way analysis of variance (ANOVA) procedure. Follow-up 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. For liver metastasis counts, pairwise comparisons were performed using the nonparametric Mann- Whitney Wilcoxon test. All p values were two-tailed and were considered significant aXp < 0.05. EXAMPLE 2
IL-27 and IL-2 synergistically enhance proliferative responses and IFN-γ production by murine splenocytes in vitro
This example demonstrates that combination of IL-27 and IL-2 interact favorably to enhance immune responsiveness. The impact of combined treatment with IL-27 and IL-2 on proliferative responses and IFN-γ production by murine splenocytes was evaluated in vitro. Surprisingly, the combination of IL-27 and IL-2 synergistically enhances proliferative responses by murine splenocytes. 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). Further, synergistic increases in IFN-γ production were observed when splenocytes were stimulated with the combination of IL-27 and IL-2 compared to either single cytokine or medium alone (medium alone:≤ 50 pg/ml, IL-2: ≤ 50 pg/ml; IL-27: 62.65 ± 12.6 pg/ml; IL-2 + IL-27: 502.5 ± 6.5 pg/ml; p < 0.001, IL-2 + IL-27 vs. either IL-2 or IL-27 or medium alone) (FIG IB). Thus, the combination of IL-2 and IL-27 appears to synergize with respect to the induction of lymphocyte proliferation and IFN-γ production in vitro.
EXAMPLE 3
Combined delivery of 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. In this model, 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). Systemic administration of IL-2 to mice bearing disseminated TBJ IL-27 tumors resulted in complete tumor regression and long-term survival in nine often mice (90%) compared to four often mice (40%) bearing TBJ-IL-27 tumors alone and zero often mice (0%) bearing TBJ FLAG tumors treated with or without systemic IL-2 alone (p = 0.018, TBJ IL-27 + IL-2 vs. TBJ IL-27 alone; p <0.0001 , TBJ FLAG treated with or without IL-2 vs. TBJ IL-27 + IL-2), (FIG 2). No significant antitumor effects were observed by IL-2 alone in this model (p = 0.0597, TBJ FLAG vs. TBJ FLAG + IL-2). Thus, combined delivery of IL-2 and IL-27 mediates synergistic antitumor effects in mice bearing disseminated neuroblastoma metastases.
EXAMPLE 4
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. Sixteen days after intravenous tumor cell injection, a marked reduction in the number of liver metastases was noted in mice bearing TBJ IL-27 tumors compared to mice bearing disseminated TBJ FLAG tumors (TBJ IL-27: 19.7 ± 5.4 macrometastases/liver vs. TBJ FLAG 270 ± 30 macrometastases/liver; p = 0.001) (FIGs 3A-B). The burden of metastatic disease in the liver of mice bearing TBJ IL-27 tumors was further inhibited by systemic administration of IL-2 (200,000 IU/injection, days 5-9, 12-15) (TBJ IL-27 + IL-2: 4.7± 1.95 macrometastases/liver) (p = 0.035; TBJ IL-27 vs. TBJ IL-27 + IL-2). At this time point, a modest but statistically insignificant inhibition of the formation of hepatic metastases was observed by IL-2 treatment in mice bearing TBJ FLAG tumors compared to control mice treated with vehicle alone (TBJ FLAG: 270 ± 30 macro-metastases/liver vs. TBJ FLAG + IL-2 : 162 ± 41 macro- metastases/liver; p = 0.105 (FIGs 3 A-B). Thus, combined delivery of IL-27 and IL-2 can mediate potent antitumor effects against TBJ neuroblastoma tumors in the liver.
EXAMPLE 5 Combined delivery of IL-27 and IL-2 completely abrogates bone marrow disease in mice bearing disseminated TBJ neuroblastoma metastases
This Example demonstrates that combined delivery of IL-27 and IL-2 completely abrogates bone marrow disease in mice bearing disseminated TBJ neuroblastoma metastases. To directly investigate the antitumor effects of combined delivery of IL-27 and IL-2 specifically in the bone marrow compartment, mice bearing disseminated TBJ neuroblastoma tumors 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. Remarkably, 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).
In complementary studies, the tumorigenicity of TBJ neuroblastoma metastases was assessed in the bone marrow of 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 1x107 nucleated bone marrow cells were injected subcutaneously into naϊve 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. In marked contrast, none of the ten (0%) mice injected with the bone marrow from mice bearing TBJ IL-27 tumors and treated with IL-2 developed tumors over 85 days post injection (p = 0.011, TBJ IL-27 + IL2 vs. TBJ IL-27 alone) (FIG 4B). Of note, with the bone marrow specimens obtained from mice bearing TBJ IL-27 tumors and treated with vehicle alone, tumors formed as noted above and then five of six tumors later regressed, consistent with the antitumor effect previously observed with local delivery of IL-27 in this model (Salcedo et al, (2004) J Immunol 173: 7170-7182).
EXAMPLE 6
Contribution of adaptive immunity to the antitumor activity of IL-27/IL-2 This Example demonstrates that CD8+T cells but not CD4+T cells or NK cells are the predominant effector cells mediating the anti-tumor effects of IL-27 and IL-2 therapy in the disseminated TBJ neuroblastoma model. Among the mice treated with combined delivery of IL-27 and IL-2, seven of nine mice (77.7%) that were cured of their original tumor rejected a subsequent rechallenge with wild-type parental TBJ tumor cells. In contrast, all mice (10/10) in the naϊve rechallenge control group died of tumor.
To directly assess the role of T and/or NK cell subsets in the antitumor mechanisms induced by combined delivery of IL-27 and IL-2, 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. Among mice bearing TBJ-IL-27 tumors treated with IL-2, complete durable tumor regression was achieved in ten often non-depleted mice (100%), eight often mice (80%) treated with normal rabbit serum, nine often mice (90%) depleted of CD4+ T cells, and nine often mice (90%) depleted of NK cells, but zero often mice depleted of CD8+ T cells (p < 0.0001, non-depleted vs. CD8+ T depleted ;p = \.0, non-depleted vs. either CD4+ T, or NK depleted; p < 0.0001 ,
CD8+ T cell depleted vs. either CD4+ T, or NK cell depleted; FIG 5). These findings clearly demonstrate that CD8+ T cells but not CD4+ T cells or NK cells are the predominant effectors cells mediating the anti-tumor effects of IL-27 and IL-2 therapy in the disseminated TBJ neuroblastoma model. EXAMPLE 7
Local delivery of IL-27 inhibits IL-2 induced expansion of CD4+CD25+Foxp3+ regulatory T cells within the microenvironment of TBJ tumors
This Example demonstrates that IL-27 modulates IL-2-induced alterations in regulatory T cell populations. IL-2 plays a critical role in the regulation of
CD4+CD25+ T cells (Malek & Bayer (2004) Nat Rev Immunol 4: 665-674), and 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). In that combined delivery of IL-27 and IL-2 nonetheless mediates potent antitumor activity in vivo, it was investigated whether 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. In this setting, IL-2 induces an increase by 2.7 fold in the number of regulatory T cells in comparison with vehicle treated control mice (IL-2: %CD4+ CD25+ Foxp3+ = 40.76 ± 4.86; vehicle control: %CD4+ CD25+ Foxp3+ = 15.18 ± 4.2; p = 0.048) (FIG 6). Notably, the up-regulation of T regulatory cells by IL-2 was substantially inhibited by concurrent delivery of IL-27 (IL-27 + IL-2: %CD4+ CD25+ Foxp3+ = 10.15 ± 4.64). 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.
EXAMPLE 8
Combined delivery of IL-27 and IL-2 synergistically enhances immunologic reactivity: IFN-y production This Example demonstrates that the combination of IL-27 and IL-2 synergistically enhances the production of IFN-γ in mice bearing metastatic neuroblastoma tumors. In light of the potent immunologic memory responses generated in mice treated with combined delivery of IL-27 and IL-2, and the ability of IL-27 to suppress IL-2 induced expansion of CD4+CD25+FoxP3+ regulatory T cells within the TIL population, the ability of this combination to modulate immunologic reactivity was investigated in vivo. 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-γ was assayed by ELISA. More than 30-fold greater production of IFN-γ was observed when splenic effector cells from mice bearing TBJ IL-27- expressing tumors were re-stimulated with irradiated TBJ IL-27 tumor cells in the presence of IL-2 versus medium alone or effector cells from mice bearing TBJ FLAG tumors and re-stimulated with irradiated TBJ FLAG cells in the presence of IL-2 or medium alone (TBJ FLAG + medium alone: <9.4 pg/ml; TBJ FLAG + IL-2 : 37 ± 22 pg/ml; TBJ IL-27+medium alone: 12 ± 3 pg/ml; TBJ IL-27 + IL2: 1301 ± 219 pg/ml; FIG 7). These observations demonstrate that the combination of IL-27 and IL-2 can synergistically enhance the production of IFN-γ in mice bearing metastatic neuroblastoma tumors.
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. To more directly investigate whether the combination of IL-27 and IL-2 could influence tumor-specific immunologic reactivity, the impact of this combination on the generation of tumor-specific cytolytic activity was investigated in mice bearing metastatic neuroblastoma tumors using the Indium ((C9H6NO)3 In-11 l)-release assay. The design of these studies permitted investigation of the relative role of IL-27 both in enhancing the in vivo priming of effector cells as well as the efferent limb of the immune response when effector cells were re-exposed to irradiated tumor cells ex vivo. 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). At the conclusion of the culture period, 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.
Lymphocytes from mice bearing disseminated TBJ FLAG tumors and re- stimulated ex vivo with irradiated TBJ FLAG tumor cells exhibited very low level CTL activity (% lysis at 50:1 E/T ratio = 10.3 ± 2.4), and this was increased by co- incubation with IL-2 (% lysis at 50: 1 E/T ratio = 24.1 ± 0.8), p < 0.001. In contrast, re-stimulation of spleen cells from mice bearing disseminated TBJ FLAG tumors with irradiated TBJ IL-27 tumor cells, enhanced tumor specific lysis by 4-fold (% lysis at 50:1 E/T ratio = 40.0 ± 1.2) and concurrent addition of IL-2 synergistically enhanced the generation of CTL reactivity (% lysis at 50:1 E/T ratio = 90.3 ± 4.5), p < 0.001, (FIG 8A).
These observations demonstrate that IL-27 and IL-2 can synergistically enhance the generation of CTL reactivity, and that IL-27 can mediate its effects at the effector phase in the generation of this response. Interestingly, re-stimulation of splenocytes with irradiated TBJ-FLAG tumor cells demonstrates substantially higher CTL reactivity using effector cells from mice bearing disseminated TBJ IL-27 tumors compared to the responses generated using effector cells from mice bearing disseminated TBJ FLAG tumors (% lysis at 50:1 E/T ratio: TBJ IL27 in vivo + TBJ FLAG ex vivo ~ 38.3 ± 2.3% versus TBJ FLAG in vivo + TBJ FLAG ex vivo = 10.3± 2.4%), p < 0.001, (FIG 8B). Similar increases in CTL reactivity are observed when splenocytes from mice bearing disseminated TBJ-FLAG tumors are re-stimulated with irradiated TBJ IL-27 tumor cells compared to those re-stimulated with irradiated TBJ FLAG tumor cells (% lysis at 50:1 E/T ratio: TBJ FLAG in vivo + TBJ IL-27 ex vivo = 40.0 ± 1.2% versus TBJ FLAG in vivo + TBJ FLAG ex vivo = 10.3 + 2.4% as noted above), p < 0.001, (FIG 8B). These observations indicate that IL-27 can also mediate important immunoregulatory effects during both the sensitization/priming and effector phases of the immune response for the generation of CTL reactivity.
These results also confirmed the specificity of the cytolytic responses observed under these conditions in that negligible reactivity was generated against irrelevant syngeneic SA-I tumor cells. These findings demonstrate 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.
EXAMPLE 10
Combined IL-2 and IL-27 have a potent immunostimulatory and anti-tumor effect
The data presented in 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. The generation of a productive adaptive immune response is coordinately regulated by potent immunoregulatory cytokines that are generated by monocyte/macrophage and dendritic cell populations during innate immune surveillance mechanisms (Trinchieri (2003) Nat Rev Immunol 3: 133-146). These include a group of heterodimeric IL- 12 related cytokines (IL- 12, IL-23 and IL- 27) ( Pflanz et al, (2002) Immunity. 16: 779-790; Stern et al, (1990)
Proc.NatlAcad.Sci. U.S.A 87: 6808-6812; Oppmann et al, (2000) Immunity 13: 715- 725), as well as IL-18 (Dinarello (1999) Methods 19: 121-132). Collectively, these cytokines potentiate the initiation, expansion and effector phases of an evolving adaptive T-cell mediated immune response that leads ultimately to the induction of IFN-γ.
In turn, these immunoregulatory cytokines including IL- 12 (Brunda et al, (1993) J.Exp.Med. 178: 1223-1230; Brunda et α/., (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. 795: 434-439) and 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) in preclinical tumor models. In addition, 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. Given the frequent occurrence of metastatic disease in patients with high-risk neuroblastoma, and the poor prognosis of these patients, it is desirable to be able to potentiate the therapeutic efficacy of IL-27 in this setting using cytokines such as IL- 2.
Thus, provided herein is the first evidence that 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. Further, there is clear synergy by combined delivery of IL-27 and IL-2 in eliminating bone marrow metastases. Marked reductions in the disease burden (as assessed by bone marrow colony counts) are noted in mice exposed to the combination of IL-27/IL-2 compared to mice treated with either IL-27 or IL-2 alone or untreated control mice.
Additionally, follow-up re-injection of bone marrow cell preparations was performed to evaluate the tumorigenicity of disease burden within the bone marrow of mice from the respective groups. Complete loss of tumor formation was noted in virtually all mice in the IL-27/IL-2 group, while tumors formed in the majority of mice exposed to IL-27 alone, IL-2 alone or untreated control groups. The ability of 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.
Consistent with previous in vitro studies using human effector cells (natural killer cells) (Pflanz et al, (2002) Immunity. 16: 779-790), disclosed herein is the finding that treatment of murine splenocytes with the combination of IL-27 and IL-2 can synergistically enhance proliferative responses and IFN-γ production in vitro. Previous studies have emphasized the functional role of IL-27 in activating naive T cell populations (Pflanz et al, (2002) Immunity. 16: 779-790; Hibbert et al, (2003) J Interferon Cytokine Res 23: 513-522), while IL-23 is thought to act predominantly on memory T cell populations (Pflanz et al, (2002) Immunity. 16: 779-790). 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.
As demonstrated herein with depletion studies, the anti-tumor mechanisms engaged by combined delivery of 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-γ in response to ex vivo re-stimulation with irradiated tumor cells. Greater than 30-fold increases in IFN-γ production are seen when splenocytes from mice bearing disseminated TBJ-IL-27 tumors are re-stimulated ex vivo with the corresponding irradiated TBJ-IL-27 tumor cells in the presence of IL-2 compared to the same effector cells re-stimulated ex vivo with irradiated TBJ-IL-27 tumors alone or effector cells from mice bearing TBJ-FLAG tumors re-stimulated ex vivo with the irradiated TBJ-FLAG in the presence or absence of IL-2.
As previously reported, delivery of IL-27 alone can enhance the generation of CTL responses to TBJ neuroblastoma tumors (Salcedo et ah, (2004) J Immunol 173: 7170-7182). Further, IL-27 can act directly on naϊve 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). As demonstrated herein, 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 naϊve T cell populations in vivo. Further, 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. The observation that IL-2 deficient mice develop autoimmunity (Horak et at, (1995) Immunol Rev 148: 35-44) and that in vivo neutralization of IL-2 results in autoimmunity with a concomitant reduction in the number of CD4+CD25+Foxp3+ cells (Malek & Bayer (2004) Nat Rev Immunol 4: 665-674), implicates IL-2 as a key modulator of CD4+ regulatory T cells. Administration of 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. This is the first report that IL-2- induced increases in tumor infiltrating regulatory T cells can be inhibited by another cytokine in vivo. 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.
Thus, the combined delivery of IL-27 and IL-2 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. Further, 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. Furthermore, 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. Together, these findings demonstrate that an IL-27-based therapeutic regimen mediates anti-tumor effects against bone marrow metastases, and that IL- 27/IL-2 therapy is particularly effective in subjects with bone marrow metastases, for example in children with high-risk neuroblastoma.
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. For instance, the term "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. The term "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. In addition to known IL-2 and IL-27 sequences, the creation of variants of these sequences is now enabled. 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. Manipulation of an IL-2 or IL-27 nucleotide sequence using standard procedures, including for instance site-directed mutagenesis or PCR, 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.
Table 1:
Original Residue Conservative Substitutions
Ala ser
Arg lys
Asn gin; his
Asp glu
Cys ser
GIn asn
GIu asp
GIy pro
His asn; gin
He leu; val
Leu ile; val
Lys arg; gin; glu
Met leu; ile Phe met; leu; tyr
Ser thr
Thr ser
Trp tyr
Tyr trp; phe
VaI ile; leu
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. The following 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).
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. Also comprehended are more closely related 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. In their most simple form, 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. Alternatively, 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. For example, because of the degeneracy of the genetic code, four nucleotide codon triplets - (GCT, GCG, GCC and GCA) code for alanine. The coding sequence of any specific alanine residue within a known IL-2 or IL-27 protein, therefore, could be changed to any of these alternative codons without affecting the amino acid composition or characteristics of the encoded protein. Based upon the degeneracy of the genetic code, 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. Thus, 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. Specific examples of IL-2 sequence variants can be found in US Patent No:
2003/0235556.
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 105 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%. In another protocol, 1 x 105 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). 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 dose was increased to 200,000 IU/injection (FIG 9C). Thus, 1 x 105 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. IL-2 treatment significantly enhanced the survival of the TBJ-IL-27 group, (p =0.2661, TBJ-IL-27 vs. TBJ-IL27 + IL-2).
Similar administration schedules can be used for administration of IL-2 and IL-27 to humans, however different doses of the interleukins will be selected. For instance, 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.
EXAMPLE 13 Methods of administration of IL-2 and IL-27 Disclosed herein is the surprising discovery that the combined delivery of IL-
27 and IL-2 stimulates an immune response (such as increased IFNγ production) and mediates a synergistic anti-tumor effect, for example against neuroblastoma, lymphoma, leukemia, melanoma, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma and lung carcinoma. This Example describes various methods that can be used to administer the IL-2 and IL- 27 to a subject.
In one embodiment of the disclosure, IL-2 and IL-27 are administered by different routes of administration. For example, in one embodiment 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. In some embodiments, both IL-2 and IL-27 are administered locally, whereas in other embodiments, both IL-2 and IL-27 are administered systemically.
An effective dose of IL-2 or IL-27 can be administered systemically in a variety of ways. For instance, 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. By way of example, one method of administration to the lungs of an individual is by inhalation through the use of a nebulizer or inhaler. For example, 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. In one embodiment, 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. In 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. In specific, non-limiting examples, 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. In some embodiments, 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. For instance, in certain embodiments 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.
In some embodiments, 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.
In other embodiments, local administration is in the form of gene therapy. In certain embodiments, 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). Other 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),
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).
Other gene therapy techniques include the use of 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.
In addition to delivery of an IL-2 or IL-27-encoding sequence to cells using viral vectors, some embodiments use non-infectious methods of delivery. For instance, 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). For instance, 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). However, 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.
In other embodiments, 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. 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.
While many small-molecule drugs are cleared from the blood by the kidneys in a matter of minutes or hours, the large, soluble antibody molecules are designed to remain in the bloodstream for long periods of time. While the antibody portion of the hybrids keeps them circulating, the small-molecule portion targets cancer cells. In the present disclosure, 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. Similarly, 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. In addition, 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.
For a detailed description of targeted delivery via alpha(v)beta(3)-targeted nanoparticles, see for example, Reynolds et al, (2003) Trends MoI Med. 9(l):2-4; and Winter et al, (2006) Arterioscler Thromb Vase Biol. 26(9):2103-9).
In still other embodiments, IL-2 and/or IL-27 is administered via adoptive transfer of T lymphocytes overexpressing IL-2 or IL-27. Adoptive immunotherapy of malignancy 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. Briefly, IL-2- or IL-27-overexpressing T lymphocytes are administered locally or systemically in dose levels ranging from about 107 to 1010 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. 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 technique. Finally, in some embodiments, local delivery of IL-2 and/or IL-27 into the primary tumor and hypoxic metastasis is accomplished using anaerobic bacteria, such as toxin-free Clostridium novyi. When spores of the anaerobic bacterium Clostridium nøvyz-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. In some embodiments, the genetically modified anaerobic bacteria are administered intravenously to subjects. For a more thourough discussion of this technique see Agrawal et al., (2004) Proc Natl Acad Sci USA. 101(42): 15172-7. For both local and systemic administration of IL-2, 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. In some embodiments, 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.
For both local and systemic administration of IL-27, 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 μg/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.
EXAMPLE 14
Specific regimens of IL-2/IL-27 administration
This Example describes particular examples of specific regimens that are useful for treating human tumors. In certain, specific examples of the methods disclosed herein, IL-2 is administered by systemic intermittent pulse administration and IL-27 is administered locally at the tumor site. In certain, particular examples, 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.
For instance, in certain examples, 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. In other embodiments, 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.
Similarly, 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.
For both local and systemic administration of IL-2, 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. In some embodiments, 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. 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.
In this particular embodiment, 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. Generally, 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. Alternatively, 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. Additionally, the combined IL-2/IL-27 therapy can be administered in conjunction with treatment with other anti-cancer agents.
EXAMPLE 15 Incorporation of IL-2 and/or IL-27 into Pharmaceutical Compositions
Pharmaceutical 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. For instance, 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. If desired, 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.
The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, 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). For 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.
In view of the many possible embodiments to which the principles of this disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure, and should not be taken as limiting the scope of the disclosure. Rather, the scope is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.

Claims

We claim:
1. A method of treating a tumor that responds to immunotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount of IL-2 and a therapeutically effective amount of IL-27, wherein the IL-2 and IL-27 interact in their effects to stimulate an immune response and treat the tumor.
2. The method of claim 1 , wherein the tumor is neuroblastoma, melanoma, leukemia, lymphoma, renal cell carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, or lung carcinoma.
3. The method of claim 1 or 2, wherein administration of the IL-2 comprises systemic administration.
4. The method of claim 3, wherein administration of the IL-2 comprises intravenous, intra-muscular, subcutaneous, intra-arterial, or intra-peritoneal administration.
5. The method of claim 1 or 2, wherein administration of the IL-2 comprises local administration.
6. The method of claim 5, wherein administration of the IL-2 comprises local injection, adoptive transfer of T-lymphocytes expressing IL-2, anaerobic bacterial administration, administration via alpha(v)beta(3)-targeted nanoparticles, or liposomal delivery of IL-2.
7. The method of any of claims 1-6, wherein administration of the IL-27 comprises systemic administration.
8. The method of claim 7, wherein administration of the IL-27 comprises intravenous, intra-muscular, subcutaneous, intra-arterial, or intra- peritoneal administration.
9. The method of any of claims 1 -6, wherein administration of the IL-27 comprises local administration.
10. The method of claim 9, wherein administration of the IL-27 comprises local injection, 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.
11. The method of claim 1 or 2, wherein administration of the IL-2 comprises systemic administration, and administration of the IL-27 comprises local administration.
12. The method of claim 1 or 2, wherein the IL-27 is constitutively expressed in the tumor and the IL-2 is intermittently administered to the subject.
13. The method of claim 12 wherein the IL-2 is intermittently administered by systemic administration of the IL-2 no more than once per day.
14. The method of any of claims 1-13, wherein the effective amount of IL-2 is from about 30,000 IU/kg to about 600,000 IU/kg.
15. The method of any of claims 1-14, wherein the effective amount of IL-27 is from about from about 30 ng/kg to about 3,000 ng/kg.
16. The method of any of claims 1-15 wherein the IL-2 and the IL-27 are administered substantially concurrently.
17. The method of any of claims 1-15 wherein the IL-27 is administered before the IL-2.
18. The method of any of claims 1-17 wherein the IL-2 is administered every eight hours.
19. The method of any of claims 1-17 wherein the IL-2 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.
20. The method of claim 19, wherein the cycle is repeated from about two to about ten times.
21. The method of any of claims 1 -20 wherein the IL-27 is administered daily.
22. The method of any of claims 1-11 and 14-21 wherein 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.
23. The method of claim 22, wherein the cycle is repeated from about two to about ten times.
24. The method of claim any of claims 1-23, wherein the subject has metastatic neuroblastoma.
25. The method of any of claims 1-24, wherein the subject is human.
26. The method of any of claims 1-24, wherein the subject is a veterinary subject.
27. A method of treating metastatic neuroblastoma in a subject, comprising administering to the subject a therapeutically effective amount of IL-2 and a therapeutically effective amount of IL-27, wherein the IL-2 is administered systemically and the IL-27 is administered locally, thereby treating the metastatic neuroblastoma.
28. A composition for use in stimulating an anti-tumor immune response, wherein the composition comprises an amount of IL-2 and IL-27 effective to stimulate an immune response and treat the tumor.
PCT/US2006/041534 2006-10-24 2006-10-24 Interaction of il-27 and il-2 for treatment of tumors WO2008051220A1 (en)

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Citations (1)

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WO2005014642A2 (en) * 2003-07-21 2005-02-17 Transgene S.A. Novel multifunctional cytokines

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Publication number Priority date Publication date Assignee Title
WO2005014642A2 (en) * 2003-07-21 2005-02-17 Transgene S.A. Novel multifunctional cytokines

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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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