WO2017091611A1 - Enhanced cancer immunotherapy using antibody-interferon fusion molecules - Google Patents

Abstract

The present invention relates to fusion molecules designed to improve the efficacy of cancer immunotherapy, comprising one or more IFN molecules attached to an antibody which specifically binds to an immune-checkpoint protein antigen. The fusion molecules of the present invention may be administered alone, or in combination with other non- fused immune-checkpoint protein antigen inhibitors or targeted immunotherapies to treat patients that initially failed to respond to previous anti-cancer therapy.

Description

ENHANCED CANCER IMMUNOTHERAPY USING ANTIBODY-INTERFERON

FUSION MOLECULES

RELATED PATENT APPLICATIONS

[001] This application claims benefit of U.S. Provisional Application No. 62/259,055, filed on November 23, 2015, incorporated in its entirety by reference herein.

BACKGROUND OF THE INVENTION

[002] Immunotherapy is the name given to cancer treatments that use the immune system to attack cancers. Systemic immunotherapy refers to immunotherapy that is used to treat the whole body and is more commonly used than local immunotherapy which is used to treat one "localized" part of the body, particularly when a cancer has spread. Although cancer cells are less immunogenic than pathogens, the immune system is clearly capable of recognizing and eliminating tumor cells, and cancer immunotherapy attempts to harness the exquisite power and specificity of the immune system for treatment of malignancy.

Unfortunately, tumors frequently interfere with the development and function of immune responses, e.g., the suppressive milieu present within established tumors inhibits effective immune responses. Thus, the challenge for immunotherapy is to use advances in cellular and molecular immunology to develop strategies which manipulates the local tumor environment to promote a pro-inflammatory environment, to promote dendritic cell activation, and to effectively and safely augment anti-tumor responses.

[003] Over the past 20 years, a broad class of extracellular "checkpoint proteins" has been found to modulate T cell responses to self-proteins. Checkpoint proteins include CTLA-4, PD-1 , LAG-3, and TIM-3 as well as several others (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012; Sharpe et al., Nat Immunol, 8:239-45, 2007). Under normal physiological conditions, immune checkpoints are crucial for the maintenance of self-tolerance (that is, the prevention of autoimmunity) and also to protect tissues from damage when the immune system is responding to pathogenic infection. It is now also clear that tumors co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012). Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors.

[004] Recent clinical data on single-agent CTLA-4 (Hodi et al., N Engl J Med, 2010) and PD-1 (Topalian et al., N Engl J Med, 366:2443-54, 2012) blockades in cancer patients demonstrates that these pathways play a critical role in the maintenance of tumor tolerance in humans, since single-agent checkpoint blockade is associated with objective tumor responses and improved overall survival. Furthermore, very recent data combining PD-1 and CTLA-4 blockade in melanoma patients showed an increased rate of objective tumor responses as compared to blocking either checkpoint alone, supporting the notion that combinatorial checkpoint blockade may result in increased clinical benefit (Wolchok et al., N Engl J Med, 366:2443-54, 2012). CTLA-4 antibodies were the first of this class of immunotherapeutics to achieve US Food and Drug Administration (FDA) approval.

[005] In models of chronic viral infection, checkpoint proteins have been

individually found to play a role in down-modulating a pathogen-specific immune response. Using the murine model of chronic infection (Lymphochoriomeningitis Virus or LCMV), a seminal study by Wherry et al. showed that non-functional antigen-specific CD8 T cells co- express multiple checkpoint proteins, including PD-1 , LAG-3, 2B4, and CD160 (Blackburn et al., Nat Immunol, 10:29-37, 2009). Of potential clinical relevance, it was noted that combination PD-1 /LAG-3 blockade was superior in terms of restoring IFN-γ secretion and viral clearance than blocking either checkpoint alone (Id.).

[006] The precise molecular pathways by which each of these checkpoint proteins signal is still not fully understood, however, pre-clinical data from studies in which multiple checkpoints were blocked simultaneously suggest that the pathways utilized by the different checkpoint proteins may be relatively unique and potentially non-redundant. This data suggests that there may be a clinical rationale for blocking multiple checkpoints to enhance anti-tumor immunity (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012; Nirschi and Drake, Clinical Cancer Research, 2013).

[007] Interferon (IFN) is an important cytokine which has multiple effects on the immune response (Theofilopoulos et al., Annu. Rev. Immunol., 23:307-336, 2005). Interferons include type 1 interferons (e.g., interferon-alpha (IFN-oc) and interferon-beta (IFN-β)) and type 2 interferons (e.g., interferon-gamma (IFN-γ)). All type 1 IFNs are recognized by a shared receptor (IFN-ocR) composed of two transmembrane proteins, IFN-ocR1 and IFN-ocR2. IFN-oc's are known to inhibit angiogenesis (Sidky YA and EC Borden, Cancer Res., 47:5155, 1987), mediate stimulation and differentiation of dendritic cells (Santini et al., J Exp Med, 191 :1777, 2000), and are important in in vivo proliferation, expansion and long-term survival of antigen specific CD8+ T cells (Tough DF et al., Science, 272:1947, 1996).

[008] Although first described for their ability to inhibit viral replication, IFN-oc's have multiple properties exhibiting anti-proliferative effects, induction of apoptosis (Rodriguez- Villanueva J and TJ McDonnell, Int J Cancer, 61 :1 10, 1995) and induction of the tumor suppressor gene, P53, in tumor cells (Takaoka A et al., Nature, 424:516, 2003). Thus, IFN-oc's were the first recombinant proteins used for the treatment of various cancers. Unfortunately, the use of IFN-a to treat cancer has been limited by its short half-life and associated systemic toxicities (Weiss K, Semin Oncol, 25:9, 1998; Jones GJ and Itri LM, Cancer, 57:1709, 2006). Because of the short in vivo half-life of IFN-a, frequent administration is required.

Pharmacokinetic (PK) studies have indicated that only 0.01 % of subcutaneously injected IFN-a reaches the target tumor site (Suzuki K et al., Gene Then, 10(9):765-773, 2003). The most common adverse events associated with IFN-a therapy are flu-like symptoms, fatigue, anorexia, and central nervous system and psychiatric reactions, and some of these side-effects may become dose-limiting (Jones GJ and Itri LM, Cancer, 57:1709, 2006). Given these limitations, it is difficult to achieve effective IFN-a concentrations at sites of malignant disease without causing systemic toxicity. The limitations of systemic IFN-a therapy have led to the exploration of alternative strategies to deliver IFN-a safely and effectively into the tumor vicinity.

[009] Immunomodulatory therapy with IFN-β has proven to be successful in reducing the severity of the underlying disease in patients with relapsing-remitting MS. FDA-approved IFN-β therapies for the treatment of relapsing-remitting MS in the United States include interferon β-l a (marketed as Avonex®, available from Biogen, Inc.), interferon^-1 b (marketed as Betaseron®, available from Chiron Corporation) and interferon β-1 a (marketed as Rebif®, available from EMD Serono and Pfizer), having combined sales exceeding three billion dollars a year. Unfortunately, each of these therapeutic agents are only partially effective in reducing the frequency and severity of relapses, slowing the rate of disease progression, or reducing the degree of brain inflammation as measured by a variety of magnetic resonance imaging (MRI) techniques. There is a continued need for more effective IFN-β products.

[010] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety. SUMMARY OF THE INVENTION

[Oil] In one aspect, the present disclosure provides fusion molecules comprising one or more interferon molecules attached to an antibody which has been specifically selected based on its ability to bind to a specific "checkpoint protein" antigen (also referred to hereinafter as "immune-checkpoint protein antigen"), wherein the fusion molecule is capable of treating a proliferative disease in a patient.

[012] In various embodiments, the proliferative disease is a cancer selected from the list including, but not limited to, a B cell lymphoma, a lung cancer, a bronchus cancer, a colorectal cancer, a prostate cancer, a breast cancer, a pancreas cancer, a stomach cancer, an ovarian cancer, a urinary bladder cancer, a peripheral nervous system cancer, an esophageal cancer, a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancer of the oral cavity or pharynx, a liver cancer, a kidney cancer, a biliary tract cancer, a small bowel or appendix cancer, a salivary gland cancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma, a chondrosarcoma, a liposarcoma, a testes cancer, and a malignant fibrous histiocytoma, a skin cancer, a head and neck cancer, lymphomas, sarcomas, multiple myeloma and leukemias.

[013] In various embodiments, the patient previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter "a recurrent cancer").

[014] In various embodiments, the patient has resistant or refractory cancer. In various embodiments, the cancer is refractory to immunotherapy treatment. In various embodiments, the cancer is refractory to treatment with a chemotherapeutic agent. In various embodiments, the cancer is refractory to targeted treatment with an immune- checkpoint protein antigen inhibitor. In various embodiments, the cancer is refractory to targeted treatment with a tumor antigen-specific, depleting antibody. In various embodiments, the cancer is refractory to targeted treatment with an immunoconjugate, antibody-drug conjugate (ADC), or fusion molecule comprising an immune-checkpoint protein antigen inhibitor (or tumor antigen-specific, depleting antibody) and a cytotoxic agent. In various embodiments, the cancer is refractory to targeted treatment with a small molecule kinase inhibitor. In various embodiments, the cancer is refractory to combination therapy involving one or more treatments selected from the group consisting of: immunotherapy treatment, treatment with a chemotherapeutic agent, treatment with an immune-checkpoint protein antigen inhibitor, targeted treatment with a tumor antigen-specific, depleting antibody, treatment with a immunoconjugate, ADC, or fusion molecule comprising an immune- checkpoint protein antigen inhibitor (or tumor antigen-specific, depleting antibody) and a cytotoxic agent, targeted treatment with a small molecule kinase inhibitor, treatment using surgery, treatment using stem cell transplantation, and treatment using radiation.

[015] In various embodiments, the fusion molecule is administered to the patient at a dosage of about 0.0001 mg/kg to about 0.9 mg/kg. In various embodiments, the fusion molecule is administered to the patient at a weekly dosage included in any of the following ranges: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, about 0.05 to about 0.06 mg/kg, about 0.06 to about 0.07 mg/kg, about 0.07 to about 0.08 mg/kg, about 0.08 to about 0.09 mg/kg, about 0.09 to about 0.1 mg/kg, about 0.1 to about 0.2 mg/kg, about 0.2 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg. In various embodiments, the fusion molecule is administered to the patient at a weekly dosage selected from the group consisting of .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .02 mg/kg, .03 mg/kg, .04 mg/kg, .05 mg/kg, .06 mg/kg, .07 mg/kg, .08 mg/kg, .09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg. In various embodiments, the fusion molecule is administered to the patient at a weekly dosage of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .02 mg/kg, .03 mg/kg, .04 mg/kg, .05 mg/kg, .06 mg/kg, .07 mg/kg, .08 mg/kg, .09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.

[016] In various embodiments, the fusion molecules comprise a type 1 interferon. In various embodiments, the fusion molecules comprise a type 2 interferon. In various embodiments, the interferon molecule is an interferon alpha (IFN-a) molecule. In various embodiments, the interferon molecule is an IFN-a mutant molecule. In various

embodiments, the interferon molecule is an interferon beta (IFN-β). In various

embodiments, the interferon molecule is an IFN-β mutant molecule. In various

embodiments, the interferon molecule is an interferon gamma (IFN-γ).

[017] In various embodiments, the fusion molecules comprise an antibody selected from a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab', a Fab₂, a Fab'₂, a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, or a diabody. In various embodiments, the antibody is a fully human monoclonal antibody. In various embodiments, the antibody is a humanized monoclonal antibody.

[018] In various embodiments, the antibody or antigen-binding fragment binds to an immune-checkpoint protein antigen with a dissociation constant (KD) of at least about 1 x10 ³ M, at least about 1 x10 ⁴ M, at least about 1 x10 ⁵ M, at least about 1 x10^-6 M, at least about 1 x10 ⁷ M, at least about 1 x10^-8 M, at least about 1 x10 ⁹ M, at least about 1 x10^_10 M, at least about 1 x10 ¹¹ M, or at least about 1 x10^-12 M.

[019] In various embodiments, the fusion molecules comprise an antibody that specifically binds an immune-checkpoint protein antigen from the list including, but not limited to, CD276, CD272, CD152, CD223, CD279, CD274, TIM-3 and B7-H4; or any immune-checkpoint protein antigen antibody taught in the art.

[020] In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti-CD276 antibody. In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti-CD272 antibody. In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti-CD152 antibody. In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti-CD223 antibody. In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti-CD279 antibody. In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti-CD274 antibody. In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti-TIM-3 antibody. In various embodiments, the fusion molecules comprise a human IFN-a molecule, or mutant molecule thereof, and an anti- B7-H4 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti-CD276 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti-CD272 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti-CD152 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti-CD223 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti-CD279 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti-CD274 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti-TIM-3 antibody. In various embodiments, the fusion molecules comprise a human IFN-β molecule, or mutant molecule thereof, and an anti- B7-H4 antibody.

[021] In various embodiments, the fusion molecules comprise an interferon molecule that is directly attached to the antibody.

[022] In various embodiments, the fusion molecules comprise an interferon molecule that is attached to the antibody via a proteolysis resistant peptide linker. In various

embodiments, the proteolysis resistant peptide linker is fewer than 20 amino acids in length. In various embodiments, the proteolysis resistant linker is selected from SGGGGS (SEQ ID NO: 20) and AEAAAKEAAAKAGS (SEQ ID NO: 21 ).

[023] In various embodiments, the fusion molecule is a recombinantly expressed fusion molecule.

[024] In another aspect, the present disclosure relates to methods of treating cancer in a patient, comprising administration of a combination of a) a therapeutically effective amount of a fusion molecule of the present disclosure; and b) a therapeutic effective amount of a second agent. In various embodiments, the second agent is an immune-checkpoint protein antigen inhibitor. In various embodiments, the second agent is an immune-checkpoint protein antigen inhibitor targeting the same immune-checkpoint protein antigen as that targeted by the fusion molecule. In various embodiments, the second agent is an immune-checkpoint protein antigen inhibitor targeting a different immune-checkpoint protein antigen from that targeted by the fusion molecule. In various embodiments, the fusion molecule and the second agent are administered simultaneously. In various embodiments, the fusion molecule and the second agent are administered sequentially. In various embodiments, the second agent is an immune-checkpoint protein antigen inhibitor selected from the list including, but not limited to, anti-CD276 Ab, anti- CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab.

[025] In various embodiments, the methods of the present disclosure may comprise one or more additional therapies selected from the group consisting of

immunotherapy, chemotherapy, targeted treatment with an immune-checkpoint protein antigen inhibitor, targeted treatment with a tumor antigen-specific, depleting antibody, targeted treatment with an immunoconjugate, antibody-drug conjugate (ADC), or fusion molecule comprising an immune-checkpoint protein antigen inhibitor (or tumor antigen-specific, depleting antibody) and a cytotoxic agent, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.

[026] In another aspect, the present disclosure provides methods of enhancing an anti-tumor immune response, or killing or inhibition of growth of tumor cells in a patient, comprising administering to the patient a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a fusion molecule of the present disclosure, wherein the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of a tumor cell.

[027] In various embodiments, the tumor cell is a cell produced by a cancer selected from the group consisting of a B cell lymphoma, a lung cancer, a bronchus cancer, a colorectal cancer, a prostate cancer, a breast cancer, a pancreas cancer, a stomach cancer, an ovarian cancer, a urinary bladder cancer, a peripheral nervous system cancer, an esophageal cancer, a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancer of the oral cavity or pharynx, a liver cancer, a kidney cancer, a biliary tract cancer, a small bowel or appendix cancer, a salivary gland cancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma, a chondrosarcoma, a liposarcoma, a testes cancer, and a malignant fibrous histiocytoma, a skin cancer, a head and neck cancer, lymphomas, sarcomas, multiple myeloma and leukemias.

[028] In another aspect, the present disclosure provides methods of enhancing an anti-tumor host immune response in a patient, comprising administering to the patient a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a fusion molecule of the present disclosure, wherein the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of an immune cell.

[029] In another aspect, the present disclosure provides a pharmaceutical composition which comprises a fusion molecule of the present disclosure as an active ingredient, in a pharmaceutically acceptable carrier or excipient. In various embodiments, the pharmaceutical composition is formulated for administration via a route selected from, e.g., subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or via infusions.

[030] In other aspects, the present disclosure provides polynucleotides that encode the fusion molecules of the present disclosure; vectors comprising polynucleotides encoding fusion molecules of the disclosure; optionally, operably-linked to control sequences recognized by a host cell transformed with the vector; host cells comprising vectors comprising polynucleotides encoding fusion molecules of the disclosure; a process for producing a fusion molecule of the disclosure comprising culturing host cells comprising vectors comprising polynucleotides encoding fusion molecules of the disclosure such that the polynucleotide is expressed; and, optionally, recovering the fusion molecule from the host cell culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

[031] Figure 1 depicts one proposed design for a genetically engineered fusion molecule of the present invention. In Figure 1 , the ovals labeled as V_L, V_H, Ci_, Cm , CH2 and CH3 represent a full length antibody (Ab) as defined herein. The oval labeled C represents a cytokine. A linker is represented by the squiggled line. As depicted in Figure 1 , C is attached to the Ab via a linker at the two CH3 sites. In alternative embodiments, C is attached to the Ab via a linker at the two V_L sites, via a linker at the two Ci_ sites, or via a linker at the two V_H sites. In alternative embodiments, C will be attached to the Ab via a linker at an internal site rather than at the CH3, V_l, Ci_ or V_H sites.

[032] Figure 2 depicts another proposed design for a genetically engineered fusion molecule of the present invention. In Figure 2, the ovals labeled as V_L, V_H, Ci_, and Cm represent a F(ab')₂ as defined herein. The oval label C represents a cytokine. A linker is represented by the squiggled line. As depicted in Figure 2, C is attached to the F(ab')₂ via a linker at the two Cm sites. In alternative embodiments, C will be attached to the F(ab')₂ via a linker at the two V_L sites, via a linker at the two V_H sites, or via a linker at the two Ci_ sites. In yet another alternative, C will be attached to the F(ab')₂ via a linker at an internal site rather than at the Cm , V_L, CL or V_H sites.

[033] Figure 3 depicts another proposed design for a genetically engineered fusion molecule of the present invention. In Figure 3, the ovals labeled as V_L, V_H, CL, and Cm represent a Fab as defined herein. The oval label C represents a cytokine. A linker is represented by the squiggled line. As depicted in Figure 3, C is attached to the Fab via a linker at the Cm site. In alternative embodiments, C will be attached to the Fab via a linker at the V_L site, via a linker at the V_H site, or via a linker at the CL site. In yet another alternative, C will be attached to the Fab via a linker at an internal site rather than at the Cm , V_L, CL or V_H sites.

DETAILED DESCRIPTION THE INVENTION [034] The present disclosure is based on inventors' unique insight that a fusion molecule comprising an interferon attached to an antibody which specifically binds to an immune-checkpoint protein antigen would provide improved, effective methods used to effectively treat cancers, including recurrent, resistant, or refractory cancers, at surprisingly low doses. Further, the fusion molecules may, among other things, possess one or more of the following properties: a) negating the suppressive action of tumors on immune cells; b) improving CD8+ CTL priming, i.e., increasing cross-presentation efficiency of tumor antigens to T cells by DCs; c) activating (e.g., directly activating) the CD8+ CTL functions; d) killing (e.g., indirect killing) of tumor cells; d), when the immune-checkpoint protein antigen is present on tumor cell surface, direct killing of the tumor cells; e) inducing the up- regulation of the co-inhibitory immune-checkpoint proteins on tumor cells and f) negating other mechanisms for immune evasion, e.g., through the major inhibitory pathways mediated by certain immune-checkpoint proteins on T cells/B cells.

[035] Accordingly, the present disclosure provides novel, non-naturally occurring fusion molecules designed to improve the efficacy of cancer immunotherapy, comprising one or more IFN molecules (the "interferon portion") attached to an antibody (the "antibody portion") which specifically binds to an immune-checkpoint protein antigen expressed on a tumor cell and/or an immune cell. In various embodiments, the immune-checkpoint protein antigen is present on the surface of a tumor cell and the fusion molecule is capable of targeting the interferon to a tumor cells. In various embodiments, the immune-checkpoint protein antigen is present on the surface of an immune cell and the fusion molecule is capable of negating the suppressive action of tumors on immune cells and activating (e.g., directly activating) an anti-tumor immune response. In various embodiments, the fusion molecule is capable of negating the suppressive action of tumors on immune cells and killing (e.g., indirectly killing) tumor cells. In various embodiments, the fusion molecule is capable of negating the suppressive action of tumors on immune cells, activating (e.g., directly activating) anti-tumor immune response, and killing (e.g., indirectly killing) tumor cells.

[036] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally,

nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Definitions

[037] The term "tumor associated antigen" (TAA) or "tumor antigen-specific" refers to, e.g., cell surface antigens that are selectively expressed by cancer cells or over-expressed in cancer cells relative to most normal cells. The terms "TAA variant" and "TAA mutant" as used herein refers to a TAA that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another TAA sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length.

[038] As used herein, a "proliferative disease" includes tumor disease (including benign or cancerous) and/or any metastases. A proliferative disease may include

hyperproliferative conditions such as hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. In various embodiments, the proliferative disease is cancer. In various embodiments, the proliferative disease is a non-cancerous disease. In various embodiments, the proliferative disease is a benign or malignant tumor. [039] "Resistant or refractory cancer" refers to tumor cells or cancer that do not respond to previous anti-cancer therapy including, e.g., chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the beginning of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the onset of treatmentor respond initially for a short period but fail to respond to treatment. Refractory tumor cells also include tumors that respond to treatment with anticancer therapy but fail to respond to subsequent rounds of therapies. For purposes of this invention, refractory tumor cells also encompass tumors that appear to be inhibited by treatment with anticancer therapy but recur up to five years, sometimes up to ten years or longer after treatment is discontinued. The anticancer therapy can employ chemotherapeutic agents alone, radiation alone, targeted therapy alone, surgery alone, or combinations thereof. For ease of description and not limitation, it will be understood that the refractory tumor cells are interchangeable with resistant tumor cells.

[040] The term "immunogenicity" as used herein refers to the ability of an antibody or antigen binding fragment to elicit an immune response (humoral or cellular) when administered to a recipient and includes, for example, the human anti-mouse antibody (HAMA) response. A HAMA response is initiated when T-cells from a subject make an immune response to the administered antibody. The T-cells then recruit B-cells to generate specific "anti-antibody" antibodies.

[041] The term "antigen presenting cells (APCs)" refers to a class of cells capable of presenting one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. APCs can be intact whole cells such as macrophages, B-cells, endothelial cells, activated T-cells, and dendritic cells; or other molecules, naturally occurring or synthetic, such as purified MHC Class I molecules complexed to β₂-Γηίο^^υΝη. In some cases, APCs can present antigens in an efficient amount to activate naive T-cells for cytotoxic T-lymphocyte (CTL) responses. While many types of cells may be capable of presenting antigens on their cell surface for T-cell recognition, only dendritic cells have the capacity to present antigens in an efficient amount to activate naive T-cells for cytotoxic T-lymphocyte (CTL) responses. The term "dendritic cells (DCs)" refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues. See Steinman, Ann. Rev. Immunol. 9:271 -296, 1991 . DCs constitute the most potent and preferred APCs in the organism. While the DCs can be differentiated from monocytes and CD34⁺ cells, they possess distinct phenotypes. For example, a particular differentiating marker, CD14 antigen, is not found in DCs but is possessed by monocytes. Also, mature DCs are not phagocytic, whereas the monocytes are strongly phagocytosing cells. It has been shown that mature DCs can provide all the signals necessary for T cell activation and proliferation.

[042] The term "immune cell" as used herein means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., an autoantigen). In various embodiments, an immune cell is an antigen presenting cell, e.g., a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, Langerhan's cells, or Kuffer cells.

[043] As used herein, the term "immunotherapy" refers to cancer treatments which include, but are not limited to, treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1 , OX-40, CD137, IDO, LAG 3, TIM-3, and VISTA, bispecific antibodies, bispecific T cell engaging antibodies such as blinatumomab, administration of cytokines such as IL-2, IL-12, IL-21 , GM- CSF and IFN-oc, cancer vaccines such as sipuleucel-T, dendritic cell vaccines, and tumor antigen peptide vaccines, chimeric antigen receptor (CAR)-T cells, CAR-NK cells, tumor infiltrating lymphocytes (TILs), adoptively transferred anti-tumor T cells (ex vivo expanded and/or TCR transgenic), and other immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG and imiquimod.

[044] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms; diminishment of extent of disease; preventing or delaying spread (e.g., metastasis, for example metastasis to the lung or to the lymph node) of disease; preventing or delaying recurrence of disease; stabilizing, delaying or slowing of disease progression; amelioration of the disease state; remission (whether partial or total); and improving quality of life. Also encompassed by "treatment" is a reduction of pathological consequence of a proliferative disease. The methods of the invention contemplate any one or more of these aspects of treatment.

[045] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to NHL and other cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably 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. An effective amount can be administered in one or more administrations.

[046] "Adjuvant setting" refers to a clinical setting in which an individual has had a history of a proliferative disease, particularly cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (such as surgical resection), radiotherapy, and chemotherapy. However, because of their history of the proliferative disease (such as cancer), these individuals are considered at risk of development of the disease.

Treatment or administration in the "adjuvant setting" refers to a subsequent mode of treatment. The degree of risk (i.e., when an individual in the adjuvant setting is considered as "high risk" or "low risk") depends upon several factors, most usually the extent of disease when first treated.

[047] The phrase "administering" or "cause to be administered" refers tothe actions taken by a medical professional (e.g., a physician), or a person controlling medical care of a patient, that control and/or permit the administration of the agent(s)/compound(s) at issue to the patient. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic regimen, and/or prescribing particular agent(s)/compounds for a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like. Where administration is described herein, "causing to be administered" is also contemplated.

[048] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine). In various embodiments, the patient can be a human {e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context.

[049] As used herein, the terms "co-administration", "co-administered" and "in combination with", referring to the fusion molecules of the invention and one or more other therapeutic agents, is intended to mean, and does refer to and include the following:

simultaneous administration of such combination of fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said individual; substantially simultaneous administration of such combination of fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said individual, whereupon said components are released at substantially the same time to said individual; sequential administration of such combination of fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said individual with a significant time interval between each administration, whereupon said components are released at substantially different times to said individual; and sequential administration of such combination of fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner whereupon they are concurrently, consecutively, and/or overlappingly released at the same and/or different times to said individual, where each part may be administered by either the same or adifferent route.

[050] The term "antibody" is used herein to refer to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes and having specificity to a tumor antigen or specificity to a molecule overexpressed in a pathological state. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as subtypes of these genes and myriad of immunoglobulin variable region genes. Light chains (LC) are classified as either kappa or lambda. Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.

[051] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, Cm , CH2 and CH3 (and in some instances, CH₄). Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or Vi_) and a light chain constant region. The light chain constant region is comprised of one domain, Ci_. The V_H and V_L 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 V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRi , CDRi , FR₂, CDR₂, FR₃, CDR₃, FR₄. The extent of the framework region and CDRs has been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass.

[052] The CDRs are primarily responsible for binding to an epitope of an antigen. The

CDRs of each chain are typically referred to as CDR1 , CDR₂, CDR₃, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a V_H CDR₃ is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a Vi_ CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[053] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. The Kabat database is now maintained online and CDR sequences can be determined, for example, see IMGT/V-QUEST programme version: 3.2.18 ., March 29, 201 1 , available on the internet and Brochet, X. et al., Nucl. Acids Res. 36, W503-508, 2008). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901 -17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc. Natl. Acad. Sci. (U.S.A.), 86:9268-9272 (1989); "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[054] The term "Fc region" is used to define the C-terminal region of an

immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes (e.g., the neonatal FcR (FcRn) binds to the Fc region of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG). Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (see, e.g., Winter et al., U.S. Patent No. 5,648,260 and 5,624,821 ).

[055] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments. Such fragments include Fab fragments, Fab' fragments, Fab₂, F(ab)'₂ fragments, single chain Fv proteins ("scFv") and disulfide stabilized Fv proteins ("dsFv"), that bind to the target antigen. A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody encompasses e.g., monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')₂ fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragments or antigen binding fragments of any of the above.

[056] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. A "Fab fragment" comprises one light chain and the Cm and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" comprises one light chain and a portion of one heavy chain that contains the V_H domain and the Cm domain and also the region between the Cm and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')₂ molecule.

[057] Pepsin treatment of an antibody yields an F(ab')₂ fragment that has two antigen- combining sites and is still capable of cross-linking antigen. A "F(ab')₂ fragment" contains two light chains and two heavy chains containing a portion of the constant region between the Cm and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')₂ fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[058] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[059] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649, U.S. Patent No. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[060] The terms "an antigen-binding fragment" and "antigen-binding protein" as used herein means any protein that binds a specified target antigen. "Antigen-binding fragment" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments. An exemplary antigen-binding fragment of an antibody is the heavy chain and/or light chain CDR(s), or the heavy and/or light chain variable region.

[061] The term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen binding protein, as used herein, is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to a given epitope. In some

embodiments, the fragments are neutralizing fragments. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain and/or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody, Fab', F(ab')₂, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life.

[062] Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises V_H and V_L regions joined by a linker that is too short to allow for pairing between two regions on the same chain, thus allowing each region to pair with a complementary region on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. (U.S.A.), 90:6444-48 (1993), and Poljak et al., Structure, 2:1 121 -23 (1994)). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

[063] Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). In various embodiments bispecific antibodies can be produced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci. (U.S.A.), 78:5807, 1981 ), by "polydoma" techniques (see, e.g., U.S. Patent No. 4,474,893), or by recombinant DNA techniques. In certain embodiments bispecific antibodies of the present disclosure can have binding specificities for at least two different epitopes at least one of which is a tumor associate antigen. In various embodiments the antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.

[064] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[065] The term "chimeric antibody" as used herein refers to an antibody which has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds targeted antigen.

[066] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR₃. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[067] The term "humanized antibody" as used herein refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

[068] The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant, combinatorial human antibody library; antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V_H and V_L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V_H and V_L sequences, may not naturally exist within the human antibody germline repertoire in vivo. All such recombinant means are well known to those of ordinary skill in the art.

[069] The term "epitope" as used herein includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, e.g., using the techniques described in the present disclosure. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind with one another, e.g., the antibodies compete for binding to the antigen.

[070] An antigen binding protein, including an antibody, "specifically binds" to an antigen if it binds to the antigen with a high binding affinity as determined by a dissociation constant (K_D, or corresponding Kb, as defined below) value of at least 1 x 10^~6 M, or at least 1 x 10^"7 M, or at least 1 x 10 ⁸ M, or at least 1 x 10 ⁹ M, or at least 1 x 10 ¹⁰ M, or at least 1 x 10^~11 M. An antigen binding protein that specifically binds to the human antigen of interest may be able to bind to the same antigen of interest from other species as well, with the same or different affinities. The term "K_D" as used herein refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[071] The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the

BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson U. et al., Ann. Biol. Clin., 51 :19-26, 1993; Jonsson U. et al., Biotechniques, 1 1 :620-627, 1991 ; Jonsson B. et al., J. Mol. Recognit., 8:125-131 , 1995; and Johnsson B. et al., Anal. Biochem., 198:268-277, 1991 . [072] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In certain embodiments, "peptides", "polypeptides", and "proteins" are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free a-amino group on an amino acid at the amino terminal of a peptide or to the a- amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.

[073] The term "recombinant polypeptide", as used herein, is intended to include all polypeptides, including fusion molecules that are prepared, expressed, created, derived from, or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell.

[074] Polypeptides of the disclosure include polypeptides that have been modified in any way and for any reason, for example, to: (1 ) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). A "conservative amino acid substitution" refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:

Alanine (A), Serine (S), and Threonine (T)

Aspartic acid (D) and Glutamic acid (E)

Asparagine (N) and Glutamine (Q)

Arginine (R) and Lysine (K)

Isoleucine (I), Leucine (L), Methionine (M), and Valine (V) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[075] The term "polypeptide fragment" and "truncated polypeptide" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to a corresponding full-length protein. In certain embodiments, fragments can be, e.g., at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids in length. In certain embodiments, fragments can also be, e.g., at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids in length. A fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein {e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence {e.g., an artificial linker sequence).

[076] The terms "polypeptide variant" and "polypeptide mutant" as used herein refers to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. In certain embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length. Variants of the present disclosure include fusion proteins.

[077] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin {e.g., human serum albumin), phosphorylation, and glycosylation.

[078] The term "% sequence identity" is used interchangeably herein with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same thing as 80% sequence identity determined by a defined algorithm, and means that a given sequence is at least 80% identical to another length of another sequence. In certain embodiments, the % identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In certain embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[079] The term "% sequence homology" is used interchangeably herein with the term

"% homology" and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence. In certain embodiments, the % homology is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In certain embodiments, the % homology is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[080] Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and T BLASTX, BLASTP and TBLASTN, publicly available on the Internet at the NCBI website. See also Altschul et al., 1990, J. Mol. Biol. 215:403-10 (with special reference to the published default setting, i.e., parameters w=4, t=17) and Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 1 1 .0, and an extended gap penalty of 1 .0, and utilize the BLOSUM-62 matrix. See id.

[081] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is, e.g., less than about 0.1 , less than about 0.01 , or less than about 0.001 .

[082] "Polynucleotide" refers to a polymer composed of nucleotide units.

Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-0- methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

[083] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[084] "Complementary" refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions. [085] "Hybridizing specifically to" or "specific hybridization" or "selectively hybridize to", refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence-dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, N.Y.; Sambrook et al., 2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3.sup.rd ed., NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[086] Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than about 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCI at 72 °C for about 15 minutes. An example of stringent wash conditions is a 0.2 x SSC wash at 65 °C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A high stringency wash can be preceded by a low stringency wash to remove background probe signal. An exemplary medium stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 1 x SSC at 45 °C for 15 minutes. An exemplary low stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 4-6 x SSC at 40 °C for 15 minutes. In general, a signal to noise ratio of 2 x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific

hybridization.

[087] "Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a

complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

[088] "Probe," when used in reference to a polynucleotide, refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In instances where a probe provides a point of initiation for synthesis of a

complementary polynucleotide, a probe can also be a primer.

[089] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), wherein additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a chosen polynucleotide.

[090] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene

Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res. 23:3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the nucleotide sequence.

[091] A "host cell" is a cell that can be used to express a polynucleotide of the disclosure. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[092] The term "isolated molecule" (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that by virtue of its origin or source of derivation (1 ) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be "isolated" from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification. [093] A protein or polypeptide is "substantially pure," "substantially homogeneous," or

"substantially purified" when at least about 60% to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as

polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[094] "Linker" refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded or otherwise severed to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers may also be cleaved by environmental cues, such as, for example, changes in temperature, pH, salt concentration, etc.

[095] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to that amount of an agent effective to produce the intended

pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21 st Ed. 2005, Mack Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[096] It is understood that aspect and embodiments of the invention described herein include "consisting" and/or "consisting essentially of" aspects and embodiments. [097] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[098] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

Immune-Checkpoint Protein Antigens and Antibodies

[099] The term "antigen" as used herein refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes. 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 three, at least five, or at least eight to ten amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

[0100] As relates to "targeted antigens" for the antibodies to be used in the preparation of the fusion molecules of present disclosure, the primary focus will be on those antigens which are immune-checkpoint protein antigens, including, but are not limited to, CD276/B7-H3

(Chapoval et al., Nature Immunol., 2:269-274, 2001 ; Sharp et al., Nature Rev. Immunol., 2:1 16- 126, 2002), CD272/BTLA (Watanabe N, et al., Nat Immunol 4 (7): 670-9, 2003), CD152/CTLA-4 (Harper K, et al., J Immunol., 147(3):1037-44, 1991 ) LAG-3/CD223 (Annunziato F, et al., FASEB J., 10(7):769-76, 1996), CD279/PD-1 (Blank C, et al., Cancer Immunol Immunother., 56(5):739-45, 2007), CD274/PD-L1 (Brahmer et al., N. Engl J Med., 366:2455-2465, 2012), TIM-3 (Hastings et al., Eur J. Immunol, 39(9):2492-2501 , 2009) and B7-H4 (Sica et al.,

Immunity, 18(6):849-861 , 2003). Each of these cited references is hereby incorporated by reference in its entirety for the specific antigens and sequences taught therein.

[0101] A number of immune-checkpoint protein antigens have been reported to be expressed on various immune cells, including, e.g., CD152 (expressed by activated CD8 T cells, CD4 T cells and regulatory T cells), CD279 (expressed on tumor infiltrating lymphocytes, expressed by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, anergic T cells, monocytes, dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, pancreatic islet cells), and CD223 (expressed by activated T cells, regulatory T cells, angergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells)(see, e.g., Pardoll, D., Nature Reviews Cancer, 12:252-264, 2012).

[0102] In various embodiments, the fusion molecules of the present disclosure comprise an immune-checkpoint protein antigen that is present on the surface of an immune cell. In various embodiments, the immune-checkpoint protein antigen is selected from the group consisting of CD276, CD272, CD152, CD223, CD279, CD274, TIM-3 and B7-H4.

[0103] In various embodiments, the immune-checkpoint protein antigen is present on the surface of a tumor cell. These include, but are not limited to, PD-L1 , B7-H3 and B7-H4.

[0104] Importantly, when the fusion molecules of the present disclosure comprise an antibody that targets an immune-checkpoint protein antigen on the surface of (e.g., expressed on or associated with) a tumor cell, the "targeting" provided by the antibody-interferon ("Ab-IFN") fusion molecule serves to increase the local concentration of IFN in the tumor microenvironment which has significant implications. For example, depending upon the antibody and/or IFN being used to prepare the fusion molecule, the increase in local concentration of IFN in the tumor microenvironment provided by the Ab-IFN fusion molecules of the invention may provide, e.g., one or more of the following advantages: 1 ) the induction of direct cell death of tumor cells by engaging IFN-ocR expressed on tumor cells; 2) negating some of the suppressive actions of tumors on dendritic cells (DCs), potentially leading to more efficient cross presentation of tumor antigens to T cells by DCs, i.e., improve CD8+ CTL priming; 3) the direct activation of the CD8+ CTL functions by the IFN which will allow efficient killing of tumor cells; 4) the direct activation of NK cell functions by the IFN which will allow efficient killing of tumor cells; 5) the induction of up- regulation of the co-inhibitory immune-checkpoint proteins expressed on or associated with tumor cells; and 6) directly negate other mechanisms for immune evasion, e.g., the major inhibitory pathways mediated by certain immune-checkpoint proteins on T cells/B cells. These effects, alone or collectively, may lead to an enhancement of an anti-tumor immune response and/or killing (either direct or indirect) of tumor cells. Accordingly, the fusion molecules would provide improved, effective methods used to effectively treat cancers, including recurrent, resistant, or refractory cancers, at surprisingly low doses.

[0105] Similarly, when the novel fusion molecules of the invention comprise an antibody that targets an immune-checkpoint protein antigen that is on the surface of (e.g., expressed on or associated with) an immune cell, the "targeting" provided by Ab-IFN fusion molecules has significant implications. For example, depending upon the antibody and/or IFN being used to prepare the fusion molecule, the Ab-IFN fusion molecules may provide, e.g., one or more of following advantages: 1 ) the inhibition or reduction of the downregulatory activity of the immune- checkpoint protein on the immune cells; 2) the stimulation and activation of immune cells in secondary lymphoid organs (e.g., draining lymph nodes, spleen); and 3) the stimulation and activation of immune cells that are present in the tumor microenvironment by directly binding to them. These effects may be mediated by the binding of the immune-checkpoint antibody to the immune-checkpoint protein antigen and blocking its ability to interact with its corresponding ligand. These effects may also be mediated by the increase in local concentration of IFN near the immune cell surface and binding of the IFN portion of Ab-IFN to its receptor IFNAR on the surface of the immune cell. This immune cell may be either the same immune cell that Ab-IFN is binding to or a nearby neighboring immune cell. These effects, alone or collectively, may lead to an enhancement of an anti-tumor host immune response and/or indirect killing of tumor cells.

[0106] Importantly, the advantages cited above open up several avenues for improving both immune-checkpoint protein targeted immunotherapies, and non-immune-checkpoint protein targeted immunotherapies. For example, the advantages provided by the Ab-IFN fusion molecules suggest an opportunity for combination therapy using an Ab-IFN fusion molecule described herein, with an immune-checkpoint protein antigen inhibitor (e.g., an anti-immune- checkpoint protein antibody). This combination therapy may serve to enhance the long-term efficacy of the Ab-IFN fusion molecule itself, and further enhance specific antitumor T cell responses at a local level within the tumor microenvironment via the action of the immune- checkpoint protein antigen inhibitor, i.e., the combination will alter the tumor microenvironment to the tumor's disadvantage. Importantly, this combination therapy may increase the overall response rates in patients that initially fail to respond to anti-immune-checkpoint protein therapy and other targeted immunotherapies, may help address intrinsic antibody resistance that is common with many current therapies, and may be effective in treating rogue, tough to treat tumors.

[0107] It is further envisioned the advantages provided by the Ab-IFN fusions described herein suggest an opportunity for combination therapy involving, e.g., other immune-checkpoint protein antigen inhibitors, biologies, small molecules, depleting antibodies, or antibody drug conjugates which are useful for anti-cancer treatment. While many proteins have been identified as immune-checkpoint proteins, the functions of these proteins may be non- overlapping. A combination therapy regimen that targets more than one checkpoint proteins thus may provide more effective therapeutic options than targeting each immune-checkpoint protein alone. The present application contemplates the combined use of any one of the fusion molecules described herein with a second immune-checkpoint inhibitor. Also contemplated are combined use of any one of the fusion molecules described herein with a second agent (such as biologies, small molecules, depleting antibodies, or antibody drug conjugates).

[0108] The fusion molecules of the present disclosure may bind one antigen or multiple antigens and the antibodies used in the preparation of the fusion molecules of the present disclosure may also be described or specified in terms of their cross-reactivity. As such, antibodies that bind immune-checkpoint protein antigens, which have at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to immune-checkpoint protein antigens described in the cited references are also included in the present disclosure.

[0109] Methods of generating antibodies that bind to antigens such as the immune- checkpoint proteins described herein are known to those skilled in the art. For example, a method for generating a monoclonal antibody that binds specifically to a targeted antigen polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the targeted antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing

hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the targeted antigen polypeptide. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to targeted antigen polypeptide. The monoclonal antibody may be purified from the cell culture. A variety of different techniques are then available for testing an antigen/antibody interaction to identify particularly desirable antibodies.

[0110] Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select recombinant antibody from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies. See e.g., Jakobovits et al., Proc. Natl. Acad. Sci. (U.S.A.), 90: 2551 -2555, 1993; Jakobovits et al., Nature, 362: 255-258, 1993; Lonberg et al., U.S. Pat. No. 5,545,806; and Surani et al., U.S. Pat. No. 5,545,807. [0111] Antibodies can be engineered in numerous ways. They can be made as single- chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab')₂ fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human.

Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.

[0112] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al., Science, 240:1041 -1043, 1988; Liu et al., Proc. Natl. Acad. Sci. (U.S.A.), 84:3439-3443, 1987; Liu et al., J. Immunol., 139:3521 -3526, 1987; Sun et al., Proc. Natl. Acad. Sci. (U.S.A.), 84:214-218, 1987; Nishimura et al., Cane. Res., 47:999-1005, 1987; Wood et al., Nature, 314:446-449, 1985; and Shaw et al., J. Natl Cancer Inst., 80:1553- 1559, 1988).

[0113] Methods for humanizing antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced from a source that is nonhuman, in addition to the nonhuman CDRs. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525, 1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S.

Patent No. 4,816,567) wherein substantially less than an intact human variable region has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0114] U.S. Patent No. 5,693,761 to Queen et al, discloses a refinement on Winter et al. for humanizing antibodies, and is based on the premise that ascribes avidity loss to problems in the structural motifs in the humanized framework which, because of steric or other chemical incompatibility, interfere with the folding of the CDRs into the binding-capable conformation found in the mouse antibody. To address this problem, Queen teaches using human framework sequences closely homologous in linear peptide sequence to framework sequences of the mouse antibody to be humanized. Accordingly, the methods of Queen focus on comparing framework sequences between species. Typically, all available human variable region sequences are compared to a particular mouse sequence and the percentage identity between correspondent framework residues is calculated. The human variable region with the highest percentage is selected to provide the framework sequences for the humanizing project. Queen also teaches that it is important to retain in the humanized framework, certain amino acid residues from the mouse framework critical for supporting the CDRs in a binding-capable conformation. Potential criticality is assessed from molecular models. Candidate residues for retention are typically those adjacent in linear sequence to a CDR or physically within 6A of any CDR residue.

[0115] In other approaches, the importance of particular framework amino acid residues is determined experimentally once a low-avidity humanized construct is obtained, by reversion of single residues to the mouse sequence and assaying antigen binding as described by Riechmann et al, 1988. Another example approach for identifying important amino acids in framework sequences is disclosed by U.S. Patent No. 5,821 ,337 to Carter et al, and by U.S. Patent No. 5,859,205 to Adair et al. These references disclose specific Kabat residue positions in the framework, which, in a humanized antibody may require substitution with the

correspondent mouse amino acid to preserve avidity.

[0116] Another method of humanizing antibodies, referred to as "framework shuffling", relies on generating a combinatorial library with nonhuman CDR variable regions fused in frame into a pool of individual human germline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). The libraries are then screened to identify clones that encode humanized antibodies which retain good binding.

[0117] The choice of human variable regions, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (framework region) for the humanized antibody (Sims et al., J. Immunol., 151 :2296, 1993; Chothia et al., J. Mol. Biol., 196:901 , 1987). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. (U.S.A.), 89:4285, 1992; Presta et al., J. Immunol.,

151 :2623, 1993).

[0118] The choice of nonhuman residues to substitute into the human variable region can be influenced by a variety of factors. These factors include, for example, the rarity of the amino acid in a particular position, the probability of interaction with either the CDRs or the antigen, and the probability of participating in the interface between the light and heavy chain variable domain interface. (See, for example, U.S. Patent Nos. 5,693,761 , 6,632,927, and 6,639,055). One method to analyze these factors is through the use of three-dimensional models of the nonhuman and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, nonhuman residues can be selected and substituted for human variable region residues in order to achieve the desired antibody characteristic, such as increased affinity for the target antigen(s).

[0119] Methods for making fully human antibodies have been described in the art. By way of example, a method for producing an anti-CD279 antibody or antigen-binding fragment thereof comprises the steps of synthesizing a library of human antibodies on phage, screening the library with CD279 or an antibody-binding portion thereof, isolating phage that bind CD279, and obtaining the antibody from the phage. By way of another example, one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci with CD279 or an antigenic portion thereof to create an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding heavy and light chains of antibodies of the disclosure from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage. Recombinant anti-CD279 antibodies of the disclosure may be obtained in this way.

[0120] Again, by way of example, recombinant human anti-CD279 antibodies of the disclosure can also be isolated by screening a recombinant combinatorial antibody library. Preferably the library is a scFv phage display library, generated using human V_L and V_H cDNAs prepared from mRNA isolated from B cells. Methods for preparing and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene SurfZAP™ phage display kit, catalog no. 240612). There also are other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91 /17271 , WO 92/20791 , WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio/Technology, 9:1370-1372 (1991 ); Hay et al., Hum. Antibod. Hybridomas, 3:81 -85, 1992; Huse et al., Science, 246:1275-1281 , 1989; McCafferty et al., Nature, 348:552-554, 1990; Griffiths et al., EMBO J., 12:725-734, 1993; Hawkins et al., J. Mol. Biol., 226:889-896, 1992; Clackson et al., Nature, 352:624-628, 1991 ; Gram et al., Proc. Natl. Acad. Sci. (U.S.A.), 89:3576-3580, 1992; Garrad et al., Bio/Technology, 9:1373-1377, 1991 ; Hoogenboom et al., Nuc. Acid Res., 19:4133-4137, 1991 ; and Barbas et al., Proc. Natl. Acad. Sci. (U.S.A.), 88:7978-7982, 1991 ), all incorporated herein by reference.

[0121] Human antibodies are also produced by immunizing a non-human, transgenic animal comprising within its genome some or all of human immunoglobulin heavy chain and light chain loci with a human IgE antigen, e.g., a XenoMouse™ animal (Abgenix, Inc./Amgen, Inc.-Fremont, Calif.). XenoMouse™ mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 , 1994 and U.S. Pat. Nos. 5,916,771 , 5,939,598, 5,985,615, 5,998,209, 6,075,181 , 6,091 ,001 , 6,1 14,598, 6,130,364, 6,162,963 and 6,150,584. XenoMouse™ mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies. In some embodiments, the XenoMouse™ mice contain approximately 80% of the human antibody V gene repertoire through introduction of megabase sized, germline configuration fragments of the human heavy chain loci and kappa light chain loci in yeast artificial chromosome (YAC). In other

embodiments, XenoMouse™ mice further contain approximately all of the human lambda light chain locus. See Mendez et al., Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J. Exp. Med., 188:483-495, 1998; and WO 98/24893.

[0122] In various embodiments, the fusion molecules of the present disclosure utilize an antibody or antigen-binding fragment thereof is a polyclonal antibody, a monoclonal antibody or antigen-binding fragment thereof, a recombinant antibody, a diabody, a chimerized or chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, a single chain antibody, an Fv, an Fd, an Fab, an Fab', or an F(ab')₂, and synthetic or semi-synthetic antibodies.

[0123] In various embodiments, the fusion molecules of the present disclosure utilize an antibody or antigen-binding fragment that binds to an immune-checkpoint protein antigen with a dissociation constant (K_D) of, e.g., at least about 1 x10 ³ M, at least about 1 x10 ⁴ M, at least about 1 x10 ⁵ M, at least about 1 x10^-6 M, at least about 1 x10 ⁷ M, at least about 1 x10^-8 M, at least about 1 x10 ⁹ M, at least about 1 x10 ¹⁰ M, at least about 1 x10 ¹¹ M, or at least about 1 x10 ¹² M. In various embodiments, the fusion molecules of the present disclosure utilize an antibody or antigen-binding fragment that binds to an immune-checkpoint protein antigen with a dissociation constant (K_D) in the range of, e.g., at least about 1 x10 ³ M to at least about 1 x10^~4 M, at least about 1 x10 ⁴ M to at least about 1 x10 ⁵ M, at least about 1 x10 ⁵ M to at least about 1 x10^-6 M, at least about 1 x10 ⁶ M to at least about 1 x10 ⁷ M, at least about 1 x10 ⁷ M to at least about 1 x10 ⁸ M, at least about 1 x10 ⁸ M to at least about 1 x10 ⁹ M, at least about 1 x10 ⁹ M to at least about 1 x10 ¹⁰ M, at least about 1 x10 ¹⁰ M to at least about 1 x10 ¹¹ M, or at least about 1 x10 ¹¹ M to at least about 1 x10^"12 M.

[0124] In various embodiments, the antibody portion of the fusion molecules exhibits one or more of the following properties: (a) high affinity binding to the immune-checkpoint protein antigen; (b) the ability to inhibit and neutralize the action of the immune-checkpoint protein antigen; (c) the ability to exhibit antibody dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing the immune-checkpoint protein antigen; (d) the ability to promote tumor cell apoptosis when the immune-checkpoint protein antigen is present on the surface of a tumor cell; and (e) the ability to localize to tumors expressing the immune-checkpoint protein antigen when the immune-checkpoint protein antigen is present on the surface of a tumor cell.

[0125] In various embodiments, the antibody portion of the fusion molecule exhibits at least two of properties (a) - (e). In various embodiments, the antibody portion of the fusion molecule exhibits at least three of properties (a) - (e). In various embodiments, the antibody or antigen-binding fragment exhibits at least four of properties (a) - (e). In various embodiments, the antibody portion of the fusion molecule exhibits all five of properties (a) - (e).

[0126] In various embodiments, the fusion molecules of the present disclosure utilize an antibody or antigen-binding fragment that cross-competes for binding to the same epitope on the immune-checkpoint protein antigen as a reference antibody which comprises the

heavy chain variable region and light chain variable region set forth in the references and sequence listings provided herein. [0127] Antibodies that bind to an antigen which is determined to be an immune- checkpoint protein are known to those skilled in the art. For example, various anti-CD276 antibodies have been described in the art (see, e.g., U.S. Pat. Public. No. 20120294796

(Johnson et al) and references cited therein); various anti-CD272 antibodies have been described in the art (see, e.g., U.S. Pat. Public. No. 20140017255 (Mataraza et al) and references cited therein); various anti-CD152/CTLA-4 antibodies have been described in the art (see, e.g., U.S. Pat. Public. No. 20130136749 (Korman et al) and references cited therein); various anti-LAG-3/CD223 antibodies have been described in the art (see, e.g., U.S. Pat.

Public. No. 201 10150892 (Thudium et al) and references cited therein); various anti-CD279/PD- 1 antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al) and references cited therein); various anti-PD-L1 antibodies have been described in the art (see, e.g., U.S. Pat. Public. No. 20130122014 (Korman et al) and references cited therein); various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Pat. Public. No. 20140044728 (Takayanagi et al) and references cited therein); and various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Pat. Public. No. 201 10085970 (Terrett et al) and references cited therein). Each of these references is hereby incorporated by reference in its entirety for the specific antibodies and sequences taught therein.

[0128] In various embodiments of the present disclosure, the antibody may be an anti-

CD276 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 1 . In various embodiments, the antibody may be an anti-CD276 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 1 . In various embodiments, the antibody is an anti-CD276 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 1 . In various embodiments, the antibody may be an anti-CD276 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 1 . In various embodiments, the antibody may be an anti-CD276 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 1 :

DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSDSSA IYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCGRGRENIYYGSRLDYWGQG TTLTVSS (SEQ ID NO: 1 )

[0129] In various embodiments of the present disclosure the antibody may be an anti-

CD276 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 2. In various embodiments, the antibody may be an anti-CD276 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 2. In various embodiments, the antibody is an anti-CD276 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 2. In various embodiments, the antibody may be an anti-CD276 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 2. In various embodiments, the antibody may be an anti-CD276 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 2:

DIAMTQSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRYSG

VPDRFTGSGSGTDFTLTINNVQSEDLAEYFCQQYNNYPFTFGSGTKLEIK

(SEQ ID NO: 2)

[0130] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 1 or 2.

[0131] In various embodiments of the present disclosure the antibody may be an anti-

CD272 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 3. In various embodiments, the antibody may be an anti-CD272 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 3. In various embodiments, the antibody is an anti-CD272 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 3. In various embodiments, the antibody may be an anti-CD272 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 3. In various embodiments, the antibody may be an anti-CD272 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 3:

QVQLQESGPGLVKPSETLSLTCTVHGGSINHYYWSWIRQPPGKGLEWIGYIYYSGST KYNPSLKSRVSISVDTSKNQFSLKLTSVTAADTAVYYCAREWPYYYYEMDVWGQG TTVTVSS (SEQ ID NO: 3)

[0132] In various embodiments of the present disclosure the antibody may be an anti-

CD272 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 4. In various embodiments, the antibody may be an anti-CD272 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 4. In various embodiments, the antibody is an anti-CD272 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 4. In various embodiments, the antibody may be an anti-CD272 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 4. In various embodiments, the antibody may be an anti-CD272 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 4:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATG

IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFRTFGQGTKVEIK

(SEQ ID NO: 4)

[0133] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 3 or 4.

[0134] In various embodiments of the present disclosure the antibody may be an anti-

CD152 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 5. In various embodiments, the antibody may be an anti-CD152 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 5. In various embodiments, the antibody is an anti-CD152 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 5. In various embodiments, the antibody may be an anti-CD152 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 5. In various embodiments, the antibody may be an anti-CD152 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 5:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDG NNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQG TLVTVSS (SEQ ID NO: 5)

[0135] In various embodiments of the present disclosure the antibody may be an anti-

CD152 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 6. In various embodiments, the antibody may be an anti-CD152 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 6. In various embodiments, the antibody is an anti-CD152 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 6. In various embodiments, the antibody may be an anti-CD152 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 6. In various embodiments, the antibody may be an anti-CD152 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 6:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

(SEQ ID NO: 6)

[0136] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 5 or 6.

[0137] In various embodiments of the present disclosure the antibody may be an anti-

CD223 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 7. In various embodiments, the antibody may be an anti-CD223 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 7. In various embodiments, the antibody is an anti-CD223 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 7. In various embodiments, the antibody may be an anti-CD223 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 7. In various embodiments, the antibody may be an anti-CD223 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 7:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHNGNT NSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGT LVTVSS (SEQ ID NO: 7)

[0138] In various embodiments of the present disclosure the antibody may be an anti-

CD223 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 8. In various embodiments, the antibody may be an anti-CD223 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 8. In various embodiments, the antibody is an anti-CD223 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 8. In various embodiments, the antibody may be an anti-CD223 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 8. In various embodiments, the antibody may be an anti-CD223 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 8:

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTHTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK (SEQ ID NO: 8)

[0139] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 7 or 8.

[0140] In various embodiments of the present disclosure the antibody may be an anti-

CD279 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody may be an anti-CD279 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody is an anti-CD279 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody may be an anti-CD279 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody may be an anti-CD279 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 9:

EVQLVQSGAEVKKPGASVKVSCKASGYRFTSYGISWVRQAPGQGLEWMGWISA YNGNTNYAQKLQGRVTMTTDTSTNTAYMELRSLRSDDTAVYYCARDADYSSG SGYWGQGTLVTVSS (SEQ ID NO: 9)

[0141] In various embodiments of the present disclosure the antibody may be an anti-

CD279 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 10. In various embodiments, the antibody may be an anti-CD279 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 10. In various embodiments, the antibody is an anti-CD279 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 10. In various embodiments, the antibody may be an anti-CD279 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 10. In various embodiments, the antibody may be an anti-CD279 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 10:

SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVMVIYKDTERP SGIPERFSGSSSGTKVTLTISGVQAEDEADYYCQSADNSITYRVFGGGTKVTVL

(SEQ ID NO: 10)

[0142] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 9 or 10.

[0143] In various embodiments of the present disclosure the antibody may be an anti-

CD274 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 1 1 . In various embodiments, the antibody may be an anti-CD274 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 1 1 . In various embodiments, the antibody is an anti-CD274 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 1 1 . In various embodiments, the antibody may be an anti-CD274 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 1 1 . In various embodiments, the antibody may be an anti-CD274 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 1 1 :

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNG NTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDYFYGMDVWGQGTT VTVSS (SEQ ID NO: 1 1 )

[0144] In various embodiments of the present disclosure the antibody may be an anti-

CD274 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 12. In various embodiments, the antibody may be an anti-CD274 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 12. In various embodiments, the antibody is an anti-CD274 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 12. In various embodiments, the antibody may be an anti-CD274 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 12. In various embodiments, the antibody may be an anti-CD274 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 12:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIP

ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK

(SEQ ID NO: 12)

[0145] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 1 1 or 12.

[0146] In various embodiments of the present disclosure the antibody may be an anti-

TIM-3 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 13. In various embodiments, the antibody may be an anti-TIM-3 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 13. In various embodiments, the antibody is an anti-TIM-3 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 13. In various embodiments, the antibody may be an anti-TIM-3 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 13. In various embodiments, the antibody may be an anti-TIM-3 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 13:

MKHLWFFLLLAAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSFSRGGYYWNW IRQPPGKGLQWIGYIYYSGSTNYNPSLKSRVTISLDTHKNQFSLKLSSVTAADTAVYYCA RHYSSSWTFDYWGQGTLVTVSS (SEQ ID NO: 13)

[0147] In various embodiments of the present disclosure the antibody may be an anti-

TIM-3 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 14. In various embodiments, the antibody may be an anti-TIM-3 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 14. In various embodiments, the antibody is an anti-TIM-3 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 14. In various embodiments, the antibody may be an anti-TIM-3 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 14. In various embodiments, the antibody may be an anti-TIM-3 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 14:

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGQRATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTF GQGTKLEIK (SEQ ID NO: 14)

[0148] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 13 or 14.

[0149] In various embodiments of the present disclosure the antibody may be an anti-

B7-H4 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 15. In various embodiments, the antibody may be an anti-B7-H4 antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 15. In various embodiments, the antibody is an anti-B7-H4 antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 15. In various embodiments, the antibody may be an anti-B7-H4 antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 15. In various embodiments, the antibody may be an anti-B7-H4 antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 15:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYFWTWIRQPPGKGLEWIGEINHSGTT NYNPSLKSRVTISADTSKNQFSLRLSSVTAADTAVYYCARLSSWSNWAFEYWGQGTLV TVSS (SEQ ID NO: 15)

[0150] In various embodiments of the present disclosure the antibody may be an anti-

B7-H4 antibody that has the same or higher antigen-binding affinity as that of the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 16. In various embodiments, the antibody may be an anti-B7-H4 antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 16. In various embodiments, the antibody is an anti-B7-H4 antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 16. In various embodiments, the antibody may be an anti-B7-H4 antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 16. In various embodiments, the antibody may be an anti-B7-H4 antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 16:

EIVLTQFPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRVLIYGASRRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK

(SEQ ID NO: 16)

[0151] In various embodiments, the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 15 or 16.

[0152] In various embodiments, the antibodies or antigen-binding fragments thereof comprise a heavy chain variable region comprising a sequence of amino acids that differs from the sequence of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NOs: 1 , 3, 5, 7, 9, 1 1 , 13 and 15 only at 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 residues, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue.

[0153] In various embodiments, the antibodies or antigen-binding fragments thereof comprise a light chain variable region comprising a sequence of amino acids that differs from the sequence of a light chain variable region having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16 only at 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 residues, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue.

Interferon and interferon mutants

[0154] In various embodiments of the present disclosure, either the N- or C- terminus of an antibody, or antigen-binding fragment heavy or light chain will be genetically constructed with one of the several contemplated interferons or interferon mutants. Interferons include type I interferons (e.g., IFN-a, IFN-β) as well as type II interferons (e.g., IFN-γ). The term "interferon" as used herein refers to a full-length interferon or to an interferon fragment (truncated interferon) or to an interferon mutant (truncated interferon and interferon mutant collectively referred to herein as 'modified interferon'), that substantially retains the biological activity of the full length wild-type interferon (e.g., retains at least 50%, for example at least about any of 60%, 70%, 80%, 90%, or more biological activity of the full length wild-type interferon). The interferon can be from essentially any mammalian species. In various embodiments, the interferon is from a species selected from the group consisting of human, equine, bovine, rodent, porcine, lagomorph, feline, canine, murine, caprine, ovine, a non-human primate, and the like.

[0155] In various embodiments, the Ab-IFN fusion molecules comprise an interferon or a modified interferon that possesses, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, of the endogenous activity of the wild-type interferon having the same amino acid sequence but not attached to an antibody.

[0156] In various embodiments, the Ab-IFN fusion molecules will comprise an interferon or a modified interferon that possesses, e.g., less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, less than 1 00%, of the endogenous activity of the wild-type interferon having the same amino acid sequence but not attached to an antibody.

[0157] In various embodiments, the Ab-IFN fusion molecules will comprise an interferon or a modified interferon that possesses, e.g., more than 5 times, more than 1 0 times, more than 15 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, more than 1 00 times, more than 1 25 times, more than 150 times, more than 1 75 times, more than 200 times, more than 250 times, more than 300 times, more than 400 times, more than 500 times, more than 750 times, and more than 1000 times, the endogenous activity of the wild-type interferon having the same amino acid sequence but not attached to an antibody.

[0158] Interferon activity can be assessed, for example, using the various anti-viral and anti-proliferative assays described in art (see, e.g., U.S. Patent No. 8,563,692, U.S. Pat. Public. No. 201 30230517, U.S. Pat. Public. No. 201 1 01 58905, PCT WO/2014/028502, and PCT WO/2013/059885) as well as the assays described in the Examples section below.

[0159] In various embodiments, the Ab-IFN fusion molecules will show at least 10, at least 100, at least 1000, at least 10,000 or at least 100,000-fold selectivity toward cells that express the immune-checkpoint protein antigen to which the Ab binds over cells that do not express the immune-checkpoint protein antigen, when compared to interferon having the same amino acid sequence not attached to an antibody.

[0160] In various embodiments of the present disclosure, the interferon mutant comprises one or more amino acid substitutions, insertions, and/or deletions. Means of identifying such modified interferon molecules are routine to those of skill in the art. In one illustrative approach, a library of truncated and/or mutated IFN-a is produced and screened for IFN-a activity. Methods of producing libraries of polypeptide variants are well known to those of skill in the art. Thus, for example, error-prone PCR can be used to create a library of mutant and/or truncated IFN-a (see, e.g., U.S. Patent No. 6,365,408). The resultant library members can then be screened according to standard methods know to those of skill in the art. Thus, for example, IFN-a activity can be assayed by measuring antiviral activity against a particular test virus. Kits for assaying for IFN-a activity are commercially available (see, e.g., I LITE™ alphabeta kit by Neutekbio, Ireland).

[0161] The use of chemically modified interferons is also contemplated. For example, in certain embodiments, the interferon is chemically modified to increase serum half-life. Thus, for example, (2-sulfo-9-fluorenylmethoxycarbony1 )₇-interferon-a2 undergoes time-dependent spontaneous hydrolysis, generating active interferon (Shechter et al., Proc. Natl. Acad. Sci., USA, 98(3): 1212-1217, 2001 ). Other modifications, include for example, N-terminal modifications in including, but not limited to the addition of PEG, protecting groups, and the like (see, e.g., U.S. Patent No. 5,824,784).

[0162] In various embodiments, the interferon contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the wildtype IFN-β-Ι a sequence provided below as SEQ ID NO: 17. In some embodiments, the mutant interferon has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 1 x, at least 1 .5x, at least 2x, at least 2.5x, or at least 3x activity of wildtype IFN-β-Ι a provided below as SEQ ID NO: 17. In some embodiments, the mutant interferon has less than any of about 70%, 75%, 80%, 85%, 90%, or 95%, activity of wildtype IFN-β-Ι a provided below as SEQ ID NO: 17:

MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKE DAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLE KEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRL TGYLRN (SEQ ID NO: 17)

[0163] In various embodiments, the interferon contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the wildtype IFN-β-Ι b sequence provided below as SEQ ID NO: 18. In some embodiments, the mutant interferon has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 1 x, at least 1 .5x, at least 2x, at least 2.5x, or at least 3x activity of wildtype IFN-β-Ι b provided below as SEQ ID NO: 18. In some embodiments, the mutant interferon has less than any of about 70%, 75%, 80%, 85%, 90%, or 95%, activity of wildtype IFN- -1 b provided below as SEQ ID NO: 18:

MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKE DAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKL EKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR LTGYLRN (SEQ ID NO: 18)

[0164] In various embodiments use of a mutated IFN-β is contemplated. A mutated

IFN-β comprising a serine substituted for the naturally occurring cysteine at amino acid 17 of IFN- -1 a has also been demonstrated to show efficacy (Hawkins et al., Cancer Res., 45:5914- 5920, 1985). Certain C-terminally truncated IFN- -1 a's have been shown to have increased activity (see, e.g., U.S. Patent Publication 2009/0025106 A1 ). Accordingly, in certain embodiments the interferons used in the fusion molecules described herein include the C- terminally truncated IFN-β described as IFN-ΔΙ , IFN-A2, IFN-A3, IFN-A4, IFN-A5, IFN-A6, IFN- Δ7, IFN-Δδ, IFN-A9, IFN-ΔΙ Ο in US 2009/0025106 A1 . This reference is incorporated by reference in its entirety herein for purposes of the interferon mutants and sequences provided therein.

[0165] In various embodiments, the interferon contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the wildtype IFN-oc2 sequence provided below as SEQ ID NO: 19. In some embodiments, the mutant interferon has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 1 x, at least 1 .5x, at least 2x, at least 2.5x, or at least 3x activity of wildtype IFN-oc2 provided below as SEQ ID NO: 19. In some embodiments, the mutant interferon has less than any of about 70%, 75%, 80%, 85%, 90%, or 95%, activity of wildtype IFN-cc2 provided below as SEQ ID NO: 19.

CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKA ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVI QGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS FSLSTNLQESLRSKE (SEQ ID NO: 19)

[0166] In various embodiments use of a mutated IFN-a is contemplated. Single point mutations contemplated for use herein include, but are not limited to, a series of mostly single point mutants (see Table 1 below) that are considered important to the binding affinity of IFN-a to IFN-aR1 based on published information on NMR structure with the assumption that a single point mutation may change the binding affinity but will not completely knock off the activity of IFN-a, therefore still retaining the anti-proliferative properties albeit at much higher

concentrations. This will potentially improve the therapeutic index of the fusion molecules comprising an antibody fused to the interferon-alpha mutants. As described herein and as depicted in Table 1 , a single mutation will be identified by the particular amino acid substitution at a specific amino acid position within the sequence of wildtype IFN-a2 provided as SEQ ID NO: 19. For example, a mutation comprising a tyrosine substituted for the full length wild type histidine at amino acid 57 is identified as H57Y.

Table 1

List of proposed IFN-a Mutant Molecules.

M5 Q61 A Decrease the IFN-a-IFN-aR1 binding affinity at Site 1 similar to M2 but only single point

M6 R149A Decrease the IFN-a-IFN-aR1 binding affinity at Site 2 based on loss of binding contacts

M7 R162A Decrease the IFN-a-IFN-aR1 binding affinity at Site 2 based on loss of binding contacts

M8 R149A, R162A Decrease the IFN-a-IFN-aR1 binding affinity at Site 2 based on loss of binding contacts

M9 L30A Decrease the IFN-a-IFN-aR1 binding affinity at Site 2 based on loss of binding contacts

M10 D35E Alter the IFN-a-IFN-aR1 binding at Site 2 based on minimal change in

structure

M1 1 E165D Alter the IFN-a-IFN-aR1 binding at Site 2 based on minimal change in

structure

M12 L26A Alter the IFN-a-IFN-aR1 binding at Site 2 based on minimal change in

structure

M13 F27A Alter the IFN-a-IFN-aR1 binding at Site 2 based on minimal change in

structure

M14 L153A Alter the IFN-a-IFN-aR1 binding at Site 2 based on minimal change in

structure

M15 A145V Alter the IFN-a-IFN-aR1 binding at Site 2 based on minimal change in

structure

[0167] Additional interferon mutants contemplated for use include those described in, e.g., PCT WO 2013/059885 (Wilson et al.), and U.S. Pat. No. 8,258,263 (Morrison et al), each of which is hereby incorporated by reference in its entirety for the interferon mutants and sequences provided therein.

Fusion Molecules

[0168] The present disclosure relates to genetically engineered fusion molecules comprising at least one antibody, or antigen-binding fragment thereof, attached to at least one interferon, or interferon mutant molecule. Generally speaking, the antibody portion and interferon molecule can be joined together in any order. Thus, for example, the interferon molecule(s) can be joined to either the amino or carboxy terminal of the antibody. Alternatively, the antibody can be joined to either the amino or carboxy terminal of the interferon molecule.

[0169] In various embodiments, the antibody and interferon molecule are linked directly to each other without an intervening peptide linker sequence and synthesized using

recombinant DNA methodology. By "linked" we mean that the first and second sequences are associated such that the second sequence is able to be transported by the first sequence to a target cell, i.e., fusion molecules in which the antibody is linked to a IFN- molecule via their polypeptide backbones through genetic expression of a DNA molecule encoding these proteins, directly synthesized proteins, and coupled proteins in which pre-formed sequences are associated by a cross-linking agent.

[0170] In various embodiments, the antibody portion is chemically conjugated to the interferon molecule. Means of chemically conjugating molecules are well known to those of skill.

[0171] The procedure for conjugating two molecules varies according to the chemical structure of the agent. Polypeptides typically contain variety of functional groups; e.g., carboxylic acid (COOH) or free amine (-NH₂) groups, that are available for reaction with a suitable functional group on the other peptide, or on a linker to join the molecules thereto. Alternatively, the antibody and/or the interferon can be derivatized to expose or attach additional reactive functional groups. The derivatization can involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford III. A bifunctional linker having one functional group reactive with a group on the antibody and another group reactive on the interferon, can be used to form the desired conjugate. Alternatively,

derivatization can involve chemical treatment of the antibody portion. Procedures for generation of, for example, free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are known (See U.S. Pat. No. 4,659,839).

[0172] Many procedures and linker molecules for attachment of various compounds including radionuclide metal chelates, toxins and drugs to proteins such as antibodies are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos. 4,671 ,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071 ; and Borlinghaus et al. (1987) Cancer Res. 47: 4071 -4075. In particular, production of various immunotoxins is well- known within the art and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982); Waldmann (1991 ) Science, 252: 1657; U.S. Pat. Nos. 4,545,985 and 4,894,443, and the like.

[0173] In various embodiments, the two molecules can be separated by a peptide linker consisting of one or more amino acids. Generally the linker will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. In certain embodiments, however, the constituent amino acids of the linker can be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. In certain embodiments, the fusion molecule is a recombinantly expressed fusion molecule and will comprise an interferon molecule attached to the antibody via a peptide linker as described herein and as depicted in, e.g., any of the Figures 1 -3. In various embodiments, the antibody portion and the interferon portion of the fusion molecule are linked (e.g., fused) without a linker.

[0174] The term "linker" is used herein to denote polypeptides comprising one or more amino acid residues joined by peptide bonds and are used to link the antibody and interferon molecules of the present disclosure. In various embodiments, the linker is capable of forming covalent bonds to both the antibody and to the interferon. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In certain embodiments, the linker(s) can be joined to the constituent amino acids of the antibody and/or the interferon through their side groups {e.g., through a disulfide linkage to cysteine). In certain preferred embodiments, the linkers are joined to the alpha carbon amino and/or carboxyl groups of the terminal amino acids of the antibody and/or the interferon. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. (U.S.A.), 90:6444, 1993; Poljak, R. J., et al., Structure, 2:1 121 , 1994). Linker length contemplated for use can vary from about 5 to 200 amino acids. In various embodiments, the linker may be a proteolysis-resistant linker of 1 to 20 amino acids in length (see, e.g., U.S. Pat. No. 8,258,263 (Morrison et al.), hereby incorporated by reference in its entirety for the proteolysis-resistant linkers and sequences provided therein).

[0175] In various embodiments, the linker is an a-helical linker. In various

embodiments, the linker is rich in G/S content (e.g., at least about 60%, 70%, 80%, 90%, or more of the amino acids in the linker are G or S. In various embodiments, the linker is rich in G/C content and is less than about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid long. In various embodiments, the linker is an a-helical linker and is less than about any of 7, 8, 9, 10, 15, 20, 25, or 30 amino acid long. In various embodiments, the linker comprises

SGGGGS (SEQ ID NO: 20). In various embodiments, the linker comprises

AEAAAKEAAAKAGS (SEQ ID NO: 21 ). In various embodiments, there is no linker.

Table 2

Examples of Proteolvsis-Resistant Linkers

GGGGS 22

SGGGGSGGGGS 23

S (GGGGS) 2 24

GGGGG 25

GAGAGAGAGA 26

AEAAAKAGS 27

GGGGGGGG 28

AEAAAKEAAAKA 29

AEAAAKA 30

GGAGG 31

S (GGGGS) 3 32

[0176] In various embodiments, the fusion molecules of the present disclosure will comprise the antibody, peptide linker, and interferon molecule combinations recited in Table 3.

Table 3

Examples of Ab-IFN Fusion Molecules

Anti-CD279 SEQ ID NO: 20 or SEQ ID NO: 21 I FN-a Mutants M1 -M15

Anti-CD274 SEQ ID NO: 20 or SEQ ID NO: 21 IFN-oc Mutants M1 -M15

Anti-TIM-3 SEQ ID NO: 20 or SEQ ID NO: 21 IFN-oc Mutants M1 -M15

Anti-B7-H4 SEQ ID NO: 20 or SEQ ID NO: 21 IFN-oc Mutants M1 -M15

Anti-CD276 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN-p's IFN-A1 -A10

US 2009/00251 06

Anti-CD272 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN-p's IFN-A1 -A10

US 2009/00251 06

Anti-CD152 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN-p's IFN-ΔΙ -ΔΙ Ο

US 2009/00251 06

Anti-CD223 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN-p's IFN-ΔΙ -ΔΙ Ο

US 2009/00251 06

Anti-CD279 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN-p's IFN-ΔΙ -ΔΙ Ο

US 2009/00251 06

Anti-CD274 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN-p's IFN-ΔΙ -ΔΙ Ο

US 2009/00251 06

Anti-TIM-3 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN-p's IFN-ΔΙ -ΔΙ Ο

US 2009/00251 06

Anti-B7-H4 SEQ ID NO: 20 or SEQ ID NO: 21 Truncated IFN- 's IFN-ΔΙ -ΔΙ Ο

US 2009/00251 06

Pharmaceutical Compositions

[0177] In another aspect, the present disclosure provides a pharmaceutical composition comprising a fusion molecule as described herein, with one or more pharmaceutically acceptable excipient(s). The pharmaceutical compositions and methods of uses described herein also encompass embodiments of combinations (co-administration) with other active agents, as detailed below.

[0178] Generally, fusion molecules of the disclosure are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s). The term 'excipient' is used herein to describe any ingredient other than the compound(s) of the disclosure. The choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. Pharmaceutical compositions of the present disclosure and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in

Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all GMP regulations of the U.S. Food and Drug Administration.

[0179] The pharmaceutical compositions of the disclosure are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to,

administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or infusions; or kidney dialytic infusion techniques.

[0180] Formulations of a pharmaceutical composition suitable for parenteral

administration typically generally comprise the active ingredient combined with a

pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile nonaqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral

administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0181] For example, in one aspect, sterile injectable solutions can be prepared by incorporating the fusion molecule in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation such as vacuum drying and freeze-drying yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0182] The fusion molecules of the disclosure can be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, or as a mixed component particle, for example, mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, as an aerosol spray from a pressurized container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, or as nasal drops.

[0183] The pressurized container, pump, spray, atomizer, or nebulizer generally contains a solution or suspension of a fusion molecule of the disclosure comprising, for example, a suitable agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent. [0184] Prior to use in a dry powder or suspension formulation, the drug product is generally micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure

homogenization, or spray drying.

[0185] Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the fusion molecule of the disclosure, a suitable powder base and a performance modifier.

[0186] Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the disclosure intended for inhaled/intranasal administration.

[0187] Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0188] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the disclosure are typically arranged to administer a metered dose or "puff" of an antibody of the disclosure. The overall daily dose will typically be administered in a single dose or, more usually, as divided doses throughout the day.

[0189] The fusion molecules of the disclosure may also be formulated for an oral administration. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids, or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.

[0190] Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents in order to provide a pharmaceutically elegant and palatable preparation. For example, to prepare orally deliverable tablets, the fusion molecule is mixed with at least one

pharmaceutical excipient, and the solid formulation is compressed to form a tablet according to known methods, for delivery to the gastrointestinal tract. The tablet composition is typically formulated with additives, e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations. To prepare orally deliverable capsules, DHEA is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsular container suitable for delivery to the gastrointestinal tract. Compositions comprising fusion molecules may be prepared as described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which is herein incorporated by reference.

[0191] In various embodiments, the pharmaceutical compositions are formulated as orally deliverable tablets containing fusion molecules in admixture with non-toxic

pharmaceutically acceptable excipients which are suitable for manufacture of tablets. These excipients may be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid, or talc. The tablets may be uncoated or they may be coated with known techniques to delay disintegration and absorption in the gastrointestinal track and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

[0192] In various embodiments, the pharmaceutical compositions are formulated as hard gelatin capsules wherein the fusion molecule is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin or as soft gelatin capsules wherein the fusion molecule is mixed with an aqueous or an oil medium, for example, arachis oil, peanut oil, liquid paraffin or olive oil.

[0193] Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules (made, for example, from gelatin or hydroxypropylmethylcellulose) and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.

[0194] Any method for administering peptides, proteins or antibodies accepted in the art may suitably be employed for administering the fusion molecules of the disclosure.

Therapeutic Methods of Use [0195] The Ab-IFN fusion molecules described herein which comprise an antibody which targets an immune-checkpoint protein antigen on the surface of (e.g., expressed on or associated with) an immune cell, but not a tumor cell, are capable of, e.g., 1 ) inhibiting or reducing the downregulatory activity of targeted immune-checkpoint protein antigen on the immune response; 2) stimulating or activating immune cells in secondary lymphoid organs (e.g., draining lymph nodes, spleen); and 3) stimulating or activating immune cells that are present in the tumor microenvironment by directly binding to them. These effects may be mediated by the binding of the immune-checkpoint antibody to the immune-checkpoint protein antigen and blocking its ability to interact with its corresponding ligand. These effects may also be mediated by the increase in local concentration of IFN near the immune cell surface and binding of the IFN portion of Ab-IFN to its receptor IFNAR on the surface of the immune cell. This immune cell may be either the same immune cell that Ab-IFN is binding to or a nearby neighboring immune cell. These effects, alone or collectively, may lead to an enhancement of an anti-tumor host immune response and/or indirect killing of tumor cells.

[0196] As such, in another aspect, the present disclosure provides methods of stimulating an immune response and/or enhancing an anti-tumor host immune response in a patient, comprising administering to the patient a therapeutically effective amount (either as monotherapy or in combination therapy) of a Ab-IFN fusion molecule comprising an antibody which targets an immune-checkpoint protein antigen, in pharmaceutically acceptable carrier. Such methods could be used, e.g., to treat a patient at risk of, or susceptible to, or having a disorder associated with aberrant immune-checkpoint protein expression or function.

[0197] The Ab-IFN fusion molecules described herein and which comprise an antibody which targets an immune-checkpoint protein antigen that is also on the surface of (e.g., expressed on or associated with) a tumor cell are further capable of, e.g., 1 ) inducing direct cell death of tumor cells by engaging IFN-ocR expressed on tumor cells; 2) negating some of the suppressive actions of tumors on dendritic cells (DCs), potentially leading to more efficient cross presentation of tumor antigens to T cells by DCs, i.e., improve CD8+ CTL priming; 3) directly activating the CD8+ CTL functions which will allow efficient killing of tumor cells; 4) the direct activation of NK cell functions by the IFN which will allow efficient killing of tumor cells; 5) inducing the up-regulation of the co-inhibitory immune-checkpoint proteins expressed on or associated with tumor cells; and 6) directly negating other mechanisms for immune evasion, e.g., the major inhibitory pathways mediated by certain immune-checkpoint proteins on T cells/B cells. [0198] As such, in another aspect, the present disclosure provides methods of treating tumor cells in a patient, comprising administering to the patient a therapeutically effective amount (either as monotherapy or combination therapy) of an Ab-IFN fusion molecule which comprises an antibody which targets an immune-checkpoint protein antigen expressed on or associated with a tumor cell, in pharmaceutically acceptable carrier.

[0199] In various embodiments, the tumor cells to be treated using the Ab-IFN fusion molecules described herein are cells derived from a cancer which includes, but is not limited to, the following: a) cancers of the breast, which include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma, lobular carcinoma in situ and metastatic breast cancer; b) proliferative diseases of lymphocytic cells, which include, but are not limited to, various T cell and B cell lymphomas, non-Hodgkins lymphoma, cutaneous T cell lymphoma, Hodgkins disease, and lymphoma of the central nervous system; (c) multiple myeloma, chronic neutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, juvenile

myelomonocytic leukemia, refractory anemia with ringed sideroblasts and without ringed sideroblasts, refractory cytopenia (myelodysplastic syndrome) with multilineage dysplasia, refractory anemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome,

myelodysplastic syndrome with t(9;12)(q22;p12), and myelogenous leukemia (e.g., Philadelphia chromosome positive (t(9;22)(qq34;q1 1 )); d) proliferative diseases of the skin, which include, but are not limited to, basal cell carcinoma, squamous cell carcinoma, malignant melanoma and Kaposi's sarcoma; e) leukemias, which include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia, f) proliferative diseases of the digestive tract, which include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach (gastric), pancreatic cancer, pancreatic cancer-Islet cell, rectal, small-intestine and salivary gland cancers; g) proliferative diseases of the liver, which include, but are not limited to, hepatocellular carcinoma,

cholangiocarcinoma, mixed hepatocellular cholangiocarcinoma, primary liver cancer and metastatic liver cancer; h) proliferative diseases of the male reproductive organs, which include, but are not limited to, prostate cancer, testicular cancer and penile cancer; i) proliferative diseases of the female reproductive organs, which include, but are not limited to, uterine cancer (endometrial), cervical, ovarian, vaginal, vulval cancers, uterine sarcoma and ovarian germ cell tumor; j) proliferative diseases of the respiratory tract, which include, but are not limited to, small cell and non-small cell lung carcinoma, bronchial adema, pleuropulmonary blastoma and malignant mesothelioma; k) proliferative diseases of the eye, which include, but are not limited to, intraocular melanoma, retinoblastoma, and rhabdomyosarcoma; I) proliferative diseases of the head and neck, which include, but are not limited to, laryngeal, hypopharyngeal,

nasopharyngeal, oropharyngeal cancers, and lip and oral cancer, squamous neck cancer, metastatic paranasal sinus cancer; m) proliferative diseases of the thyroid, which include, but are not limited to, thyroid cancer, thymoma, malignant thymoma, medullary thyroid carcinomas, papillary thyroid carcinomas, multiple endocrine neoplasia type 2A (MEN2A),

pheochromocytoma, parathyroid adenomas, multiple endocrine neoplasia type 2B (MEN2B), familial medullary thyroid carcinoma (FMTC) and carcinoids; n) proliferative diseases of the urinary tract, which include, but are not limited to, bladder cancer; o) sarcomas, which include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma; p) proliferative diseases of the kidneys, which include, but are not limited to, renal cell carcinoma, clear cell carcinoma of the kidney; and renal cell adenocarcinoma; q) precursor B-lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia), B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B- cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma, extranodal marginal zone B-cell lymphoma of MALT type, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, diffuse large B-cell lymphoma, mediastinal large B-cell lymphoma, primary effusion lymphoma and Burkitt's lymphoma/Burkitt cell leukemia; r) precursor T- lymphoblastic lymphoma/leukemia (precursor T-cell acute lymphoblastic leukemia), T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell lymphoma/leukemia (HTLV-1 ), extranodal NK/T-cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides/Sezary syndrome, anaplastic large-cell lymphoma, T/null cell, primary cutaneous type, peripheral T-cell lymphoma, not otherwise characterized, angioimmunoblastic T-cell lymphoma, anaplastic large-cell lymphoma, T/null cell, and primary systemic type; s) nodular lymphocyte-predominant Hodgkin's lymphoma, nodular sclerosis Hodgkin's lymphoma (grades 1 and 2), lymphocyte-rich classical Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma, and lymphocyte depletion Hodgkin's lymphoma; and t) AML with t(8;21 )(q22;q22), AML1 (CBF-alpha)/ETO, acute promyelocytic leukemia (AML with t(15;17)(q22;q1 1 -12) and variants, PML/RAR-alpha), AML with abnormal bone marrow eosinophils (inv(16)(p13q22) or t(16;16)(p13;q1 1 ), CBFb/MYH1 1 .times.), and AML with 1 1 q23 (MLL) abnormalities, AML minimally differentiated, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroid leukemia, acute megakaryocytic leukemia, acute basophilic leukemia, and acute panmyelosis with myelofibrosis.

[0200] In various embodiments, the cancer is immunogenic which includes, but is not limited to melanomas, renal, lung, prostate, breast, neuroblastomas, and ovary.

[0201] In various embodiments, the patient is a human. In various embodiments, the patient is immunocompromised. In various embodiments, the patient is at least about any of 65, 70, 75, or 80 years old. In some embodiments, the individual is resistant to treatment with an anti-immune-checkpoint protein antibody not fused to an interferon.

[0202] A therapeutically effective dose can be estimated initially from cell culture assays by determining an IC₅o- A dose can then be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅o as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC. The exact composition, route of

administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0203] Dosage regimens can be adjusted to provide the optimum desired response

(e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeat or maintenance) can be administered over time and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure will be dictated primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0204] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure.

[0205] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed

composition. Further, the dosage regimen with the compositions of this disclosure may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular antibody employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present disclosure encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

[0206] For administration to human subjects, the total monthly dose of the fusion molecules of the invention can be in the range of 0.0002-500 mg per patient, 0.0002-400 mg per patient, 0.0002-300 mg per patient, 0.0002-200 mg per patient, 0.0002-100 mg per patient, 0.0002-50 mg per patient, 0.0006-500 mg per patient, 0.0006-400 mg per patient, 0.0006-300 mg per patient, 0.0006-200 mg per patient, 0.0006-100 mg per patient, 0.0006-50 mg per patient, 0.002-500 mg per patient, 0.002-400 mg per patient, 0.002-300 mg per patient, 0.002- 200 mg per patient, 0.002-100 mg per patient, 0.002-50 mg per patient, 0.006-500 mg per patient, 0.006-400 mg per patient, 0.006-300 mg per patient, 0.006-200 mg per patient, 0.006- 100 mg per patient, 0.006-50 mg per patient, 0.02-500 mg per patient, 0.02-400 mg per patient, 0.02-300 mg per patient, 0.02-200 mg per patient, 0.02-100 mg per patient, 0.02-50 mg per patient, 0.06-500 mg per patient, 0.06-400 mg per patient, 0.06-300 mg per patient, 0.06-200 mg per patient, 0.06-100 mg per patient, 0.06-50 mg per patient, 0.2-500 mg per patient, 0.2- 400 mg per patient, 0.2-300 mg per patient, 0.2-200 mg per patient, 0.2-100 mg per patient, 0.2- 50 mg per patient, 0.6-500 mg per patient, 0.6-400 mg per patient, 0.6-300 mg per patient, 0.6- 200 mg per patient, 0.6-100 mg per patient, or 0.6-50 mg per patient, 2-500 mg per patient, 2- 400 mg per patient, 2-300 mg per patient, 2-200 mg per patient, 2-100 mg per patient, 2-50 mg per patient, 6-500 mg per patient, 6-400 mg per patient, 6-300 mg per patient, 6-200 mg per patient, 6-100 mg per patient, or 6-50 mg per patient, depending, of course, on the mode of administration. The total monthly dose can be administered in single or divided doses and can, at the physician's discretion, fall outside of the typical ranges given herein.

[0207] An exemplary, non-limiting weekly dosing range for a therapeutically effective amount of the fusion molecules of the invention can be about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, about 0.05 to about 0.06 mg/kg, about 0.06 to about 0.07 mg/kg, about 0.07 to about 0.08 mg/kg, about 0.08 to about 0.09 mg/kg, about 0.09 to about 0.1 mg/kg, about 0.1 to about 0.2 mg/kg, about 0.2 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg.

[0208] In various embodiments, the fusion molecule is administered to the patient at a weekly dosage selected from the group consisting of .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .02 mg/kg, .03 mg/kg, .04 mg/kg, .05 mg/kg, .06 mg/kg, .07 mg/kg, .08 mg/kg, .09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.

[0209] In various embodiments, the fusion molecule is administered to the patient at a weekly dosage of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .02 mg/kg, .03 mg/kg, .04 mg/kg, .05 mg/kg, .06 mg/kg, .07 mg/kg, .08 mg/kg, .09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.

[0210] In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.0001 mg/kg body weight. In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.0003 mg/kg body weight. In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.001 mg/kg body weight. In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.003 mg/kg body weight. In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.01 mg/kg body weight. In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.03 mg/kg body weight. In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.1 mg/kg body weight. In various embodiments, the weekly dose for a therapeutically effective amount of a fusion molecule of the invention will be 0.3 mg/kg body weight. In various embodiments the fusion molecules will be administered via intravenous (IV) infusion for up to three cycles of eight once weekly doses.

[0211] In various embodiments, the fusion molecule is administered to the patient at a weekly dosage included in any of the following ranges: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, about 0.05 to about 0.06 mg/kg, about 0.06 to about 0.07 mg/kg, about 0.07 to about 0.08 mg/kg, about 0.08 to about 0.09 mg/kg, about 0.09 to about 0.1 mg/kg, about 0.1 to about 0.2 mg/kg, about 0.2 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg. In various embodiments, the fusion molecule is administered to the patient at a weekly dosage selected from the group consisting of .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .02 mg/kg, .03 mg/kg, .04 mg/kg, .05 mg/kg, .06 mg/kg, .07 mg/kg, .08 mg/kg, .09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg. In various embodiments, the fusion molecule is administered to the patient at a weekly dosage of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .02 mg/kg, .03 mg/kg, .04 mg/kg, .05 mg/kg, .06 mg/kg, .07 mg/kg, .08 mg/kg, .09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.

[0212] Toxicity and therapeutic index of the pharmaceutical compositions of the invention 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 ED₅o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effective dose is the therapeutic index and it can be expressed as the ratio LD₅o/ED₅o. Compositions that exhibit large therapeutic indices are generally preferred.

[0213] In various embodiments, single or multiple administrations of the pharmaceutical compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the fusion molecules disclosed herein to effectively treat the patient. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. [0214] The dosing frequency of the administration of the fusion molecule pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The patient can be treated at regular intervals, such as weekly or monthly, until a desired therapeutic result is achieved. Exemplary dosing frequencies include, but are not limited to: once weekly without break; once weekly, every other week; once every 2 weeks; once every 3 weeks;

weakly without break for 2 weeks, then monthly; weakly without break for 3 weeks, then monthly; monthly; once every other month; once every three months; once every four months; once every five months; or once every six months, or yearly.

[0215] In another aspect, the present disclosure provides for methods of inhibiting the growth of cancer cells in a patient comprising administering an effective amount of the fusion molecule to the patient. These methods may inhibit or prevent the growth of the cancer cells of said patient, such as for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. As a result, where the cancer is a solid tumor, the modulation may reduce the size of the solid tumor by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0216] The inhibition of the cancer cell proliferation can be measured by cell-based assays, such as bromodeoxyuridine (BRDU) incorporation (Hoshino et al., Int. J. Cancer 38, 369, 1986; Campana et al., J. Immunol. Meth. 107:79, 1988; [³H]-thymidine incorporation (Chen, J., Oncogene 13:1395-403, 1996; Jeoung, J., J. Biol. Chem. 270:18367-73, 1995; the dye Alamar Blue (available from Biosource International) (Voytik-Harbin et al., In Vitro Cell Dev Biol Anim 34:239-46, 1998). The anchorage independent growth of cancer cells is assessed by colony formation assay in soft agar, such as by counting the number of cancer cell colonies formed on top of the soft agar (see Examples and Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989).

[0217] The inhibition of cancer cell growth in a subject may be assessed by monitoring the cancer growth in a subject, for example in an animal model or in human subjects. One exemplary monitoring method is tumorigenicity assays. In one example, a xenograft comprises human cells from a pre-existing tumor or from a tumor cell line. Tumor xenograft assays are known in the art and described herein (see, e.g., Ogawa et al., Oncogene 19:6043-6052, 2000). In another embodiment, tumorigenicity is monitored using the hollow fiber assay, which is described in U.S. Patent No. 5,698,413, which is incorporated herein by reference in its entirety. [0218] The percentage of the inhibition is calculated by comparing the cancer cell proliferation, anchorage independent growth, or cancer cell growth under modulator treatment with that under negative control condition (typically without modulator treatment). For example, where the number of cancer cells or cancer cell colonies (colony formation assay), or PRDU or [³H]-thymidine incorporation is A (under the treatment of modulators) and C (under negative control condition), the percentage of inhibition would be (C-A)/Cx100%.

[0219] Examples of tumor cell lines derived from human tumors and available for use in the in vitro and in vivo studies include, but are not limited to, leukemia cell lines (e.g., CCRF- CEM, HL-60(TB), K-562, MOLT-4, RPM1 -8226, SR, P388 and P388/ADR); non-small cell lung cancer cell lines (e.g., A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522 and LXFL 529); small cell lung cancer cell lines (e.g., DMS 1 14 and SHP- 77); colon cancer cell lines (e.g., COLO 205, HCC-2998, HCT-1 16, HCT-15, HT29, KM12, SW- 620, DLD-1 and KM20L2); central nervous system (CNS) cancer cell lines (e.g., SF-268, SF- 295, SF-539, SNB-19, SNB-75, U251 , SNB-78 and XF 498); melanoma cell lines (e.g., LOX I MVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, RPMI-7951 and M19-MEL); ovarian cancer cell lines (e.g., IGROV1 , OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8 and SK-OV-3); renal cancer cell lines (e.g., 786-0, A498, ACHN, CAKI-1 , RXF 393, SN12C, TK-10, UO-31 , RXF-631 and SN12K1 ); prostate cancer cell lines (e.g., PC-3 and DU- 145); breast cancer cell lines (e.g., MCF7, NCI/ADR-RES, MDA-MB-231/ATCC, HS 578T, MDA-MB-435, BT-549, T-47D and MDA-MB-468); and thyroid cancer cell lines (e.g., SK-N- SH).

Combination Therapy

[0220] As used herein, the terms "co-administration", "co-administered" and "in combination with", referring to the fusion molecules of the disclosure and one or more other therapeutic agents, is intended to mean, and does refer to and include the following:

simultaneous administration of such combination of fusion molecules of the disclosure and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient; substantially simultaneous administration of such combination of fusion molecules of the disclosure and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient; sequential administration of such combination of fusion molecules of the disclosure and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and sequential administration of such combination of fusion molecules of the disclosure and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner whereupon they are concurrently, consecutively, and/or overlappingly released at the same and/or different times to said patient, where each part may be administered by either the same or a different route.

[0221] In another aspect, the present disclosure relates to methods of treating cancer in a patient, comprising administration of a combination of a) a therapeutically effective amount of a fusion molecule of the present disclosure; and b) a second agent. This combination therapy may be particularly effective against a cancer that is resistant or refractory to treatment using the second agent not fused to an interferon.

[0222] In various embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a fusion molecule that comprises IFN-a attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti- CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab.

[0223] In various embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a fusion molecule that comprises IFN-a attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD138 Ab, anti- Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab.

[0224] In various embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a fusion molecule that comprises IFN-a attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent is selected from the group consisting of an anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD138 Ab, anti-Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab, and wherein the second Ab is fused to an IFN molecule.

[0225] In various embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a Ab-IFN fusion molecule that comprises IFN-β attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti- CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab.

[0226] In various embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a Ab-IFN fusion molecule that comprises IFN-β attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD138 Ab, anti- Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab.

[0227] In various embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a Ab-IFN fusion molecule that comprises IFN-β attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent is selected from the group consisting of an anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD138 Ab, anti-Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab, and wherein the second antibody is fused to an IFN molecule.

[0228] In various embodiments, the combination therapy comprises administering the fusion molecule composition and the second agent composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, fusion molecule composition and the second agent composition are administered sequentially, i.e., the fusion molecule composition is administered either prior to or after the administration of the second agent composition.

[0229] In various embodiments, the administrations of the fusion molecule composition and the second agent composition are concurrent, i.e., the administration period of the fusion molecule composition and the second agent composition overlap with each other.

[0230] In various embodiments, the administrations of the fusion molecule composition and the second agent composition are non-concurrent. For example, in various embodiments, the administration of the fusion molecule composition is terminated before the second agent composition is administered. In various embodiments, the administration second agent composition is terminated before the fusion molecule composition is administered.

[0231] In various embodiments, the dose of the fusion molecule to be administered and the dose of the second agent to be administered is the same. In various embodiments, the dose of the fusion molecule to be administered is less than the dose of the second agent to be administered, e.g., at least 2-fold less, at least 5-fold less, at least 10-fold less, at least 20-fold less, at least 30-fold less, at least 40-fold less, at least 50-fold less, at least 60-fold less, at least 70-fold less, at least 80-fold less, at least 90-fold less, or at least 100-fold less. In various embodiments, the dose of the fusion molecule to be administered is more than the dose of the second agent to be administered, e.g., at least 2-fold more, at least 5-fold more, at least 10-fold more, at least 20-fold more, at least 30-fold more, at least 40-fold more, at least 50-fold more, at least 60-fold more, at least 70-fold more, at least 80-fold more, at least 90-fold more, or at least 100-fold more.

[0232] In various embodiments, the methods described herein may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the fusion molecules described herein.

[0233] A wide array of conventional compounds has been shown to have anti-neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant T-cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.

[0234] When the fusion molecule disclosed herein is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such fusion molecule may enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an antineoplastic agent in resistant T-cells.

[0235] When used in combination with a second agent (such as any one of the second agents described herein, e.g., an anti-immune-checkpoint protein antigen inhibitor, which may or may target the same or different immune-checkpoint proteins as the fusion molecule; or an anti-immune-checkpoint protein antigen antibody as described herein, which may or may target the same or different immune-checkpoint proteins as the fusion molecule), the fusion molecule and the second agent can be administered using the same route of administration or different routes of administration. In various embodiments (for both simultaneous and sequential administrations), the fusion molecule and the second agent are administered at a predetermined ratio. For example, in various embodiments, the ratio by weight of the fusion molecule and the second agent is about 1 to 1 . In various embodiments, the weight ratio may be between about

0.001 to about 1 and about 1000 to about 1 , or between about 0.01 to about 1 and 100 to about

1 . In various embodiments, the ratio by weight of the fusion molecule and the second agent is less than about any of 100:1 , 50:1 , 30:1 , 10:1 , 9:1 , 8:1 , 7:1 , 6:1 , 5:1 , 4:1 , 3:1 , 2:1 , and 1 :1 . In various embodiments, the ratio by weight of the fusion molecule and the second agent is more than about any of 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 30:1 , 50:1 , 100:1 . Other ratios are contemplated. In various embodiments, the molar ratio of the fusion molecule and the second agent is about 1 to 1 . In various embodiments, the molar ratio may be between about 0.001 to about 1 and about 1000 to about 1 , or between about 0.01 to about 1 and 100 to about 1 . In various embodiments, the molar ratio of the fusion molecule and the second agent is less than about any of 100:1 , 50:1 , 30:1 , 10:1 , 9:1 , 8:1 , 7:1 , 6:1 , 5:1 , 4:1 , 3:1 , 2:1 , and 1 :1 In various embodiments, the molar ratio of the fusion molecule and the second agent is more than about any of 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 30:1 , 50:1 , 100:1 . Other ratios are contemplated.

Nucleic acid Molecules and Fusion Molecule Expression

[0236] The present application further provides nucleic acid molecules comprising nucleotide sequences encoding the recombinant, genetically engineered fusion molecules described herein. Because of the degeneracy of the genetic code, a variety of nucleic acid sequences encode each fusion molecule amino acid sequence. The application further provides nucleic acid molecules that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to nucleic acid molecules that encode a fusion molecule. Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6xSSC at about 45 °C followed by one or more washes in 0.2xSSC/0.1 % SDS at about 50-65 °C, highly stringent conditions such as hybridization to filter-bound DNA in 6xSSC at about 45 °C followed by one or more washes in 0.1 xSSC/0.2% SDS at about 60 °C, or any other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, F. M. et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1 , Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

[0237] The nucleic acid molecules may be obtained, and the nucleotide sequence of the nucleic acid molecules determined by, any method known in the art. For example, if the nucleotide sequence of the fusion molecule is known, a nucleic acid molecule encoding the fusion molecule may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242, 1994), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated

oligonucleotides by PCR. In one embodiment, the codons that are used comprise those that are typical for human or mouse (see, e.g., Nakamura, Y., Nucleic Acids Res. 28: 292, 2000).

[0238] A nucleic acid molecule encoding a fusion molecule may also be generated from nucleic acid from a suitable source. For example, if a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[0239] In one embodiment of the present disclosure, nucleic acid sequences encoding the appropriate antibody framework are optionally cloned and ligated into appropriate vectors (e.g., expression vectors for, e.g., prokaryotic or eukaryotic organisms). Additionally, nucleic acid sequences encoding the appropriate interferon molecule are optionally cloned into the same vector in the appropriate orientation and location so that expression from the vector produces an antibody-interferon molecule fusion molecule. Some optional embodiments also require post-expression modification, e.g., assembly of antibody subunits, etc. The techniques and art for the above (and similar) manipulations are well known to those skilled in the art.

Pertinent instructions are found in, e.g., Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.

(supplemented through 1999).

[0240] The present disclosure is also directed to host cells that express the fusion molecules of the disclosure. Host cells suitable for replicating and for supporting recombinant expression of fusion protein are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the protein for clinical applications. Such cells may include prokaryotic microorganisms, such as E. coli; various eukaryotic cells, such as Chinese hamster ovary cells (CHO), NSO, 293; HEK Yeast; insect cells; hybridomas; human cell lines; and transgenic animals and transgenic plants, and the like. Standard technologies are known in the art to express foreign genes in these systems. The recombinant protein gene is typically operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.

[0241] To express an antibody-IFN fusion molecule recombinantly, a host cell is transformed, transduced, infected or the like with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and/or heavy chains of the antibody and attached interferon such that the light and/or heavy chains are expressed in the host cell. The heavy chain and the light chain may be expressed independently from different promoters to which they are operably-linked in one vector or, alternatively, the heavy chain and the light chain may be expressed independently from different promoters to which they are operably- linked in two vectors one expressing the heavy chain and one expressing the light chain.

Optionally, the heavy chain and light chain may be expressed in different host cells. [0242] Additionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody light and/or heavy chain from a host cell. The antibody light and/or heavy chain gene can be cloned into the vector such that the signal peptide is operably- linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Preferably, the recombinant antibodies are secreted into the medium in which the host cells are cultured, from which the antibodies can be recovered or purified.

[0243] An isolated DNA encoding a HCVR can be converted to a full-length heavy chain gene by operably-linking the HCVR-encoding DNA to another DNA molecule encoding heavy chain constant regions. The sequences of human, as well as other mammalian, heavy chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained e.g., by standard PCR amplification. The heavy chain constant region can be of any type, (e.g., IgG, IgA, IgE, IgM or IgD), class (e.g., IgGi , lgG₂, lgG₃ and lgG₄) or subclass constant region and any allotypic variant thereof as described in Kabat (supra).

[0244] An isolated DNA encoding a LCVR region may be converted to a full-length light chain gene (as well as to a Fab light chain gene) by operably linking the LCVR-encoding DNA to another DNA molecule encoding a light chain constant region. The sequences of human, as well as other mammalian, light chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0245] Additionally, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and one or more selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (dhfr) gene (for use in dhfr-minus host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and glutamine synthetase (GS) in a GS-negative cell line (such as NSO) for selection/amplification.

[0246] For expression of the light and/or heavy chains with attached interferon, the expression vector(s) encoding the heavy and/or light chains is introduced into a host cell by standard techniques e.g. electroporation, calcium phosphate precipitation, DEAE-dextran transfection, transduction, infection and the like. Although it is theoretically possible to express the antibodies of the disclosure in either prokaryotic or eukaryotic host cells, eukaryotic cells and most specifically mammalian host cells, are more typical because such cells are more likely to assemble and secrete a properly folded and immunologically active antibody. Mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) [including dhfr minus CHO cells, as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-20, 1980, used with a DHFR selectable marker, e.g. as described in Kaufman and Sharp, J. Mol. Biol. 159:601 -21 , 1982], NSO myeloma cells, COS cells, and SP2/0 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown under appropriate conditions known in the art. Antibodies can be recovered from the host cell and/or the culture medium using standard purification methods.

[0247] Once expressed, the intact antibodies, individual light and heavy chains, or other immunoglobulin forms of the present disclosure can be purified according to standard procedures of the art, including ammonium sulfate precipitation, ion exchange, affinity (e.g., Protein A), reverse phase, hydrophobic interaction column chromatography, hydroxyapatite chromatography, gel electrophoresis, and the like. Standard procedures for purification of therapeutic antibodies are described, for example, by Feng L1 , Joe X. Zhou, Xiaoming Yang, Tim Tressel, and Brian Lee in an article entitled "Current Therapeutic Antibody Production and Process Optimization" (BioProcessing Journal, September/October 2005), for example.

Additionally, standard techniques for removing viruses from recombinantly expressed antibody preparations are also known in the art (see, for example, Gerd Kern and Mani Krishnan, "Viral Removal by Filtration: Points to Consider" (Biopharm International, October 2006)). The effectiveness of filtration to remove viruses from preparations of therapeutic antibodies is known to be at least in part dependent on the concentration of protein and/or the antibody in the solution to be filtered. The purification process for antibodies of the present disclosure may include a step of filtering to remove viruses from the mainstream of one or more

chromatography operations. Preferably, prior to filtering through a pharmaceutical grade nanofilter to remove viruses, a chromatography mainstream containing an antibody of the present disclosure is diluted or concentrated to give total protein and/or total antibody concentration of about 1 g/L to about 3 g/L. Even more preferably, the nanofilter is a DV20 nanofilter (e.g., Pall Corporation; East Hills, N.Y.). Substantially pure immunoglobulins of at least about 90%, about 92%, about 94% or about 96% homogeneity are preferred, and about 98 to about 99% or more homogeneity most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the sterile antibodies may then be used therapeutically, as directed herein.

[0248] In view of the aforementioned discussion, the present disclosure is further directed to a fusion molecule obtainable by a process comprising the steps of culturing a host cell including, but not limited to a mammalian, plant, bacterial, transgenic animal, or transgenic plant cell which has been transformed by a nucleic acid molecule or a vector comprising nucleic acid molecules encoding antibodies of the disclosure so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture medium.

Kits

[0249] In certain embodiments, this disclosure provides for kits for the treatment of cancer and/or in an adjunct therapy. Kits typically comprise a container containing a fusion molecule of the present disclosure. The fusion molecule can be present in a pharmacologically acceptable excipient.

[0250] In addition, the kits can optionally include instructional materials disclosing means of use of the fusion molecule to treat a cancer. The instructional materials may also, optionally, teach preferred dosages, counter-indications, and the like.

[0251] The kits can also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, and additionally comprise means for disinfecting a wound, for reducing pain, for attachment of a dressing, and the like.

[0252] While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and

communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

Exemplary Embodiments

[0253] In one embodiment, there is provided a fusion molecule comprising an interferon

(IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen, wherein the fusion molecule is capable of treating a cancer. [0254] In some embodiments, the fusion molecule comprises a type 1 interferon molecule, or mutant molecule thereof.

[0255] In some embodiments, the fusion molecule comprises an interferon alpha (IFN- ) molecule.

[0256] In some embodiments, the fusion molecule comprises an IFN- mutant molecule.

[0257] In some embodiments, the fusion molecule comprises an IFN-a mutant molecule having the amino acid sequence of SEQ ID NO: 19 wherein the arginine at amino acid residue position 149 has been replaced with an alanine and the arginine at amino acid residue position 162 has been replaced with an alanine.

[0258] In some embodiments, the fusion molecule comprises an interferon beta (IFN-β), or mutant molecule thereof.

[0259] In some embodiments, the fusion molecule comprises an interferon-gamma (IFN-

Y) molecule.

[0260] In some embodiments, the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of an immune cell.

[0261] In some embodiments, the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of a tumor cell.

[0262] In some embodiments, the fusion molecule comprises an antibody selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding fragment, Fab, Fab', Fab₂, Fab'₂, IgG, IgM, IgA, IgE, scFv, dsFv, dAb, nanobodies, unibodies, and diabodies.

[0263] In some embodiments, the fusion molecule comprises a fully human monoclonal antibody.

[0264] In some embodiments, the fusion molecule comprises a humanized monoclonal antibody.

[0265] In some embodiments, the fusion molecule comprises an antibody or antigen- binding fragment that binds to an immune-checkpoint protein antigen with a dissociation constant (KD) of at least about 1 x10 ³ M, at least about 1 x10 ⁴ M, at least about 1 x10 ⁵ M, at least about 1 x10 ⁶ M, at least about 1 x10 ⁷ M, at least about 1 x10 ⁸ M, at least about 1 x10 ⁹ M, at least about 1 x10 ¹⁰ M, at least about 1 x10 ¹¹ M, or at least about 1 x10 ¹² M.

[0266] In some embodiments, the antibody specifically binds an antigen selected from the group consisting of CD276, CD272, CD152, CD223, CD279, CD274, TIM-3 and B7-H4 antigen, and the interferon molecule is an interferon alpha (IFN-a) molecule, or mutant molecule thereof.

[0267] In some embodiments, the antibody is an anti-CD276 antibody, and the interferon molecule is an interferon alpha (IFN-a) molecule.

[0268] In some embodiments, the antibody is an anti-CD272 antibody, and the interferon molecule is an interferon alpha (IFN-a) molecule.

[0269] In some embodiments, the antibody is an anti-CD152 antibody, and the interferon molecule is an interferon alpha (IFN-a) molecule.

[0270] In some embodiments, the antibody is an anti-CD223 antibody, and the interferon molecule is an interferon alpha (IFN-a) molecule.

[0271] In some embodiments, the antibody is an anti-CD279 antibody, and the interferon molecule is an interferon alpha (IFN-a) molecule.

[0272] In some embodiments, the antibody is an anti-CD274 antibody, and the interferon molecule is an interferon alpha (IFN-a) molecule.

[0273] In some embodiments, the antibody is an anti-B7-H4 antibody, and the interferon molecule is an interferon alpha (IFN-a) molecule.

[0274] In some embodiments, the antibody specifically binds an antigen selected from the group consisting of CD276, CD272, CD152, CD223, CD279, CD274, TIM-3 and B7-H4 antigen, and the interferon molecule is an interferon alpha (IFN-β) molecule, or mutant molecule thereof.

[0275] In some embodiments, the antibody is an anti-CD276 antibody, and the interferon molecule is an interferon alpha (IFN-β) molecule.

[0276] In some embodiments, the antibody is an anti-CD272 antibody, and the interferon molecule is an interferon alpha (IFN-β) molecule.

[0277] In some embodiments, the antibody is an anti-CD152 antibody, and the interferon molecule is an interferon alpha (IFN-β) molecule.

[0278] In some embodiments, the antibody is an anti-CD223 antibody, and the interferon molecule is an interferon alpha (IFN-β) molecule. [0279] In some embodiments, the antibody is an anti-CD279 antibody, and the interferon molecule is an interferon alpha (IFN-β) molecule.

[0280] In some embodiments, the antibody is an anti-CD274 antibody, and the interferon molecule is an interferon alpha (IFN-β) molecule.

[0281] In some embodiments, the antibody is an anti-B7-H4 antibody, and the interferon molecule is an interferon alpha (IFN-β) molecule.

[0282] In some embodiments, the fusion molecule comprises an interferon molecule directly attached to the antibody.

[0283] In some embodiments, the fusion molecule comprises an interferon molecule attached to the antibody via a proteolysis resistant peptide linker that is fewer than 20 amino acids in length.

[0284] In some embodiments, the peptide linker has the sequence set forth in SEQ ID

NO: 20.

[0285] In some embodiments, the peptide linker has the sequence set forth in SEQ ID

NO: 21 .

[0286] In some embodiments, the fusion molecule is a recombinantly expressed fusion molecule.

[0287] In a further embodiment, there is provided a method of treating a proliferative disease in a patient, comprising administering to the patient a therapeutically effective amount of a fusion molecule comprising an interferon (IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen.

[0288] In some embodiments, the proliferative disease is a cancer selected from the group consisting of a B cell lymphoma, a lung cancer, a bronchus cancer, a colorectal cancer, a prostate cancer, a breast cancer, a pancreas cancer, a stomach cancer, an ovarian cancer, a urinary bladder cancer, a peripheral nervous system cancer, an esophageal cancer, a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancer of the oral cavity or pharynx, a liver cancer, a kidney cancer, a biliary tract cancer, a small bowel or appendix cancer, a salivary gland cancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma, a chondrosarcoma, a liposarcoma, a testes cancer, and a malignant fibrous histiocytoma, a skin cancer, a head and neck cancer, lymphomas, sarcomas, multiple myeloma and leukemias.

[0289] In some embodiments, the patient has a recurrent cancer.

[0290] In some embodiments, the patient has a resistant or refractory cancer. [0291] In some embodiments, the cancer is refractory to a therapy selected from the group consisting of immunotherapy, chemotherapy, targeted treatment with an immune- checkpoint protein antigen inhibitor, targeted treatment with a tumor antigen-specific, depleting antibody, targeted treatment with an immunoconjugate, antibody-drug conjugate (ADC), or fusion molecule comprising an immune-checkpoint protein antigen inhibitor (or tumor antigen-specific, depleting antibody) and a cytotoxic agent, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.

[0292] In some embodiments, the cancer is refractory to combination therapy involving two or more therapies selected from the group consisting of immunotherapy, chemotherapy, targeted treatment with an immune-checkpoint protein antigen inhibitor, targeted treatment with a tumor antigen-specific, depleting antibody, targeted treatment with an immunoconjugate, antibody-drug conjugate (ADC), or fusion molecule comprising an immune-checkpoint protein antigen inhibitor (or tumor antigen-specific, depleting antibody) and a cytotoxic agent, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.

[0293] In some embodiments, the fusion molecule is administered to the patient at a dosage selected from the group consisting of: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, about 0.05 to about 0.06 mg/kg, about 0.06 to about 0.07 mg/kg, about 0.07 to about 0.08 mg/kg, about 0.08 to about 0.09 mg/kg, about 0.09 to about 0.1 mg/kg, about 0.1 to about 0.2 mg/kg, about 0.2 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg.

[0294] In some embodiments, the fusion molecule is administered to the patient at a dosage selected from the group consisting of: about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, about 0.05 to about 0.06 mg/kg, about 0.06 to about 0.07 mg/kg, about 0.07 to about 0.08 mg/kg, about 0.08 to about 0.09 mg/kg, and about 0.09 to about 0.1 mg/kg.

[0295] In a further embodiment, there is provided a method of treating melanoma in a patient, comprising administering to the patient an effective amount of an anti-CD274 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD274-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-274-IFN-alpha fusion molecule is administered weekly.

[0296] In a further embodiment, there is provided a method of treating non-small cell lung cancer in a patient, comprising administering to the patient an effective amount of an anti- CD274 Ab-IFN-alpha fusion molecule. In some embodiments, the fusion molecule is

administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD274-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-274-IFN-alpha fusion molecule is

administered weekly.

[0297] In a further embodiment, there is provided a method of treating renal cell carcinoma in a patient, comprising administering to the patient an effective amount of an anti- CD274 Ab-IFN-alpha fusion molecule. In some embodiments, the fusion molecule is

administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD274-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-274-IFN-alpha fusion molecule is

administered weekly.

[0298] In a further embodiment, there is provided a method of treating breast cancer in a patient, comprising administering to the patient an effective amount of an anti-CD274 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD274-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-274-IFN-alpha fusion molecule is administered weekly.

[0299] In a further embodiment, there is provided a method of treating ovarian cancer in a patient, comprising administering to the patient an effective amount of an anti-CD274 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD274-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-274-IFN-alpha fusion molecule is administered weekly.

[0300] In a further embodiment, there is provided a method of treating bladder cancer in a patient, comprising administering to the patient an effective amount of an anti-CD274 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD274-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-274-IFN-alpha fusion molecule is administered weekly.

[0301] In a further embodiment, there is provided a method of treating melanoma in a patient, comprising administering to the patient an effective amount of an anti-CD279 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD279-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-279-IFN-alpha fusion molecule is administered weekly.

[0302] In a further embodiment, there is provided a method of treating non-small cell lung cancer in a patient, comprising administering to the patient an effective amount of an anti- CD279 Ab-IFN-alpha fusion molecule. In some embodiments, the fusion molecule is

administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD279-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-279-IFN-alpha fusion molecule is

administered weekly.

[0303] In a further embodiment, there is provided a method of treating renal cell carcinoma in a patient, comprising administering to the patient an effective amount of an anti- CD279 Ab-IFN-alpha fusion molecule. In some embodiments, the fusion molecule is

administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD279-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-279-IFN-alpha fusion molecule is

administered weekly.

[0304] In a further embodiment, there is provided a method of treating breast cancer in a patient, comprising administering to the patient an effective amount of an anti-CD279 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD279-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-279-IFN-alpha fusion molecule is administered weekly.

[0305] In a further embodiment, there is provided a method of treating ovarian cancer in a patient, comprising administering to the patient an effective amount of an anti-CD279 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD279-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-279-IFN-alpha fusion molecule is administered weekly.

[0306] In a further embodiment, there is provided a method of treating bladder cancer in a patient, comprising administering to the patient an effective amount of an anti-CD279 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD279-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-279-IFN-alpha fusion molecule is administered weekly.

[0307] In a further embodiment, there is provided a method of treating melanoma in a patient, comprising administering to the patient an effective amount of an anti-CD223 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD223-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-223-IFN-alpha fusion molecule is administered weekly. In some embodiments, the method further comprises administration of a non-fused anti-CD279 Ab. In some embodiments, the non-fused anti-CD279 Ab is selected from the group consisting of pembrolizumab and nivolumab.

[0308] In a further embodiment, there is provided a method of treating non-small cell lung cancer in a patient, comprising administering to the patient an effective amount of an anti- CD223 Ab-IFN-alpha fusion molecule. In some embodiments, the fusion molecule is

administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD223-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-223-IFN-alpha fusion molecule is

administered weekly. In some embodiments, the method further comprises administration of a non-fused anti-CD279 Ab. In some embodiments, the non-fused anti-CD279 Ab is selected from the group consisting of pembrolizumab and nivolumab.

[0309] In a further embodiment, there is provided a method of treating renal cell carcinoma in a patient, comprising administering to the patient an effective amount of an anti- CD223 Ab-IFN-alpha fusion molecule. In some embodiments, the fusion molecule is

administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD223-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-223-IFN-alpha fusion molecule is administered weekly. In some embodiments, the method further comprises administration of a non-fused anti-CD279 Ab. In some embodiments, the non-fused anti-CD279 Ab is selected from the group consisting of pembrolizumab and nivolumab.

[0310] In a further embodiment, there is provided a method of treating breast cancer in a patient, comprising administering to the patient an effective amount of an anti-CD223 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD223-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-223-IFN-alpha fusion molecule is administered weekly. In some embodiments, the method further comprises administration of a non-fused anti-CD279 Ab. In some embodiments, the non-fused anti-CD279 Ab is selected from the group consisting of pembrolizumab and nivolumab.

[0311] In a further embodiment, there is provided a method of treating ovarian cancer in a patient, comprising administering to the patient an effective amount of an anti-CD223 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD223-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-223-IFN-alpha fusion molecule is administered weekly. In some embodiments, the method further comprises administration of a non-fused anti-CD279 Ab. In some embodiments, the non-fused anti-CD279 Ab is selected from the group consisting of pembrolizumab and nivolumab.

[0312] In a further embodiment, there is provided a method of treating bladder cancer in a patient, comprising administering to the patient an effective amount of an anti-CD223 Ab-IFN- alpha fusion molecule. In some embodiments, the fusion molecule is administered to the patient at a dosage of about 0.001 to about 0.1 mg/kg, including for example about 0.001 to about 0.002 mg/kg, about 0.002 to about 0.003 mg/kg, about 0.003 to about 0.004 mg/kg, about 0.004 to about 0.005 mg/kg, about 0.005 to about 0.006 mg/kg, about 0.006 to about 0.007 mg/kg, about 0.007 to about 0.008 mg.kg, about 0.008 to about 0.009 mg/kg, about 0.009 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, or about 0.05 to about 0.1 mg/kg. In some embodiments, the anti-CD223-IFN-alpha fusion molecule is administered intravenously. In some embodiments, the anti-CD-223-IFN-alpha fusion molecule is administered weekly. In some embodiments, the method further comprises administration of a non-fused anti-CD279 Ab. In some embodiments, the non-fused anti-CD279 Ab is selected from the group consisting of pembrolizumab and nivolumab.

[0313] In a further embodiment, there is provided a method of treating a proliferative disease in a patient, comprising administrating to the patient: a) a therapeutically effective amount of a fusion molecule comprising an interferon (IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen; and b) a therapeutically effective amount of a second agent.

[0314] In some embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a fusion molecule that comprises IFN-a attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti- CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab.

[0315] In some embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a fusion molecule that comprises IFN-a attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD138 Ab, anti- Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab.

[0316] In some embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a fusion molecule that comprises IFN-β attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-CD276 Ab, anti-CD272 Ab, anti-CD152 Ab, anti-CD223 Ab, anti- CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab.

[0317] In some embodiments, the combination therapy comprises a) administration of a therapeutically effective amount of a fusion molecule that comprises IFN-β attached to an antibody selected from the group consisting of anti-CD276 Ab, anti-CD272 Ab, anti-CD1 52 Ab, anti-CD223 Ab, anti-CD279 Ab, anti-CD274 Ab, anti-TIM-3 Ab and anti-B7-H4 Ab; and b) administration of a therapeutically effective amount of a second agent selected from the group consisting of a non-fused anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD1 38 Ab, anti- Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab.

[0318] In some embodiments, the antibody portion of the fusion molecule and the second antibody agent specifically bind to the same antigen. In some embodiments, the antibody portion of the fusion molecule and the second antibody agent specifically bind to a different antigen.

[0319] In some embodiments, the fusion molecule and the second agent are administered simultaneously. In some embodiments, the fusion molecule and the second agent are administered sequentially.

[0320] In a further embodiment, the method may comprise one or more additional therapies selected from the group consisting of immunotherapy, chemotherapy, targeted treatment with an immune-checkpoint protein antigen inhibitor, targeted treatment with a tumor antigen-specific, depleting antibody, targeted treatment with an immunoconjugate, antibody-drug conjugate (ADC), or fusion molecule comprising an immune-checkpoint protein antigen inhibitor (or tumor antigen-specific, depleting antibody) and a cytotoxic agent, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.

[0321] In a further embodiment, there is a provided a method of enhancing an antitumor immune response, or killing or inhibiting growth of tumor cells in a patient, comprising administering to the patient a therapeutically effective amount of a fusion molecule comprising an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of an immune cell.

[0322] In a further embodiment, there is a provided a method of enhancing an antitumor immune response, or killing or inhibiting growth of tumor cells in a patient, comprising administering to the patient a therapeutically effective amount of a fusion molecule comprising an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of a tumor cell.

[0323] In a further embodiment, there is provided a pharmaceutical composition comprising a fusion molecule of the present disclosure in a pharmaceutically acceptable carrier.

[0324] In a further embodiment, the pharmaceutical composition is formulated for administration via a route selected from the group consisting of subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection and infusions.

[0325] In a further embodiment, there is provided a fusion molecule comprising an interferon (IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen for the preparation of a medicament for treatment, prophylaxis and/or prevention of cancer in a patient in need thereof.

[0326] In a further embodiment, there is provided a fusion molecule comprising an interferon (IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen for the preparation of a medicament for stimulating an immune response and/or enhancing a host anti-tumor immune response in a patient in need thereof.

[0327] In a further embodiment, there is provided a vector comprising a nucleic acid that encodes a fusion molecule comprising an interferon (IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen of the present disclosure.

[0328] In a further embodiment, there is provided a host cell comprising a vector comprising a nucleic acid that encodes a fusion molecule comprising an interferon (IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen of the present disclosure.

[0329] The following examples are provided to describe the disclosure in further detail.

Example 1

[0330] This example generally describes the preparation of genetically engineered fusion molecules comprising an interferon attached to an anti-immune-checkpoint protein antibody, wherein the interferon is attached to the antibody via proteolysis resistant peptide linker. The fusion molecules were initially constructed as depicted in Figure 1 , with the interferon molecule attached via a linker to the C-terminus of heavy chain of the antibody. All of the fusion molecules described in these examples are prepared using recombinant DNA methods and techniques that are well known and understood by one of ordinary skill in the art.

[0331] The preparation of the Ab-IFN fusion molecules for evaluation and testing as described in the Examples below can be generally described as follows: a vector encoding the heavy chain of the selected antibody is recombinantly engineered with an interferon, or interferon mutant, at the carboxy-terminus using a proteolysis resistant peptide linker, e.g., SEQ ID NO: 20. After verifying that the vector has the correct fusion molecule nucleotide sequence, it is transfected, along with a vector encoding the light chain of the selected antibody into mammalian cells, e.g., NSO, CHO, or HEK293. Transfectants are screened by ELISA for the production of the complete fusion molecule. The clone giving the highest signal is expanded and following sub-cloning is grown in roller bottles. Conditioned medium is collected, concentrated, and the protein of interest purified using a single Protein A affinity chromatography step or appropriate alternative chromatography methods. The final product is formulated in a desired buffer and at a desired concentration (the protein concentration is confirmed by UV absorption). The purity of the final product is determined by SDS-PAGE both under reducing and non- reducing conditions. Western blot analysis was used to confirm the expected size of the molecule.

[0332] The fusion molecules prepared as described above are evaluated and tested using the various in vitro functional assays and in vivo assays described in the Examples below.

Example 2

[0333] This Example describes the evaluation and testing of various Ab-IFN fusion molecules listed in Table 4. Each of the targeted immune-checkpoint protein antigens for the antibodies listed in Table 4 are on the surface of various immune cells. As such, in the assays used for the evaluation of a particular fusion molecule, immune cells known to have the targeted antigen on its surface are used, e.g., immune cells having CD276 on its surface are used to evaluate an anti-CD276-IFN fusion molecule.

Table 4

Anti-CD152 SEQ ID NO: 20 wtlFN-a (SEQ ID NO 19)

Anti-CD223 SEQ ID NO: 20 wtlFN-a (SEQ ID NO 19)

Anti-CD279 SEQ ID NO: 20 wtlFN-a (SEQ ID NO 19)

Anti-CD274 SEQ ID NO: 20 wtlFN-a (SEQ ID NO 19)

Anti-TIM-3 SEQ ID NO: 20 wtlFN-a (SEQ ID NO 19)

Anti-B7-H4 SEQ ID NO: 20 wtlFN-a (SEQ ID NO 19)

[0334] The evaluation and testing of each of these Ab-IFN fusion molecules

demonstrate the significant advantages provided by them including, e.g., but not limited to, the ability to: inhibit or reduce the downregulatory activity of targeted immune-checkpoint protein antigen on the immune response; stimulate or activate immune cells in secondary lymphoid organs (e.g., draining lymph nodes, spleen); and stimulate or activate immune cells that are present in the tumor microenvironment by directly binding to them.

A. Evaluation of the ability of the fusion molecule to bind cells expressing

the targeted antigen.

[0335] To assess the ability of the Ab-IFN fusion molecule to bind to the targeted antigen, immune cells having the targeted antigen on its surface are incubated with the Ab-IFN fusion molecule or the control reagents. The binding of the Ab-IFN fusion molecule is confirmed and then compared to binding of naked Ab. Cells are reacted with biotinylated rat anti-human IgG (BD Biosciences), followed by PE-labeled streptavidin (BD Biosciences) and then analyzed by flow cytometry using a FACScan (Becton Dickinson) and analyzed using FlowJo software (TreeStar Inc).

B. Evaluation of the IFN bioactivity of the fusion molecules

[0336] To assess the anti-viral activity of the Ab-IFN fusion molecules, WISH cells

(transformed human cell line of epitheloid morphology) are seeded at 2 X 10⁵ cells/ml and treated with two-fold serial dilutions of the fusion molecules or recombinant human interferon alpha-2, or recombinant human interferon beta-1 b (or 1 b) for 24 hrs. Cells are then infected with VSV (vesicular stomatitis virus) at a concentration of 4000 pfu/100 μ I. After 72 hrs, cells are stained with 0.1 % crystal violet. Protection against viral infection is determined either by quantitating the cells surviving the infection by staining with 0.1 % crystal violet and determining the amount of dye in each well using a spot densitometer of by counting the number of plaques. C. Evaluation of Ab-IFN Fusion Molecule to Inhibit or Reduce the Downregulatory Activity of Targeted Immune-Checkpoint Protein Antigen on the Immune Response

[0337] To assess the effect of an Ab-IFN fusion molecule on proliferation in a mixed lymphocyte reaction, T cells are tested for proliferation, IFN-γ secretion and IL-2 secretion in the presence or absence of an Ab-IFN fusion molecule. For example, human CD4+ T-cells are purified from PBMC using a CD4+ positive selection kit (Dynal Biotech). Dendritic cells are derived from purified monocytes cultured with 1000 U/ml of IL-4 and 500 U/ml of GM-CSF (R&D Biosystems) for seven days. Monocytes are prepared using a monocyte negative selection kig (Mitenyi Biotech). Ab-IFN fusion molecules, naked Ab, or control reagents are added to each culture at different concentrations. The cells are cultured for 5 days at 37°C. After day 5, 100 μΙ of medium is taken from each culture for cytokine measurement. The levels of IFN-γ and IL-2 are measured using OptEIA ELISA kits (BD Biosciences). The cells are labeled with ³H- thymidine, cultured for another 18 hours, and analyzed for cell proliferation.

[0338] T regulatory cells (CD4+, CD25+) are lymphocytes that suppress the immune response. To assess the effect of an Ab-IFN fusion molecule on T regulatory cells, the effect of the addition of T regulatory cells on proliferation and IFN-γ secretion in the allogeneic dendritic cell and T cell MLR in the presence or absence of an Ab-IFN fusion molecule s tested. For example, T regulatory cells are purified from PBMC using a CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec). T regulatory cells are added into a mixed lymphocyte reaction (see above) containing purified CD4+CD25- T cells and allogeneic dendritic cells in a 2:1 ratio of CD4+CD25- to T regulatory cells. An Ab-IFN fusion molecule is added to each culture at a concentration of 10 μg/ml. The cells are cultured for 5 days at 37°C at which time the

supernatants were analyzed for IFN-γ secretion using a Beadlyte cytokine detection system (Upstate). The cells are labeled with ³H-thymidine, cultured for another 18 hours, and analyzed for cell proliferation.

D. Evaluation of Ab-IFN Fusion Molecule to Stimulate Immune Cells

[0339] To assess the ability of the Ab-IFN fusion molecule to stimulate an antigen- specific T cell response, a 3A9 T Cell Peptide Stimulation Assay (see e.g., Workman et al.

(2003) J. Immunol. 169:5392-5395; Workman et al. (2002) Eur. J. Immunol. 32:2255-2263) may be used. Example 3

[0340] This Example describes the evaluation and testing of various Ab-IFN fusion molecules listed in Table 5. Each of the targeted immune-checkpoint protein antigens for the antibodies listed in Table 5 are on the surface of various tumor cells. As such, in the assays used for the evaluation of a particular fusion molecule, tumor cells known to have the targeted antigen on its surface are used, e.g., tumor cells having CD274 on its surface are used to evaluate an anti-CD274-IFN fusion molecule.

Table 5

[0341] The evaluation and testing of each of these Ab-IFN fusion molecules serves to identify and demonstrate the significant advantages provided by them, including, e.g., the ability to: induce direct cell death of tumor cells by engaging IFN-aR expressed on tumor cells; negate some of the suppressive actions of tumors on dendritic cells (DCs), potentially leading to more efficient cross presentation of tumor antigens to T cells by DCs, i.e., improve CD8+ CTL priming; directly activate the CD8+ CTL functions which will allow efficient killing of tumor cells; directly activating NK cell functions by the IFN which will allow efficient killing of tumor cells; induce the up-regulation of the co-inhibitory immune-checkpoint proteins expressed on or associated with tumor cells; and directly negate other mechanisms for immune evasion, e.g., the major inhibitory pathways mediated by certain immune-checkpoint proteins on T cells/B cells.

[0342] In addition to the assays described in Example 2, the following additional assays may be performed.

E. In vitro anti-proliferative effects

[0343] To assess the ability of the fusions molecules to inhibit/kill tumor cells, freshly isolated tumor cell lines which have the targeted immune-checkpoint protein antigen on its surface are incubated with various concentrations of interferon, naked antibody or fusion molecule for 72 hrs and growth inhibition assessed using the CellTiter 96 Aqueous cell proliferation assay. Alternatively, tumor cells having the targeted antigen on its surface are incubated with various concentrations of interferon, naked antibody or fusion molecule for 72 hrs and then stained with Annexin V and propidium iodide (PI) and analyzed by flow cytometry.

F. Determination of Apoptosis

[0344] Freshly isolated tumor cells which have the targeted antigen on its surface are treated with various amounts of the Ab-IFN fusion molecules, or control antibody. After 3-4 days of culture, apoptosis of tumor cells will be determined by washing the cells with ice-cold PBS, and the Annexin V/propidium iodide (PI) assay will be conducted using the Vybrant Apoptosis Assay Kit #2 following procedures suggested by the manufacturer (Molecular Probes). The percentage of apoptotic cells is calculated as the sum of the percentages of early apoptotic cells and late apoptotic cells.

G. In vivo Efficacy

[0345] The in vivo efficacy of the of the fusions molecules may be evaluated in, e.g., murine xenograft models that are well known and described in the art, or in in vivo mice assays generally described as follows (here using an anti-CD274 Ab-IFN-a fusion molecule as an example):

[0346] Mice (groups of 4) are injected subcutaneously with CD274-expressing tumor cells on day zero. On days 1 , 2 and 3 they are treated intravenously with phosphate buffered saline (PBS) or 0.4 μg, 2 μg, or 10 μg of anti-CD274 Ab-IFN-α fusion molecules and tumor growth monitored.

[0347] Mice are inoculated with CD274-expressing tumor cells on day 0. On days 5, 6 and 7 they are treated with PBS or 10 μg of anti-CD274 Ab-IFN-α fusion molecules. They were monitored for tumor growth and survival.

[0348] Mice are inoculated with CD274-expressing tumor cells on day 0 and treated on days 5, 6 and 7 with 10 μg of anti-PD-1 Ab-lgG3, or 10 μg of anti-CD274 Ab-IFN-α fusion molecules and followed for tumor growth and survival.

Example 4 [0349] This Example describes the use of the Ab-IFN fusion molecules described herein to treat a patient at risk for, or afflicted with, cancer. It is anticipated that the collection of significant advantages provided by the Ab-IFN fusion molecules as described above, e.g., the ability to: inhibit or reduce the downregulatory activity of targeted immune-checkpoint protein antigen on the immune response; stimulate or activate immune cells in secondary lymphoid organs (e.g., draining lymph nodes, spleen); stimulate or activate immune cells that are present in the tumor microenvironment by directly binding to them; induce direct cell death of tumor cells by engaging IFN-ocR expressed on tumor cells; negate some of the suppressive actions of tumors on dendritic cells (DCs), potentially leading to more efficient cross presentation of tumor antigens to T cells by DCs, i.e., improve CD8+ CTL priming; directly activate the CD8+ CTL functions which will allow efficient killing of tumor cells; directly activate NK cell functions by the IFN which will allow efficient killing of tumor cells; induce the up-regulation of the co-inhibitory immune-checkpoint proteins expressed on or associated with tumor cells; and directly negate other mechanisms for immune evasion, e.g., the major inhibitory pathways mediated by certain immune-checkpoint proteins on T cells/B cells, will provide for optimized and improved cancer immunotherapy.

[0350] To test the clinical efficacy of Ab-IFN fusion molecules in humans, individuals with various cancers are identified and randomized to a treatment group. Treatment groups include a placebo group and one to three groups treated with an Ab-IFN fusion molecule. In certain treatment groups, the individuals would also be treated with an immune-checkpoint protein antigen inhibitor. Individuals are followed prospectively for one to three years. It is anticipated that individuals receiving treatment would exhibit an improvement. It is anticipated that the treatment may be particularly beneficial to an individual who has previously failed to respond to immune-checkpoint protein antigen inhibitor monotherapy.

[0351] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the invention. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Sequence Listings

[0352] The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter code for amino acids, as defined in 37 C.F.R. 1 .822.

[0353] SEQ ID NO: 1 is the amino acid sequence of the heavy chain variable region of an anti-CD276 antibody. SEQ ID NO: 2 is the amino acid sequence encoding the light chain variable region of an anti-CD276 antibody.

[0354] SEQ ID NO: 3 is the amino acid sequence of the heavy chain variable region of an anti-CD272 antibody. SEQ ID NO: 4 is the amino acid sequence encoding the light chain variable region of an anti-CD272 antibody.

[0355] SEQ ID NO: 5 is the amino acid sequence of the heavy chain variable region of an anti-CD152 antibody. SEQ ID NO: 6 is the amino acid sequence encoding the light chain variable region of an anti-CD152 antibody.

[0356] SEQ ID NO: 7 is the amino acid sequence of the heavy chain variable region of an anti-CD223 antibody. SEQ ID NO: 8 is the amino acid sequence encoding the light chain variable region of an anti-CD223 antibody.

[0357] SEQ ID NO: 9 is the amino acid sequence of the heavy chain variable region of an anti-CD279 antibody. SEQ ID NO: 10 is the amino acid sequence encoding the light chain variable region of an anti-CD279 antibody.

[0358] SEQ ID NO: 1 1 is the amino acid sequence of the heavy chain variable region of an anti-CD274 antibody. SEQ ID NO: 12 is the amino acid sequence encoding the light chain variable region of an anti-CD274 antibody.

[0359] SEQ ID NO: 13 is the amino acid sequence of the heavy chain variable region of an anti-CD274 antibody. SEQ ID NO: 14 is the amino acid sequence encoding the light chain variable region of an anti-TIM-3 antibody.

[0360] SEQ ID NO: 15 is the amino acid sequence of the heavy chain variable region of an anti-B7-H4 antibody. SEQ ID NO: 16 is the amino acid sequence encoding the light chain variable region of an anti-B7-H4 antibody.

[0361] SEQ ID NO: 17 is the amino acid sequence of a human wildtype ΙΡΝ-β-l a molecule.

[0362] SEQ ID NO: 18 is the amino acid sequence of a human wildtype IFN-β-Ι b molecule.

[0363] SEQ ID NO: 19 is the amino acid sequence of a human wildtype IFN-oc2 molecule.

[0364] SEQ ID NOS: 20 - 32 are the amino acid sequences of various peptide linkers.

Claims

What is claimed is:

1 . A fusion molecule comprising an interferon (IFN) molecule attached to an antibody (Ab) which targets an immune-checkpoint protein antigen, wherein the fusion molecule is capable of treating a proliferative disease.

2. A fusion molecule according to claim 1 , wherein the IFN molecule is a type 1 interferon molecule selected from the group consisting of an interferon alpha (IFN- ) molecule, an IFN- mutant molecule, an interferon beta (IFN-β) molecule and an IFN-β mutant molecule.

3. A fusion molecule according to any one of claims 1 -2, wherein the antibody is an antibody selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding fragment, Fab, Fab', Fab₂, Fab'₂, IgG, IgM, IgA, IgE, scFv, dsFv, dAb, nanobodies, unibodies, and diabodies.

4. A fusion molecule according to any one of claims 1 -3, wherein the antibody binds to an immune-checkpoint protein antigen with a dissociation constant (K_D) of at least about 1 x10 ³ M, at least about 1 x10 ⁴ M, at least about 1 x10 ⁵ M, at least about 1 x10^-6 M, at least about 1 x10 ⁷ M, at least about 1 x10^-8 M, at least about 1 x10 ⁹ M, at least about 1 x10^_10 M, at least about 1 x10 ¹¹ M, or at least about 1 x10^-12 M.

5. A fusion molecule according to any one of claims 1 -4, wherein the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of an immune cell.

6. A fusion molecule according to any one of claims 1 -4, wherein the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of a tumor cell.

7. A fusion molecule according to any one of claims 1 -6, wherein the antibody specifically binds an antigen selected from the group consisting of CD276, CD272, CD152, CD223, CD279, CD274, TIM-3 and B7-H4 antigen.

8. A fusion molecule according to any one of claims 1 -7, wherein the interferon molecule is directly attached to the antibody.

9. A fusion molecule according to any one of claims 1 -8, wherein the interferon molecule is attached to the antibody via a proteolysis resistant peptide linker that is fewer than 20 amino acids in length.

10. A fusion molecule of claim 9 wherein the peptide linker has the sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 20 and SEQ ID NO: 21 .

1 1 . A fusion molecule according to any one of claims 1 -10, wherein the fusion molecule is a recombinantly expressed fusion molecule.

12. A method of treating a proliferative disease in a patient, comprising administering to the patient a therapeutically effective amount of a fusion molecule of according to any one of claims 1 -1 1 .

13. A method according to claim 12, wherein the proliferative disease is a cancer selected from the group consisting of a B cell lymphoma, a lung cancer, a bronchus cancer, a colorectal cancer, a prostate cancer, a breast cancer, a pancreas cancer, a stomach cancer, an ovarian cancer, a urinary bladder cancer, a peripheral nervous system cancer, an esophageal cancer, a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancer of the oral cavity or pharynx, a liver cancer, a kidney cancer, a biliary tract cancer, a small bowel or appendix cancer, a salivary gland cancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma, a chondrosarcoma, a liposarcoma, a testes cancer, and a malignant fibrous histiocytoma, a skin cancer, a head and neck cancer, lymphomas, sarcomas, multiple myeloma and leukemias.

14. A method according to any one of claims 12-13, wherein the fusion molecule is administered to the patient at a dosage selected from the group consisting of: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, about 0.05 to about

0.06 mg/kg, about 0.06 to about 0.07 mg/kg, about 0.07 to about 0.08 mg/kg, about 0.08 to about 0.09 mg/kg, about 0.09 to about 0.1 mg/kg, about 0.1 to about 0.2 mg/kg, about 0.2 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg.

15. A method according to any one of claims 12-14, wherein the fusion molecule is administered to the patient at a dosage selected from the group consisting of: about 0.01 to about 0.02 mg/kg, about 0.02 to about 0.03 mg/kg, about 0.03 to about 0.04 mg/kg, about 0.04 to about 0.05 mg/kg, about 0.05 to about 0.06 mg/kg, about 0.06 to about 0.07 mg/kg, about 0.07 to about 0.08 mg/kg, about 0.08 to about 0.09 mg/kg, and about 0.09 to about 0.1 mg/kg.

16. A method according to any one of claims 12-15, wherein the patient has a recurrent cancer.

17. A method according to any one of claims 12-15, wherein the patient has a resistant or refractory cancer.

18. A method of treating a proliferative disease in a patient, comprising administrating to the patient: a) a therapeutically effective amount of a fusion molecule according to any one of claims 1 -1 1 ; and b) a therapeutically effective amount of a second agent.

19. A method according to claim 18, wherein the second agent is an immune-checkpoint protein antigen inhibitor selected from the group consisting of an anti-CD276 Ab, an anti-CD272 Ab, an anti-CD152 Ab, an anti-CD223 Ab, an anti-CD279 Ab, an anti-CD274 Ab, an anti-TIM-3 Ab and an anti-B7-H4 Ab.

20. A method according to claim 18, wherein the second agent is selected from the group consisting of a non-fused anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD138 Ab, anti- Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab.

21 . A method according to claim 18, wherein the second agent is selected from the group consisting of an anti-HER2/neu Ab, anti-CD20 Ab, anti-CD33 Ab, anti-CD138 Ab, anti-Grp94 (endoplasmin) Ab, anti-CD276 Ab, and anti-CD70 Ab, and wherein the Ab is fused to an IFN molecule.

22. A method of enhancing an anti-tumor immune response, or killing or inhibiting growth of tumor cells in a patient, comprising administering to the patient a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a fusion molecule according to any one of claims 1 -1 1 , wherein the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of an immune cell.

23. A method of enhancing an anti-tumor immune response, or killing or inhibiting growth of tumor cells in a patient, comprising administering to the patient a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a fusion molecule according to any one of claims 1 -1 1 , wherein the fusion molecule comprises an interferon molecule attached to an antibody which targets an immune-checkpoint protein antigen on the surface of a tumor cell.

24. A pharmaceutical composition comprising a fusion molecule according to any one of claims 1 -1 1 in a pharmaceutically acceptable carrier.

25. A pharmaceutical composition of claim 24, wherein the composition is formulated for administration via a route selected from e.g., subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or infusions.