PD-1 ANTAGONIST AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/359073, filed July 6, 2016. The content of the aforementioned application is hereby incorporated by reference.

BACKGROUND

The National Cancer Institute has estimated that in the United States alone, 1 in 3 people will be struck with cancer during their lifetime. Moreover, approximately 50% to 60% of people contracting cancer will eventually succumb to the disease. Once tumor cells escape from the primary site they passage through the lymphatic and/or circulatory system and ultimately a few establish at distant sites to give metastases, and 95% of deaths from solid tumors in the developed world are due to metastasis.

The widespread occurrence of cancer tumors underscores the need for improved anticancer regimens. However, despite advances in multimodal therapy, increases in overall survival in cancer patients have been limited. Accordingly, it is an object of the present invention to provide improved methods for treating subjects with such tumors.

SUMMARY OF THE INVENTION

The present inventors have discovered for the first time that co-administration of a TIM-4 antagonist (e.g., an antibody) and an anti-PD- 1 antagonist (e.g., an antibody) effectively inhibits tumor growth in vivo, even synergistically. Accordingly, it is an object of the present invention to provide improved methods for treating subjects with cancer.

Specifically, it is an object of the invention to provide efficacious combination treatment regimens wherein a TIM-4 antagonist is combined with an anti-PD- 1 antagonist for the treatment of cancer.

In one aspect, the present invention provides a method for the treatment of cancer in a subject by co-administering an effective amount of a PD-1 antagonist and a TIM-4 antagonist. Suitable TIM-4 antagonists for use in the methods of the invention, include, without limitation, ligands, antibodies (e.g., monoclonal antibodies and bispecific antibodies) and multivalent agents. The TIM-4 antagonist can be a non-activating ligand or ligand binding partner (e.g., a small molecule, engineered PS emulator or soluble TIM-1, or other binding protein). In one embodiment, the TIM-4 antagonist is an anti-TIM4 antibody. In one embodiment, the TIM-4 antagonist is an antibody which binds to an epitope within the IgV domain of TIM-4. In another embodiment, the TIM-4 antagonist is an antibody such as 9F4, RMT4-53, RMT4-54, F31-563 or 21T112.

An exemplary anti-TIM-4 antibody contains heavy and light chains comprising the heavy and light chain CDRs of 9F4, RMT4-53, RMT4-54, F31-563 or 21T112, and optionally comprises a framework region with at least about 90% amino acid sequence identity with the framework region of the corresponding antibody. Anti-TIM-4 antibodies may also comprise heavy and light chain variable domains that are at least about 90% , 95% or 99% identical with that of antibody 9F4, RMT4-53, RMT4-54, F31-563 or 21T112. In certain embodiments, the anti-TIM-4 antagonist antibody competes for binding with, and/or binds to the same epitope on TIM-4 as, 9F4, RMT4-53, RMT4-54, F31-563 or 21T112.

Suitable PD-1 antagonists for use in the methods of the invention, include, without limitation, ligands, antibodies (e.g., monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-Ll antibody. In another embodiment, the PD-1 antagonist is an antibody, such as MK-3475 or CT-011. In one embodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusion protein, such as AMP-244.

An exemplary anti-PD-1 antibody is 5C4 (referred to as 5C4 in WO 2006/121168; also known as MDX-1106, ONO-4538, and nivolumab) comprising heavy and light chains having the sequences shown in SEQ ID NOs: 11 and 12, respectively, or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions (VRs) of 5C4. Accordingly, in one embodiment, the antibody comprises the CDRl, CDR2, and CDR3 domains of the VH region of 5C4 having the sequence shown in SEQ ID NO: 13, and the CDRl, CDR2 and CDR3 domains of the VL region of 5C4 having the sequence shown in SEQ ID NO: 15. In another embodiment, the antibody comprises the heavy chain CDRl, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively, and the light chain CDRl, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively. In another embodiment, the antibody comprises VH and/or VL regions having the amino acid sequences set forth in SEQ ID NO: 13 and/or SEQ ID NO: 15, respectively. In another embodiment, the antibody comprises the heavy chain variable (VH) and/or light chain variable (VL) regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 14 and/or SEQ ID NO: 16, respectively. In another embodiment, the antibody competes for binding with, and/or binds to the same epitope on PD-1 as, the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90% , 95% or 99% variable region identity with SEQ ID NO: 13 or SEQ ID NO: 15). In one embodiment, the PD-1 antagonist is an anti-PD-Ll antibody, such as

MEDI4736 (also known as Anti-B7-Hl) or MPDL3280A (also known as RG7446). An exemplary anti-PD-Ll antibody is 12A4 (referred to as 12A4 in WO 2007/005874 and US Patent No. 7,943,743). In one embodiment, the antibody comprises the heavy and light chain CDRs or VRs of 12A4. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of 12A4 having the sequence shown in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains of the VL region of 12A4 having the sequence shown in SEQ ID NO: 3. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively. In another embodiment, the antibody comprises VH and/or VL regions having the amino acid sequences set forth in SEQ ID NO: 1 and/or SEQ ID NO: 3, respectively. In another embodiment, the antibody comprises the heavy chain variable (VH) and/or light chain variable (VL) regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 2 and/or SEQ ID NO: 4, respectively. In another embodiment, the antibody competes for binding with, and/or binds to the same epitope on PD-L1 as, the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above- mentioned antibodies (e.g., at least about 90% , 95% or 99% variable region identity with SEQ ID NO: 1 or SEQ ID NO: 3). In one embodiment, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a PD-1 antagonist and a TIM-4 antagonist, wherein (a) the PD-1 antagonist is an anti-PD-1 antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 15; and

(b) the TIM-4 antagonist is an antibody.

In another embodiment, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a PD-1 antagonist and a TIM-4 antagonist, wherein

(a) the PD-1 antagonist is an anti-PD-Ll antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 3; and

(b) the TIM-4 antagonist is an antibody.

The efficacy of the treatment methods provided herein can be assessed using any suitable means. In one embodiment, the treatment produces at least one therapeutic effect selected from the group consisting of reduction in size of a tumor, reduction in number of metastasic lesions over time, complete response, partial response, and stable disease. In another embodiment, administration of a PD-1 antagonist and a TIM-4 antagonist results in at least a 1, 1.25, 1.50, 1.75, 2, 2.25, 2.50, 2.75, 3, 3.25, 3.5, 3.75, or 4-fold reduction in tumor volume, e.g., relative to treatment with the PD-1 antagonist or TIM-4 antagonist alone, or relative to tumor volume before initiation of treatment. In another embodiment, administration of the PD-1 antagonist and TIM-4 antagonist results in at least a 1-fold, 2-fold, or more preferably a 3 -fold reduction in tumor volume, e.g., relative to treatment with the PD-1 antagonist or TIM-4 antagonist alone, or relative to tumor volume before initiation of treatment. In a further embodiment, administration of a PD-1 antagonist and a TIM-4 antagonist results in tumor growth inhibition of at least 50%, 60%, 70% or 80%, e.g., relative to treatment with the PD-1 antagonist or TIM-4 antagonist alone, or relative to tumor volume before initiation of treatment. In certain embodiments, tumor volume is reduced by 50%, 60%, 70%, 80%, 90% or more, e.g., relative to tumor size before initiation of the treatment.

The PD-1 antagonist and TIM-4 antagonist can be administered accordingly to a suitable dosage, route (e.g., intravenous, intraperitoneal, intramuscular, intrathecal or subcutaneous). The antagonist and agonist can also be administered according to any suitable schedule. For example, the antagonist and agonist can be simultaneously administered in a single formulation. Alternatively, the antagonist and agonist can be formulated for separate administration, wherein they are administered concurrently or sequentially. In one embodiment, the PD- 1 antagonist is administered prior to administration of the TIM-4 antagonist. In another embodiment, the TIM-4 antagonist is administered prior to administration of the PD-1 antagonist. In a further embodiment, the TIM-4 antagonist and the PD-1 antagonist are administered simultaneously.

In one embodiment, the cancer is a cancer selected from the group consisting of carcinoma, sarcoma, blastoma, lymphoma and leukemia. In one embodiment, the cancer is selected from the group consisting of small-cell lung cancer, non- small cell lung cancer, gastrointestinal cancer, colorectal cancer, stomach cancer, colon carcinoma, and

glioblastoma. In another embodiment, the cancer is selected from the group consisting of chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CML). Additional agents and therapies can be administered in combination with the agonists and antagonists described herein. In one embodiment, the methods comprise administration of an additional therapeutic agent (e.g., a cyotoxin or chemotherapeutic agent.

Also provided herein are compositions comprising a PD-1 antagonist and a TIM-4 antagonist. In one embodiment, the antagonist is a ligand, antibody (e.g., monoclonal antibody or bispecific antibody) or multivalent agent. In another embodiment, the PD-1 antagonist is an anti-PD- 1 antibody comprising the heavy and light chain CDRs or VRs of 5C4. In another embodiment, the PD-1 antagonist is an anti-PD-Ll antibody comprising the heavy and light chain CDRs or VRs of 12A4.

Further provided are kits for treating a cancer in a subject, the kit comprising:

(a) a dose of a PD-1 antagonist;

(b) a dose of a TIM-4 antagonist; and (c) instructions for using the PD-1 antagonist and TIM-4 antagonist in the methods described herein. In one embodiment, the TIM-4 antagonist is an antibody. In another embodiment, the PD-1 antagonist is an antibody. In a particular embodiment, the PD-1 antagonist is an anti-PD- 1 antibody comprising the CDRl, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 13, and the CDRl, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 15. In another particular embodiment, the PD-1 antagonist is an anti-PD-Ll antibody comprising antibody comprises the CDRl, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 1, and the CDRl, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 3.

Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts an alignment of the amino acid sequences of human TIM-4 isoforms 1 and 2.

Figure 2 depicts the alignment of the amino acid sequences of cynomologous monkey, mouse and human TIM-4 orthologs. Figure 3A-E are graphs depicting the myeloid cell populations in spleen and tumors from mice after administration of a control, a TIM-4 antagonist antibody, a PD-1 antagonist antibody, or a combination of an anti-PD- 1 antibody and an anti-TIM-4 antibody.

Figure 4A-F are graphs depicting the CD8+ cell populations in spleen and tumors from mice after administration of a control, an TIM-4 antagonist antibody, an anti-PD- 1 antibody, or a combination of an anti-PD- 1 antibody and an anti-TIM-4 antibody.

Figure 5A-F are graphs depicting the CD4+ populations in spleen and tumors from mice after administration of a control, an TIM-4 antagonist antibody, an anti-PD- 1 antibody, or a combination of an anti-PD- 1 antibody and an anti-TIM-4 antibody.

Figure 6A-D are graphs depicting tumor volume (mm ³ ) in individual mice after administration of a control, an TIM-4 antagonist antibody, an anti-PD- 1 antibody, or a combination of an anti-PD- 1 antibody and an anti-TIM-4 antibody. Figure 7A-B are graphs depicting the mean and median tumor volume (mm ³ ) in mice after administration of a control, an TIM-4 antagonist antibody, an anti-PD- 1 antibody, or a combination of an anti-PD-1 antibody and an anti-TIM-4 antibody.

Figure 8 is a graph depicting the tumor volumes at day 28 in mice (mm ³ ) in mice after administration of a control, an TIM-4 antagonist antibody, an anti-PD- 1 antibody, or a combination of an anti-PD-1 antibody and an anti-TIM-4 antibody.

Figure 9 (A) is a graph depicting the survival rates of mice after administration of a control, an TIM-4 antagonist antibody, an anti-PD- 1 antibody, or a combination of an anti- PD-1 antibody and an anti-TIM-4 antibody in the CT26 animal model; and (B) is a table indicating the median survival rate of each treatment group.

Figure 10 is a graph depicting the percentage progression free survival rates of mice after administration of a control, an TIM-4 antagonist antibody, an anti-PD- 1 antibody, or a combination of an anti-PD-1 antibody and an anti-TIM-4 antibody in the MC38 animal model. Figure 11 shows the mean tumor volume relative to the days post implant in the mice that were in the experiment shown in Figure 10.

Figure 12A-C shows the individual tumor volumes relative to the days post implant in the mice that were in the experiment shown in Figures 10 and 11.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, the invention is based on the discovery that co-administration of a TIM-4 antagonist (e.g., an antibody) and a PD- 1 antagonist (e.g., an antibody) effectively inhibits tumor growth in vivo, even synergistically. Accordingly, the present invention provides a method for the treatment of cancer in a subject which comprises administering to a subject (e.g., human) an effective amount of a PD- 1 antagonist and a TIM-4 antagonist.

I. Definitions

In order that the present description may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, and conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The use of "or" or "and" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as

"include", "includes", and "included", is not limiting.

The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + 20% or + 10%, more preferably + 5%, even more preferably + 1%, and still more preferably + 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, etc., used herein are to be understood as being modified by the term "about".

As used herein, the term "subject" or "patient" are used interchangeably herein and refer to a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig and the like (e.g., a patient having cancer).

A "solid tumor" includes, for example, sarcoma, melanoma, carcinoma, prostate carcinoma, lung carcinoma, colon carcinoma, or other solid tumor cancer.

The terms "cancer", "cancerous", or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, the term includes pre-malignant as well as malignant cancers. Examples of cancer include, for example, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particular examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CML). The term "carcinoma" as used herein refers to a type of cancer that develops from epithelial cells. Specifically, a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that generally arises from cells originating in the endodermal or ectodermal germ layer during embryogenesis. Various subtypes of

carcinomas include, adenocarcinoma (featuring microscopic glandular-related tissue cytology, tissue architecture, and/or gland-related molecular products, e.g., mucin), squamous cell carcinoma (observable features and characteristics indicative of squamous differentiation, e.g., intercellular bridges, keratinization, squamous pearls), adenosquamous carcinoma (a mixed tumor containing both adenocarcinoma and squamous cell carcinoma, wherein each of these cell types comprise at least 10% of the tumor volume), anaplastic or undifferentiated carcinoma (a heterogeneous group of high-grade carcinomas that feature cells lacking distinct histological or cytological evidence of any of the more specifically differentiated

neoplasnms), large cell carcinoma (composed of large, monotonous rounded or overtly polygonal- shaped cells with abundant cytoplasm), small cell carcinoma (cells are usually round and are less than approximately 3 times the diameter of a resting lymphocyte and little evident cytoplasm).

As used herein, the term "immune cell" refers to cells that play a role in the immune response, including lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. The term "immune response" as used herein refers to a cellular immune response, including T cell mediated and/or B cell mediated immune responses such as stimulation of T lymphocytes, macrophages, and/or natural killer cells.

The terms "myeloid-derived suppressor cell" or "MDSC" are used interchangeably herein to refer to a heterogenous group of immune cells from the myeloid lineage (a family of cells that originate from bone marrow stem cells), to which dendritic cells, macrophages and neutrophils also belong. MDSCs strongly expand in pathological situations such as chronic infections and cancer, as a result of an altered hematopoiesis. MDSCs are further classified into two subtypes, monocytic MDSCs and granulocytic MDSCs. MDSC suppressor function lies in their ability to inhibit T cell proliferation and activation. For example, under chronic inflammatory conditions (viral and bacterial infections) or cancer, myeloid differentiation is skewed towards the expansion of MDSCs. These MDSCs infiltrate inflammation sites and tumors, where they inhibit immune responses by inhibiting T cells (e.g., CD8+ T cells) and NK cells, for example. MDSCs also accelerate angiogenesis, tumor progression and metastasis through the expression of cytokines and factors such as TGF-beta.

The terms "damage-associated molecular patterns" or "DAMPs" are used

interchangeably herein to refer to intracellular molecules released by injured tissues. DAMPs are molecules that have a physiological role inside the cell, but acquire additional functions when exposed to the extracellular environment. For example, DAMPs alert the body about danger, stimulate an inflammatory response, and finally promote the regeneration process. Beside their passive release by dead cells, some DAMPs can be secreted or exposed by living cells undergoing a life-threatening stress. DAMPs have been linked to inflammation and related disorders. DAMPs include, but are not limited to, histones, genomic DNA, HMGB 1, ILla, IL33, ATP, F-actin, cyclophilin A, HSPs, uric acid crystals, S lOOs, mitochondrial DNA, mitochondrial transcription factor A, alreticulin.

The term "efferocytosis" refers to the process by which dying/dead cells (e.g. , apoptotic or necrotic) are removed by phagocytic cells (e.g., neurtrophils, monocytes, macrophages, mast cells and dendritic cells). During efferocytosis, phagocytes accumulate to the sites of apoptotic cells and the cell membrane of phagocytic cells engulfs the apoptotic cell, forming a large fluid-filled vesicle, the "effersome" containing the dead cell. The effect of efferocytosis is that dead cells are removed before their membrane integrity is breached and their contents leak into the surrounding tissue. Efferocytosis triggers specific downstream intracellular signal transduction pathways, for example resulting in anti-inflammatory, anti- protease and growth-promoting effects. Conversely, impaired efferocytosis has been linked to autoimmune disease and tissue damage.

"Autophagy" or "autophagocytosis" is the natural, destructive mechanism that disassembles, through a regulated process, unnecessary or dysfunctional cellular components. Autophagy allows the orderly degradation and recycling of cellular components. During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome. The autophagosome then fuses with a lysosome and the contents are degraded and recycled. There are three different forms of autophagy that are commonly described, namely macroautophagy, microautophagy and chaperone-mediated autophagy. In the context of disease, autophagy has been seen as an adaptive response to stress which promotes survival, whereas in other cases it appears to promote cell death and morbidity. The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent or combination of active agents to the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.

As used herein, "effective treatment" or "positive therapeutic response" refer to a treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder, e.g., cancer. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method. For example, a beneficial effect can take the form of slowing, stabilizing, stopping or reversing the progression of a cancer in a subject at any clinical stage, as evidenced by a decrease or elimination of a clinical or diagnostic symptom of the disease, or of a marker of cancer. Effective treatment may, for example, decrease in tumor size, decrease the presence of circulating tumor cells, reduce or prevent metastases of a tumor, slow or arrest tumor growth and/or prevent or delay tumor recurrence or relapse.

The term "effective amount" or "therapeutically effective amount" refer to an amount of an agent or combination of agents that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to solid tumors, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor development. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and may stop tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. In one example, an "effective amount" is the amount of a PD-1 antagonist (e.g., an antibody) and TEVI-4 antagonist antibody (e.g., an antibody), in combination, to effect a significant decrease in cancer or slowing of progression of cancer, such as an advanced solid tumor. An effective amount of the combination therapy is administered according to the methods described herein in an "effective regimen" which refers to a combination of the PD-1 antagonist and the TEVI-4 antagonist, wherein the order of administration and dosage frequency is adequate to effect treatment.

"Optimal biologic dose (OBD)" is defined as the minimum dose of an agent or combination of agents that gives the most optimal and lasting in vivo response without clinically unacceptable toxicity. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. As used herein, the terms "synergy", "therapeutic synergy", and "synergistic effect" refer to a phenomenon where treatment of patients with a combination of therapeutic agents (e.g., PD-1 antagonist in combination with TIM-4 antagonist) manifests a therapeutically superior outcome to the outcome achieved by each individual constituent of the combination used when used alone (see, e.g., T. H. Corbett et al., 1982, Cancer Treatment Reports, 66, 1187). In this context a therapeutically superior outcome includes one or more of the following (a) an increase in therapeutic response that is greater than the sum of the separate effects of each agent alone at the same dose as in the combination; (b) a decrease in the dose of one or more agents in the combination without a decrease in therapeutic efficacy; (c) a decrease in the incidence of adverse events while receiving a therapeutic benefit that is equal to or greater than the monotherapy of each agent at the same dose as in the combination, (d) a reduction in dose-limiting toxicities while receiving a therapeutic benefit that is greater than the monotherapy of each agent; (e) a delay or minimization of the induction of drug resistance. In xenograft models, a combination, used at its maximum tolerated dose, in which each of the constituents will be present at a dose generally not exceeding its individual maximum tolerated dose, manifests therapeutic synergy when decrease in tumor growth achieved by administration of the combination is greater than the value of the decrease in tumor growth of the best constituent when the constituent is administered alone. Synergism of a drug combination may be determined, for example, according to the combination index (CI) theorem of Chou-Talalay (Chou et al, Adv. Enzyme Regul. 1984; 22:27-55; Chou, Cancer Res. 2010;70(2):440-446).

"Free of cancer" or "disease free" or NED (No Evidence of Disease) means that the patient has demonstrated a clinical response induced by treatment with the current standard of care therapies. By "clinical response," it is meant that the clinical signs, radiological signs, and symptoms of cancer have been significantly diminished or have disappeared entirely based on clinical diagnostics, although cancerous cells can still exist in the body. Thus, it is contemplated that clinical response encompasses partial and complete response. The presence of residual cancer cells can be enumerated by assays such as CTC (Circulating Tumor Cells) and can be predictive of recurrence.

"Relapse" or "recurrence" or "resurgence" are used interchangeably herein, and refer to the radiographic diagnosis of return, or signs and symptoms of return of cancer after a period of improvement or response.

As used herein, the term "antagonist" refers to a molecule which blocks (e.g., reduces or prevents) a biological activity. The term "inhibit" or "inhibition" means to reduce by a measurable amount.

As used herein, the term "ligand" refers to a molecule that forms a complex with a biomolecule (e.g., a receptor) to serve a biological purpose. In a narrower sense, is a signal triggering molecule, binding to a site on a target protein. The binding occurs by

intermolecular forces, such as ionic bonds, hydrogen bonds and van der Waals forces. The docking (association) is usually reversible (dissociation). Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems. Ligand binding to a receptor (receptor protein) alters its chemical conformation (three dimensional shape). The conformational state of a receptor protein determines its functional state. The "level" of a protein refers to the amount of protein in a sample as determined using any method known in the art for measuring protein levels, including electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotmetric assays, flow cytometry, immmunodiffusion, solution phase assay, Immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),

immunofluorescent assays and electrochemiluminescence immunoassays. The term "sample" refers to a collection of fluids, cells or tissues isolated from a subject. Biological fluids are typically liquids at physiological temperatures and may include naturally occurring fluids present in, withdrawn from, expressed or otherwise extracted from a subject or biological source. Examples of biological fluids include blood, serum, serosal fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, cystic fluid, tear drops, feces, sputum, mucosal secretions, vaginal secretions, gynecological fluids, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like.

The term "control sample", as used herein, refers to any clinically relevant control sample, including, for example, a sample from a healthy subject or a sample made at an earlier time point from the subject to be assessed. For example, the control sample can be a sample taken from the subject prior to onset of cancer, at an earlier stage of disease, or before the administration of treatment or of a portion of treatment.

As used herein, the term "antibody" includes whole antibodies and any antigen binding fragment (i.e. , "antigen-binding fragments" (also known as "antigen-binding portions")). Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" also encompasses chimeric antibodies, humanized antibodies, fully human antibodies, as well as multimeric forms of antibodies, such as minibodies, bis-scFv, diabodies, triabodies, tetrabodies and chemically conjugated Fab' multimers. The term "antibody fragment" (also referred to as "antigen-binding fragment" or "antigen-binding portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen- binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment is essentially a Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the VH and CHI domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et ah , (1989) Nature 341 :544-546), which consists of a VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody (also known as a single-domain antibody (sdAb)), which is a heavy chain variable region containing a single variable domain and two constant domains. Single domain antibodies include VHH fragments (single-domain antibodies engineered from heavy-chain antibodies found in camelids, as well as VNAR fragments (single-domain antibodies obtained from heavy-chain antibodies (IgNAR,

'immunoglobulin new antigen receptor') of cartilaginous fishes).

"Antigen binding scaffolds" are proteins that bind specifically to a target (or antigen) or epitope, such as proteins comprising an Ig fold or an Ig-like fold. Antibodies or antigen binding fragments thereof are also antigen binding scaffolds. Antigen binding scaffolds can be monovalent, multivalent, e.g., bivalent, trivalent, tetravalent, or bind 5, 6 or more epitopes. Multivalent antigen binding scaffolds can be monospecific or multispecific, i.e., binding to multiple (at least 2, 3, 4 or 5) epitopes that are different from one another. For example, a multivalent monospecific antigen binding scaffold is a protein that binds to at least 2, 3, 4 or 5 identical epitopes, and may be a protein comprising at least 2, 3, 4 or 5 identical antigen binding portions. For example, TIM-4 binding scaffolds may comprise 2- 10, e.g., 2-6, 2-5, 2- 4 or 2-3 TIM-4 binding portions, which may be the same or different from one another.

A multivalent antibody includes antibodies comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigen binding portions of antibodies, which antigen binding portions may comprise a portion of a heavy chain and a portion of a light chain. An antigen binding portion may be on a single polypeptide or comprise more than one polypeptide. For example, a multivalent antibody may comprise from 2- 10 antigen binding portions, which may be the same or different from each other. A multivalent antibody may be monospecific or multispecific. A multispecific antibody may be bispecific, trispecific, tetraspecific or bind to 5 or more different epitopes.

Furthermore, although the two domains of the Fv fragment, V _L and V¾ are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V _L and V _H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen- binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

As used herein, an antigen binding scaffold that "specifically binds" to an antigen or epitope thereof is an antigen binding scaffold that binds to the antigen or epitope thereof with a K _D of 10 ^"7 M, 5 x 10 ^"8 M, 10 ^"8 M, 5 x 10 ^"9 M, 10 ^"9 M, 5 x 10 ⁴⁰ M, 10 ⁴⁰ M, 5 x 10 ⁴¹ M, 10 ^{" 11} M, 5 x 10 ⁴² M, 10 ⁴² M or less. For example, an antigen binding scaffold that specifically binds to TIM-4 is an antigen binding scaffold that binds to TEVI-4 with a K _D of 10 ^"7 M, 5 x 10 ^"8 M, 10 ^"8 M, 5 x 10 ^"9 M, 10 ^"9 M, 5 x 10 ⁴⁰ M, 10 ⁴⁰ M, 5 x 10 ⁴¹ M, 10 ⁴¹ M, 5 x 10 ⁴² M, 10 ⁴² M or less. For example, an antibody that "specifically binds to human PD-1" or "specifically binds to human PD-L1" is intended to refer to an antibody that binds to human PD-1 or PD-L1, respectively, with a K _D of 10 ^"7 M, 5 x 10 ^"8 M, 10 ^"8 M, 5 x 10 ^"9 M, 10 ^"9 M, 5 x 10 ⁴⁰ M, 10 ⁴⁰ M, 5 x 10 ⁴¹ M, 10 ⁴¹ M, 5 x 10 ⁴² M, 10 ⁴² M or less. An antigen binding scaffold that comprises 2 or more regions binding to an antigen or epitope may bind specifically to the antigen or epitope even it has a lower affinity of binding to the antigen or epitope than the ranges provided above, as it will bind to the antigen or epitope with increased avidity.

A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al, J. Immunol. 148, 1547-1553 (1992).

The term "monoclonal antibody" as used herein, refers to refers to an antibody from a population of substantially homogeneous antibodies that display a single binding specificity and affinity for a particular epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Accordingly, the term "human monoclonal antibody" or "monoclonal antibody composition" refers to an antibody which has variable and optional constant regions derived from human germline immunoglobulin sequences. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies described herein may be made by a variety of techniques. In one embodiment, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography, HDX-MS and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term "epitope mapping" refers to the process of identification of the molecular determinants for antibody- antigen recognition.

The term "binds to the same epitope," with reference to two or more antibodies, means that the antibodies compete for binding to an antigen and bind to the same, overlapping, or encompassing continuous or discontinuous segments of amino acids. Those of skill in the art understand that the phrase "binds to the same epitope" does not necessarily mean that the antibodies bind to exactly the same amino acids. The precise amino acids to which the antibodies bind can differ. For example, a first antibody can bind to a segment of amino acids that is completely encompassed by the segment of amino acids bound by a second antibody. In another example, a first antibody binds one or more segments of amino acids that significantly overlap the one or more segments bound by the second antibody. For the purposes herein, such antibodies are considered to "bind to the same epitope." Two antibodies "bind to the same epitope" as determined by a given method, e.g., HDX-MS, crystallography or target mutational analysis, requires that the same amino acids are identified for both antibodies by one or more given methods.

Accordingly, also, encompassed by the present invention are antibodies that bind to an epitope which comprises all or a portion of an epitope recognized by the particular antibodies described herein (e.g., the same or an overlapping region or a region between or spanning the region) or that bind to the same epitope as determined by a given method, e.g., HDX-MS, crystallography or target mutational analysis.

Also encompassed by the present invention are antibodies that compete for binding with the antibodies described herein, and optionally bind the same epitope. Antibodies that compete for binding can be identified using routine techniques. Such techniques include, for example, an immunoassay, which shows the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen. Numerous types of competitive binding assays are known, for example: Biacore, flow cytometry, solid phase direct or indirect

radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et ah, Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et ah, J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et ah, Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et ah, Virology 176:546 (1990)); and direct labeled RIA.

(Moldenhauer et ah, Scand. J. Immunol. 32:77 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70- 75% or more. Epitope mapping methods also include x-ray analyses of crystals of antigen: antibody complexes which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display or yeast display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed which have been shown to map conformational discontinuous epitopes.

Chimeric molecules (or fusion molecules) comprising an antigen binding domain, or equivalent, fused to another polypeptide or molecule are also encompassed by the present invention. For example, the polypeptides may be fused or conjugated to an antibody Fc region, or portion thereof (e.g., an Fc fusion protein). The antibody portion fused to a polypeptide may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5, 112,946; EP 307,434; EP 367,166; PCT Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535- 10539 (1991); Zheng et al., J. Immunol.,

154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA, 89: 11337- 11341 (1992).

As used herein, the term "immunoconjugate" refers to an antibody linked to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g. , kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g. , methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. , mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,

streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g. , daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. , dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g. , vincristine and vinblastine). Antibodies use in the present invention can be conjugated to a radioisotope, e.g. , radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating cancer.

Immunoconjugates can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL- l "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g. , Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in

Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303- 16 (Academic Press 1985), and Thorpe et al.,

"The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982). As used herein, the term "multivalent" refers to a recombinant molecule that incorporates more than two biologically active segments. The protein fragments forming the multivalent molecule optionally may be linked through a polypeptide linker which attaches the constituent parts and permits each to function independently. As used herein, "functional equivalents" refer to peptides or polypeptides (e.g., antibodies or antigen binding portion thereof) which maintain a substantial amount of their original immunological activity. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. It is thus contemplated that various changes may be made in the amino acid sequences of the disclosed compositions without appreciable loss of their biological utility or activity. Amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art, and such immunologically functional equivalents are also encompassed within the present invention.

"Percent (%) amino acid sequence identity" herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by a sequence alignment program, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR), in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. II. PD-1 Antagonists

As used herein, the terms "Protein PD-1," "PD-1," PD1," "PDCD1" are used interchangeably with "Programmed Death 1," "Programmed Cell Death 1." The complete human PD-1 sequence can be found under GenBank Accession No. U64863 (SEQ ID NO:23). As used herein, the terms "PD-L1", " PDL1", "PDCD1L1", "PDCD1LG1",

"CD274", "B7 homolog 1", "B7-H1", "B7-H", and "B7H1" are used interchangeably with "Programmed Cell Death 1 Ligand 1." The complete human PD-L1 amino acid sequence - isoform a precursor - can be found under GenBank Accession No. NP_054862.1 (SEQ ID NO:24). The complete human PD-L1 amino acid sequence -isoform b precursor - can be found under GenBank Accession No. NP_001254635.1 (SEQ ID NO:25).

The protein Programmed Death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, that also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8). The initial members of the family, CD28 and ICOS, were discovered by functional effects on augmenting T cell proliferation following the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260). PD-1 was discovered through screening for differential expression in apototic cells (Ishida et al. (1992) EMBO J U_:3887-95). The other members of the family, CTLA-4 and BTLA, were discovered through screening for differential expression in cytotoxic T lymphocytes and THl cells, respectively. CD28, ICOS and CTLA-4 all have an unpaired cysteine residue allowing for homodimerization. In contrast, PD-1 is suggested to exist as a monomer, lacking the unpaired cysteine residue characteristic in other CD28 family members.

The PD-1 protein is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72). PD-1 contains a membrane proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane distal tyrosine - based switch motif (ITS M) (Thomas, M.L. (1995) J Exp Med 181: 1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91). Although structurally similar to CTLA-4, PD-1 lacks the MYPPPY motif (SEQ ID NO: 27) that is critical for B7-1 and B7-2 binding.

Consistent with PD- 1 being an inhibitory member of the CD28 family, PD- 1 deficient animals develop various autoimmune phenotypes, including autoimmune cardiomyopathy and a lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity

11: 141-51; Nishimura et al. (2001) Science 291:319-22). Additionally, PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus- host disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78; Prokunina and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004) Lupus 13:510). In a murine B cell tumor line, the ITSM of PD-1 was shown to be essential to block BCR-mediated Ca ²⁺ -flux and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98: 13866-71).

Two ligands for PD-1 have been identified, PD-Ll and PD-L2, that have been shown to downregulate T cell activation upon binding to PD- 1 (Freeman et al. (2000) J Exp Med 192: 1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J

Immunol 32:634-43). Both PD-Ll and PD-L2 are B7 homologs that bind to PD-1, but do not bind to other CD28 family members. PD-Ll is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-Ll results in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-Ll, and the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et al. (2002) Proc. Nat' I. Acad. Sci. USA

99: 12293-7; Brown et al. (2003) J. Immunol. 170: 1257-66).

The methods of the present invention involve the use of a PD-1 antagonist {e.g., an antibody) in combination with a TIM-4 antagonist {e.g., an antibody), for treating cancer. Accordingly, PD-1 antagonists of the invention bind to ligands of PD-1 and interfere with, reduce, or inhibit the binding of one or more ligands to the PD- 1 receptor, or bind directly to the PD-1 receptor, without engaging in signal transduction through the PD-1 receptor. In one embodiment, the PD-1 antagonist binds directly to PD-1 and blocks PD-1 inhibitory signal transduction. In another embodiment the PD-1 antagonist binds to one or more ligands of PD-1 (e.g., PD-Ll and PD-L2) and reduces or inhibits the ligand(s) from triggering inhibitory signal transduction through the PD- 1. In one embodiment, the PD- 1 antagonist binds directly to PD-L1, inhibiting or preventing PD-L1 from binding to PD-1, thereby blocking PD-1 inhibitory signal transduction.

PD-1 antagonists used in the methods and compositions of the present invention include PD-1 binding scaffold proteins and include, but are not limited to, PD-1 ligands, antibodies and multivalent agents. In a particular embodiment, the antagonist is a fusion protein, such as AMP-224. In another embodiment, the antagonist is an anti-PD-1 antibody ("PD-1 antibody"). Anti-human-PD-1 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the invention can be generated using methods well known in the art. Alternatively, art recognized anti-PD-1 antibodies can be used. For example, antibodies nivolumab (OPDIVO™), MK-3475 (pembrolizumab (KEYTRUDA™), PDR001 or CT-011 can be used. Additionally, monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168, the teachings of which are hereby incorporated by reference, can be used. Antibodies that compete with any of these art-recognized antibodies for binding to PD-1 also can be used.

An exemplary anti-PD- 1 antibody is 5C4 comprising heavy and light chains having the sequences shown in SEQ ID NOs: 11 and 12, respectively, or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of 5C4. Accordingly, in one embodiment, the antibody comprises the CDRl, CDR2, and CDR3 domains of the VH of 5C4 having the sequence set forth in SEQ ID NO: 13, and the CDRl, CDR2 and CDR3 domains of the VL of 5C4 having the sequences set forth in SEQ ID NO: 15. In another embodiment, the antibody comprises CDRl, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively, and CDRl, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively. In another embodiment, the antibody comprises VH and/or VL regions having the amino acid sequences set forth in SEQ ID NO: 13 and/or SEQ ID NO: 15, respectively. In another embodiment, the antibody comprises the heavy chain variable (VH) and/or light chain variable (VL) regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 14 and/or SEQ ID NO: 16, respectively. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD- 1 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95% or 99% variable region identity with SEQ ID NO: 13 or SEQ ID NO: 15).

In certain embodiments, the PD1 antibodies exhibit one or more desirable functional properties, such as high affinity binding to PD-1, e.g., binding to human PD-1 with a K _D of 10 ^"7 M or less; lack of significant cross-reactivity to other CD28 family members, e.g., CD28, CTLA-4 and ICOS; the ability to stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) assay; the ability to increase IFN-γ and/or IL-2 secretion in an MLR; the ability to inhibit binding of one or more PD-1 ligands (e.g., PD-L1 and/or PD-L2) to PD-1; the ability to stimulate antigen- specific memory responses; the ability to stimulate antibody responses and/or the ability to inhibit growth of tumor cells in vivo.

In another embodiment, the PD-1 antagonist is an anti-PD-Ll antibody. Anti-human- PD-L1 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the invention can be generated using methods well known in the art. Alternatively, art recognized anti-PD-Ll antibodies can be used. For example, MEDI4736 (also known as Anti-B7-Hl ; durvalumab (IMFINZI™)), MPDL3280A (atezolizumab (TECENTRIQ™) also known as RG7446), and avelumab (BAVENCIO™) and can be used. Additionally, monoclonal antibodies 12A4, 3G10, 10A5, 5F8, 10H10, IB 12, 7H1, 11E6, 12B7, and 13G4 described in WO 2007/005874 and US Patent No. 7,943,743, the teachings of which are hereby incorporated by reference, can be used. Antibodies that compete with any of these art-recognized antibodies for binding to PD-L1 also can be used.

An exemplary anti-PD-Ll antibody is 12A4 (WO 2007/005874 and US Patent No. 7,943,743). In one embodiment, the antibody comprises the heavy and light chain CDRs or VRs of 12A4. Accordingly, in one embodiment, the antibody comprises the CDRl, CDR2, and CDR3 domains of the VH region of 12A4 having the sequence shown in SEQ ID NO: 1 and the CDRl, CDR2 and CDR3 domains of the VL region of 12A4 having the sequence shown in SEQ ID NO: 3. In another embodiment, the antibody comprises the heavy chain CDRl, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the light chain CDRl, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively. In another embodiment, the antibody comprises VH and/or VL regions having the amino acid sequences set forth in SEQ ID NO: 1 and/or SEQ ID NO: 3, respectively. In another embodiment, the antibody comprises the heavy chain variable (VH) and/or light chain variable (VL) regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 2 and/or SEQ ID NO: 4, respectively. In another embodiment, the antibody competes for binding with, and/or binds to the same epitope on PD-Ll as, the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90% , 95% or 99% variable region identity with SEQ ID NO: 1 or SEQ ID NO: 3).

Anti-PD-1 or anti-PD-Ll antibodies may bind to PD-1 or PD-Ll, respectively, with a K _D of 10 ^"7 M, 5 x 10 ^"8 M, 10 ^"8 M, 5 x 10 ^"9 M, 10 ^"9 M, 5 x 10 ^"10 M, 10 ^"10 M or less.

In certain embodiments, an anti-PD-1 or anti-PD-Ll antibody is an IgG antibody, such as an IgGl, IgG2 or IgG4 antibody. In specific embodiments, an anti-PD-1 or PD-11 antibody has an effectorless constant region. Anti-PD-1 or PD-Ll antibodies may be IgG4 antibodies, e.g., IgG4 antibodies having an S228P mutation.

III. TIM-4 antagonists

As used herein, the terms "TIM-4" and "TIMD-4", also known as "T-cell

immunoglobulin and mucin domain-containing protein 4", "T-cell immunoglobulin mucin receptor 4," "T-cell membrane protein 4" and "SMUCKLER" are used interchangeably. The complete amino acid and nucleotide sequences of isoform- 1 of human TIM-4 can be found under GenBank Accession Nos. NP_612388.2 and NM_138379.2, respectively (SEQ ID NOs: 26-27). The complete amino acid and nucleotide sequences of isoform-2 of human TEVI-4 can be found under GenBank Accession Nos. NP_001140198.1 and

NM_001146726.1, respectively (SEQ ID NOs: 28-29) (Jones et al, Int Arch Allergy

Immunol. 2006; 141(4):331-6). An alignment of the two human isoforms is depicted in

Figure 1.

The amino acid sequences of TIM-4 isoforms from cynomologus monkey can be found under GenBank Accession Nos. XP_005558436 and EHH54702, and the amino acid sequences for murine TIM-4 isoforms can be found under GenBank Accession Nos.

NP_848874 and NP_599009. An alignment of the amino acid sequences of mouse, cynomolgus monkey and human TIM-4 polypeptides is provided in Figure 2, and the percent identity of the full-length and IgV regions of human, cynomolgus and murine orthologs are set forth in Table 1. Table 1. TIM-4 Orthologs

hTIM4-FL hTIM4 IgV

The protein "TIM-4" is type I membrane protein that is a member of T-cell immunoglobulin and mucin domain-containing (TIM) family. The human TIM family contains three members (TIM- 1, TIM-3 and TIM-4) on human chromosome 5q33.2, located in a chromosomal region that has been linked with asthma, allergy and autoimmunity. TIM proteins are type I cell-surface glycoproteins with common structural features including an N- terminal immunoglobulin (IgV)-like domain with highly conserved cysteine residues, a threonine-rich mucin domain with O-linked and N-linked glycosylations, a single

transmembrane domain, and a cytoplasmic region. While the cytoplasmic domain of human TIM- 1 and TIM-3 contain tyrosine phosphorylation motif(s), TIM-4 lacks a phosphotyrosine motif.

TIM-4 is expressed on myeloid cells, including dendritic cells (DCs) and

macrophages from spleen, lymph nodes, or peritoneal cavity. TIM-4 has been demonstrated to bind to TIM- 1, MerTK, Integrin ανβ3 and phosphatidyl serine via the IgV domain.

MerTK and Integrin ανβ3 may play a role in certain TEVI4 mediated biological activities (Nishi et al. (2014) Mol Cell Biol. 34(8): 1512-20 and Toda et al. (2012) Mol Cell Biol.

32(1): 118-25). TIM-4 differentially regulates T cell homeostasis by inhibiting naive T cells during the induction phase of an immune response and enhancing T cell responses at the effector phase (Rodriguez-Manzanet et al., supra). For example, TIM-4 binds to TIM-1, which is present on activated T cells, and co-stimulates T cell proliferation. TEVI-4 binds to the phosphatidylserine (PS) through the FG-CC binding cleft in the

N-terminal immunoglobulin variable (IgV) domain. TIM-4 is upregulated following the release of DAMPs, and enhances efferocytosis of apoptotic cells by macrophages

(Kobayashi, 2007; Albacker, et al., J. Immunol. 2010; 185:6839-6849; Mizui et al. Int.

Immunol. 2008; 20:695-708). In contrast, TIM-4 knockout mice have defects in lysosomal degradation, antigen presentation and cross-priming (Miyanishi 2012, Rodrigues-Manzaneta, 2010).

TIM-4 has been demonstrated to be expressed on macrophages and DCs in the tumor microenvironment (TME), and may modulate the interaction between myeloid cells and antigen-specific cytotoxic T cells within tumors. Bahgdadi et al. {supra) reported that apoptotic tumor cells were ingested by TIM-4+ bone-marrow derived macrophages

(BMDMs), suggesting that TIM-4 contributes to immune tolerance by promoting excessive lysosomal degradation of ingested tumor cells, leading to impaired tumor antigen presentation by TIM-4+ tumor-associated macrophages (TAMs). Inhibition of this function appears to promote antitumor activity following monotherapy with an anti-TIM-4 blocking antibody and demonstrates synergistic antitumor activity when combined with an anti-TIM-3 blocking antibody in the context of the vaccine therapy model (Baghadadi 2013).

The term "antagonist" as used with reference to TIM-4 refers to any molecule that partially or fully inhibits one or more biological activities of TIM-4, in vitro, in situ, or in vivo. Examples of such biological activities include binding of TIM-4 to PS, efferocytosis of tumor cells, and suppression of tumor antigen presentation, as well as those further reported in the literature. TIM-4 antagonists may function in a direct or indirect manner. For example, the TIM-4 antagonist may function to partially or fully inhibit one or more biological activities of TIM-4, in vitro, in situ, or in vivo as a result of blocking direct binding to TIM-4 to PS and/or blocking the interaction of TIM-4 to other PS receptors and reducing cellular ability to engage in efferocytosis of tumor cells. The antagonist may also function by interfering with TIM-4 interaction with TIM-1 thereby preventing direct T cell regulatory activity. The TIM-4 antagonist may also function indirectly to partially or fully inhibit one or more biological activities of TIM-4, in vitro, in situ, or in vivo as a result of, e.g., inhibiting another molecule which then blocks TIM-4 activation or expression. It is contemplated that an antagonist may act as a molecule which functions indirectly to block, inhibit or decrease TIM-4 expression or activity.

A TIM-4 antagonist may be any molecule that directly or indirectly inhibits or decreases the activity of TIM-4 and reduces tumor growth, whether on its own or in combination with another treatment, such as a PD-1 antagonist. Exemplary TIM-4 antagonists include TIM-4 binding scaffolds, such as anti-TIM-4 antibodies ("TIM-4 antibodies"), e.g., chimeric, humanized or fully human antibodies, an antigen binding portion thereof, or molecules that are based on or derived from any of these. TIM-4 antagonists may also be non-antibody proteins. For example, TIM-4 antagonist also include modified TIM-4 ligands or binding proteins, e.g., TIM-1 and molecules that are derived from or based on PS. In addition, TEVI-4-regulated biology may be interfered with by fusion proteins such as TIM- 4: Ig. A TIM-4 antagonist may be monovalent or multivalent. In certain embodiments, a TIM-4 antagonist is bivalent, trivalent, tetravalent, or binds to 5, 6, 7, 8, 9, 10 or more TIM-4 epitopes, which may be the same or different TIM-4 epitopes. In certain embodiments, a TIM-4 antagonist is a multivalent monospecific TIM-4 binding scaffold, e.g., a protein comprising a TIM-4 binding scaffold that comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more regions that specifically bind to the same TIM-4 epitope, which binding regions may be composed of the same or a different amino acid sequence. For example, a TIM-4 antagonist may be a TIM-4 binding scaffold comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the same TIM-4 binding region, e.g., the N-terminal IgV region or portion thereof containing the FG-CC binding cleft of the IgV region.

In certain embodiments, a TIM-4 antagonist binds specifically to TIM-4, but does not bind significantly or specifically to other members of the TIM family, such as TIM- 1 or TIM- 3. In other embodiments, a TIM-4 antagonist binds specifically to TIM-4 and TIM- 1.

In another embodiment, the TIM-4 antagonist is an antibody, e.g., an antibody that binds to human TIM-4 with a K _D of 10 ^"7 M, 5 x 10 ^"8 M, 10 ^"8 M, 5 x 10 ^"9 M, 10 ^"9 M, 5 x 10 ^"10 M, 10 ^"10 M or less, wherein the antibody inhibits tumor growth, and/or increases tumor antigen specific CTLs. Antibodies binding to human TIM-4 are known in the art. Any of these antibodies may be used in combination with a PD- 1 antagonist, provided that their combination results in inhibition of tumor growth or reduction in tumor size, e.g., in a subject having cancer. Exemplary antibodies include those which bind specifically to the N-terminal IgV domain of TIM-4 (e.g., the FG-CC" binding cleft). Anti-TIM-4 antibodies include those which bind human TIM-4, e.g., 9F4 (BioLegend). Anti-mouse anti-TIM4 antibodies include RMT4-53 (BioXCell, GeneTex), RMT4-54 (BioLegend) F31-5G3 and 21H12 (BioLegend, BD Biosciences, respectively). Variants of these antibodies, such as antibodies comprising the CDRs of these antibodies, or antibodies that compete for binding to human or murine TIM-4 with one of these antibodies or antibodies that bind to the same or similar epitope on TIM-4 as one of these antibodies may be used in combinations with a PD- 1 antagonist.

In certain embodiments, an anti-TIM4 antibody for use in the methods described herein binds to a region on human TIM-4 that is not the IgV domain, and can be, e.g., the stem domain or any other region in the extracellular domain of TIM-4, provided that the antibody antagonizes the biological activity of TIM-4, and has at least an additive effect with a PD- 1 antagonist when used for treating cancer (relative to either agent alone). In another embodiment, the TIM-4 antagonist is a multivalent agent, such as a multimer (e.g., a polypeptide construct with trimerizing domain and a polypeptide that binds TIM-4).

Agents, which compete for binding to TIM-4 with any of the exemplary agents listed herein, and which inhibit tumor growth or reduces tumor size may also be used. Antibodies having VH and VL chains comprising an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to those of any of the anti-TIM-4 antibodies listed herein may be used.

Administration of a TIM-4 antagonist may be monitored by imaging with a phosphatidylserine (PS)-binding agent (e.g. annexin V peptides, TIM-4 binding fragments), where the PS binding agent may be labeled for imaging, e.g. PET, SPECT, fluorescence, etc. The binding agent is brought into contact with the target tumor cells, and the presence of bound agent is indicative of PS being present and able to interact with TIM-4.

For example, Annexin V is a ubiquitous intracellular protein in humans that has a nanomolar affinity for the membrane -bound constitutive anionic phospholipid

phosphatidylserine (PS), which is selectively expressed on the surface of apoptotic or physiologically stressed cells. As such, radiolabeled forms of annexin V have been used in both animal models and human Phase I and Phase II trials for utilizing the tracer as an early surrogate marker of therapeutic efficacy (e.g., Blankenberg et al. Proc. Am. J. Thoracic. Soc. 2009; 6:469-476). For example, the in vivo monitoring of PS within the tumor

microenvironment may be performed with anti- annexin V linked to a label radiotracer (for PET or SPECT), or with a TIM-4 fusion protein linked to a label or radiotracer. Labels could include q-dots for near IR imaging, or other more conventional labels. pK/Kd studies may be performed with radiolabeled antibodies.

IV. Compositions

In one aspect, the present invention provides composition comprising a PD- 1 antagonist and a TIM-4 antagonist (e.g., formulated together in a single composition or separately formulated). In one embodiment, the PD-1 antagonist is nivolumab,

pembrolizumab, durvalumab, atezolizumab, avelumab or PDR001. In one embodiment, the composition comprises a PD- 1 antagonist and a TIM-4 antagonist, wherein (a) the PD- 1 antagonist is an anti-PD- 1 antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 15; and (b) the TIM-4 antagonist is an antibody. In another embodiment, the composition comprises a PD-1 antagonist and a TIM-4 antagonist, wherein (a) the PD-1 antagonist is an anti-PD-Ll antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 3 and (b) the TIM-4 antagonist is an antibody.

Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, e.g., in a liquid carrier, or suitable for reconstitution into liquid solution or suspension for intravenous administration. In general, such compositions typically comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or aqueous solution saline and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravenous, intraperitoneal, intramuscular, intrathecal and subcutaneous.

For oral use, the pharmaceutical compositions of the present invention, may be administered, for example, in the form of tablets or capsules, powders, dispersible granules, or cachets, or as aqueous solutions or suspensions. In the case of tablets for oral use, carriers which are commonly used include lactose, corn starch, magnesium carbonate, talc, and sugar, and lubricating agents such as magnesium stearate are commonly added. For oral

administration in capsule form, useful carriers include lactose, corn starch, magnesium carbonate, talc, and sugar. When aqueous suspensions are used for oral administration, emulsifying and/or suspending agents are commonly added. In addition, sweetening and/or flavoring agents may be added to the oral

compositions. For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient(s) are usually employed, and the pH of the solutions should be suitably adjusted and buffered. For intravenous use, the total concentration of the solute(s) should be controlled in order to render the preparation isotonic.

For preparing suppositories according to the invention, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted, and the active ingredient is dispersed homogeneously in the wax, for example by stirring. The molten homogeneous mixture is then poured into conveniently sized molds and allowed to cool and thereby solidify.

Liquid preparations include solutions, suspensions and emulsions. Such preparations are exemplified by water or water/propylene glycol solutions for parenteral injection. Liquid preparations may also include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas.

Also included are solid preparations which are intended for conversion, shortly before use, to liquid preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

V. Patient Populations

Provided herein are effective methods for treating cancer in a patient, e.g., using a combination of a TIM-4 antagonist and PD- 1 antagonist. In one embodiment, the patient suffers from a cancer selected from the group consisting of carcinoma, sarcoma, blastoma, leukemia and lymphoma. In another embodiment, the patient suffers from a cancer selected from the group consisting of non- small cell lung cancer, small cell lung cancer,

gastrointenstinal cancer, colorectal cancer, stomach cancer, colon carcinoma and

glioblastoma. In another embodiment, the patient suffers from a cancer selected from the group consisting of chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non- small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CML).

VI. Additional Agents/Therapies

The combinations of the present invention (e.g., PD-1 antagonist in combination with TEVI-4 antagonist) may also be used in conjunction with other well known therapies that are selected for their particular usefulness against the cancer that is being treated. Combinations of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when inappropriate.

For example, the PD- 1 antagonists and TIM-4 antagonist described herein can further be used in combination (e.g., simultaneously or separately) with an additional treatment, such as irradiation, chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, doxorubicin, 5-fu, or camptothecin + apo21/TRAIL (a 6X combo)), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2' (bcl-xl inhibitor), AT- 101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX- 15-070 (obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein- 1) antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g., smac7, smac4, small molecule smac mimetic, synthetic smac peptides (see Fulda et al, Nat Med 2002;8:808-15), ISIS23722 (LY2181308), or AEG- 35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents targeting

VEGF and VEGFR, synthetic triterpenoids (see Hyer et al., Cancer Research 2005;65:4799- 808), c-FLIP (cellular FLICE-inhibitory protein) modulators (e.g., natural and synthetic ligands of PPARy (peroxisome proliferator-activated receptor γ), 5809354 or 5569100), kinase inhibitors (e.g., Sorafenib), and/or genotoxic drugs. The PD-1 antagonists and TIM-4 antagonists described herein can further be used in combination with one or more anti-proliferative cytotoxic agents. Classes of compounds that may be used as anti-proliferative cytotoxic agents include, but are not limited to, the following:

Alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,

Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for use in the methods of the invention, include, without limitation, taxanes, paclitaxel (paclitaxel is commercially available as

TAXOL.RTM.), docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone Bl, [17]-dehydrodesoxyepothilone B,

[18]dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16- desmethylepothilone B, epothilone B 10, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX- 651 (tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoid immunoconjugates (DM-1), MKC-1, ABT-751, Tl-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin, 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-l,3,5(10)-trien-3 -ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)- discodermolides, and cryptothilone 1, in addition to other microtubuline stabilizing agents known in the art.

In cases where it is desirable to render aberrantly proliferative cells quiescent in conjunction with or prior to treatment with the chemotherapeutic methods of the invention, hormones and steroids (including synthetic analogs), such as 17a-Ethinylestradiol,

Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEX™, can also be administered to the patient. When employing the methods or compositions of the present invention, other agents used in the modulation of tumor growth or metastasis in a clinical setting, such as antimimetics, can also be administered as desired. Methods for the safe and effective administration of chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA); the disclosure of which is incorporated herein by reference thereto.

A PD- 1 antagonist and a TJJVI4 antagonist may also be combined with one or more immunotherapy agent that stimulates the immune system, e.g., an agent that binds to human CTLA-4, LAG- 3, GITR, OX40, IDO, CSF-1R etc. The chemotherapeutic agent(s) and/or radiation therapy can be administered according to therapeutic protocols well known in the art. It will be apparent to those skilled in the art that the administration of the chemotherapeutic agent(s) and/or radiation therapy can be varied depending on the disease being treated and the known effects of the

chemotherapeutic agent(s) and/or radiation therapy on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents on the patient, and in view of the observed responses of the disease to the administered therapeutic agents.

VII. Treatment Protocols

Suitable treatment protocols for treating cancer in a patient include, for example, administering to the patient an effective amount of a PD-1 antagonist (e.g., antibody) and a TEVI-4 antagonist (e.g., antibody).

As used herein, adjunctive or combined administration (co-administration) includes simultaneous administration of the two antagonists in the same or different dosage form, or separate administration of the two antagonist (e.g., sequential administration). Thus, the PD-1 antagonist (e.g., antibody) and TIM-4 antagonist (e.g., antibody) can be simultaneously administered in a single formulation. Alternatively, the PD-1 antagonist and TIM-4 antagonist can be formulated for separate administration and are administered concurrently or sequentially. For example, the PD-1 antagonist can be administered first followed by (e.g., immediately followed by) the administration of the TIM-4 antagonist, or vice versa. In one embodiment, the PD- 1 antagonist is administered prior to administration of the TIM-4 antagonist, e.g., the PD-1 antagonist is infused into the patient first, followed from 10 minutes to 3 hours later by an infusion of the TIM-4 antagonist. In one embodiment, the TIM-4 antagonist is administered prior to administration of the PD-1 antagonist, e.g., the TIM-4 antagonist is infused into the patient first, followed from 10 minutes to 3 hours later by an infusion of the PD- 1 antagonist. Such concurrent or sequential administration preferably results in both antagonists being simultaneously present in treated patients. In another embodiment, the TIM-4 antagonist and the PD-1 antagonist are administered simultaneously.

In one embodiment, a subject is administered a single dose of a TIM-4 antagonist and a single dose of the PD-1 antagonist, e.g., an anti-PD- 1 or anti-PD-Ll antibody. In certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of a TIM-4 antagonist and multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of a PD-1 antagonist are

administered to a subject in need of treatment. Administration of the TIM-4 antagonist and the PD- 1 antagonist may be on the same day, or alternatively, the TIM-4 antagonist may be administered 1 or more days before or after the PD- 1 antagonist.

In one embodiment, administrations of a TIM-4 antagonist and a PD- 1 antagonist may be done weekly or monthly, in which regimen, they may be administered on the same day (e.g., simultaneously), or one after the other (e.g., one or more minutes, hours or days before or after one another). In one embodiment, the TIM-4 antagonist and PD- 1 antagonist are administered every three days.

In one embodiment, the dose of the PD-1 antagonist and/or TIM-4 antagonist is varied over time. For example, the PD- 1 antagonist and/or TIM-4 antagonist may be initially administered at a high dose and may be lowered over time. In another embodiment, the PD-1 antagonist and/or TIM-4 antagonist is initially administered at a low dose and increased over time.

In another embodiment, the amount of the PD-1 antagonist and/or TIM-4 antagonist administered is constant for each dose. In another embodiment, the amount of the PD-1 antagonist and/or TIM-4 antagonist varies with each dose. For example, the maintenance (or follow-on) dose of the antagonist can be higher or the same as the loading dose which is first administered. In another embodiment, the maintenance dose of the antagonist a can be lower or the same as the loading dose. A clinician may utilize preferred dosages as warranted by the condition of the patient being treated. The dose may depend upon a number of factors, including stage of disease, etc. The specific dose that should be administered based upon the presence of one or more of such factors is within the skill of the artisan. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small amounts until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. Intermittent therapy (e.g., one week out of three weeks or three out of four weeks) may also be used.

In one embodiment, the TIM-4 antagonist (e.g., antibody) is administered at a dose of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight. In another embodiment, the PD-1 antagonist (e.g., antibody) is administered at a dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight. Generally, 200 μg/mouse is approximately 10 mg/kg and 100 μg/mouse is approximately 5 mg/kg. Therefore, based on the experiments described herein, one or more doses of 1-20 mg/kg body weight, 1-10 mg/kg body weight, 5-20 mg/kg body weight or 5-10 mg/kg body weight of a TIM-4 antagonist and PD- 1 antagonist may be administered to a subject. In certain embodiments, a dose of 0.3 mg/kg to 10 mg/kg body weight of a TIM-4 antagonist is used and a dose of at least 1 mg/kg, e.g., 1-10 mg/kg body weight of a PD-1 antagonist is used.

VIII. Outcomes

Patients, e.g., humans, treated according to the methods disclosed herein preferably experience improvement in at least one sign of cancer. In one embodiment, improvement is measured by a reduction in the quantity and/or size of measurable tumor lesions. In another embodiment, lesions can be measured on chest x-rays or CT or MRI films. In another embodiment, cytology or histology can be used to evaluate responsiveness to a therapy.

In one embodiment, the patient treated exhibits a reduction in size of a tumor, reduction in number of metastasic lesions over time, complete response, partial response, and stable disease. In another embodiment, the patient treated experiences tumor shrinkage and/or decrease in growth rate, i.e., suppression of tumor growth. In another embodiment, unwanted cell proliferation is reduced or inhibited. In yet another embodiment, one or more of the following can occur: the number of cancer cells can be reduced; tumor size can be reduced; cancer cell infiltration into peripheral organs can be inhibited, retarded, slowed, or stopped; tumor metastasis can be slowed or inhibited; tumor growth can be inhibited; recurrence of tumor can be prevented or delayed; one or more of the symptoms associated with cancer can be relieved to some extent.

In another embodiment, the methods of treatment produce a comparable clinical benefit rate (CBR = CR (complete response), PR (partial response) or SD (stable disease) > 6 months) better than that achieved by a PD-1 (e.g., antibody) or TIM-4 antagonist (e.g., antibody) alone. In other embodiments, the improvement of clinical benefit rate is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, e.g., compared to treatment with a PD-1 antagonist or TIM-4 antagonist alone or relative to tumor growth on the first day of treatment or immediately before initiation of treatment. In another embodiment, administration of a PD-1 antagonist and a TIM-4 antagonist results in at least a three-fold reduction (e.g., a 3.5-fold reduction) in tumor volume, e.g., relative to treatment with the PD-1 antagonist or the TIM-4 antagonist alone or relative to tumor growth on the first day of treatment or immediately before initiation of treatment.

In a further embodiment, administration of a PD- 1 antagonist and a TIM-4 antagonist results in tumor growth inhibition of at least 80%, e.g., relative to treatment with the PD-1 antagonist or TIM-4 antagonist alone or relative to tumor growth on the first day of treatment or immediately before initiation of treatment.

In certain embodiments, administration of a PD-1 antagonist and a TIM-4 antagonist reduces tumor mass by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% relative to the tumor mass prior to initiation of the treatment or on the first day of treatment. In some embodiment, the tumor mass is no longer detectable following treatment as described herein. In some embodiments, a subject is in partial or full remission. In certain

embodiments, a subject has an increased overall survival, median survival rate, and/or progression free survival. IX. Kits and Unit Dosage Forms

Also provided herein are kits which include a pharmaceutical composition containing (a) a PD-1 antagonist and (b) a TIM-4 antagonist and a pharmaceutically-acceptable carrier, in a therapeutically effective amount adapted for use in the preceding methods. In one embodiment, the PD-1 antagonist is an antibody (e.g., 5C4 or 12A4, respectively). In another embodiment, the TIM-4 antagonist is an antibody. The kits optionally also can include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to a patient having cancer. The kit also can include a syringe.

Optionally, the kits include multiple packages of the single-dose pharmaceutical compositions each containing an effective amount of the PD-1 antagonist and the TIM-4 antagonist for a single administration in accordance with the methods provided above.

Instruments or devices necessary for administering the pharmaceutical composition(s) also may be included in the kits. For instance, a kit may provide one or more pre-filled syringes containing an amount of the PD- 1 antagonist and the TIM-4 antagonist.

In one embodiment, the present invention provides a kit for treating cancer in a patient, the kit comprising:

(a) a dose of a PD-1 antagonist;

(b) a dose of a TIM-4 antagonist; and

(c) instructions for using the PD-1 antagonist and TIM-4 antagonist in the methods described herein.

In one embodiment, the present invention provides a kit for treating cancer in a patient, the kit comprising:

(a) one or more doses of a PD-1 antagonist;

(b) one or more doses of a TIM-4 antagonist; and

(c) instructions for using the PD-1 antagonist and TIM-4 antagonist in the methods described herein.

In certain embodiments, the TIM-4 antagonist is an antibody. In certain

embodiments, the PD-1 antagonist is an antibody. In particular embodiments, the PD-1 antagonist is an anti-PD-1 antibody comprising the CDRl, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 13, and the CDRl, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 15. In another particular embodiment, the PD-1 antagonist is an anti-PD-Ll antibody comprising antibody comprises the CDRl, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 1, and the CDRl, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 3. The following examples are merely illustrative and should not be construed as limiting the scope of this disclosure in any way as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure.

The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES

Materials and Methods

Animals Ten to eleven-week-old female C57/BL6 mice (Harlan) were used in the studies.

Mice received food and water ad libitum and were maintained in a controlled environment according to Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International regulations. All animal studies have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Antibodies

Anti-mouse PD-1 mAb (anti-mPD-1 mAb) clone 4H2, mouse IgGl isotype was produced and purified by Bristol-Myers Squibb (Biologies Discovery, CA). The anti-TIM-4 mAb, RMT4-53 has been previously described (Yeung et al., J. Immunol. 2009; 191:4447- 4455). Briefly, Sprague Dawley rats were immunized with a TIM-4-Ig fusion protein containing the extracellular domain of murine TIM-4 (aa 1-288) to the Fc portion of mouse IgG2a, and fusing LN cells with P3U1 myeloma cells. RMT4-53 reacts with TIM-4/NRK cells but not with parental NRK or other transfected TIM family (TIM-1 B6/NRK, TIM-1 BALB/NRK, TIM-2/NRK, TIM-3 B6/NRK, TIM-3 B ALB /NRK) cells. Antibodies were certified to have <0.5 EU/mg endotoxin levels, >95% purity and <5% high molecular weight species. Stock solutions of anti-mPD-1 mAb (clone 4H2; mouse IgGl) and anti-mTIM-4 antibody (clone RM 4-53; rat IgG2a), were kept at 4°C prior to use. Dosing solutions of anti- mPD-1 mAb and anti-mTIM-4 mAb were prepared in sterile phosphate buffered saline (pH 7.0) and maintained at 4°C. Cell Lines

CT-26 is an undifferentiated colon carcinoma cell line with a fibroblast morphology (ATCC). Mice inoculated subcutaneously develop lethal tumors at 80% frequency with 10 ³ cells and at 100% with 10 ⁴ cells. Pulmonary metastases develop when mice are inoculated, intravenously, with 10 ⁴ cells (Wang et al. J. Immunol. 1995; 154:4685-4692). The MC38 cell line was derived from C57BL6 murine colon adenocarcinoma cells (CD44+ALDH1+) with an epithelial morphology.

Expression of TIM-4

Expression of TIM-4 on mouse peritoneal macrophages and TAMs was confirmed by flow cytometry.

Immunohistochemistry

Immunohistochemical analysis using Rabbit anti-TIM4 (Atlas Antibodies,

HPA015625) demonstrated expression of TIM-4 on tissue resident macrophages present in human tonsil, lung and liver. Expression was demonstrated on fixed tissue was sectioned and applied to Superfrost Plus glass slides at a thickness of 5 microns. Sections were allowed to dry overnight at room temperature. Tissues were then deparaffinized in xylene followed by hydration to distilled water in graded series of ethanol. Antigen Retrieval was performed using HIER with Biocare Medical EDTA, pH 8.2 (IX) at approximately 115°C for 1 minute and then cooled for 20 minutes at RT. Sections were rinsed in TNT Buffer (TBS + 0.5% Tween) and isolated using a PAP pen. Blocking was performed with Biocare Medical

Background Sniper for 30 minutes at RT, followed by IX TNT Buffer rinse, incubation in Dako Peroxidase Block for 10 minutes at RT and 2X TNT Buffer rinses. Primary Antibody staining was performed using a 1/1000 dilution in Biocare Medical Renaissance antibody diluent and background reducer for 60 minutes at RT and 3X TNT Buffer rinses. Detection antibody was applied using appropriate number of drops to cover section with Biocare

Medical MACH3 Rabbit polymer-HRP and incubating for 30 minutes at RT followed by and 3X TNT Buffer rinses. Samples were incubated with Biocare Medical Betazoid DAB and reaction stopped by placing slides in water). Sections were counterstained in Hematoxylin, Leica for 30 seconds, washed in tap water, incubated in Bluing Solution for 45 seconds to 1 minute, washed 2X in water, dehydrated in graded series of alcohol, and Xylene and then mounted. Example 1; Inhibition of Efferocytosis by anti-TIM-4 Antibody

Thymocytes were isolated from BALB/c mice and cultured with or without dexamethasone for 4 hours to induce apoptosis. Cells were then incubated with pHrodo dye for 5-10 minutes according to manufacturer's instructions (Thermofisher Scientific). Briefly, 2 xlO ⁷ cells were washed in media without serum 1 x and resuspended in 1 mL of Diluent C (Catalog Number G8278). Cells were then stained by adding 1 mL of 2x dye solution (by adding 4 mL of the PKH26 ethanolic dye solution (Catalog Number P9691) to 1 mL of Diluent C) and mixing sample by pipetting. Incubation was for 5 minutes with periodic mixing. Staining reaction was stopped by adding an equal volume of serum and incubating 1 minute. Cells were then washed 1 x in media without serum and 2x in PBS.3 x 10 ⁶ pHrodo- labeled cells were injected i.p. into BALB/c mice and peritoneal macrophages were collected by lavage by injecting 3 - 5 mL of ice cold PBS+ 2mM EDTA into the peritoneal cavity of euthanized mice, palpating the abdomen and then collecting the solution via needle aspiration. Experimental animals were injected with RMT4-53 anti-TIM4 antibody 1 hour prior to injection of the pHrodo-labeled thymocytes T.

The uptake of apoptotic cells by peritoneal macrophages was measured using flow cytometry. Briefly, peritoneal macrophages were washed 2x in PBS, resuspended in 100 μΐ _^ viability dye (eBioscience) and incubated on ice 15 minutes. Cells were washed 1 x in PBS and incubated in FcBlock (BioLegend) for 15 minutes on ice. Cells were then stained with antibodies to CD45 (BD), Tim-4 (BioLegend), CD 1 lb (BioLegend), CD 206 (BioLegend), and F4/80+ (BioLegend), for 30 minutes on ice. Cells were then washed 1 x and resuspended in Perm/Fix. Samples were then collected on a LSR Fortessa X20 (BD). To identify cells that engaged in efferocytosis, CD45 +/CD1 lb+ myeloid cells from the live cell population were gated on. Out of these cells, macrophages that engaged in efferocytosis were defined as F4/80+, OPKH26+. The results are set forth in Table 2.

Table 2.

The results demonstrate an increase in efferocytosis of apoptotic cells (50-70%) relative to non-apoptotic cells (25-40%). The addition of anti-TIM-4 blocking anybody greatly reduced uptake of both viable and apoptotic cells. This demonstrates that TIM-4 is an important mediator of cellular uptake.

Example 2: Inhibition of Tumor Growth In Vivo in the CT26 model by Combination Treatment with Anti-TIM-4 Antibody and Anti-PD-1 Antibody

An experiment was conducted in a murine tumor model to test the hypothesis that the combination of anti-TIM-4 and anti-PD-1 would potentiate anti-tumor efficacy. SC CT-26 mice, a colon adenocarcinoma tumor model (TGM-1438), were evaluated for tumor growth after treatment with the anti-TIM4 antibody, RTM-453, alone or in combination with an anti- PD1 antibody, IgGl D265A.

Female BALB/c mice (Harlan; approximately 8-9 wk old) were subcutaneously implanted with 10 ⁶ CT-26 cells on day 0. Sixty mice were evaluated by body weight and tumor measurements after 2X/wk dosing according to the following schedule:

Table 3: Dosing Schedule:

On the days of dosing, the two antibodies were combined and a total of ΙΟΟμί of combined antibody was injected into the mice. Spleens and tumors from five mice per group were collected and processed for flow cytometry analysis of tumor infiltrating lymphocytes (TILs), and spleen immune cells on day 16 according to the following protocol:

Briefly, cells were resuspended in PBS and aliquoted into plates (spleens 2 x 10 ⁶ /well, all tumor cells/well) and then were washed 2x in PBS, backspace. Cells were then

resuspended in 100 viability dye (eBioscience) and incubated on ice 15 minutes. Cells were washed 1 x in PBS and incubated in 50 FcBlock (BioLegend) for 15 minutes on ice and then stained with 50 μΐ _^ of antibody stain mix as described below for 30 minutes. Cells were then washed 1 x with FACS buffer and resuspended in Perm/Fix. Samples were then collected on a LSR Fortessa X20 (BD).

T cell Panel

Ki-67+ to assess proliferating cells;

CD44/CD62L analysis to determine subset T cells into naive, activated, and memory T cell subsets;

FoxP3 to assess population of Tregs (FoxP3+CD4+ T cells);

CD8+CD107a+ to assess degranulating CD8+ T cells (Antigen/Tumor- specific CTL)

Myeloid Panel

Myeloid Cell subsetting for TAMs, MDSCs, NK cells, B cells

CD80, CD86 staining for activated APCs

The myeloid populations in spleen and tumors are shown in Figure 3. The CD8+ T cell populations in spleen and tumor infiltrating lymphocytes are shown in Figure 4. The CD4+ cell populations in spleen and tumor infiltrating lymphocytes are shown in Figure 5. The data demonstrate a shift in myeloid cell populations, and an increase in activated T cells in tumor infiltrating lymphocytes.

The remaining ten mice per group were monitored for tumor growth. Tumor size and body weights were measured twice weekly. Tumor size (measured as mm ) was calculated by multiplying the tumor length by the square of the tumor width divided by 2. Treatments were initiated when subcutaneous tumors reached a median size of 200 mm (established model).

Anti-TIM-4 antibody administered in combination with anti-PD- 1 antibody provided enhanced anti-tumor activity above the activity of either agent alone. The combination demonstrated decreased tumor growth rates (Figures 6-8), and an increased overall survival rate (Figure 9). In sum, the combination of the TIM-4 mAb and the PD-1 mAb resulted in synergistic activity compared to the activity elicited by single agents alone. Therefore, results from this study demonstrate that a combination regimen of anti-mTIM-4 mAb and PD-1 mAb is well-tolerated and result in marked antitumor activity.

Example 3: Inhibition of Tumor Growth In Vivo in the MC38 model by Combination

Treatment with Anti-TIM-4 Antibody and Anti-PD-1 Antibody

MC38 mice, a colon adenocarcinoma tumor model, were evaluated for tumor growth after treatment with the anti-TIM4 antibody, RTM-453, alone or in combination with an anti- PD 1 antibody, IgGl D265A. Female C57/BL6 mice (Harlan; approximately 8-9 wk old) were subcutaneously implanted with 10 ⁶ MC38 cells on day 0. Mice were dosed beginning on Day 6. Three doses at 200μg/injection were administered IP every 4th day. When the two antibodies were administered to a mouse, the antibodies were first combined, and administered together into the mouse. Tumors were measured using calipers and tumor volumes calculated using the formula (L2xW)/2. Progression Free Survival defined as # of days for tumor to reach 4x initial tumor volume.

The results are provided in Figures 10, 11 and 12, and Table X. Figure 10, which provides the percentage of progression free survival relative to the days post implant, show that administration of the combination of the anti-PD- 1 and anti-TIM4 antibodies results in a higher percentage of progression free survival relative to each antibody separately and relative to the isotype control. Table X shows that the combination treatment provided more complete regressions relative to each antibody separately and relative to the isotype control. Figure 11, which shows the MC38 mean tumor volume relative to the days post implant, indicates that the combination treatment reduced the mean tumor volume more relative to each antibody separately and relative to the isotype control. Figure 12A-C shows the MC38 individual tumor volumes, and confirms the results shown in the Figures 10, 11 and Table X.

Table 5: Number of mice with complete regressions of tumors

Thus, administration of a combination of an anti-TIM4 antibody with an anti-PD-1 antibody in the CT26 and MC39 animal models resulted in a stronger anti-tumor effect relative to each antibody alone.

SUMMARY OF THE SEQUENCE LISTING

SEQ SEQUENCE

ID NO:

Heavy Chain CDR3 Amino Acid Sequence

Anti-PD-Ll mAb (12A4; 12A4 in WO 2007/005874 and US Patent No. 7, 943, 743)

KFHFVSGSPFGMDV

Light Chain CDR1 Amino Acid Sequence

Anti-PD-Ll mAb (12A4; 12A4 in WO 2007/005874 and US Patent No. 7, 943, 743)

RASQSVSSYLA

Light Chain CDR2 Amino Acid Sequence

Anti-PD-Ll mAb (12A4; 12A4 in WO 2007/005874 and US Patent No. 7, 943, 743)

DASNRAT

0 Light Chain CDR3 Amino Acid Sequence

Anti-PD-Ll mAb (12A4; 12A4 in WO 2007/005874 and US Patent No. 7, 943, 743)

QQRSNWPT

1 Heavy Chain Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(variable region underlined; constant region bold)

QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGL

EWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLFLQMNSLRAEDT

AVYYCATNDDYWGQGTLVTVSSASTKGPSVFPIiAPCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTIiMISRTPEVTCVWDVSQEDPEVQFNWYVDGV

EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK

2 Light Chain Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(variable region underlined; constant region bold)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI

YDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQSSNWPR

TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

3 Heavy Chain Variable Region (VH) Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:4 from WO 2006/121168)

QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAV IWYDGSKRYYADSVKGRFTI SRDNSKNTLFLQMNSLRAEDTAVYYCATND DYWGQGTLVTVSS

4 Heavy Chain Variable Region (VH) Nucleotide Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168) SEQ SEQUENCE

ID NO:

(SEQ ID NO: 60 from WO 2006/121168)

cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tec ctg aga etc gac tgt aaa gcg tct gga ate acc ttc agt aac tct ggc atg cac tgg gtc cgc cag get cca ggc aag ggg ctg gag tgg gtg gca gtt att tgg tat gat gga agt aaa aga tac tat gca gac tec gtg aag ggc cga ttc acc ate tec aga gac aat tec aag aac acg ctg ttt ctg caa atg aac age ctg aga gee gag gac acg get gtg tat tac tgt gcg aca

aac gac gac tac tgg ggc cag gga acc ctg gtc acc gtc tec

tea

5 Light Chain Variable Region (VL) Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:ll from WO 2006/121168)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQSSNWPRTFGQ GTKVEIK

6 I Light Chain Variable Region (VL) Nucleotide Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:67 from WO 2006/121168)

gaa att gtg ttg aca cag tct cca gec acc ctg tct ttg tct cca ggg gaa aga gec acc etc tec tgc agg gee agt cag agt gtt agt agt tac tta gec tgg tac caa cag aaa cct ggc cag get ccc agg etc etc ate tat gat gca tec aac agg gec act ggc ate cca gee agg ttc agt ggc agt ggg tct ggg aca gac ttc act etc acc ate age age eta gag cct gaa gat ttt gca gtt tat tac tgt cag cag agt age aac tgg cct egg acg ttc

ggc caa ggg acc aag gtg gaa ate aaa

I Heavy Chain CDRl Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:18 from WO 2006/121168)

NSGMH

Heavy Chain CDR2 Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:25 from WO 2006/121168)

VIWYDGSKRYYADSVKG

I Heavy Chain CDR3 Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:32 from WO 2006/121168)

NDDY

0 I Light Chain CDRl Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:39 from WO 2006/121168)

RASQSVSSYLA SEQ SEQUENCE

ID NO:

1 Light Chain CDR2 Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:46 from WO 2006/121168)

DASNRAT

2 Light Chain CDR3 Amino Acid Sequence

Anti-PD-1 mAb (5C4 in WO 2006/121168)

(SEQ ID NO:53 from WO 2006/121168)

QQSSNWPRT

3 Complete PD-1 sequence (GenBank Accession No.: U64863)

agtttccctt ccgctcacct ccgcctgagc agtggagaag gcggcactct ggtggggctg ctccaggcat gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgg gctggcggcc aggatggttc ttagactccc cagacaggcc ctggaacccc cccaccttct tcccagccct gctcgtggtg accgaagggg acaacgccac cttcacctgc agcttctcca acacatcgga gagcttcgtg ctaaactggt accgcatgag ccccagcaac cagacggaca agctggccgc cttccccgag gaccgcagcc agcccggcca ggactgccgc ttccgtgtca cacaactgcc caacgggcgt gacttccaca tgagcgtggt cagggcccgg cgcaatgaca gcggcaccta cctctgtggg gccatctccc tggcccccaa ggcgcagatc aaagagagcc tgcgggcaga gctcagggtg acagagagaa gggcagaagt gcccacagcc caccccagcc cctcacccag gccagccggc cagttccaaa ccctggtggt tggtgtcgtg ggcggcctgc tgggcagcct ggtgctgcta gtctgggtcc tggccgtcat ctgctcccgg gccgcacgag ggacaatagg agccaggcgc accggccagc ccctgaagga ggacccctca gccgtgcctg tgttctctgt ggactatggg gagctggatt tccagtggcg agagaagacc ccggagcccc ccgtgccctg tgtccctgag cagacggagt atgccaccat tgtctttcct agcggaatgg gcacctcatc ccccgcccgc aggggctcag ccgacggccc tcggagtgcc cagccactga ggcctgagga tggacactgc tcttggcccc tctgaccggc ttccttggcc accagtgttc tgcagaccct ccaccatgag cccgggtcag cgcatttcct caggagaagc aggcagggtg caggccattg caggccgtcc aggggctgag ctgcctgggg gcgaccgggg ctccagcctg cacctgcacc aggcacagcc ccaccacagg actcatgtct caatgcccac agtgagccca ggcagcaggt gtcaccgtcc cctacaggga gggccagatg cagtcactgc ttcaggtcct gccagcacag agctgcctgc gtccagctcc ctgaatctct gctgctgctg ctgctgctgc tgctgctgcc tgcggcccgg ggctgaaggc gccgtggccc tgcctgacgc cccggagcct cctgcctgaa cttgggggct ggttggagat ggccttggag cagccaaggt gcccctggca gtggcatccc gaaacgccct ggacgcaggg cccaagactg ggcacaggag tgggaggtac atggggctgg ggactcccca ggagttatct gctccctgca ggcctagaga agtttcaggg aaggtcagaa gagctcctgg ctgtggtggg cagggcagga aacccctccc acctttacac atgcccaggc agcacctcag gccctttgtg gggcagggaa gctgaggcag taagcgggca ggcagagctg gaggcctttc aggccagcca gcactctggc ctcctgccgc cgcattccac cccagcccct cacaccactc gggagaggga catcctacgg tcccaaggtc aggagggcag ggctggggtt gactcaggcc cctcccagct gtggccacct gggtgttggg agggcagaag tgcaggcacc tagggccccc catgtgccca ccctgggagc tctccttgga acccattcct gaaattattt aaaggggttg gccgggctcc caccagggcc tgggtgggaa ggtacaggcg ttcccccggg gcctagtacc cccgcgtggc ctatccactc ctcacatcca cacactgcac ccccactcct ggggcagggc caccagcatc caggcggcca gcaggcacct gagtggctgg gacaagggat cccccttccc tgtggttcta ttatattata attataatta aatatgagag catgct

4 Human PD-L1 amino acid sequence - isoform a precursor (GenBank Accession No. NP_054862.1)

MRIFAVFIFM TYWHLLNAFT VTVPKDLYW EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNI IQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMI SYGG SEQ SEQUENCE

ID NO:

ADYKRITVKV NAPYNKINQR ILWDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET

25 Human PD-L1 amino acid sequence - isoform b precursor (GenBank

Accession No. NP_001254635.1)

MRIFAVFIFM TYWHLLNAPY NKINQRILW DPVTSEHELT CQAEGYPKAE VIWTSSDHQV LSGKTTTTNS KREEKLFNVT STLRINTTTN EIFYCTFRRL DPEENHTAEL VIPELPLAHP PNERTHLVIL GAILLCLGVA LTFIFRLRKG RMMDVKKCGI QDTNSKKQSD THLEET

6 Human TIM-4 amino acid sequence - isoform 1

(GenBank Accession No. NP_612388.2)

MSKEPLILWLMIEFWWLYLTPVTSETWTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCPY SGCKEALIR TDGMRVTSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEVPGWFNDVKINVRLN LQRASTTTHR TATTTTRRTTTTSPTTTRQMTTTPAALPTTWTTPDLTTGTPLQMTTIAVFTTANTCLSLT PSTLPEEAT GLLTPEPSKEGPILTAESETVLPSDSWSSVESTSADTVLLTSKESKVWDLPSTSHVSMWK TSDSVSSPQP GASDTAVPEQNKTTKTGQMDGIPMSMKNEMP I SQLLMI IAPSLGFVLFALFVAFLLRGKLMETYCSQKHT RLDYIGDSKNVLNDVQHGREDEDGLFTL 7 Homo sapiens T-cell (TIMD4), transcript variant 1, mRNA

(GenBank Accession No. NM_138379.2)

ATAAGAGGTTGGGCTTTGGATAGATAGACAGACTCCTGGGTCCGGTCAACCGTCAAAATG TCCAAAGAAC CTCTCATTCTCTGGCTGATGATTGAGTTTTGGTGGCTTTACCTGACACCAGTCACTTCAG AGACTGTTGT GACGGAGGTTTTGGGTCACCGGGTGACTTTGCCCTGTCTGTACTCATCCTGGTCTCACAA CAGCAACAGC ATGTGCTGGGGGAAAGACCAGTGCCCCTACTCCGGTTGCAAGGAGGCGCTCATCCGCACT GATGGAATGA GGGTGACCTCAAGAAAGTCAGCAAAATATAGACTTCAGGGGACTATCCCGAGAGGTGATG TCTCCTTGAC CATCTTAAACCCCAGTGAAAGTGACAGCGGTGTGTACTGCTGCCGCATAGAAGTGCCTGG CTGGTTCAAC GATGTAAAGATAAACGTGCGCCTGAATCTACAGAGAGCCTCAACAACCACGCACAGAACA GCAACCACCA CCACACGCAGAACAACAACAACAAGCCCCACCACCACCCGACAAATGACAACAACCCCAG CTGCACTTCC AACAACAGTCGTGACCACACCCGATCTCACAACCGGAACACCACTCCAGATGACAACCAT TGCCGTCTTC ACAACAGCAAACACGTGCCTTTCACTAACCCCAAGCACCCTTCCGGAGGAAGCCACAGGT CTTCTGACTC CCGAGCCTTCTAAGGAAGGGCCCATCCTCACTGCAGAATCAGAAACTGTCCTCCCCAGTG ATTCCTGGAG TAGTGTTGAGTCTACTTCTGCTGACACTGTCCTGCTGACATCCAAAGAGTCCAAAGTTTG GGATCTCCCA TCAACATCCCACGTGTCAATGTGGAAAACGAGTGATTCTGTGTCTTCTCCTCAGCCTGGA GCATCTGATA CAGCAGTTCCTGAGCAGAACAAAACAACAAAAACAGGACAGATGGATGGAATACCCATGT CAATGAAGAA TGAAATGCCCATCTCCCAACTACTGATGATCATCGCCCCCTCCTTGGGATTTGTGCTCTT CGCATTGTTT GTGGCGTTTCTCCTGAGAGGGAAACTCATGGAAACCTATTGTTCGCAGAAACACACAAGG CTAGACTACA TTGGAGATAGTAAAAATGTCCTCAATGACGTGCAGCATGGAAGGGAAGACGAAGACGGCC TTTTTACCCT CTAACAACGCAGTAGCATGTTAGATTGAGGATGGGGGCATGACACTCCAGTGTCAAAATA AGTCTTAGTA GATTTCCTTGTTTCATAAAAAAGACTCACTTATTCCATGGATGTCATTGATCCAGGCTTG CTTTAGTTTC ATGAATGAAGGGTACTTTAGAGACCACAACTTCTCTGTCAAAAA

Human TIM-4 amino acid sequence isoform 2

(GenBank Accession No. NP_001140198.1)

MSKEPLILWLMIEFWWLYLTPVTSETWTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCPY SGCKEALIR TDGMRVTSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEVPGWFNDVKINVRLN LQRASTTTHR TATTTTRRTTTTSPTTTRQMTTTPAALPTTWTTPDLTTGTPLQMTTIAVFTTANTCLSLT PSTLPEEAT GLLTPEPSKEGP ILTAESETVLPSDSWSSVESTSADTVLLTSKASDTAVPEQNKTTKTGQMDGIPMSMKN EMP I SQLLMI IAPSLGFVLFALFVAFLLRGKLMETYCSQKHTRLDYIGDSKNVLNDVQHGREDEDGLFTL SEQ SEQUENCE

ID NO:

Homo sapiens T-cell (TIMD4), transcript variant 2, mRNA

(GenBank Accession No. NM_001146726.1)

ATAAGAGGTTGGGCTTTGGATAGATAGACAGACTCCTGGGTCCGGTCAACCGTCAAAATG TCCAAAGAAC CTCTCATTCTCTGGCTGATGATTGAGTTTTGGTGGCTTTACCTGACACCAGTCACTTCAG AGACTGTTGT GACGGAGGTTTTGGGTCACCGGGTGACTTTGCCCTGTCTGTACTCATCCTGGTCTCACAA CAGCAACAGC ATGTGCTGGGGGAAAGACCAGTGCCCCTACTCCGGTTGCAAGGAGGCGCTCATCCGCACT GATGGAATGA GGGTGACCTCAAGAAAGTCAGCAAAATATAGACTTCAGGGGACTATCCCGAGAGGTGATG TCTCCTTGAC CATCTTAAACCCCAGTGAAAGTGACAGCGGTGTGTACTGCTGCCGCATAGAAGTGCCTGG CTGGTTCAAC GATGTAAAGATAAACGTGCGCCTGAATCTACAGAGAGCCTCAACAACCACGCACAGAACA GCAACCACCA CCACACGCAGAACAACAACAACAAGCCCCACCACCACCCGACAAATGACAACAACCCCAG CTGCACTTCC AACAACAGTCGTGACCACACCCGATCTCACAACCGGAACACCACTCCAGATGACAACCAT TGCCGTCTTC ACAACAGCAAACACGTGCCTTTCACTAACCCCAAGCACCCTTCCGGAGGAAGCCACAGGT CTTCTGACTC CCGAGCCTTCTAAGGAAGGGCCCATCCTCACTGCAGAATCAGAAACTGTCCTCCCCAGTG ATTCCTGGAG TAGTGTTGAGTCTACTTCTGCTGACACTGTCCTGCTGACATCCAAAGCATCTGATACAGC AGTTCCTGAG CAGAACAAAACAACAAAAACAGGACAGATGGATGGAATACCCATGTCAATGAAGAATGAA ATGCCCATCT CCCAACTACTGATGATCATCGCCCCCTCCTTGGGATTTGTGCTCTTCGCATTGTTTGTGG CGTTTCTCCT GAGAGGGAAACTCATGGAAACCTATTGTTCGCAGAAACACACAAGGCTAGACTACATTGG AGATAGTAAA AATGTCCTCAATGACGTGCAGCATGGAAGGGAAGACGAAGACGGCCTTTTTACCCTCTAA CAACGCAGTA GCATGTTAGATTGAGGATGGGGGCATGACACTCCAGTGTCAAAATAAGTCTTAGTAGATT TCCTTGTTTC ATAAAAAAGACTCACTTATTCCATGGATGTCATTGATCCAGGCTTGCTTTAGTTTCATGA ATGAAGGGTA CTTTAGAGACCACAACTTCTCTGTCAAAAA