Anti-CD-137 Monoclonal Antibodies with Distinct FcyR Binding Abilities for Treatment of

Cancer or Autoimmunity

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This Application claims the benefit of priority to U.S. Provisional Application 62/008,714 filed on June 06, 2014, which is incorporated herein by reference in its entity.

TECHNICAL FIELD

[0002] Embodiments of the disclosure concern at least the fields of immunology, cell biology, molecular biology, and medicine.

BACKGROUND OF THE INVENTION

[0003] CD137 (4-1BB) is an inducible co-signaling molecule expressed by T cells, natural killer (NK) cells, dendritic cells (DC), B cells, and other cells of the immune system (Wang, et ah, 2009). CD 137 ligation, by either its ligand (CD137L) or agonistic monoclonal antibodies (mAbs), mediates cellular activation and prevents activation induced cell death (Wang, et ah, 2009). Administration of agonistic antibodies to CD 137 results in tumor resolution in a CD8 ⁺ T cell dependent fashion, with additional contributions from CD4 ⁺ T cells, NK cells and/or DCs dependent upon the tumor model (Wilcox, et ah, 2002; Choi, et ah, 2007; Melero, et ah, 1997; Murillo, et ah, 2009). To date, the majority of the functional responses to anti-CD 137 stimulation have been attributed to Fab-CD 137 interactions. However, the contribution of interactions between the Fc fragments of anti-CD 137 mAbs with Fc gamma receptors (FcyRs) to the anti-tumor activity of anti-CD 137 mAbs remains unknown.

[0004] Mice have three activating and one inhibitory FcyR, which are expressed alone or in combination on individual cell populations (Bruhns, et ah, 2012). The activating receptors, FcyRI, FcyRIII, and FcyRIV, each consists of a unique cc-chain and a common Fcy-chain, containing the immune receptor tyrosine-based activating motif (IT AM) in the cytoplasmic tail. In contrast, the cytoplasmic tail of the inhibitory receptor, FcyRIIB, is comprised of an immune receptor tyrosine-based inhibitory motif (ITIM) (Bruhns, et ah, 2012). IT AM signaling results in the secretion of pro-inflammatory cytokines, initiation of phagocytosis and other effector functions, whereas ITIM signaling abrogates these effector responses (Bruhns, et ah, 2012). Importantly, FcyRIIB and FcyRIII have limited ability to bind to naturally occurring circulating IgG homodimers, but are able to bind aggregated IgG complexes, like those formed after Fab engagement of polyvalent targets (Bruhns, et ah, 2012). Therefore, Fab-CD 137 interactions have the potential to enhance and localize Fc mediated signaling through low affinity FcyRs, and possibly alter the functional outcomes of CD 137 stimulation.

[0005] Recent studies have characterized the import of Fc-FcyR interactions on the function of immunomodulatory mAbs. Collectively, these studies indicate that the relative functional import of Fc interactions with individual FcRs is dependent upon the cosignaling moiety targeted by the Fab fragment. For example, anti-CD40 mAbs require engagement of FcyRIIB for therapeutic efficacy (White, et ah, 2011; Li, et ah, 2011). For other immune receptor targets, such as CD 152 (CTLA-4) and the anti-glucocorticoid- induced TNFR-related protein (GITR), engagement of FcyRIV and FcyRIII, respectively, are functionally relevant. Specifically, these interactions mediate depletion of tumor infiltrating T regulatory cells (Tregs), thus increasing the ratio of CD8 ⁺ T cells to Tregs, with a resultant improvement in the anti-tumor immune response (Simpson, et ah, 2013; Bulliard, et ah, 2013). These data underscore the need to define how Fc interactions with each of the cognate FcyRs contribute to the biologic function of monoclonal antibodies targeting cosignaling pathways.

[0006] It has been previously demonstrated that ligation of FcyRIII on human NK cells results in the up regulation of CD137 (Lin, et ah, 2008). Based on these findings, it was considered that Fc-FcyR interactions would augment anti-CD 137 mediated anti-tumor immunity. Using an anti-CD 137 mAb sensitive EL4E7 lymphoma tumor model, in mice deficient for individual FcyRs, it is shown herein that anti-CD 137 mediated anti-tumor immunity against EL4E7 is enhanced in FcyRIII ^"7" mice (Wilcox, et ah, 2002). This effect is associated with increases in CD8 ⁺ T cells and CD1 lc ⁺ major histocompatibility II (MHCII) ⁺ populations.

These data indicate that FcyRIII stimulation impairs anti-CD 137 mediated anti-tumor immunity against EL4E7.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention is directed to methods and/or compositions that provide immunotherapy to individuals in need of the immunotherapy. In particular embodiments, the disclosure concerns monoclonal antibodies that have been engineered to have certain fragment crystallizable (Fc) regions that have the function of being able to bind a specific group of Fc receptors or that have the function of either not being able to or having reduced absolute and/or relative potential to bind individual and/or a specific group of Fc receptors. Such binding pattern provides for appropriate recruitment of the immune system to attack target cells or provides for appropriate abrogation of immune system defenses to reduce an autoimmune reaction, depending on the particular binding pattern. Thus, where certain embodiments of the disclosure concern particular monoclonal antibodies that recruit the immune system to kill a target cell, such as a target cancer cell, other embodiments of the disclosure concern particular monoclonal antibodies that abrogate the immune system to treat one or more autoimmune diseases.

[0008] In specific embodiments for applications enhancing the immune system's ability to target cancer cells, the anti-CD 137 monoclonal antibodies are engineered such that their Fc portion particularly does or does not bind certain Fc receptors, and/or has relatively increased or decreased ability to bind specific Fc receptors, alone and/or in combination, such as FcyRIII and/or FcyRIIA. Therefore, in particular embodiments, there are methods of enhancing anticancer autoimmunity in an individual in need thereof by administering a therapeutically effective amount of anti-CD 137 monoclonal antibodies to the individual, wherein the antibodies comprise an Fc region that has reduced or abolished ability to bind to FcyRIII, FcyRIIA, or both. Methods of treatment of cancer are encompassed in the disclosure.

[0009] In embodiments for applications for autoimmune disorders, the anti-CD 137 monoclonal antibodies are engineered such that their Fc portion particularly does bind, or has enhanced or reduced binding to, certain Fc receptors, such as FcyRIII and/or FcyRIIA. Thus, in certain embodiments there are methods of treating one or more autoimmune disorders in an individual in need thereof by administering a therapeutically effective amount of anti-CD 137 monoclonal antibodies to the individual, wherein the antibodies comprise an Fc region that has the ability (or enhanced and/or reduced ability) to bind to FcyRIII, FcyRIIA, or both

[0010] In one embodiment, there is a method of enhancing anti-cancer immunity in an individual in need thereof, comprising the step of administering a therapeutically effective amount of anti-CD 137 monoclonal antibodies or CD137-targeting fusion proteins to the individual, wherein the antibodies or fusion proteins respectively comprise an Fc region that has reduced or abolished ability to bind to FcyRIII, FcyRIIA, or both. In specific embodiments, the antibodies were designed to comprise the Fc region that has reduced or abolished ability to bind to FcyRIII, FcyRIIA, or both. [0011] The method of claim 1, wherein the antibodies were determined empirically whether they comprised the Fc region that has reduced or abolished ability to bind to FcyRIII, FcyRIIA, or both. In additional embodiments, the method comprises the step of determining whether or not the antibodies or the Fc region of the antibodies has the ability to bind with FcyRIII, RcyRIIA, or both. The individual may have a type of cancer that is known to be immunologically responsive to anti-CD 137 antibodies. The cancer may be lymphoma, melanoma, renal cell carcinoma, ovarian cancer, sarcoma, breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, colon cancer, skin cancer, parotid gland cancer, sarcoma, bone, connective tissue, leukemia, myeloma, brain cancer, bone cancer, head and neck cancer, esophageal cancer, stomach cancer, salivary gland cancer, pancreatic cancer, bile duct cancer or liver cancer. In some embodiments, the individual is given an additional cancer therapy, such as immunotherapy, chemotherapy, hormone therapy, radiation, surgery, ultrasound therapy, light therapy, gene therapy, nanoparticle therapy, or a combination thereof.

[0012] In one embodiment, there is a method of treating an autoimmune disease in an individual in need thereof, comprising the step of administering a therapeutically effective amount of anti-CD 137 monoclonal antibodies or CD137-targeting fusion proteins to the individual, wherein the antibodies or fusion proteins respectively comprise an Fc region that has the ability to bind with FcyRIII, FcyRIIA, or both, or has enhanced ability to bind with FcyRIII, FcyRIIA, or both. In specific embodiments, the antibodies were designed to comprise the Fc region that has the ability to bind with FcyRIII, FcyRIIA, or both, or has enhanced ability to bind with FcyRIII, FcyRIIA, or both. In specific embodiments, the antibodies were determined empirically whether they comprised the Fc region that has the ability to bind with FcyRIII, FcyRIIA, or both, or has enhanced ability to bind with FcyRIII, FcyRIIA, or both. In some embodiments, the method comprises the step of determining whether or not the antibodies or the Fc region of the antibodies has the ability to bind with FcyRIII, FcyRIIA, or both. The autoimmune disease is known to be immunologically responsive to anti-CD 137 antibodies, in certain embodiments, and the autoimmune disease may be rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Addison's disease, celiac disease, dermatomyositis, Graves' disease, multiple sclerosis, myasthenia gravis, asthma, inflammatory bowel disease, psoriasis, Hashimoto's thyroiditis, vasculitis, scleroderma, Guillain-Barre syndrome, Chronic

Inflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic IgM demyelinating Polyneuropathy, Lambert-Eaton myasthenic syndrome, Myasthenia gravis, Multifocal Motor Neuropathy, Lower Motor Neuron Syndrome associated with anti-GMl antibodies, Demyelination, Multiple Sclerosis and optic neuritis, Stiff Man Syndrome, Paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis, Myelitis, Myelopathy especially associated with Human T-cell lymphotropic virus- 1, Autoimmune Diabetic Neuropathy, Acute Idiopathic Dysautonomic Neuropathy, Kawasaki's disease, Rheumatoid arthritis, Felty's syndrome, ANCA-positive Vasculitis, Spontaneous Polymyositis, Dermatomyositis,

Antiphospholipid syndromes, Recurrent spontaneous abortions, Systemic Lupus Erythematosus, Juvenile idiopathic arthritis, Raynaud's, CREST syndrome, Uveitis, Epidermal Necrolysis, Gangrene, Granuloma, Autoimmune skin blistering diseases including Pemphigus vulgaris, Bullous Pemphigoid, and Pemphigus foliaccus, Vitiligo, Streptococcal toxic shock syndrome, Scleroderma, systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, Atopic dermatitis, steroid dependent Atopic dermatitis, Inclusion Body Myositis, Necrotizing fasciitis, Inflammatory Myopathies, Myositis, Anti-Decorin (BJ antigen) Myopathy,

Paraneoplastic Necrotic Myopathy, X-linked Vacuolated Myopathy, Penacillamine-induced Polymyositis, Atherosclerosis, Coronary Artery Disease, Cardiomyopathy, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, Celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis, Reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis, or sclerosing cholangitis.

[0013] In one embodiment, there is a method of producing anti-CD 137 monoclonal antibodies, comprising the step of generating anti-CD 137 monoclonal antibodies that have been designed specifically to comprise one of a) or b): a) an Fc region that has reduced or no ability to bind with FcyRIII, FcyRIIA, or both; or b) an Fc region that has the ability to bind with FcyRIII, FcyRIIA, or both, or has enhanced ability to bind with FcyRIII, FcyRIIA, or both.

[0014] In one embodiment, there is a composition of matter comprising isolated anti- CD 137 monoclonal antibodies or CD137-targeting fusion proteins that comprise an Fc region that has reduced or no ability to bind with FcyRIII, RcyRIIA, or both. The antibodies were designed to comprise the Fc region that has reduced or abolished ability to bind to FcyRIII, FcyRIIA, or both, in certain embodiments.

[0015] In an embodiment, there is a kit comprising the cancer therapy composition, said composition housed in a suitable container. In specific embodiments, the kit further comprises a second composition, wherein said second composition is a cancer therapy, such as one or more of abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, fuldarabine, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, and vinblastine.

[0016] In specific embodiment, there is a composition of matter that comprises isolated anti-CD 137 monoclonal antibodies or CD137-targeting fusion proteins that comprise an Fc region that has the ability or enhanced ability to bind with FcyRIII, RcyRIIA, or both. The antibodies were designed to comprise the Fc region that has the ability or enhanced ability to bind with FcyRIII, RcyRIIA, or both, in specific embodiments and the disclosure encompasses kits with the composition(s) housed in a suitable container. In specific embodiment, the kits further comprise a second composition, wherein said second composition is an autoimmune disorder therapy. The kit may further comprise a therapeutic for rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Addison's disease, celiac disease, dermatomyositis, Graves' disease, multiple sclerosis, myasthenia gravis, asthma, inflammatory bowel disease, psoriasis, Hashimoto's thyroiditis, vasculitis, or scleroderma. In specific embodiments, the kit may further comprise a second composition, wherein said second composition is CTLA-4Ig, anti-TNF, anti- anti-IL-17 (need to list other cytokines), CD20, anti-CD3, methotrexate, gold, or a combination thereof.

[0017] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:

[0019] FIGS. 1A-1B - 2A binds to mouse FcyRIIB, FcyRIII, and FcyRIV but not to FcyRI. FIG. 1A Mouse IgG2a, Rat IgG2a, and 2A were bound to a SPR chip at ΙΟμ^πύ. Recombinant mouse FcyRs were serially diluted from ΙμΜ to less than InM with running buffer (HBS-EP), injected for 120sec at 20μ1/ηιίη over all FCs, and followed by a 120sec dissociation step. FIG. IB The KD values (nM) and the Chi ² in parenthesis.

[0020] FIGS. 2A-2C - 2A treatment preferentially enhances survival of EL4E7 tumor bearing FcRy ^"7" mice. Mice were inoculated with 8X10 ⁶ EL4E7 tumor cells on Day 0. Survival and growth curves for mice, FIG. 2A FcyRIIB ^"7"* WT 2A VS WT IgG p<0.0001 and, ^† FcyRIIB ^"7" 2A VS FcyRIIB ^"7" IgG p=0.0128, and FIG. 2B Fey ^{"7" *} WT 2A vs WT IgG p<0.0001, ^† FcRy ^"7" 2A vs FcRy ^"7" IgG p<0.0001, and * FcRy ^"7" 2A vs WT 2A p=0.002, treated with lOOug of either Rat IgG or 2A on Day 7 and Day 10. FIG. 2C Survival and growth curves for FcRy ^"7" mice treated with lOOug of either Rat IgG or 2A on Day 10 and Day 13, WT 2A vs WT IgG p= 0.0063, ^† FcRy ^"7" 2A vs FcRy ^"7" IgG p= 0.0027, and * FcRy ^"7" 2A vs WT 2A p= 0.0161.

Numbers in parentheses indicate the number of mice. Statistical significance determined by Log- rank Test.

[0021] FIGS. 3A-3B - 2A treatment preferentially enhances survival of EL4E7 tumor bearing FcyRIII ^"7" mice. Mice were inoculated with 8X10 ⁶ EL4E7 tumor cells on Day 0. On Day 10 and Day 13 mice were treated with either 100μg of 2A or Rat IgG. FIG. 3A FcyRIII ^{" 7"} ; ^* WT IgG vs WT 2A p=0.0023, ^† FcyRIH ^"7" IgG vs FcyRIII ^"7" 2A p<0.0001, *WT IgG vs FcyRIII ^"7" IgG p=0.0311, and WT 2A vs FcyRIII ^"7" 2A p=0.0006. FIG. 3B FcyRIV ^"7" ; ^* WT IgG vs WT 2A p=0.0484 and ^† FcyRIV ^"7" IgG vs FcyRIV ^"7" 2A p=0.0217. Number in parenthesis indicates the number of mice. Both figures are the results of two pooled experiments. Statistical significance was determined by the Log-rank Test.

[0022] FIGS. 4A-4C - The 3H3 mediated anti-EL4E7 response is enhanced in FcyRIH ^"7" mice. FIG. 4A Mice were inoculated with 8xl0 ⁶ EL4E7 tumor cells on Day 0. On Day 10 and Day 13 mice were treated with either lOC^g of anti-CD 137 mAb or Rat IgG.

Numbers in parentheses indicate the number of mice. Statistical significance was determined by the Log-rank Test: * p< 0.05, ** p< 0.01, *** p< 0.001, **** p<0.0001. Survival curve and growth curves represent the sum of two experiments. FIG. 4B HEK-mCD137L expressing cells incubated with anti-CD 137 mAb clone 2A or 3H3. FIG. 4C HEK-mCD137L expressing cells incubated with recombinant mCD137L at 31ng/ml (2 ^nd panel). Cells were initially incubated with anti-CD 137 mAb at concentrations listed in FIG. 4A, followed by secondary incubation with mCD137L at 31ng/ml.

[0023] FIGS. 5A-5C - 2A treated tumor bearing FCYRIII ^"7" mice have an increase in CD8 ⁺ CDllc ⁺ T cells. FIG. 5A Mice were inoculated with 8X10 ⁶ EL4E7 tumor cells on Day 0. On Day 7 and Day 10 mice received either 100μg of 2A or Rat IgG. Spleens were harvested from 2- 3 mice per group on Days 7, 12, 16, and 21. Representative data from one experiment repeated three times. FIG. 5B Pearson's correlation of CD8 ⁺ CD1 lc ⁺ CD103 ^" absolute cell number versus tumor volume. Data are combined from two experiments for a final n=6 or 7. FIG. 5C Cells isolated by positive selection from spleens of naive WT and FcyRIir ^7" mice, and stimulated with anti-CD3 and anti-CD28 at ^g/ml for 48 hours. CD 137 expression was determined by flow cytometry. Representative data from one experiment repeated four times.

[0024] FIGS. 6A-6D - 2A treated tumor bearing FCYRIII ^"7" mice have an increase in CDllc ⁺ MHCi cells with an APC phenotype due to the FcyRIII deficiency. FIG. 6A

Mice were inoculated with 8X10 ⁶ EL4E7 tumor cells on Day 0. On Day 7 and Day 10 mice received either 100μg of 2A or Rat IgG. Spleens were harvested from 2- 3 mice per group on Days 7, 12, 16, and 21. Representative data from one experiment repeated three times. FIG. 6B The expression of CD40, CD80, and CD86 on CD1 lc ⁺ MHClf cells from spleen on Day 16. Data combined from two experiments for a final n=6 or 7. A significance difference determined by one way ANOVA and Tukey's multiple comparison test * p< 0.05, ** p< 0.01, *** p< 0.001, **** p<0.0001. FIG. 6C Pearson's correlation of CDl lc ⁺ MHCII ⁺ absolute cell number versus tumor volume. Data combined from two experiments for a final n=6 or 7. FIG. 6D Cells were isolated by positive selection from spleens of naive WT and FcyRIIl ' mice. CDl lc ⁺ cells were stimulated with 5 g/ml of LPS for 48 hours. CD137 and CD137L expression were determined by flow cytometry. Representative data from one experiment repeated four times.

[0025] FIGS. 7A-7C - Host expression of CD137 is required for the anti-tumor effects of 2 A. FIG. 7A Mice inoculated with EL4E7 tumors on Day 0 and treated with lOOmg of either Rat IgG or 2A on Day 7 and Day 10. Statistical significance determined by Log-rank Test: * p< 0.05, ** p< 0.01, *** p< 0.001, **** p<0.0001. Survival and growth curves represent the sum of two experiments. FIG. 7B First panel, CD 137 expression on HEK-CD137 transfected cells; second panel, CD137 expression on EL4E7 ex vivo. Gray histogram is the IgG control. FIG. 7C EL4E7 cells incubated with anti-FcgRIIB/FcgRIII, followed with a secondary antibody against Rat IgG. The gray shaded region is secondary antibody only.

[0026] FIG. 8 - Treatment with anti-CD137 mAb does not elicit antibody dependent cellular cytotoxicity (ADCC). Effector (E) were NK cells isolated from WT and FcgRIII-/- mice, Splenocytes were stimulated with ConA for 48 hours to induce CD 137. 2 A opsonized splenocyte targets (T) were labeled with chromium, and cocultured with the NK cells for 16 hours. Cytotoxicity was determined by the following calculation: lysis= [experimental cpm-sponteneous cpm) x 100}/(maximum cpm- spontaneous cpm). YAC-1 is a murine NK sensitive cell line used as a positive control for cytotoxicity.

[0027] Other and further objects, features, and advantages will be apparent from the following description of the presently preferred embodiments of the invention, which are given for the purpose of disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The use of the word "a" or "an" when used in conjunction with the term

"comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." I. [0029] General Embodiments

[0030] The present disclosure encompasses immunotherapeutic methods and compositions that employ specific anti-CD 137 monoclonal antibodies, the design of which directly affects the efficacy of the antibodies for particular desired therapeutic applications. Antibodies that target the CD137 (4-1BB; TNFRSF-9) co-signaling molecule display diverse therapeutic effects, serving to both enhance the anti-tumor immune response in mice and treat mouse models of autoimmunity. Until the present disclosure, the biological basis for these disparate therapeutic effects was an enigma.

[0031] The inventors have discovered that anti-CD 137 monoclonal antibodies (mAbs) containing Fc regions that can interact with Fc receptor subtype FcyRIII (and with mouse Fc receptor subtype FcyRIV, or its human analogue, FcyRIIA, in specific embodiments)

paradoxically dampen CD137-mediated anti-tumor immunity. Accordingly, anti-CD 137 mAbs containing Fc regions that lack the ability to interact with FcyRIII (and with FcyRIIA, in specific embodiments) can be administered to potentiate anti-tumor immunity in patients in need thereof. Furthermore, anti-CD 137 mAbs having an Fc region that interacts with FcyRIII and/or FcyRIIa can be administered to ameliorate autoimmunity in patients in need thereof.

[0032] In embodiments, particular CD 137 monoclonal antibodies are selected by choice because of one or more properties of the particular Fc fragment and, in specific embodiments, its specific ability to interact with specific FcRs. In particular aspects, the selection of the Fc fragment also is directed by the desire to reduce the potential or risk for inflammation at non- tumor sites such as the liver.

[0033] In specific embodiments, particular monoclonal antibodies that target CD 137 are selected because of the following: 1) a specific desired interaction of the Fc region of the monoclonal antibody and a specific Fc receptor; or 2) because of a specific inability for interaction between the Fc of the monoclonal antibody and a specific Fc receptor. Such a selection between 1) and 2) depends on the intended use for the antibody.

[0034] In certain embodiments, Fc:FcR interactions are manipulated to affect CD 137- mediated tumor immunity in vivo or in contrasting ways to improve autoimmunity disease treatment in vivo. [0035] In embodiments, the therapeutic efficacy of the desired CD 137 monoclonal antibodies occurs in the absence of, or to a reduced extent of, antibody-dependent cell mediated cytotoxicity (ADCC) mechanisms.

II. [0036] Cancer Embodiments

[0037] Certain anti-CD 137 mAbs described herein can be used to treat any type of cancer that would be treated by administering anti-CD 137 antibodies. Such cancers include but are not limited to lymphoma, melanoma, renal cell carcinoma, ovarian carcinoma, sarcoma, and more generally, any epithelial carcinoma (e.g. but not limited to breast, lung, colon, prostate, ovary, cervical, colon, skin, parotid gland, and the like), as well as any sarcoma (e.g. but not limited to bone, connective tissue, and the like), and any cancer of the hematopoietic or immune system (e.g. but not limited to leukemia, lymphoma, myeloma, and the like). In particular embodiments, the cancer is bladder cancer, breast cancer, colon or rectal cancer, endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, head and neck cancer, esophageal cancer, stomach cancer, salivary gland cancer, pancreatic cancer, bile duct cancer and liver cancer.

[0038] Thus, embodiments of the disclosure concern antibody-based cancer

therapeutics. Embodiments provide improved CD 137 monoclonal antibodies over known such antibodies in the art. Specific embodiments of the disclosure provide evidence that both the Fab '-CD 137 and Fc-FcR interactions have an impact on defining the tumor activity for a specific CD 137 monoclonal antibody.

[0039] Embodiments of the disclosure concern selection for CD 137 monoclonal antibodies for improved protective and therapeutic tumor immunity. In specific embodiments, the improvement stems from the ability of the Fc-FcR interaction to induce CD 137 expression on the surface of one or more types of immune cells, such as effector cell types, including at least NK cells, γΔΤ cells and NKT cells.

[0040] Embodiments of the disclosure concern optimization of one or more particular CD 137 antibodies for stimulating tumor immunity. [0041] In particular embodiments, the disclosure concerns agonistic antibodies specific for CD- 137 and their methods of use and manufacturing. Provided herein are methods of improving the therapeutic efficacy and toxicity profiles of monoclonal antibodies that target the costimulatory molecule, CD137, which is useful for cancer immunotherapy. The selected antibodies may be used alone as a cancer therapy and/or prevention or with one or more other types of cancer therapies, including other cancer immunotherapies, where appropriate. The additional cancer therapy may be of any kind, including immunotherapy, vaccines, hormone therapy, drug therapy, chemotherapy, surgery, radiation, and a combination thereof. The additional therapy may be given at the same time or at a different time as the cancer

immunotherapy of the disclosure. The additional therapy may be given to the individual before, during and/or after the time that the therapy of the disclosure is provided to the individual. In cases wherein the therapeutic antibodies are employed with one or more chemotherapeutics, all classes of chemotherapeutic agents may be employed, including alkylating agents,

antimetabalites, plant alkaloids, antibiotics, hormonal agents, and miscellaneous anticancer drugs. Specific agents include, for example, abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine,

mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, fuldarabine, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5- fluorouracil, vincristin, and vinblastin, or any analog or derivative variant of the foregoing and also combinations thereof.

[0042] More than one dose of the antibodies may be provided to the individual. More than one dose of the antibodies may be provided to the individual, and the subsequent dosings may be spaced in time on the order of minutes, hours, days, weeks, or months from each other. Optimization of dosing regimens is routine in the art of immunotherapy.

[0043] In specific embodiments, the individual is known to have the cancer, although in some embodiments the methods include determination of cancer in the individual. The individual may be susceptible to the cancer, such as having a personal or family history, being a smoker, having exposure to aging, tobacco, sun exposure, radiation exposure, chemicals and other substances, some viruses and bacteria, certain hormones, alcohol, poor diet, lack of physical activity, or being overweight, or a combination thereof. The individual may be genetically predisposed to the cancer because of having one or more genetic markers. The cancer may have been resected but have a high risk of recurrence, in certain cases. The cancer may be refractory to one or more other therapies. The cancer may be metastatic. The cancer may or may not be solid.

III. [0044] Autoimmune Embodiments

[0045] The disclosure provides autoimmune therapies in the form of certain anti- CD 137 antibodies having Fc regions that are capable of binding FcyRIII and/or FcyRIIA or having enhanced binding thereof. The antibodies may or may not be designed to have such Fc regions that are capable of binding FcyRIII and/or FcyRIIA or having enhanced binding thereof.

[0046] Other embodiments of the disclosure concern optimization of one or more particular CD 137 antibodies for reducing autoimmune responses, such as those that directly or indirectly are related to autoimmune disease.

[0047] Certain anti-CD 137 mAbs described herein can be used to treat any

inflammatory condition or autoimmune disease that is amenable to treatment by administration of anti-CD 137 antibodies, including but not limited to rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Addison's disease, celiac disease, dermatomyositis, Graves' disease, multiple sclerosis, myasthenia gravis, asthma, inflammatory bowel disease, psoriasis, Hashimoto's thyroiditis, vasculitis, scleroderma, Guillain-Barre syndrome, Chronic

Inflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic IgM demyelinating Polyneuropathy, Lambert-Eaton myasthenic syndrome, Myasthenia gravis, Multifocal Motor Neuropathy, Lower Motor Neuron Syndrome associated with anti-GMl antibodies,

Demyelination, Multiple Sclerosis and optic neuritis, Stiff Man Syndrome, Paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis, Myelitis, Myelopathy especially associated with Human T-cell lymphotropic virus- 1, Autoimmune Diabetic Neuropathy, Acute Idiopathic Dysautonomic Neuropathy, Kawasaki's disease, Rheumatoid arthritis, Felty's syndrome, ANCA-positive Vasculitis, Spontaneous Polymyositis, Dermatomyositis,

Antiphospholipid syndromes, Recurrent spontaneous abortions, Systemic Lupus Erythematosus, Juvenile idiopathic arthritis, Raynaud's, CREST syndrome, Uveitis, Epidermal Necrolysis, Gangrene, Granuloma, Autoimmune skin blistering diseases including Pemphigus vulgaris, Bullous Pemphigoid, and Pemphigus foliaccus, Vitiligo, Streptococcal toxic shock syndrome, Scleroderma, systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, Atopic dermatitis, steroid dependent Atopic dermatitis, Inclusion Body Myositis, Necrotizing fasciitis, Inflammatory Myopathies, Myositis, Anti-Decorin (BJ antigen) Myopathy,

Paraneoplastic Necrotic Myopathy, X-linked Vacuolated Myopathy, Penacillamine-induced Polymyositis, Atherosclerosis, Coronary Artery Disease, Cardiomyopathy, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, Celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis, Reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis, sclerosing cholangitis, Graft Versus Host Disease, Antibody-mediated rejection of the graft, Post-bone marrow transplant rejection, Post-infectious disease inflammation, Lymphoma, Leukemia, Neoplasia, Asthma, Type 1 Diabetes mellitus with anti-beta cell antibodies, Sjogren's syndrome, Mixed Connective Tissue Disease, Addison's disease, Vogt- Koyanagi-Harada Syndrome, Membranoproliferative glomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis,

micropolyarterits, Churg-Strauss syndrome, Polyarteritis nodosa or Multisystem organ failure.

[0048] More than one dose of the antibodies may be provided to the individual, and the subsequent dosings may be spaced in time on the order of minutes, hours, days, weeks, or months from each other. Optimization of dosing regimens is routine in the art of

immunotherapy.

[0049] Any autoimmune disorder may be treated with embodiments of the disclosure and the disorder may result in the destruction of body tissue; abnormal growth of an organ; and/or changes in organ function. The autoimmune disorder may affect one or more organ and/or tissue types, including blood vessels; connective tissues; endocrine glands, such as the thyroid or pancreas; joints; muscles; red blood cells; skin; and so forth. The individual may experience one or more symptoms depending on the type and location of the faulty immune response, such as fatigue; fever; malaise; joint pain; rash; and so forth. The autoimmune disease may be diagnosed in any manner, and in specific embodiments part of certain methods of the disclosure encompass a diagnosis step, such as using antinuclear antibody tests; autoantibody tests; CBC panel; comprehensive metabolic panel; C-reactive protein (CRP) test; erythrocyte sedimentation rate (ESR); urinalysis; and a combination thereof. [0050] In particular embodiments, methods and/or compositions of the disclosure can be used in combination with other modalities to treat autoimmune disease and in specific embodiments are synergistic with other treatment(s).

IV. [0051] Antibodies and Fusion Proteins of the Disclosure and Generation Thereof

[0052] The disclosure provides particular antibodies for enhancement or abrogation of their ability to mediate cellular cytotoxic effector functions (such as Ab-dependent cell-mediated cytotoxicity (ADCC) and Ab-dependent cell-mediated phagocytosis (ADCP)) that are directed by engagement of the Fc region with the Fey receptors. All FcyRs bind the same region on IgG Fc, but have differing affinities: high (FcyRI) and low (FcyRII and FcyRIII). (For review, see Nimmerjahn et al., 2015.)

[0053] One particular group of IgG Fc receptors related to embodiments of the disclosure include FcyR expressed on immune effector cells; three classes of FcyR include FcyRI (CD64); FcyRII (CD32); and FcyRIII (CD16). In humans, FcyRII receptors are subdivided into FcyRIIA and FcyRIIB, and FcyRIII receptors are subdivided into FcyRIIIA, FcyRIIIB, and FcyRIIIC. The skilled artisan recognizes that comprehensive mapping of the binding site on human IgG for the different FcyR (Shields et al., 2001) is known in the art and, therefore, one can utilize this knowledge for modulating the Fc region to either abrogate or enhance immune recruitment via FcyR. Examples of applying such information to generate desired Fc-engineered antibodies is known in the art (Horton et al., 2010; Lazar et al., 2006).

[0054] For example, as described in Shields et al. (2001), a common set of IgGl residues is involved in binding to all FcyR, and FcyRII and FcyRIII also utilize particular residues in addition to the common set. Furthermore, residues that abrogated binding to one or more Fc receptors were identified and also certain positions that improved binding to specific receptors of one type yet reduced binding to another type In embodiments wherein there is desired reduced or abolished binding to FcyRIII or FcyRII, one may make one or more of the following amino acid substitutions in the Fc region: D270A; Q295A; and A327S. In

embodiments wherein there is desired reduced or abolished binding to FcyRIIIA yet no impact on binding to FcyRII, one may make one or more of the following amino acid substitutions in the Fc region: S239A; E269A; E293A; Y296F; V303A; A327G; K338A; and D376A. In embodiments wherein there is desired reduced or abolished binding to FcyRII yet no impact on binding to FcyRIIIA, one may make one or both of the following amino acid substitutions in the Fc region: R292A and K414A. In specific embodiments, one or more of the aforementioned mutations may be utilized in the design of particular anti-CD 137 monoclonal antibodies for potentiating anti-tumor auto immunity.

[0055] In embodiments wherein there is desired improved binding to FcyRIIIA or FcyRII, one may make one or both of the following amino acid substitutions in the Fc region: T256A and K290A. In embodiments wherein there is desired improved binding to FcyRII yet no effect on FcyRIIIA, one may make one or more of the following amino acid substitutions in the Fc region: R255A; E258A; S267A; E272A; N276A; D280A; H285A; N286A; T307A; L309A; N315A; K326A; P331A; S337A; A378Q; and E430A. In embodiments wherein there is desired improved binding to FcyRIIIA yet no effect on FcyRII, one may make one or more of the following amino acid substitutions in the Fc region: E333A; K334A; and A339T. In specific embodiments, one or more of the aforementioned mutations may be utilized in the design of particular anti-CD 137 monoclonal antibodies for ameliorating autoimmunity.

[0056] Antibodies that have their sugars removed cannot bind canonical Fc receptors and, in specific embodiments, aglycosylated antibodies are employed in methods of the disclosure. In specific embodiments, the antibody can be modified to be an aglycosylated form, for example by mutating an amino acid to which the sugar binds to another amino acid to which a sugar would not bind (such as Asn to Gin; see Lin et al., 2008).

[0057] In particular embodiments, the anti-Cdl37 monoclonal antibodies comprise an Fc region that has reduced or no ability to bind with FcyRIII, FcyRIIA, or both, and as used herein "reduced" ability to bind with FcyRIII, FcyRIIA, or both refers to either an absolute or relative change in affinity of the Fc molecule for these receptors versus either FcyRIIB and/or FcyRI and/or FcRL5 and/or CD22.

[0058] In particular embodiments, the anti-Cdl37 monoclonal antibodies comprise an Fc region that has the ability or an enhanced ability to bind with FcyRIII, FcyRIIA, or both, and as used herein "enhanced" ability to bind with FcyRIII, FcyRIIA, or both refers to either an absolute or relative change in affinity of the Fc molecule for these receptors versus either FcyRIIB and/or FcyRI and/or FcRL5 and/or CD22. [0059] In particular embodiments, the antibodies of the disclosure are Fc-engineered, and as used herein the term refers to the Fc region being modified directly or indirectly by the hand of man to have an altered Fc region compared to the Fc region in a natural state. In particular, Fc-engineered refers to a specifically designed antibody that as a result of the engineering binds to certain sub-groups of FcyR receptors or, alternatively, is not able to bind to certain sub-groups of FcyR receptors. In specific embodiments, the antibodies are non-natural.

[0060] In alternative embodiments, anti-CD 137 monoclonal antibodies are employed that were not specifically engineered for differential binding to different FcyRs but that have been determined empirically to bind to certain classes of FcyRs more or less than others. One of skill in the art recognizes that there are routine methods to ascertain the binding to various FcyRs, such as measuring cell-based assays, for example ADCC or ADCP (see at least Lazar et a/., 2006).

[0061] There are known anti-CD 137 antibodies in the art, and one of skill in the art recognizes generally how to produce such types of antibodies. Specific examples include at least the following: 1) Anti-Human CD137 (4-1BB) FITC (Also known as: Ly-63, TNFRSF9, ILA); Clone: 4B4 (4B4-1); 2) Hybridoma (17b5), Producing Monoclonal Antibody Specific For Mouse 4-1BB; 3) 4-1BB Ligand / CD137L Antibody, PE conjugate (TKS-1); 4-1BB Ligand / CD137L Monoclonal Antibody (Life Technologies); 4) CD137 / TNFRSF9 Antibody (BBK-2); CD137 / TNFRSF9 Monoclonal Antibody (Thermo Scientific); 5) 1D8; 6) 3H3; 7) 2A; 8) BMS-663513 (Bristol Myers Squibb); and 9) PF-2566 (Pfizer).

[0062] In particular embodiments, instead of an antibody that targets CD137, non- antibodies, such as fusion proteins, may be employed. For example, one can utilize a fusion protein that has a moiety capable of binding and activating CD137 (for example, 4-1BBL) and that also has a moiety of the Fc region having reduced ability to bind FcyRIIA and/or FcyRIII. The moiety that binds CD 137 may comprise part or all of SEQ ID NO: l. The moiety that binds CD137 may have sequence identity to part or all of SEQ ID NO: l, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The moieties may be in any order and the fusion protein may also comprise additional moieties. In specific embodiments, such a fusion protein produces an effect substantially equivalent to an antibody. Routine methods of generating fusion proteins are known in the art. V. [0063] Pharmaceutical Preparations

[0064] Pharmaceutical compositions of the present invention comprise an effective amount of one or more anti-CD 137 monoclonal antibodies having Fc regions with particular Fc receptor-binding capabilities, wherein the antibodies are dissolved or dispersed in a

pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one anti- CD 137 monoclonal antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21 ^st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0065] In specific embodiments, the dosage range of antibodies is 0.1- 10 mg/kg/dose, including 0.1-7.5 mg/kg/dose; 0.1-5 mg/kg/dose; 0.1-2.5 mg/kg/dose; 0.1- 1 mg/kg/dose; 0.5-10 mg/kg/dose; 0.5-7.5 mg/kg/dose; 0.5-5 mg/kg/dose; 0.5-2.5 mg/kg/dose; 0.5- 1 mg/kg/dose;. 1- 10 mg/kg/dose; 1-7.5 mg/kg/dose; 1-5 mg/kg/dose; 1-2.5 mg/kg/dose; 2.5- 10 mg/kg/dose; 2.5-7.5 mg/kg/dose; 2.5-5 mg/kg/dose; 5-10 mg/kg/dose; 5-7.5 mg/kg/dose; 7.5- 10 mg/kg/dose; and so forth.

[0066] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0067] The antibodies may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0068] The antibodies may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

[0069] Further in accordance with the present invention, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0070] In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.

[0071] In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0072] In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include the antibodies and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

[0073] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the antibodies may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0074] The actual dosage amount of a composition of the present invention

administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0075] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0076] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500

microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

A. Parenteral Compositions and Formulations

[0077] In further embodiments, antibodies may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract.

Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).

[0078] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0079] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of

Biologies standards.

[0080] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.

B. Alimentary Compositions and Formulations

[0081] In one embodiment of the present invention, the antibodies are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of

administration in which the composition is in direct contact with the alimentary tract.

Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. [0082] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et ah, 1997; Hwang et ah, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or

combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and

propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0083] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

[0084] Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

C. Miscellaneous Pharmaceutical Compositions and Formulations

[0085] In other preferred embodiments of the invention, the active compound antibodies may be formulated for administration via various miscellaneous routes, for example, topical (i.e. , transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.

[0086] Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial

preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time. [0087] In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et ah, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

[0088] The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.

VI. [0089] Kits of the Disclosure

[0090] Any of the compositions described herein may be comprised in a kit. In a non- limiting example, anti-CD 137 monoclonal antibodies having particular Fc regions or CD 137- targeting fusion proteins may be comprised in a kit. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the antibodies and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. [0091] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The antibody compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.

[0092] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0093] Irrespective of the number and/or type of containers, the kits of the invention may also comprise, and/or be packaged with, an instrument for assisting with the

injection/administration and/or placement of the ultimate composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, and/or any such medically approved delivery vehicle, for example.

[0094] In particular embodiments, the kit may also comprise one or more cancer therapeutics or one or more autoimmune disorder therapeutics.

EXAMPLES

[0095] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow present techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1

ANTI-CD- 137 MONOCLONAL ANTIBODIES WITH DISTINCT FCyR BINDING

ABILITIES FOR TREATMENT OF CANCER OR AUTOIMMUNITY

Overview

[0096] Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD 137 to function as an analogue for CD137L. Although binding of CD 137 by anti-CD 137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcyR), the relative import of such Fc-FcyR interactions in mediating anti- CD 137 associated anti-tumor immunity is unknown. Provided herein are studies of the ability of a rat anti-mouse CD 137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcyR ^"7" strains. 2A treated FcRy ^"7" mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcyRIIT ^7" animals. These improved anti-tumor responses were associated with increased splenic CD8 T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. The results demonstrate an unexpected inhibitory role for FcyRIII in the anti-tumor function of anti-CD 137, and underscore the need to consider antibody isotype when engineering therapeutic mAbs.

Methods

Mice, cell lines and therapeutic antibodies

[0097] C57BL/6 and B6.129P2- cgr3 ^im/¾ 7SjvJ (FcyRIir ⁷ ) were purchased from Jackson Laboratory. Fcerlg (FcRy ⁷ ) and Fcgr2b (FcyRIIB ⁷ ) were purchased from Taconic Farms. CD137 ^"7" and FcyRrV ^"7" mice were generous gifts from Dr. Lieping Chen and Dr. Jeffery Ravetch, respectively. Breeding colonies for FcyRIIT ^7" , FcyRIV ^"7" , and CD137 ^"7" were maintained in the animal facility at the University of Maryland School of Medicine. Female mice from eight to twelve weeks of age were used in the experiments. The Institutional Animal Care and Use Committee at the University of Maryland, School of Medicine approved all experiments. [0098] The EL4E7 cell line, a gift from Dr. Chen, was passaged in vivo four times to generate a cell line for the tumor experiments. B16-F10 cells were a gift from Dr. Paul Antony. The EL4E7 and B16-F10 cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamine (Invitrogen), 1% penicillin- streptomycin (Invitrogen), and 1% HEPES buffer (Corning).

[0099] Rat anti-mouse CD 137 mAb clone 2A (IgG2a) and clone 3H3 (IgG2a), were gifts from Dr. Chen and Dr. Robert Mittler, respectively. The antibodies were purified from hybridoma supernatant using affinity chromatography on a protein G sepharose column (GE Healthcare). Antibodies were verified to have <1 IU/ml of endotoxin using the Limulus

Amebocyte Lysate Test (Charles Rivers Laboratories). Rat IgG control was purchased from Sigma or Innovative Research.

Flow cytometry and antibodies

[0100] Antibodies against CD8 FITC (ebioH35-17.2), CD103 PE (2E7), CD40 APC (1C10), CD80 APC (16-10A1), CD86 APC (GL1), CD137L (TKS-1) PE, Armenian hamster IgG PE, rat IgG2a PE and rat IgG2a APC were purchased from eBioscience. Antibodies against CD1 lc PercP-Cy5.5 (HL3), hamster IgG PercP-Cy5.5, and mouse IgG APC were purchased from BD Biosciences. Antibodies against MHCII/I-A ^b APC (AF6-120.1), CD137 biotin (17B5), Syrian hamster biotin, and TruStain fcX were purchased from Biolegend. Anti-polyhistidine PE was purchased from R&D systems. Recombinant mouse CD137L containing a His tag was purchased from R&D systems. Data were acquired using an LSRII instrument (BD Biosciences) and were analyzed using FlowJo (Tree Star).

Measurement of mouse FcyRs binding to rat IgG2a by Surface Plasmon Resonance (SPR)

[0101] Antibodies (2A, rat IgG2a (eBioscience), and mouse IgG2a (eBioscience)) at 10μg/mL in Acetate pH4.5 were immobilized to a CM5 chip in a BIAcore 3000 (GE

Healthcare). A response of 600RU was the immobilization target level. Actual immobilization levels were 680RU, 670RU and 640RU, respectively, on flow cell (FC) 2, FC3 and FC4. FC1 was used as a blank reference. Mouse FcyRI, FcyRIIB, FcyRIII and FcyRIV were serially diluted from ΙμΜ to less than InM with running buffer (HBS-EP) and injected for 120sec at 20μ1/ηιίη over all FCs followed by a 120 sec dissociation step. Full regeneration was achieved using a 15- 30sec injection of 1M MgCl at ΙΟΟμΙ/min. FCs were washed with a 30sec injection of running buffer at 20μ1/ηιίη. KDs were generated using a 1: 1 Langmuir binding model. Kinetics were run in duplicate.

Tumor growth models

[0102] Mice were inoculated subcutaneously with 8 x 10 ⁶ EL4E7 tumor cells in the flank. Tumor volume was determined by measuring the width (w) and length (1) of the tumor and using the following formula: (w *1)*0.5. Mice bearing tumors with a length of 5mm on Day 7 and 8mm on Day 10 were randomized into two groups that received a first injection of 100μg of either Rat IgG or 2A by intraperitoneal injection on Day 7 or Day 10 and a second injection, three days later. Tumor growth was monitored until mice appeared moribund or the diameter of the tumors reached 25 mm. Mice were sacrificed, based on these endpoints, and this was recorded as the date of death for survival studies. Mice with complete tumor resolution were challenged with EL4E7 and B16-F10 tumor cells. Tumor growth was monitored until the combined tumor volume reached approximately 20% of the mouse's total body weight, and then the mice were sacrificed.

[0103] To evaluate immune cell kinetics, mice bearing tumors with an average length of 5mm on Day 7 were randomized into two groups, which received 100μg of either Rat IgG or 2A, by intraperitoneal injection on Days 7 and 10. Two or three mice per group were euthanized on Day 7, Day 12, Day 16, and Day 21 and spleens were processed. Cell counts from individual spleens were taken manually or with the Countess automated cell counter (Invitrogen) using trypan blue exclusion to count live cells. Total cell numbers were calculated by multiplying the cell count by the lymphocyte gate and then by the appropriate gate of the cell population of interest.

Competition assay for CD137L binding

[0104] HEK-293 cells were stably transfected with mouse CD 137 using lipofectamine (Invitrogen) transfection with a pcDNA3.3 vector containing CD 137. The HEK-mCD137 cells were incubated with Rat IgG or anti-CD137 mAb (clone 2A or 3H3) at 0. lug/ml, lug/ml, lOug/ml or lOOug/ml; followed by incubation with 31ng/ml of His-tagged recombinant mouse CD137L (R&D systems). Finally, cells were incubated with anti-polyhistidine PE (R&D systems) to determine CD137L binding. Data were acquired on an LSRII instrument (BD Biosciences) and analyzed using FlowJo (TreeStar). Statistical Analysis

[0105] All Kaplan-Meier survival curves were compared using the log rank test.

Statistical significance was determined using a one-way Anova with Tukey' s multiple comparison test. All statistical analyses were performed using GraphPad (Prism). A p value < 0.05 was considered significant.

Exemplary Results

Rat anti-mouse CD137 binds to mouse FcyRIIB, FcyRIII, and FcyRIV.

[0106] The majority of anti-CD137 mAbs used to study the impact of CD137 ligation on anti-tumor immunity are of rat origin (Wilcox, et ah, 2002; Kim, e al., 2001; Shuford, et ah, 1997). To characterize the potential of the Fc fragments of these rat mAbs to impact the antitumor immune response in mice, the interactions between rat IgG2a and mouse FcyRs were evaluated by surface plasmon resonance. Mouse FcyRIIB, FcyRIII, and FcyRrV, but not FcyRI, bound to immobilized rat anti-CD137 (clone 2A)(FIG. 1). FcyRIIB and FcyRIII bound comparably to both immobilized 2A and control mouse IgG2a (anti-CD 137 mAb: FcyRIIB KD= 528nM, FcyRIII KD= 238 nM; mouse IgG2a: FcyRIIB KD= 501nM, FcyRIII KD= 138 nM). In contrast, the interactions between FcyRIV and 2A (KD= 3180nM) were dramatically weaker than correlate interactions between FcyRIV and control mouse IgG2a (KD= 8.63nM). These data indicate that rat IgG2a, the isotype of the anti-CD 137 mAbs used in this study, are useful to interact with select murine FcyRs.

Anti-CD137 has enhanced activity against EL4E7 in mice lacking the common Fcy-chain.

[0107] The absolute requirement was validated for host expression of CD 137 on the anti-tumor activity of 2A. As anticipated, 2A failed to mediate tumor regression in CD137 ^"7" mice, while retaining its therapeutic efficacy in wild type (WT) animals (FIG. 7A, WT 2A vs WT IgG p<0.0001; WT 2A vs CD137 ^"7" 2A p=0.0013). In order to rule out the possibility that EL4E7 might express CD 137 and/or FcyRs, and thus confound the findings, the levels were determined of CD 137 and FcyRs on the EL4E7 tumors. The ex vivo EL4E7 tumor samples lacked expression of CD 137 (FIG. 7B). Furthermore, the EL4E7 tumor cells expressed exceptionally low levels of FcyRIII and/or FcyRIIB (FIG. 7C). These data confirm that the EL4E7 tumor is an appropriate model for determining the role of Fc-FcyR interactions on the anti-tumor function of anti-CD 137, independent of direct antibody- tumor interactions. [0108] As an initial step in defining the potential role of Fc-FcyR interactions on the anti-tumor activity of anti-CD 137, Fc common γ-chain (FcRy) and FcyRIIB deficient mice were employed. The FcRy ^"7" mice do not express activating FcyRs, yet maintain expression of

FcyRIIB; whereas the FcyRIIB ^"7" mice only express the activating FcyRs (Takai, et ah, 1994). The FcRy ^"7" and FcyRIIB ^"7" mice were challenged with EL4E7 tumors and treated with 2 A on days 7 and 10 post tumor inoculation. Unexpectedly, the both FcyRIIB ^"7" and WT mice had a similar anti-tumor response to 2A, with both experiencing progressive disease (Fig. 2A, FcyRIIB ^{" 7"} 2A vs FcyRIIB ^"7" IgG p=0.0128, WT 2A vs WT IgG p<0.0001). In contrast, the FcRy ^"7" mice had improved survival compared to both WT animals treated with 2A and control mice treated with rat IgG, (Fig. 2B, FcRy ^"7" 2A vs FcRy ^"7" IgG p<0.0001, FcRy ^"7" 2A vs WT 2A p=0.002, WT 2A vs WT IgG p<0.0001). These data indicate FcyRIIB engagement is not involved in enhancing the 2A mediated anti-tumor immune response. The enhanced anti-tumor response was further assessed in the 2A treated FcRy ^"7" mice by delaying treatment with 2A to days 10 and 13 post tumor inoculation. The 2A treated FcRy ^"7" mice had significantly improved anti-tumor responses compared to the 2A treated WT mice and IgG controls (Fig. 2C, FcRy ^"7" 2A vs FcRy ^"7" IgG p= 0.0027; FcRy ^"7" 2A vs WT 2A p= 0.0161, WT 2A vs WT IgG p= 0.0063). The FcRy ^"7" mice which enjoyed initial 2A mediated tumor rejection were immune to distant challenge with EL4E7 but not B16 melanoma (data not shown). Taken in concert, these data indicate the expression of the Fey chain impairs the 2A-mediated immune response against EL4E7.

Anti-CD137 has enhanced activity against EL4E7 in FcyRIII ^"7" mice.

[0109] Next, the individual activating FcyR contributing to the improved 2A mediated anti-tumor immunity in FcRy ^"7" mice was investigated. The contribution of FcyRIII and FcyRIV was studied, excluding FcyRI based on the lack of binding to rat IgG2a (FIG. 1). 2A treatment significantly improved survival of tumor bearing FcyRIII ^"7" , but not FcyRIV ^"7" mice versus WT controls (FIG. 3A, FcyRIII ^"7" 2A vs FcyRIII ^"7" IgG p< 0.0001; WT 2A vs WT IgG p= 0.0023; FcyRIII ^"7" IgG vs WT IgG p= 0.0311, and FcyRIII ^"7" 2A vs WT 2A p= 0.0006 and, 3B, FcyRIV ^"7" 2A vs FcyRIV ^"7" IgG p=0.0217; WT 2A vs WT IgG p= 0.0484; and FcyRIV ^"7" 2A vs WT 2A p= 0.1062). FcyRIII ^"7" mice, which enjoyed initial 2A mediated tumor rejection, were immune to second challenge with EL4E7 but not B16-F10 melanoma. These data identify FcyRIII as the receptor involved in the modulation of the anti-CD 137 mediated anti-tumor response against EL4E7. [0110] To exclude the possibility that the observations were 2A specific, a second clone of anti-CD 137 mAb, 3H3 (rat IgG2a), was evaluated in FcyRIir ^7" mice. 3H3 preferentially prolonged survival of EL4E7 tumor bearing FcyRIH ^"7" mice, but, unlike 2A, did not result in complete tumor resolution (Fig. 4A, FcyRIir ^7" 3H3 vs FcyRIir ^7" IgG p= 0.015, and FcyRIir ^7" 3H3 vs WT 3H3 p= 0.0010). Differences in the absolute efficacy of these individual mAbs may relate to the fact that at higher doses, 3H3, but not 2A, blocks naturally occurring CD137- CD137L interactions (FIG. 4B and 4C). These data demonstrate that FcyRIII impairs anti- CD 137 mediated anti-tumor immunity with antibody dependent variation in therapeutic benefit.

[0111] To determine if the modulation of the 2A mediated anti-tumor response in the FcyRIir ^7" mice could be recapitulated in another tumor type, the MC38 colon cancer model was utilized, which has a weak therapeutic response to 2A treatment (Dubrot et al., 2010). Both WT and FcyRIir ^7" mice experienced equivalent MC38 tumor growth in response to 2A. This result suggests that FcyRIII deficiency is not sufficient for further enhancement of the 2A mediated anti-tumor response in the MC38 model.

Anti-CD137 therapy preferentially enhances Οϋ8β ⁺ T cell and CDllc ⁺ dendritic cell populations in FcyRIir ^7" mice.

[0112] To understand the mechanisms underlying the improved anti-tumor efficacy of anti-CD137 in FcyRIir ^7" mice, a detailed kinetic analysis of CD8 ⁺ CDl lc ⁺ T cells and DCs was performed, both of which are implicated in 2A mediated tumor rejection (Murillo et al., 2009; Ju et al., 2007). As anticipated, anti-CD137 treatment resulted in expansion of the CD8 ⁺ T cell population in WT and FcyRIir ^7" mice (FIG. 5A). However, the increase of the CD8 ⁺ cell population began at an earlier time point and was dramatically more pronounced in the spleens of FcyRIir ^7" mice (FIG. 5A). Next, based on reports that anti-CD137 treatment mediates CD103 up regulation on CD8 ⁺ CD1 lc ⁺ cells and that CD103 expression is associated with CD8 ⁺ regulatory T cell function, CD 103 was employed as a phenotypic marker of suppressor activity (Uss et al,. 2006; Myers et al, 2005; Vinay et al, 2009). Remarkably, in anti-CD137 treated FcyRIir ^7" mice, while the ratio of CD 103 negative to positive T cells remained stable, there was almost a 1 log order difference in absolute numbers of CD 103 negative to positive T cells in the spleen (FIG. 5A). Direct comparison of the number of CD8 ⁺ CDl lc ⁺ CD103 ^" T cells to tumor volume using Pearson's correlation coefficient suggested that the CD8 ⁺ CD1 lc ⁺ CD103 ^" T cells were involved in tumor resolution (FIG. 5B). Importantly, the expansion of the CD8 ⁺ T cells and tumor resolution were not due to differences in expression of CD 137 or CD137L in the WT and FcyRIir ^7" mice (FIG. 5C). Finally, there was no antibody dependent cellular cytoxicity (ADCC) detected using either WT or FcyRIIT ^7" NK incubated with 2A opsonized ConA stimulated T cells, suggesting the enhanced CD8 ⁺ T cells in the 2A treated FcyRIIT ^7" mice was not due to ADCC mediated T cell depletion in the WT mice (FIG. 8). These data indicate the FcyRIIT ^7" mice produce a more rapid and robust CD8 ⁺ CD1 lc ⁺ T cell response after 2A treatment in the EL4E7 tumor model.

[0113] DCs have been implicated in the anti-tumor function of anti-CD 137 treatment (Bruhns, et ah, 2012). FcyRIII is not expressed on CD8 ⁺ T cells, suggesting FcyR ⁺ DCs are likely responsible for the regulation of the CD8 ⁺ T cell response. Interestingly, the

CDl lc ⁺ MHCII ⁺ cell population was preferentially expanded in the spleens of 2A treated

FcyRIir ^7" mice (FIG. 6A). Three lines of evidence support the role of these CDl lc ⁺ MHCII ⁺ cells in mediating the enhanced anti-tumor function of 2A in FcyRIir ^7" mice. First, these FcyRIir ^7" CDl lc ⁺ MHCII ⁺ cells evidenced increased expression of CD40, CD80, and CD86, suggesting their potential for enhanced antigen presentation (FIG. 6B). From a functional perspective, the enhanced antigen presentation capacity of these cells was indirectly supported by observed increases in the numbers of OT-I transferred T cells in 2A treated FcyRIir ^7" mice (data not shown). Second, similar to the T cell data, a significant positive Pearson's correlation was found between the CDl lc ⁺ MHCII ⁺ cell population and the tumor volume in the 2A treated FcyRIir ^7" mice (FIG. 6C). Third, the increased numbers of CDl lc ⁺ MHCII ⁺ cells in FcyRIir ^7" treated mice corresponded with the presence of disease, suggesting that CDl lc ⁺ MHCII ⁺ cells are involved tumor regression. Importantly, the expansion of the CDl lc ⁺ MHCII ⁺ cell population was not due to differences in expression of CD 137 or CD137L in the WT and FcyRIir ^7" mice (FIG. 6D). Taken together with the increase in the CD8 T cell population, the data suggest a paradigm in which 2A treatment preferentially enhances DC numbers and function in FcyRIIT ^7" mice, with resultant secondary expansion of effector T cells, at least in the EL4E7 tumor model.

Significance of Certain Embodiments

[0114] The present disclosure provides the contributions of Fc-FcyR interactions to the anti-tumor activity of anti-CD 137. The data indicate that anti-CD 137 treated FcyRIir ^7" mice have improved responses against EL4E7, which are associated with an increase in CD8 ⁺ T cells and phenotypically activated DCs in the spleen. The results indicate a paradigm in which anti- CD 137 treatment preferentially enhances DC function in tumor bearing FcyRIIT ^~ mice, with resultant increases in effector T cell activity against EL4E7.

[0115] The anti-tumor function of anti-CD 137 was defined in preclinical mouse models using rat-anti mouse Abs (Wilcox, et ah, 2002; Melero, et ah, 1997; Shuford, et ah, 1997).

Despite the recognized cross reactivity between mouse FcyRs and rat IgG, little is known about the binding affinity and kinetics of rat Fc-mouse FcyR interactions. The data indicate that mouse FcyRIIB and FcyRIII interact with rat IgG2a in a manner similar to control mouse IgG2a;

whereas FcyRI and FcyRIV, the high affinity receptors, have different binding affinities and kinetics for rat and mouse IgG2a (FIG. 1). The binding prolife for rat IgG2a to mouse FcyRs is similar that of mouse IgGl (White, et ah, 2011). This similarity in binding profiles between rat IgG2a and mouse IgGl is anticipated based on their structural homology (Bruggemann, et ah, 1986). Collectively, these data and findings from other investigators, suggest that rat IgG2a interacts with mouse FcyRs in a manner similar to mouse IgGl, in specific aspects.

[0116] The import of Fc-FcyR interactions in mediating the anti-tumor activity of immunomodulatory mAbs has been acknowledged in recent publications. For example, engagement of activating FcyRs is required for the anti-tumor function of anti-GITR and anti- CTLA-4 mAbs (Simpson, et ah, 2013; Bulliard, et ah, 2013). Interestingly, agonistic mAbs against CD40 are functionally dependent on interactions with FcyRIIB (White, et ah, 2011; Li, et a/. , 2011). The data adds to this growing body of literature, showing that Fc-FcyRIII interactions dampen the anti-CD 137 mediated anti-tumor response against EL4E7.

[0117] The exact mechanism by which FcyRIII attenuates anti-CD 137 immunity against EL4E7 is uncertain. FcyRIII is associated with both pro and anti-inflammatory responses (Aloulou, et al., 2012; Ivashkiv, et al., 2009; Siragam, et al., 2006; Sutterwala, et al., 1998; Erdinc, et ah, 2013). For example, FcyRIII is associated with inhibitory IT AM signaling resulting from tonic Fc-FcyR interactions that initiate inhibitsome formation (Aloulou, et al. , 2012; Pfirsch-Maisonnas, et ah, 2011). A recent publication demonstrates anti-CD137 mAb is rapidly internalized after Fab-CD 137 interactions, which would limit Fc exposure to FcyRIII (Martinez-Forero, et ah, 2013). Therefore, the combined tonic Fc interaction with FcyRIII may inhibit the NF-κΒ signaling induced by anti-CD 137 mAb interactions resulting in a reduction of CD 137 stimulation (Martinez-Forero, et ah, 2013). Besides the induction of inhibitory signaling, FcyRIII is involved in the clearance of apoptotic cells (Erdinc Sunay, et al., 2013). The reduction in the clearance of apoptotic cells would allow for enhanced tumor antigen exposure. Taken in concert with the data, these published findings indicate that the improved anti-tumor activity of anti-CD 137 in FcyRIII ^"7" is likely a result of several non-mutually exclusive and potentially overlapping mechanisms.

[0118] The data does not rule out the potential role of FcyRIV in modulating anti- CD 137 mAb tumor responses. FcyRIV expression is limited to Ly6C ^lQ monocytes,

macrophages, and neutrophils, which are common tumor infiltrating leukocytes (Mancardi, et al., 2008; Biburger, et al., 2011). CD137 expression is highest on tumor infiltrating lymphocytes, potentially enabling enhanced localization of Fc-FcyRIV interactions at the tumor site (Palazon, et al., 2012). These expression patterns of CD 137 and FcyRIV in vivo may surmount the weaker interaction of FcyRIV to rat IgG2a by increasing the concentration of 2A at the tumor and may explain the trend towards improvement of the anti-tumor response in 2A treated FcyRIV ^"7" mice.

[0119] To understand the mechanisms underlying the improved anti-tumor efficacy of anti-CD 137 mAb in FcyRIII ^"7" mice, detailed kinetic analysis was performed of CD8 ⁺ T cells and DCs, which are implicated in 2A mediated tumor rejection (Melero, et al., 1997; Murillo, et al., 2009; John, et al, 2012; Ito, et al, 2004; Cuadros, et al, 2005; Houot, et al, 2009; Murillo, et al., 2008; Kim, et al., 2009). Following anti-CD 137 therapy, there was a profound increase in the CD8 ⁺ T cell and DC populations, which was dramatically enhanced in the FcyRIII ^"7" mice. CD8 ⁺ T cells do not express FcyRs suggesting a model in which FcyRIII deficiency on DCs is modulating the anti-tumor immune response elicited by anti-CD 137 in the EL4E7 tumor model (Bruhns, et al., 2012).

[0120] DCs are considered professional antigen presenting cells, and improved antigen presentation would result in an enhanced CD8 ⁺ T cell response (Banchereau, et al., 1998). The 2A treated FcyRIII ^"7" mice had increases in numbers of DCs expressing costimulatory molecules associated with antigen presentation. CD137 stimulation likely contributed to this phenotype as: (1) FcyRIII ^"7" DCs have comparable antigen presentation capabilities as WT DCs, and (2) anti- CD 137 stimulation enhances CD40 stimulation of DCs corresponding to an increase in tumor infiltrating T cells (French, et al, 2007; Miller, et al, 2002). The lack of potential Fc-FcyRIII interactions is also functionally relevant as the loss of IT AM signaling on DCs is recognized to improve toll-like receptor stimulation by increasing costimulatory molecule expression and the production of pro-inflammatory cytokines (Chu, et ah, 2008). When taken in combination with the findings, these studies indicate that antibody binding to CD137, in the absence of FcyRIII interactions, results in enhanced antigen presentation capabilities that promote the secondary expansion of CD8 ⁺ T cells against EL4E7, in at least some embodiments.

[0121] The data indicates that anti-CD 137 mAb interactions with FcyRIII inhibit antitumor immune responses to EL4E7 tumors. When taken in combination with the rapidly evolving literature in this field, the studies indicate that the functional role of Fc-FcyR

interactions on antibody mediated immune modulatory therapy is defined by both patterns of receptor and relevant FcR expression. These data indicate that Fc-FcyR interactions play a critical role in the biology of antibodies targeting co signaling molecules, and that manipulation of these interactions allows for improved therapeutic efficacy, in certain embodiments.

[0122] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0123] Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. REFERENCES

[0124] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in their entirety.

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[0125] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the invention as defined by the claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one will readily appreciate from the disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized. Accordingly, the claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.