Antigen binding proteins targeting PD-1

Abstract

The present disclosure provides antigen binding proteins that specifically bind PD-1. The present disclosure further provides mouse anti-mouse PD-1 blocking antibodies, engineered cells, and methods of making and using the antigen binding proteins, anti-PD-1 antibodies, and cells of the disclosure. Aspects of the disclosure further relate to a polypeptide comprising any one of the complementary determining regions of the present disclosure.

Description

TITLE OF THE INVENTIONANTIGEN BINDING PROTEINS TARGETING PD-1CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No. 63 / 503,632, filed May 22, 2023, the entire disclosure of which is incorporated herein by reference.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named “MDCC010WO_ST26.xml” which is 15.2 kilobytes (measured in MS-Windows®) and created on May 20, 2024, and comprises 16 sequences, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of cancer immunotherapy, and more specifically to compositions and methods for the treatment of cancer.BACKGROUND OF THE INVENTION

[0004] Programmed cell death protein 1 , also known as PD- 1 is a cell surface receptor protein expressed on certain immune cells. PD-1 functions as an “immune checkpoint” inhibitor that regulates immune self-tolerance and protects against autoimmunity. PD-1 limits the expansion and activity of self-reactive T-cells and may also have a positive role in expanding immunosuppressive regulatory T-cells (Treg). The PD-1 ligands PD-L1 and PD-L2 negatively regulate T-cell responses following receptor engagement. PD-L1 is upregulated on macrophages and dendritic cells in response to inflammation, and on T-cells and B-cells following immune cell activation. PD-L2 is also expressed on myeloid cells when induced by a range of inflammatory stimuli such as cytokine exposure. PD-L1 and / or PD-L2 are also highly expressed in many cancers, including breast cancer, lung cancer, brain cancer, skin cancer, gastric cancer, colorectal cancer, esophageal cancer, pancreatic cancer, thymic cancer, cervical cancer, bladder cancer, thyroid cancer, ovarian cancer, leukemia, melanoma, thymoma, adrenocortical carcinoma, head and neck squamous cancer, oropharyngeal squamous cell carcinoma, glioma, mesothelioma, merkel cell carcinoma, hepatocellular carcinoma, intrahepatic cholangiocarcinoma, renal cell carcinoma, and nasopharyngealcarcinoma. Antibodies which block the PD-1 signaling pathway and thus activate and derepress host anti-tumor immune responses are promising therapeutics for the treatment of cancer.

[0005] Immunocompetent mouse models have been used to study immunotherapy and, in particular, immune checkpoint inhibitors in a preclinical setting for more than 20 years. Currently available anti-mouse PD-1 blocking antibodies for use in such studies are derived from other rodent species, such as rat or hamster, and repeated administration of such antibodies to an immunocompetent mouse produces a host anti-drug antibody response. As a result, currently available anti-mouse PD-1 blocking antibodies can only be administered for a very short time in vivo. This severely limits the capacity to model human anti-PD-1 therapy, which is often administered for years, in murine model systems. There is thus a continuing need in the art for novel PD-1 specific antibodies for use in preclinical and clinical cancer treatment.SUMMARY OF THE INVENTION

[0006] In one aspect the present disclosure provides an isolated antigen binding protein that specifically binds programmed death cell protein 1 (PD-1) comprising: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC-CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC- CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein the variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof. In one embodiment, the HC-CDR1 region comprises the amino acid sequence of SEQ ID NO:1, the HC-CDR2 region comprises the amino acid sequence of SEQ ID NO:2, and the HC-CDR3 region comprises the amino acid sequence of SEQ ID NO:3; and the LC-CDR1 region comprises the amino acid sequence of SEQ ID NO:5, the LC-CDR2 region comprises the amino acid sequence of SEQ ID NO:6, and a LC-CDR3 region comprises the amino acid sequence of SEQ ID NO:7. In another embodiment, the antigen binding protein is selected from the group consisting of a monoclonal antibody, an antigen binding fragment,a chimeric antibody, a bispecific antibody, and a chimeric antigen receptor. The antigen binding protein, in yet another embodiment, specifically binds mouse PD- 1. In still yet another embodiment, the heavy chain variable region further comprises an amino acid sequence having at least about 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4. In one embodiment, the light chain variable region further comprises an amino acid sequence having at least about 80% amino acid sequence identity to SEQ ID NO:8. The antigen binding protein, in another embodiment, is a monoclonal antibody, and the monoclonal antibody is a murine, a rodent, a rabbit, a chimeric, a humanized, or a human antibody. In yet another embodiment, the monoclonal antibody is a murine antibody. The monoclonal antibody, in still yet another embodiment, is a humanized antibody. In one embodiment, the antigen binding protein comprises a single-chain variable fragment (scFv) fragment, a fragment antigenbinding region (Fab) fragment, Fab' fragment, a F(ab’)2 fragment, or a Fv fragment. In another embodiment, a composition comprising an antigen binding protein of the present disclosure is provided.

[0007] In another aspect, the present disclosure provides an antibody-drug conjugate comprising an isolated antigen binding protein that specifically binds programmed death cell protein 1 (PD-1) comprising: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC-CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; andb) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC-CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein the variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof, and an anticancer drug or a detectable label. In one embodiment, the anticancer drug or detectable label is conjugated to the isolated antigen binding protein through a cleavable linker. In another embodiment, the anticancer drug is a chemotherapeutic agent, an immunotherapeutic agent, or a radiotherapeutic agent. In yet another embodiment, the detectable label is a paramagnetic ion, a radioactive isotope, a fluorochrome, an NMR-detectable agent, or an X-ray imaging agent.

[0008] In yet another aspect, the present disclosure provides a recombinant polynucleotide molecule comprising a nucleotide sequence that encodes an antigen binding protein thatspecifically binds programmed death cell protein 1 (PD- 1 ), wherein the antigen binding protein comprises: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC-CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b)a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC-CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein the variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof. In one embodiment, a cell comprising a recombinant polynucleotide molecule of the present disclosure is provided. In another embodiment, the present disclosure provides a method of producing an engineered cell, the method comprising introducing a recombinant polynucleotide molecule of the present disclosure into a cell.

[0009] In still yet another aspect, the present disclosure provides a hybridoma or an engineered cell that produces an antigen binding protein that specifically binds programmed death cell protein 1 (PD-1), wherein the antigen binding protein comprises: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC-CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC-CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein the variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof. In one embodiment, the present disclosure provides a method of producing an antigen binding protein, the method comprising culturing a hybridoma or engineered cell of the present disclosure.

[0010] In one aspect, the present disclosure provides a recombinant antigen binding protein comprising: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, a HC-CDR2 region comprising theamino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC-CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC-CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, whereinthe variant of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof. In one embodiment, the recombinant antigen binding protein is selected from the group consisting of a recombinant antibody, a bispecific antibody, a chimeric antibody, a chimeric antigen receptor, and antigen binding fragments thereof.

[0011] In another aspect, the present disclosure provides, a composition comprising an antigen binding protein comprising: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC-CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; andb) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC-CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein the variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof. In one embodiment, the composition is serum-free, endotoxin-free, or sterile.

[0012] In yet another aspect, the present disclosure provides a method of treating cancer, the method comprising administering an isolated antigen binding protein, a composition, a hybridoma, an engineered cell, an antibody-drug conjugate, or a recombinant antigen binding protein to a subject in need thereof. In one embodiment, the cancer comprises a cancer cell that expresses a PD-1 ligand. In another embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, a lung cancer cell, a brain cancer cell, a skin cancer cell, a gastric cancer cell, a colorectal cancer cell, a esophageal cancer cell, a pancreatic cancer cell, a thymic cancer cell, a cervical cancer cell, a bladder cancer cell, a thyroid cancer cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a thymoma cell, an adrenocortical carcinoma cell, a head and neck squamous cancer cell, an oropharyngeal squamous cellcarcinoma cell, a glioma cell, a mesothelioma cell, a merkel cell carcinoma cell, a hepatocellular carcinoma cell, an intrahepatic cholangiocarcinoma cell, a renal cell carcinoma cell, and a nasopharyngeal carcinoma cell. In yet another embodiment, the subject is a rodent subject, a murine subject, or a human subject. In still yet another embodiment, the methods of the present disclosure may further comprise administering a second therapy to the subject. The second therapy, in one embodiment, is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, or surgery.

[0013] In still yet another aspect, the present disclosure provides a method of detecting PD-1 in a sample, the method comprising: a) contacting the sample with the isolated monoclonal antibody or antigen binding protein of claim 1; and b) detecting binding of the isolated monoclonal antibody or antigen binding protein to the sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIG. 1 demonstrates the results of an ELISA assay in which the supernatant from selected antibody clones was tested to analyze antibody binding to mouse PD-1.

[0016] FIG. 2 demonstrates the results of an ELISA assay in which selected purified antibodies were tested to analyze antibody binding to mouse PD-1.

[0017] FIG. 3 demonstrates the results of an assay to measure the ability of selected antibodies to reduce mouse PD-L1-FC binding to mouse PD-1 by about 2.5-fold.

[0018] FIG. 4 demonstrates the results of an experiment performed to determine whether selected antibodies were able to reduce tumor burden (FIG. 4A) or increased survival (FIG. 4B) in an immunocompetent mouse model of colorectal cancer.

[0019] FIG. 5 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of antibody 384-35.

[0020] FIG. 6 shows the nucleotide sequence and amino acid sequence of the light chain variable region of antibody 384-35.BRIEF DESCRIPTION OF THE SEQUENCES

[0021] SEQ ID NO: 1 - The amino acid sequence of the CDR1 of the heavy chain of antibody 384-35.

[0022] SEQ ID NO:2 - The amino acid sequence of the CDR2 of the heavy chain of antibody 384-35.

[0023] SEQ ID NO:3 - The amino acid sequence of the CDR3 of the heavy chain of antibody 384-35.

[0024] SEQ ID NO:4 - The amino acid sequence of the heavy chain variable region of antibody 384-35.

[0025] SEQ ID NO:5 - The amino acid sequence of the CDR1 of the light chain of antibody 384-35.

[0026] SEQ ID NO:6 - The amino acid sequence of the CDR2 of the light chain of antibody 384-35.

[0027] SEQ ID NO:7 - The amino acid sequence of the CDR3 of the light chain of antibody 384-35.

[0028] SEQ ID NO:8 - The amino acid sequence of the light chain variable region of antibody 384-35.

[0029] SEQ ID NO:9 - A nucleotide sequence that encodes SEQ ID NO:1.

[0030] SEQ ID NO: 10 - A nucleotide sequence that encodes SEQ ID NO:2.

[0031] SEQ ID NO: 11 - A nucleotide sequence that encodes SEQ ID NO:3.

[0032] SEQ ID NO: 12 - A nucleotide sequence that encodes SEQ ID NO:4.

[0033] SEQ ID NO: 13 - A nucleotide sequence that encodes SEQ ID NO:5.

[0034] SEQ ID NO: 14 - A nucleotide sequence that encodes SEQ ID NO:6.

[0035] SEQ ID NO: 15 - A nucleotide sequence that encodes SEQ ID NO:7.

[0036] SEQ ID NO: 16 - A nucleotide sequence that encodes SEQ ID NO:8.DETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure provides antibodies and antigen binding fragments which specifically bind epitopes of PD-1. In particular embodiments, the antibodies and antigenbinding fragments specifically block PD-1 from engaging with PD-1 ligands. The present disclosure further provides hybridomas and engineered cells, encoding nucleotide sequences, expression constructs, and associated methods of treating cancer which comprise or encode the antibodies and antigen binding fragments of the present disclosure.

[0038] Immunocompetent mouse models have been used to study immunotherapy and, in particular, immune checkpoint inhibitors in a preclinical setting for several years. Currently available anti-mouse PD-1 blocking antibodies for use in such studies are derived from other rodent species, such as rat or hamster, and repeated administration of such antibodies to an immunocompetent mouse produces a host anti-drug antibody response. As a result, currently available anti-mouse PD-1 blocking antibodies can only be administered for a very short time in vivo. There is a continuing need in the art for novel PD-1 specific antibodies for use in preclinical and clinical cancer treatment, and the present disclosure provides antibodies and antigen binding fragments that meets this need.A. Antibodies and Antigen Binding Fragments

[0039] Antibodies and antigen binding fragments are both members of the broader genus that includes all antigen binding proteins. The term “antibody” as used herein refers to an intact immunoglobulin of any isotype or an antibody fragment that can compete with an intact antibody for specific binding to the target antigen. An “antigen binding fragment” as used herein refers to refers to a portion of a protein which is capable of binding specifically to an antigen. The term “antigen binding protein” as used herein refers to any protein that binds a specified target antigen. In some embodiments of the present disclosure the specified target antigen is the PD-1 protein or fragment thereof. An antigen binding protein includes but is not limited to antibodies and antigen binding fragments. Antibodies of the present disclosure, may include but are not limited to chimeric, humanized, fully human, and bispecific antibodies. An intact antibody may comprise, in certain embodiments, two full-length heavy chains and two full-length light chains. In other embodiments, however, an antibody may include fewer chains. For example, antibodies naturally occurring in camelids can comprise only heavy chains. Antibodies can be derived from a single source or may be chimeric. As used herein the term “chimeric antibody” refers to an antibody that comprises portions that are derived from two different antibodies or an antibody variable region derived from one species paired with a constant region from a different species. The antigen binding proteins, antibodies, and binding fragments of the present disclosure may be produced using any technique known in the art. Non-limiting examples of such techniques include production in hybridomas,production by recombinant DNA techniques, and production by enzymatic or chemical cleavage of intact antibodies. An antibody or antigen binding fragment may include, in many embodiments, two full-length heavy chains and two full-length light chains. In some embodiments, an antibody, antigen binding fragment, or an antigen binding protein may include an antibody derivative, an antibody variant, an antibody fragment, or an antibody mutant. Non-limiting examples of antibodies, antigen binding fragments, and antigen binding proteins include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, peptibodies, and fragments thereof.

[0040] In some embodiments, an antigen binding fragment may be derived from an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. In certain embodiments, the antigen binding fragment is not derived from an antibody but rather is derived from a receptor. Examples of antigen binding fragment include but are not limited to a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody, a single domain antibody (sdAb), a camelid antibody or a nanobody, a domain antibody, and a bivalent domain antibody. An antigen binding fragment, in many embodiments, is capable of binding to the same antigen to which the parent antibody binds. In particular embodiments, an antigen binding fragment may comprise one or more CDRs from a particular antibody grafted onto a framework region of one or more different antibodies. The antigen binding fragment, in certain embodiments, may be derived from a receptor. In particular embodiments, an antigen binding fragment may comprise one or more amino acid substitutions, additions, deletions, or mutations. In certain embodiments, an antigen binding fragment does may not bind to the natural ligand of the receptor from which the antigen binding fragment is derived. The term “Fab fragment” as used herein refers to an antigen binding protein that comprises one light chain and the CHi and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The term “Fab' fragment” as used herein refers to an antigen binding protein that comprises one light chain and a portion of one heavy chain that contains the VH domain and the CHi domain and also the region between the CHi and CH2 domains, such that an interchain disulfide bond may be formed between two heavy chains of two Fab' fragments to form an F(ab')2 molecule. A “F(ab')2 fragment” as used herein refers toan antigen binding protein that comprises two light chains and two heavy chains comprising a portion of the constant region between the CHi and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')z fragment thus comprises two Fab' fragments with a disulfide bond between the two heavy chains.

[0041] A single chain variable fragment (scFv) is a fusion protein produced by the fusion of the variable regions of the heavy and light chains. In certain embodiments, the heavy and light chains are linked using a short linker sequence. In one embodiment, the linker sequence comprises helix-tum-helix promoting amino acid residues. In another embodiment, the linker sequence comprises the amino acids alanine, serine, or glycine. This chimeric molecule retains the antigen specificity, despite removal of the constant regions and the introduction of a linker peptide. Single chain variable fragments may be produced using any method known in the art, including but not limited to phage display and subcloning of heavy and light chains derived from a hybridoma. Single chain variable fragments may be purified or immobilized using any method known in the art, including but not limited to Protein L purification or immobilization.

[0042] A “Fc region” as used herein refers to a portion of an antibody comprising two heavy chain fragments comprising the CHi and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions. The “Fv region” as used herein refers to a portion of an antibody comprising the variable regions from both the heavy chain and the light chain. The Fv region does not comprise the constant regions of an antibody. A “single-chain antibody” as used herein refers to an Fv molecule in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain that forms an antigen binding region. Single chain antibodies are described in International Patent Application Publication No. WO 88 / 01649, U.S. Pat. No. 4,946,778, and U.S. Pat. No. 5,260,203, the disclosures of which are incorporated by reference. A “domain antibody” as used herein refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some embodiments, a domain antibody may comprise two or more VH regions which are covalently joined with a peptide linker to create a bivalent domain antibody. In certain embodiments, the two VH regions of a bivalent domain antibody can target the same or different antigens.

[0043] Naturally occurring antibodies are usually produced as a tetramer. Each tetramer typically comprises two identical pairs of polypeptide chains, each pair comprising one full- length light chain and one full-length heavy chain. In some embodiments, the light chain ofan antibody is about 25 kDa in size, and the heavy chain of an antibody is about 50-70 kDa in size. The amino-terminal portion of each chain typically includes an antigen binding variable region of about 100 to 110 amino acids that is responsible for antigen specific binding. The carboxy-terminal portion of each chain typically includes a constant region that may be responsible for effector function. In some embodiments, light chains may be classified as kappa light chains or lambda light chains. Heavy chains, in particular embodiments, may be classified as mu, delta, gamma, alpha, or epsilon. Heavy chain classification, in many embodiments, defines isotype of the antibody as IgM, IgD, IgG, IgA, or IgE. The IgG isotype has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. The IgM isotype has subclasses including, but not limited to, IgMl and IgM2. The IgA isotype is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. In many embodiments, the variable and constant regions of a full-length heavy chain and the variable and constant regions of a full-length light chain are joined by a “J” region of about 12 or more amino acids. In certain embodiments, the variable regions and the constant regions of a heavy chain may also be joined by a “D” region of about 10 more amino acids in addition to the “J” region. See, e.g., Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. 1989) (incorporated herein by reference). The variable regions of each light / heavy chain pair typically form the antigen binding site of any antibody or antigen binding fragment.

[0044] The term “variable region” or “variable domain” as used herein refers the variable portion of the heavy and / or light chain of an antibody, antigen binding fragment, or antigen binding protein. In some embodiments, the variable region may include about 120 to about 130 amino-terminal amino acids of the heavy chain and about 100 to 110 amino-terminal amino acids of the light chain. The variable region of different antibodies, antigen binding fragments, or antigen binding proteins, in particular embodiments, may differ extensively in amino acid sequence, even when the different antibodies are of the same species. The variable region of an antibody, antigen binding fragment, or antigen binding protein typically determines antigen binding specificity. The variable regions of an antibody, antigen binding fragment, or antigen binding protein may, in a number of embodiments, exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the light chain and the heavy chain, in particular embodiments, are aligned by the framework regions, allowing for epitope specific binding. From N-terminal to C-terminal, both light and heavy chain variable regions may comprise the domains FR1 , CDR1 , FR2, CDR2, FR3 , CDR3 ,and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987) or Chothia et al., Nature, 342:878-883 (1989).

[0045] In certain embodiments, an antibody heavy chain may specifically bind to an antigen in the absence of an antibody light chain. In some embodiments, an antibody light chain may specifically bind to an antigen in the absence of an antibody heavy chain. An antibody binding region, in particular embodiments, may specifically bind to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region may specifically bind to an antigen in the absence of an antibody heavy chain. An individual variable region, in some embodiments, specifically binds to an antigen in the absence of other variable regions.

[0046] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. This can be accomplished, in some embodiments, by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In particular embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, IMGT definition, and the contact definition. The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition considers 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 (USA), 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 other 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). The IMGT definition uses a unique numbering system that combines the definitionof framework (FR) and CDR regions, structural data from X-ray diffraction studies, and the characterization of the hypervariable loops, as described in by Lefranc M-P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol 27:55-77 (2003). In one embodiment, the CDR sequences are based on IMGT definition. In some embodiments, the CDR regions in the heavy chain are typically referred to as Hl, H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. In particular embodiments, the CDR regions in the light chain are typically referred to as LI, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. In the present disclosure, the CDR regions of the light chain variable region are also designated as LC-CDR1 , LC-CDR2, and LC- CDR3, and the CDR regions of the heavy chain variable region are designated as HC-CDR1, HC-CDR2, and HC-CDR3.

[0047] The term “light chain” as used herein refers to a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain, in many embodiments, is located at the aminoterminus of the polypeptide. Non-limiting examples of light chains include kappa chains and lambda chains. The term “heavy chain” as used herein refers to a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CHi, CH2, and CH3. The VH domain, in a number of embodiments, is located at the amino terminus of the polypeptide. In many embodiments, the CH domains are located at the carboxyl-terminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE.

[0048] The term “bispecific antigen binding protein” as used herein refers to a molecule with two antigen binding domains, which may bind the same antigen or may bind different antigens. In some embodiments, the two binding sites of a bispecific antigen binding protein or antibody may bind to two different epitopes, which can reside on the same or different protein targets. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab ' fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992). A “bivalent antigen binding protein” or “bivalent antibody” as used herein refers to an antigenbinding protein comprising two antigen binding sites. In certain embodiments, the two binding sites of a bivalent antigen binding protein have the same antigen specificity. In particular embodiments, bivalent antigen binding proteins and bivalent antibodies can be bispecific.

[0049] The phrase “specifically (or selectively) binds” or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein or complex, often in a heterogeneous population of proteins or complexes. For example, the antigen binding proteins of the present disclosure specifically bind PD-1. Thus, under typical immunoassay conditions, a specified antigen binding protein may bind to a particular protein or complex at least two times the background. In specific embodiments, a specified antigen binding protein may bind a particular protein or complex at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, 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, or at least 100 times background, including any range derivable therebetween. Specific binding to an antigen binding protein under such conditions requires an antigen binding protein that is selected by virtue of its specificity for a particular protein or complex. A variety of assay formats known in the art may be used to select antigen binding protein specifically immunoreactive with a particular protein or complex and any such assay may be used to select an antigen binding protein of the present disclosure. The antigen binding protein of the present disclosure may specifically bind, in particular embodiments, to PD-1. In one embodiment, the antigen binding protein of the present disclosure may specifically bind mouse PD-1. In another embodiment, the antigen binding protein of the present disclosure may specifically bind PD-1 and block PD-1 from engaging with PD-1 ligands. In some embodiments, an antigen binding protein of the present disclosure may crossreact with a small number of highly similar antigens. In some embodiments, the antigen binding protein of the present disclosure binds PD-1 and has a dissociation constant (Kd) of less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 0.1 nM, less than about 0.01 nM, or less than about 0.001 nM, including all ranges derivable therebetween. The term “compete” as used herein in the context of antigen binding proteins that compete for the same epitope refers to the competition between antigen binding proteins as determined by an assay in which the antigen binding protein being tested prevents or reduces specific binding of a reference antigen binding protein to a common antigen. Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.l37:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, etal., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol.32:77-82). In certain embodiments, antigen binding proteins identified by a competition assay (competing antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein, and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. In particular embodiments, when a competing antigen binding protein is present in excess, the competing antigen binding protein will reduce specific binding of a reference antigen binding protein to a common antigen by at least about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 85%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%, including all ranges derivable therebetween.

[0050] “Binding affinity” as used herein refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In some embodiments, the term binding affinity may refer to the intrinsic binding affinity reflecting a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Binding affinity can be measured by any number of common methods known in the art, and any such method may be used according to the embodiments of the present disclosure. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.

[0051] The term “antigen” as used herein refers to a substance capable of inducing an adaptive immune response. Antigen binding proteins associated with an adaptive immune response specifically bind to their target antigen. In some embodiments, an antigen may be a molecule that binds to antigen-specific receptors but cannot induce an immune response alone. Nonlimiting examples of antigens include proteins, polysaccharides, and lipids. Antigens, inparticular embodiments, may include but are not limited to parts of bacteria (coats, capsules, cell walls, flagella, fimbria, and toxins), viruses, and other microorganisms. In some embodiments, antigens also include tumor antigens that antigens generated by mutations in tumors. Antigens may also include immunogens and haptens.

[0052] The term “epitope” as used herein refers to the specific group of atoms or amino acids of an antigen to which an antigen binding protein specifically binds. In some embodiments, an epitope may be a linear epitope or a conformational epitope. A linear epitope, in particular embodiments, may be formed by a continuous sequence of amino acids of the antigen. A conformational epitope, in certain embodiments, may be comprised of discontinuous sections of the amino acid sequence of an antigen. A linear epitope many interact with an antigen binding protein, in particular embodiments, based on primary structure. A conformational epitope may interact with an antigen binding protein, in certain embodiments, based on the 3D structure of the antigen. An epitope, in some embodiments, may be about 3 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, or about 5 to about 6 amino acids in length, including all ranges derivable therebetween. In particular embodiments, two antigen binding proteins may bind the same epitope if they exhibit competitive binding for the antigen.

[0053] The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificially constructed hybrid protein or polypeptide containing an antigen binding protein linked to a domain that activates an immune cell. In certain embodiments, a chimeric antigen reception may comprise a single chain variable fragment (scFv)) and a T-cell signaling, T-cell activation, or NK cell activation domain (see, e.g., Kershaw et al., supra, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993), and Sadelain et al., Curr. Opin. Immunol. 21(2): 215-223 (2009)). CARs are capable of directing an immune response against a selected target in a non- MHC-restricted manner.

[0054] The term "isolated " as used herein in reference to, for example, an antibody, antigen binding fragment, an antigen binding protein, a polypeptide, or polynucleotide molecule, refers to a molecule that is not associated with naturally associated components that accompany the molecule in its native state. In some embodiments, an isolated molecule is substantially free of other molecules from the same species, is expressed by a cell from a different species, or is expressed by a different cell type that the cell type in which it is expressed in nature. A molecule that is chemically synthesized or that is expressed in a cellular system different from the cell from which it naturally originates, will be isolated from its naturally associated components. A molecule, in certain embodiments, may also be rendered substantially free ofnaturally 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.

[0055] The terms “linked” or “conjugated” as used herein refer to the association of one molecule with another molecule through intramolecular interactions or intermolecular interactions. Non-limiting example of such intramolecular interactions include covalent bonds, metallic bonds, and ionic bonds. Non-limiting examples of intermolecular interactions include hydrogen bonds and noncovalent bonds.

[0056] In some aspects, the present disclosure provides recombinant polynucleotide molecules that encode an antibody, an antigen binding fragment, or an antigen binding protein of the present disclosure. The present disclosure also provides recombinant polypeptides and recombinant or engineered cells comprising an antibody, an antigen binding fragment, or an antigen binding protein of the present disclosure.

[0057] As used herein, the term “recombinant” refers to a polynucleotide molecule, protein, or cell that is not naturally present, or is not naturally present in the same form or structure and was created by human intervention. In one embodiment, a recombinant polynucleotide may be a DNA molecule or may be an RNA molecule. A recombinant polynucleotide molecule or a recombinant polypeptide molecule or protein may comprise, in certain embodiments, a combination of two or more polynucleotide or polypeptide sequences that do not naturally occur together in the same manner, such as a polynucleotide molecule or protein that comprises at least two polynucleotide or protein sequences that are operably linked but heterologous with respect to each other. As used herein the term “heterologous” refers to a polynucleotide molecule or protein that is not naturally present or is not naturally present in the same form or structure and was created by human intervention. For example, a heterologous polynucleotide molecule or protein may not naturally occur in the cell being transformed or may be expressed in a manner or genomic context that differs from the natural expression pattern or genomic context found in the cell being transformed. The heterologous polynucleotide molecule or protein, in some embodiments, may be overexpressed in the cell being transformed. In certain embodiments, a recombinant polynucleotide molecule, protein, construct, or vector may comprise any combination of two or more polynucleotide or protein sequences in the samemolecule which are heterologous to one another, such that the combination is man-made and not normally found in nature. As used herein, the phrase “not normally found in nature” means not found in nature without human intervention. A recombinant polynucleotide or protein molecule, may comprise, for example, polynucleotide or protein sequences that are separated from other polynucleotide or protein sequences that exist in proximity to each other in nature. A recombinant polynucleotide or protein molecule may also comprise, for example, polynucleotide or protein sequences that are adjacent to or contiguous with other polynucleotide or protein sequences that are not naturally in proximity with each other. Such a recombinant polynucleotide molecule, protein, or expression construct may also refer to a polynucleotide or protein molecule or sequence that has been genetically engineered or constructed outside of a cell. For example, a recombinant polynucleotide molecule may comprise any engineered or man-made plasmid, vector, or expression construct, and may include a linear or circular DNA molecule. Such plasmids, vectors, and expression constructs may comprise, for example, various maintenance elements including, but not limited to, a heterologous promoter sequence, a prokaryotic origin of replication, or a selectable marker.

[0058] The present disclosure further provides engineered antigen binding proteins, which comprise one or more HC-CDRs or LC-CDRS of the disclosure. As used herein the term “engineered” when used in reference to an antibody, antigen binding fragment, or antigen binding protein refers to an antibody, antigen binding fragment, or antigen binding protein which comprises a recombinant protein or is encoded by a recombinant polynucleotide molecule of the present disclosure. In some embodiments, an engineered antigen binding protein may comprise a chimeric polypeptide comprising an antigen binding fragment of the present disclosure and a heterologous antibody constant region. In particular embodiments, the LC-CDRs and the HC-CDRs of the present disclosure are used with framework region sequences from a different mammalian species, for example a primate. The framework sequences, in certain embodiments may be humanized or human framework sequences, which may be used to create an antibody that specifically binds PD-1. In some embodiments, the light chain variable region comprises four light chain framework regions, designated as FRLCI, FRLC2, FRLC3, and FRLC4. The LC-CDRs of the present disclosure may be organized, in particular embodiments, as follows from the NHito the COOH direction: FRLC1-LC-CDR1- FRLC2-LC-CDR2-FRLC3,-LC-CDR3-FRLC4. In certain embodiments, the heavy chain variable region comprises four heavy chain framework regions, designated as FRHCI, FRHC2, FRHC3, and FRHC4. The HC-CDRs of the present disclosure may be organized, in certainembodiments, as follows from the NH2 to the COOH direction: FRHC1-HC-CDR1-FRHC2-HC- CDR2-FRHC3-HC-CDR3-FRHC4. In some embodiments, the framework regions of a parent light chain variable region may be replaced with framework regions of human light chain variable regions to form humanized light chain variable regions. In particular embodiments, the framework regions of a parent heavy chain variable region may be replaced with framework regions of human heavy chain variable regions to form humanized heavy chain variable regions.

[0059] The engineered antigen binding proteins of the present disclosure may, in certain embodiments, specifically bind to the proteins and complexes described herein. The engineered antigen binding proteins of the present disclosure may specifically bind, in particular embodiments, a PD-1. In one embodiment, the engineered antigen binding proteins of the present disclosure may specifically mouse PD-1 or human PD-1. In another embodiment, the antigen binding proteins of the present disclosure may specifically bind PD- 1 and block PD-1 from engaging with PD-1 ligands. In some embodiments, the engineered antigen binding proteins of the present disclosure may cross-react with a small number of highly similar antigens. In some embodiments, the polynucleotide or protein sequences of the engineered antigen binding proteins of the present disclosure may comprise heterologous sequences that are used for cloning, enhanced expression, detection, secretion, or for therapeutic control of the recombinant polynucleotide molecule or protein that are not present in endogenous antigen binding proteins. Non-limiting examples of such heterologous sequences include, multiple cloning sites, linkers, hinge sequences, modified hinge sequences, modified transmembrane sequences, a polynucleotide or protein molecule used for detection, or therapeutic controls that allow for selection or screening of cells comprising the antigen binding protein. In some aspects of the present disclosure, the engineered antigen binding protein may comprise non-antibody sequences. Accordingly, certain aspects of the present disclosure relate to engineered antigen binding proteins comprising sequences that are not naturally found in antibodies. In certain embodiments, the engineered antigen binding protein is chimeric, and thus comprises, for example, sequences which are found normally found or encoded by an antibody gene.

[0060] In some aspects, a cell specifically recognized by an antigen binding protein of the present disclosure may be any cell which expresses PD-1. In one embodiment, the cell is an immune cell. In another embodiment, the cell may be a T-cell, a B cell, or a monocyte.

[0061] In some aspects, the engineered antigen binding proteins provided by the present disclosure may comprise an amino acid sequence or be encoded by a nucleotide sequence comprising a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs:l-16 or fragments thereof, including any range derivable therebetween. In certain embodiments, the engineered antigen binding proteins of the present disclosure comprise an antigen binding fragment which comprises or is encoded by a fragment of a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs:l-16, or fragments thereof, including any range derivable therebetween. The antigen binding fragment may comprise, for example, at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275,300, 325, 340, or 350, nucleotides or amino acid residues of a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs:l-16, including any range derivable therebetween.

[0062] The terms “% identity” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI web site found at ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then referred to as “substantially identical.” This definition also refers to, or applies to, the compliment of a particular sequence. The definition may also include sequences that have deletions, additions, and / or substitutions. Likewise, this definition also is intended to apply to complementarity between sequences.

[0063] In some aspects, a polynucleotide or polypeptide molecule provided by the present disclosure may comprise, may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, or 350 contiguous amino acid or nucleic acid residues of any one of SEQ ID NOS: 1-16, including all ranges derivable therebetween.

[0064] The polypeptides of the present disclosure may comprise, in certain embodiments, one or more amino acid substitutions. Any amino acid provided or encoded by any one of SEQ ID NOs:l-16 may be substituted for any other amino acid.

[0065] In particular aspects, the engineered antigen binding proteins of the present disclosure comprise an amino acid sequence or are encoded by a nucleotide sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 sequences having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs:l-16, or fragments thereof, including all ranges derivable therebetween. In certain embodiments, the engineered antigen binding proteins of the present disclosure comprise an antigen binding fragment which comprises or is encoded by a sequencecomprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 sequences having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs:l-16, or fragments thereof, including all ranges derivable therebetween.

[0066] In some embodiments, the recombinant polynucleotide or protein molecules of the present disclosure are based on a sequence found in mice or in humans. In one embodiment, the recombinant polynucleotide or protein molecules of the present disclosure comprise sequences or a combination of sequences which are not found in nature.B. Proteins and Protein Compositions

[0067] The present disclosure provides a number of polypeptide sequences that may be present, for example, in an antigen binding protein as described herein. As used herein, the term “wild-type” refers to the endogenous version of a molecule that naturally occurs in an organism. In some aspects of the present disclosure, a wild-type version of a protein or polypeptide may be employed. In many aspects of the present disclosure, however, a recombinant protein or polypeptide is employed to generate an immune response. The terms recombinant protein and recombinant polypeptide may be used interchangeably with the terms modified protein, modified polypeptide, and variant. In certain embodiments, a recombinant protein refers to a protein having an altered chemical structure or amino acid sequence compared to the wild-type protein. In some aspects, a recombinant protein may have at least one modified activity or function compared to the wild-type protein. As is known in the art, proteins may have multiple activities or functions. In some embodiments, the function of a recombinant protein may be altered with respect to one activity or function but retain the activity or function of the wild-type protein in other respects, such as immunogenicity. In certain embodiments, the proteins of the present disclosure may include those which comprise a mutation compared to the wild-type protein. In one embodiment, the mutation may comprise an insertion, a deletion, a truncation, or at least one amino acid substitution.

[0068] Where a protein is specifically mentioned herein, it may refer to a wild-type protein or to a recombinant protein. In some embodiments, a protein of the present disclosure may comprise a heterologous signal sequence or may have had its signal sequence removed. Aprotein of the present disclosure may be isolated from the organism in which it naturally occurs, produced by recombinant expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods known in the art.

[0069] The nucleotide and protein sequences for various genes have been previously disclosed, and may be found in computerized databases known in the art. Two such databases are the National Center for Biotechnology Information’s GenBank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified or expressed using techniques known in the art or those disclosed herein.

[0070] The present disclosure provides compositions comprising the polypeptides of the present disclosure. In some embodiments, the compositions may comprise, for example, about 0.001 mg / ml to about 10.0 mg / ml of total polypeptide. The concentration of polypeptide may be, for example, 0.01 mg / ml to about 10.0 mg / ml, about 0.05 mg / ml to about 9.5 mg / ml, about 0.10 mg / ml to about 9.0 mg / ml, about 0.20 mg / ml to about 8.5 mg / ml, about 0.3 mg / ml to about 8.0 mg / ml, or at least 0.001 mg / ml, 0.010 mg / ml, 0.050 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.3mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 5.5 mg / ml, 6.0 mg / ml, 6.5 mg / ml, 7.0 mg / ml, 7.5 mg / ml, 8.0 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, 10.0 mg / ml, including all ranges derivable therebetween. In certain embodiments, the composition may be serum-free, endotoxin-free, mycoplasma-free, and / or sterile.

[0071] As is known in the art, amino acid residues may be changed in a polypeptide sequence to create an equivalent, or even improved, second-generation variant polypeptide. For example, certain amino acids may be substituted for other amino acids in a polypeptide sequence without appreciable loss of binding capacity or specificity to structures such as, for example, binding sites on substrate molecules. Since the binding capacity, the binding specificity, and the nature of a protein define the functional activity of a protein, certain amino acid substitutions can be made in a protein sequence, and / or in its corresponding DNA coding sequence, such that the resultant variant protein comprises similar or desirable properties of the original protein. Thus, in some embodiments, the polynucleotide and polypeptide sequences of the present disclosure may comprise various amino acid or nucleic acid substitutions, deletions, and / or insertions without appreciable loss of biological utility or activity. As used herein the term “functionally equivalent codon” refers to codons that encodethe same amino acid, such as the six different codons known in the art which code for arginine. As used herein, the terms “neutral substitutions” and “neutral mutations” refer to a change in a polypeptide sequence, or the encoding nucleotide sequence, such that the sequence comprises or encodes a biologically equivalent amino acid compared to that found in the original sequence. In certain embodiments, any amino acid of any polypeptide described herein may be substituted with any biologically equivalent amino acid. Biologically equivalent amino acids are known in the art.

[0072] Nucleic acid or amino acid sequence variants of the disclosure may comprise, in some embodiments, a substitution, an insertion, or a deletion. A polypeptide variant of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the polypeptide, as compared to the referenced polypeptide or to the wild-type polypeptide, including any range derivable therebetween. A variant polypeptide may comprise, for example, an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a sequence comprising or encoded by any one of SEQ ID NOs:l-16 or fragments thereof, including all ranges derivable therebetween. A polypeptide may include, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[0073] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5’ or 3’ sequences, respectively, and yet still be essentially identical to the sequences provided by the present disclosure. Such essentially identical sequences, in some embodiments, may maintain the biological activity described herein for the sequences of the present disclosure. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5’ or 3’ portions of the coding region.

[0074] Deletion variants typically lack one or more amino acid residues compared to the protein from which the variant was derived, the native protein, or the wild-type protein. In certain embodiments, individual amino acid residues may be deleted, or a number of contiguous amino acids may be deleted. In one embodiment, a stop codon may be introduced, for example by substitution or insertion, into an encoding nucleic acid sequence to generate a truncated protein variant.

[0075] Insertional variants typically involve the addition of one or more amino acid residues at a non-terminal point of a polypeptide. Terminal additions may also be generated and can include fusion proteins. Non-limiting examples of such fusion proteins include multimers or concatemers of one or more polypeptides provided by the present disclosure.

[0076] Substitutional variants typically comprise the exchange of one amino acid for another at one or more sites within the polypeptide. Substitutional variants may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties of the polypeptide. Amino acid substitutions may be conservative amino acid substitutions. As used herein, the term “conservative amino acid substitution” refers to an amino acid substitution wherein one amino acid is replaced with another amino acid having similar chemical properties. Conservative amino acid substitutions may involve, for example, the exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may, in some embodiments, encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0077] Alternatively, substitutions maybe non-conservative. As used herein, the term “nonconservative amino acid substitution” refers to an amino acid substitution that affects a function of the polypeptide. Non-conservative amino acid substitutions typically involve substituting an amino acid residue with one that is chemically dissimilar. Non-conservative amino acid substitutions may include, for example, the substitution of a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may also include, for example, the substitution of a member of one of the amino acid classes for a member from another class.

[0078] One skilled in the art can determine suitable polypeptide variants, as set forth herein, using well-known techniques. For example, one skilled in the art may identify suitable areasof the polypeptide molecule that may be changed without affecting activity by targeting regions not believed to be critical for activity. The skilled artisan will also be able to identify amino acid residues and portions of the polypeptide molecules that are conserved among similar proteins or polypeptides. In some embodiments, regions of a polypeptide molecule that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or adversely affecting the protein structure.

[0079] When making conservative or non-conservative amino acid substitutions, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain aspects, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the disclosure, those that are within ±1 are included, and in other aspects of the disclosure, those within ±0.5 are included.

[0080] As is known in the art, the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with the immunogenicity and antigen binding properties ofthe protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In some embodiments, amino acid substitutions based upon similar hydrophilicity values may include the substitution of amino acids whose hydrophilicity values are within ±2 of each other. In one embodiment, the substitution of amino acids whose hydrophilicity values are within ±1 or within ±0.5 are included. In some embodiments, a person of skill in the art may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitopic core regions." As is known in the art, an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0081] Additionally, it is well within the knowledge of a person of ordinary skill in the art to review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such amino acid residues predicted to be important.

[0082] One skilled in the art can also analyze the three-dimensional structure of a protein along with the amino acid sequence in relation to that structure in similar proteins. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antigen binding protein with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments. This information may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other substitutions. Various tools available to determine secondarystructure can be found on the world wide web, for example, at expasy.org / proteomic s / protein structure.

[0083] In some aspects of the present disclosure, amino acid substitutions may be made that: (1 ) reduce susceptibility to proteolysis; (2) reduce susceptibility to oxidation; (3) alter binding affinity for forming protein complexes; (4) alter ligand or antigen binding affinities; and / or (5) confer or modify other physicochemical or functional properties of the polypeptides provided herein. For example, single or multiple amino acid substitutions may be made in the naturally occurring sequence. In one embodiment, the single or multiple amino acid substitutions may be conservative amino acid substitutions. Substitutions, in some embodiments, may be made in the portion of an antigen binding protein that lie outside the domain(s) that form intermolecular contacts. In certain embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein. For example, one or more amino acid substitutions may be made that do not disrupt the secondary structure that characterizes the antigen binding site of the antigen binding protein.C. Nucleic Acids

[0084] In certain aspects, the present disclosure provides polynucleotide molecules that encode the polypeptide molecules describes herein. Non-limiting example of such polynucleotide molecules include isolated polynucleotide segments, recombinant vectors, and recombinant polynucleotide molecules. The polynucleotide molecules may comprise, for example, sequences which encode one or more chains of an antigen binding protein or a fragment thereof, sequences which encode a derivative, mutant, or variant of an antigen binding protein or a fragment of thereof, or hybridization probes, PCR primers, or sequencing primers for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide. Polynucleotide molecules of the present disclosure may also include, for example, anti-sense polynucleotide molecules for inhibiting expression of a polynucleotide. Polynucleotide molecules that encode certain epitopes to which the antigen binding proteins described herein bind are also provided. Polynucleotide molecules encoding fusion proteins that include the polypeptides described herein are also provided. Polynucleotide molecules may be, in some embodiments, single-stranded or double-stranded, and may comprise RNA and / or DNA, or artificial variants thereof, for example peptide nucleic acids.

[0085] A nucleic acid molecule is the “complement” of another nucleic acid molecule if they exhibit complete complementarity. As used herein, two molecules exhibit “complete complementarity” if when aligned every nucleotide of the first molecule is complementary to every nucleotide of the second molecule. Two molecules are “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molecules are “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a doublestranded structure.

[0086] Appropriate stringency conditions that promote DNA hybridization, for example, 6.0 x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.

[0087] By convention, the DNA sequences of the present disclosure and fragments thereof are disclosed with reference to only one strand of the two complementary DNA sequence strands. By implication and intent, the complementary sequences of the sequences provided here (the sequences of the complementary strand), also referred to in the art as the reverse complementary sequences, are within the scope of the disclosure and are expressly intended to be within the scope of the subject matter claimed. Thus, as used herein reference to any one of SEQ ID NOs:9-16 and fragments thereof include and refer to the sequence of the complementary strand and fragments thereof.

[0088] A polynucleotide molecule of the present disclosure may, in some embodiments, comprise a contiguous nucleic acid sequence that encodes all or part of a polypeptide described herein. In certain embodiments, a polypeptide described herein may be encoded by variant nucleic acid sequences that encode the same or a substantially similar protein.

[0089] The polynucleotide molecules and fragments thereof provided by the present disclosure may, in some embodiments be combined with other polynucleotide molecules which comprise elements, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that the overall length of the polynucleotide molecule may vary considerably. The polynucleotide molecule provided by the present disclosure can be any length. In some cases, a nucleic acid sequence may encode a polypeptide sequence that comprises additional heterologous coding sequences. Such additional heterologous coding sequences may, for example, allow for purification, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy.D. Mutation

[0090] Changes introduced by mutation into a polynucleotide molecule may result in changes in the amino acid sequence of an encoded polypeptide, such as an antigen binding protein. Mutations can be introduced using any technique known in the art. In one embodiment, one or more selected amino acid residues may be changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more random amino acid residues may be changed using, for example, a random mutagenesis protocol. Any resulting mutant polypeptide can be expressed and screened for desired properties, regardless of how it was created.

[0091] In certain embodiments, mutations may be introduced into a polynucleotide sequence without significantly altering the biological activity of its encoded polypeptide. For example, nucleotide substitutions may be made which result in amino acid substitutions at non-essential amino acid residues. In certain embodiments, one or more mutations may be introduced into a polynucleotide sequence that selectively change the biological activity of the encoded polypeptide (Romain Studer, et al., Biochem. J. 449:581-594 (2013)). In one embodiment, the mutation may quantitatively or qualitatively alter the biological activity of the encoded polypeptide. Non-limiting examples of such quantitative changes include increasing, reducing, or eliminating the activity of the encoded polypeptide. Non-limiting examples of such qualitative changes include altering the antigen specificity of an antigen binding protein.

[0092] In some embodiments, the heavy chain constant region of any antibody may comprise one or more mutations which alter the properties of the Fc constant region. For example, such mutations may alter antibody stability, glycosylation, or Fc receptor binding. In certainembodiments, the present disclosure provides an anti-PD-1 antibody that has been modified to alter at least one constant region-mediated biological effector function relative to an unmodified antibody. A non-limiting example of such a constant region-mediated effector function includes binding to one or more Fc receptors (FcyR), such as FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA and / or FcyRIIIB. In certain embodiments, FcyR binding may be reduced by mutating the immunoglobulin constant region segment of the antibody at particular regions necessary for FcyR interactions (see, e.g., Canfield and Morrison, 1991, J. Exp. Med.l73:1483-1491; and Lund et al., 1991, J. Immunol.147:2657-2662). By reducing the FcyR binding ability of the antibody other effector functions which rely on FcyR interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity ("ADCC"). In particular embodiments, the heavy chain constant region of an antibody may be modified to increase FcyR binding (see, e.g., US Publication No.2006 / 0134709). In certain embodiments, antigen binding proteins of the present disclosure may have increased or decreased opsonization, phagocytosis, or ADCC. Modifications in antibodies that reduce ADCC activity are described in U.S. Patent No.5,834,597. Additional substitutions that can modify FcyR binding and / or ADCC effector function include the K322A substitution or the L234A and L235A double substitution in the Fc region (see, e.g., Hezareh, et al. J. Virol., 2001, 75 (24): 12161- 12168. In some embodiments, the antibodies of the present disclosure comprise low levels of, or lack, fucose. Antibodies lacking fucose have been correlated with enhanced ADCC activity (see, e.g., Shields et al., J. Biol. Chem., 2002, 277:26733-26740; Shinkawa et al., J. Biol. Chem., 2003, 278:3466-73). Methods of preparing fucose-less antibodies are known in the art and any such methods may be used according to the embodiments of the present disclosure.

[0093] In some embodiments, an antigen binding protein of the present disclosure can comprise a modified CH2 domain or a modified Fc domain that includes amino acid substitutions that increase binding to FcyRIIB and / or reduced binding to FcyRIIIA as compared to the binding of a corresponding wild-type CH2 or Fc region. Such variant CH2 and Fc domains have been described in U.S. Patent Publication 2014 / 0377253 and Vonderheide, et al., Clin. Cancer Res., 19(5), 1035-1043 (2013), which are incorporated herein by reference in their entirety. In some embodiments, the antigen binding proteins of the present disclosure may include modifications that increase or decrease their binding affinities to the fetal Fc receptor, FcRn (see, e.g., WO 2005 / 123780 and U.S. PatentNo.7,217,797, which are incorporated herein by reference in their entirety). Mutations that increase binding to FcRn protect the antibodies from degradation and increase half-life.E. Probes

[0094] In another aspect, the present disclosure provides polynucleotide molecules that are suitable for use as primers or hybridization probes for the detection of the polynucleotide molecules described herein. A polynucleotide molecule serving as a primer or probe, may comprise, in certain embodiments, only a portion or fragment of a nucleic acid sequence encoding a full-length polypeptide. Such primers or probes may be used, in certain embodiments, as probes or PCR primers for specific antigen binding protein sequences. For instance, a nucleic acid molecule probe may be used in diagnostic methods. In certain embodiments, a nucleic acid molecule PCR primer may be used to amplify regions of DNA that could be used to isolate nucleic acid sequences for use in producing antigen binding proteins. See, e.g., Gaily Kivi, e / al., BMC Biotechnol. 16:2 (2016). In one embodiment, the nucleic acid molecules are oligonucleotides. In another embodiment, the oligonucleotides are from the variable regions of the heavy and light chains of an antigen binding protein of the present disclosure. In still yet another embodiment, the oligonucleotides encode all or part of one or more of the CDRs or antigen binding proteins.

[0095] Probes based on the desired sequence of a nucleic acid molecule can be used to detect that nucleic acid molecule or similar nucleic acid molecules, for example, transcripts encoding a polypeptide of interest. Probes may comprise, in certain embodiments, a label, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. In particular embodiments, the probes described herein may be used to identify a cell that expresses a polypeptide of interest.F. Methods of Monoclonal Antibody Production

[0096] Methods for producing monoclonal antibodies are well known in the art and any such method may be used according to the embodiments of the present disclosure. In some embodiments, the first step in a method for producing a monoclonal antibody is immunization of an appropriate host. As is well known in the art, a given composition for immunization may vary in its immunogenicity. In certain embodiments, it may therefore be necessary to conjugate a peptide immunogen to a carrier molecule to increase immunogenicity. Nonlimiting examples of such carrier molecules include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, mouse serum albumin, and rabbit serum albumin.Methods and compositions for conjugating a peptide to a carrier molecule are well known in the art any such method may be used according to the embodiments of the present disclosure. Non-limiting examples of such compositions include glutaraldehyde, m-maleimidobencoyl- N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Non-limiting examples of such adjuvants include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants, and aluminum hydroxide adjuvant.

[0097] The amount of an immunogen composition used during immunization of an appropriate host may vary based upon the nature of the immunogen and the host used for immunization. Immunization may be performed using a variety of routes of administration, non-limiting examples of which include subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal administration. The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various time points following immunization. In certain embodiments, a second, booster immunization may also be administered. This process of immunization and determining antibody titer, in some embodiments, may be repeated until a desired antibody titer is achieved. In particular embodiments, once a desired antibody titer is achieved, the immunized host many be bled and the serum isolated and stored.

[0098] In some embodiments, the immunized animal may be used to generate monoclonal antibodies. In particular embodiments, monoclonal antibodies may be produced from B cells collected from the biopsied spleens, lymph nodes, or circulating blood of immunized animals. These B cells may then, in certain embodiments, be fused with cells of an immortal myeloma cell. In one embodiment, the immortal myeloma cell is from the same species as the animal that was immunized, a human myeloma cell, or a human / mouse chimeric cell. Myeloma cell lines suitable for use in hybridoma-producing fusion methods, in certain embodiments, may be non-antibody-producing, have high fusion efficiency, and comprise enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Numerous myeloma cell lines are known in the art and any such cell line may be used according to the embodiments of the present disclosure. Methods for generating hybridomas are well known in the art and any such method may be used according to the embodiments of the present disclosure to create a population ofhybridomas from which specific hybridomas are selected. In certain embodiments, the selection of hybridomas may be performed by single cell dilution followed by culturing the single cell clones in microtiter plates, followed by testing of individual clonal supernatants for the desired reactivity. Non-limiting examples of assays that may be used for testing individual clonal supernatants include radioimmunoassays, enzyme immunoassays, cytotoxicity assays, and plaque assays dot immunobinding assays. In particular embodiments, selected hybridomas may then be serially diluted or single-cell sorted and cloned into individual antibody-producing cell lines. These antibody producing cell lines may then be propagated indefinitely to provide monoclonal antibodies.

[0099] In many embodiments, monoclonal antibodies may be purified or isolated using any method known in the art. Non-limiting examples of such methods include filtration, centrifugation, and various chromatographic methods such as FPLC and affinity chromatography. In a number of embodiments, fragments of a monoclonal antibody of the present disclosure may be obtained from a purified monoclonal antibody by any method known in the art, non-limiting examples of which include digestion with enzymes, such as pepsin or papain, and cleavage of disulfide bonds by chemical reduction. In one embodiment, antigen binding proteins of the present disclosure may be synthesized using an automated peptide synthesizer.

[0100] In some embodiments, a molecular cloning approach may be used to generate the antigen binding proteins of the present disclosure. For example, RNA may be isolated from a hybridotna line of interest, and the antibody genes of the line may be amplified and cloned into an immunoglobulin expression vector. In one embodiment, combinatorial immunoglobulin phagemid libraries may be prepared from RNA isolated from a hybridoma cell line of interest and phagemids expressing the desired antibody selected by panning using viral antigens.G. Polypeptide Expression

[0101] In some aspects, the present disclosure provides nucleic acid molecules encoding polypeptides described herein. In some embodiments, these nucleic acid molecules may encode an antigen binding protein or an antigenic peptide as described herein. These nucleic acid molecules may be generated by any method known in the art. In some embodiments, the nucleic acids may be isolated from the organism in which they naturally occur or produced using phage display. In certain embodiments, nucleic acid molecules may be expressed inany suitable recombinant expression system and allowed to assemble to form molecules which comprise an antigen binding protein or an antigenic peptide as described herein. The nucleic acid molecules may be used to express large quantities of polypeptides. If the nucleic acid molecules are derived from a non-human animal, the nucleic acid molecules may be used for humanization of the antibody genes.I. Vectors

[0102] In some aspects, the present disclosure provides expression vectors comprising a nucleic acid molecule encoding a polypeptide of a desired sequence or a portion thereof. In particular embodiments, the nucleic acid molecule may encode a fragment comprising one or more CDRs or one or more variable region domains. Expression vectors comprising the nucleic acid molecules may encode a light chain, a heavy chain, or an antigen binding variable region thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antigen binding proteins, or fragments thereof. In certain embodiments, the expression vectors described herein may comprise control sequences that govern transcription and translation, as well as nucleic acid sequences that serve other functions. In particular embodiments, polypeptides or peptides of the disclosure may be expressed when polynucleotide molecules of the present disclosure are inserted into expression vectors such that the coding sequence is operably linked to transcriptional and translational control sequences. As used herein, the term “operably linked” refers to at least two nucleotide molecules arranged or linked in a manner so that one can affect the function of the other. The two nucleotide molecules can be part of a single contiguous nucleotide molecule and can be adjacent or separated. In some aspects, the present disclosure provides a vector that encodes a functionally complete antigen binding protein sequence with appropriate restriction sites engineered so that any variable region or CDR sequences can be inserted and expressed. In particular embodiments, the present disclosure provides a vector that encodes a functionally complete human or mouse light chain or heavy chain sequence with appropriate restriction sites engineered so that any variable sequence, or any CDR1, CDR2, and / or CDR3 encoding sequence can be inserted and expressed. Expression vectors may be used in any host cell and may, in some embodiments, contain sequences for plasmid or virus maintenance, or for cloning and expression of exogenous nucleotide sequences. In certain embodiments, such sequences, collectively referred to as “flanking sequences” may include one or more of the following operably linked nucleotide sequences: a promoter, an enhancer sequence, an origin of replication, a transcriptional termination sequence, a completeintron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a “polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, or a selectable marker element. Such sequences and methods of using the same are well known in the art.IL Expression Systems

[0103] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use to produce nucleic acid sequences provided by the present disclosure, or their encoded polypeptides, proteins, and peptides. Commercially and widely available systems include but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its encoded polypeptide, protein or peptide using an appropriate expression system.III. Methods of Gene Transfer

[0104] Suitable methods for nucleic acid delivery to a host cell, tissue, or organism are well- known in the art, and any such methods may be used to introduce a nucleic acid provided by the present disclosure. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987 ; Rippe, et al. , 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer, et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley, et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler, et al., 1990; U.S. Patents 5,302,523 and 5,464,765, eachincorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh, et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation / inhibition mediated DNA uptake (Potrykus, et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.IV. Host Cells

[0105] In another aspect, the present disclosure provides host cells comprising an expression vector as described herein. Antigen binding proteins, antibodies, and antibody fragments can be expressed in a variety of cell types, and the nucleic acids and proteins of the present disclosure may be expressed in any cell type known in the art. In some embodiments, an expression construct encoding antigen binding protein may be transfected into any host cell known in the art using any of the variety of methods known in the art and described herein. Expression vectors may be introduced, in some embodiments, into prokaryotic or eukaryotic cells using any transformation or transfection technique known in the art. Some expression vectors may comprise control sequences that allow for replication and / or expression in both prokaryotic and eukaryotic cells. Conditions under which to incubate host cells in order to maintain them and to permit replication of a vector are well-known in the art. Also understood and known in the art are techniques and conditions that allow for large-scale vector, nucleic acid, and polypeptide production, and any such techniques may be used to produce the vectors, polynucleotide molecules, and polypeptides described herein.

[0106] As is known in the art, mammalian cells may be stably transfected with a nucleic acid molecule of interest. During stable transfection, in certain embodiments, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these stable transfectants, a selectable marker may be introduced into the host cells along with the gene of interest. In one embodiment, the selectable marker may be a gene that confers resistance to antibiotics. Stable transfectants may, in certain embodiments, be identified using drug selection or by any other method known in the art.I. Pharmaceutical and Therapeutic Compositions

[0107] In certain aspects, the present disclosure provides pharmaceutical and therapeutic compositions comprising the polynucleotide molecules, antigen binding proteins, antibodies, antigen binding fragments, and / or cells of the present disclosure. In some embodiments, thepolynucleotide molecules, antigen binding proteins, and / or cells of the present disclosure may be combined with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier,” “pharmaceutically acceptable adjuvant,” or “adjuvant” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with the polynucleotide molecules, polypeptide molecules, cells, and / or or other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit / risk ratio. Also included may be an agent that modifies the effect of other agents and is useful in preparing a therapeutic compound or composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable. Such an agent may be added to a therapeutic composition to modify the immune response of a subject by boosting the response such as to give a higher amount of polynucleotide molecules, polypeptide molecules, and or cells and longer-lasting protection from degradation. Such an agent may include any excipient, diluent, carrier, or adjuvant that is acceptable for pharmaceutical use. Such an agent may be non-naturally occurring, or may be naturally occurring, but not naturally found in combination with other agents in the immunogenic composition.

[0108] As used herein, a “therapeutic compound” or “therapeutic composition” refers to a composition comprising an antigen binding protein, polynucleotide molecule, or a cell of the present disclosure. In some embodiments, a therapeutic composition has the activity of specifically binding an antigenic peptide as described herein. In one embodiment, the composition is capable of eliciting an immune response. Such a compound or composition is meant to encompass a composition suitable for administration to a subject, such as a mammal, particularly a mouse subject or a human subject. In general, a therapeutic composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the immunogenic composition is pharmaceutical grade). Therapeutic compositions may be designed for administration to subjects in need thereof via a number of different routes of administration including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalational, and the like. The appropriate dosage of a composition, as described herein, may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, clinical history, response to the treatment, and the discretion of the attending physician. In someembodiments, therapeutic compositions provided by the present disclosure may include various “unit doses.” A unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0109] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing. In some embodiments, an antigen binding protein of the present disclosure may be administered at a dose of about 0.0001 mg / kg body weight to about 100 mg / kg body weight. The dose of antigen binding protein administered may vary widely in view of the variety of antigen binding protein or antibody compositions available and the differing efficacies that may be associated with a particular chosen route of administration. The dose administered, in certain embodiments, may be about 0.001 mg / kg body weight to about 75 mg / kg body weight; about 0.005 mg / kg body weight to about 50 mg / kg body weight; about 0.01 mg / kg body weight to about 30 mg / kg body weight; or about 0.01 mg / kg body weight to about 5 mg / kg body weight, including all ranges derivable therebetween.

[0110] In certain embodiments the present disclosure provides antibody-drug conjugates which comprise the antigen binding proteins of the present disclosure. As used herein the term “antibody-drug conjugate” refers to a composition comprising an antigen binding protein conjugated to a drug or to a detectable label. Any drug or detectable label known in the art may be conjugated to an antigen binding protein of the present disclosure. Non-limiting examples of drugs that may be conjugated to an antigen binding protein of the present disclosure include a chemotherapeutic agent, an immunotherapeutic agent, or a radiotherapeutic agent. In certain embodiments, antibody-drug conjugates may be utilized as an in vitro or an in vivo diagnostic agent. Numerous appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for example, U.S. Patent Nos. 5,021,236, 4,938,948, and 4,472,509). Non-limiting examples of imaging agents that may be used according to certain embodiments of the present disclosure include paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.In particular embodiments, an antibody-drug conjugate of the present disclosure may comprise a cleavable linker, which conjugates the antigen binding protein to the drug or detectable label. A variety of cleavable linkers and methods for use in the production of antibody-drug conjugates are known in the art and any such linker or method may be used according to the embodiments of the present disclosure. Non-limiting methods for producing antibody-drug conjugates include those which use a metal chelate complex utilizing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; or tetrachloro-3a- 6a- diphenylglycouril-3, those which use an enzyme and a coupling agent such as glutaraldehyde or periodate, and those which utilize a reaction with an isothiocyanate. Nonlimiting examples of linkers with may be used according to certain embodiments of the present disclosure include methyl-p-hydroxybenzimidate and N-succinimidyl-3-(4- hydroxyphenyljpropionate. In particular embodiments, antibody-drug conjugates may be produced by selectively introducing sulfhydryl groups into the Fc region of an immunoglobulin, or by site-specific attachment of effector or reporter molecules to a carbohydrate residue in the Fc region.

[0111] As used herein, “subject” or “patient” refers to animals, including humans, who are treated with the therapeutic compounds or compositions or in accordance with the methods described herein. For diagnostic or research applications, a wide variety of mammals may be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs and the like. In particular embodiments, a subject in need of therapy may be any subject who comprises a cell that expresses a PD-1 ligand. In one embodiment, the cell is a cancer cell. In another embodiment, the cancer cell may be selected from the group consisting of: The method of claim 23, wherein the cancer cell is selected from the group consisting of a breast cancer cell, a lung cancer cell, a brain cancer cell, a skin cancer cell, a gastric cancer cell, a colorectal cancer cell, a esophageal cancer cell, a pancreatic cancer cell, a thymic cancer cell, a cervical cancer cell, a bladder cancer cell, a thyroid cancer cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a thymoma cell, an adrenocortical carcinoma cell, a head and neck squamous cancer cell, an oropharyngeal squamous cell carcinoma cell, a glioma cell, a mesothelioma cell, a merkel cell carcinoma cell, a hepatocellular carcinoma cell, an intrahepatic cholangiocarcinoma cell, a renal cell carcinoma cell, and a nasopharyngeal carcinoma cell.

[0112] A composition, as described herein, may include, in particular embodiments, a combination of therapeutic agents. In some embodiments, a composition as described here may be administered as a single composition or as more than one composition. Different compositions as provided herein, in certain embodiments, may be administered by the same route of administration or by different routes of administration.

[0113] In certain embodiments, the compositions and methods for treating an individual described herein may be combined with any other composition or method of treatment known in the art. The compositions and methods may be administered in any suitable manner known in the art. For example, a first and a second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, a first and a second cancer treatment may be administered in separate compositions. In certain embodiments, a first and a second cancer treatment may be administered in the same composition.

[0114] Non-limiting examples of additional treatment modalities that may be included in combination with the compositions and methods provided herein include immunotherapy, chemotherapy, radiation therapy, and surgery. Types of immunotherapy that may be used in combination with the compositions and methods provided by the present disclosure include, but are not limited, to: (a) checkpoint inhibitors, for example, inhibitors of PD-1, PDL1, PDL2, and CTLA-4; (b) inhibitors of co-stimulatory molecules, for example, inhibitors of B7-I, B7-2 CD28, ICOS, 0X40, 4-1BB, CD137, CD40L, and GITR;(c) dendritic cell therapy; (d) T-cell therapy directed to the same or to a different antigen; (e) cytokine therapy. Nonlimiting types of chemotherapy that may be used in combination with the compositions and methods provided by the present disclosure include (a) alkylating agents; (b) antimetabolites; (c) natural products, for example, vinca alkaloids, enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-a); and (d) other agents, for example, platinum coordination complexes, substituted ureas, and taxols.J. Detection and Therapeutic Agents

[0115] In some aspects, the present disclosure provided methods and compositions for detection and therapeutic labeling of the antigen binding proteins, antibodies, antigen binding fragments, fusion proteins, and / or cells provided by the present disclosure. Methods for labeling and detection of polypeptide molecules are well-known in the art, and any such method known in the art may be used to label or detect the polypeptides described herein. As a non-limiting example, polypeptides may be labeled with a detectable moiety, such as aradioactive atom, a chromophore, a fluorophore, or the like, and then detected using methods known in the art. Such labeled polypeptides may be used, in some embodiments, for in vivo or in vitro diagnostic techniques.

[0116] As used herein, the term “label” refers to a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected. In certain embodiments, a polynucleotide molecule, protein, or cell may be labeled to generate a labeled composition. In particular embodiments, labeled compositions also include sequences which are conjugated a polynucleotide molecule that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. Labels may be suitable for small scale detection or for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected or may be quantified. In certain embodiments, labels that may be quantified provide numerically reportable value. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.K. Formulations and Culture of the Cells

[0117] In particular aspects, the cells of the disclosure may be cultured in a particular medium. The medium, in certain aspects, may be prepared using a medium used for culturing mammalian cells as their basal medium. Non-limiting examples of such a medium include such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer’s media, as well as any combinations thereof. In one embodiment, the medium may be xeno-free or chemically defined.

[0118] The medium, in particular embodiments, may be a serum-containing, serum-free, or xeno-free. In some embodiments, serum may be derived from the same animal as that of the cells being cultured to prevent contamination with heterogeneous animal-derived components. Serum-free medium refers to a medium with no unprocessed or unpurifiedserum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0119] In certain embodiments, the medium may contain or may not contain a serum alternative. Alternatives to serum may include, in one embodiment, one or more of the following: albumin (such as lipid-rich albumin, bovine albumin, recombinant albumin, humanized albumin, plant starch, dextran, or protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3’- thioglycerol, or equivalents thereof. The alternatives to serum can be prepared by the method disclosed in International Publication No. WO 98 / 30679, for example, (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. Such commercially available materials include, but are not limited to, Knockout Serum Replacement (KSR), Chemically defined Lipid Concentrated (Gibco), and Glutamax (Gibco).

[0120] In certain aspects, the medium may comprise at least of the following: (1) vitamins such as biotin, DL alpha-tocopherol acetate, DL alpha-tocopherol, or vitamin A; (2) proteins such as BSA (bovine serum albumin), human albumin, fatty acid free fraction V, catalase, human recombinant insulin, human transferrin, or superoxide dismutase; or (2) other components such as corticosterone, D-galactose, ethanolamine HC1, glutathione (reduced), L- camitine HC1, linoleic acid, progesterone, putrescine 2HC1, sodium selenite, or T3 (triodo-I- thyronine). In specific aspects, one or more of the components listed above maybe explicitly excluded.

[0121] In some aspects, the medium comprises vitamins. In particular embodiments, the medium comprises at least one of the following: biotin, DL alpha-tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin Bl 2, or the medium may include combinations thereof or salts thereof. In some aspects, the medium comprises or consists essentially of biotin, DL alpha-tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamins include or consist essentially of biotin, DL alpha-tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises proteins. In certain embodiments, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof.In some aspects, the medium further comprises one or more of the following: corticosterone, D-dalactose, ethanolamine, glutathione, L-camitine, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, or combinations thereof. In one embodiment, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In another embodiment, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In yet another embodiment, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In still yet another embodiment, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof.

[0122] In certain embodiments, one or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, pg / ml, mg / ml, including any range derivable therebetween.

[0123] In specific aspects, the cells of the disclosure are specifically formulated. In one embodiment, the cells may or may not be formulated as a cell suspension. In another embodiment, the cells may be formulated in a single dose form. In certain embodiments, the cells may be formulated for systemic or local administration. In particular embodiments, the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO). In one embodiment, the cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. In another embodiment, the cells may be formulated specifically for intravenous administration. In specific embodiments, the cells are formulated for intravenous administration over less than one hour. In particular aspects the cells are in a formulated cell suspension that is stable at room temperature for at least about 1, 2, 3, or 4 hours from time of thawing.

[0124] In particular aspects, the cells of the disclosure comprise or produce an exogenous antigen binding protein, which may be of a defined antigen specificity. In certain embodiments, the endogenous antibody genes of a cell have been modified by genome editing so that they do not express a protein. Methods of gene editing such as methods using the CRISPR / Cas9 system are known in the art and described herein.

[0125] In some aspects, the cells of the disclosure further comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which a CAR may be directed include at least 5T4, 8H9, av(36 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPC AM, EphA2, folate receptor-a, FAP, FBP, fetal AchR, FRa, GD2, G250 / CAIX, GD3, Glypican-3 (GPC3), Her2, IL-13Ra2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Muc 1, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE- 1, PAME, SSX-2, Melan-A / MART-1, GPlOO / pmel 17, TRP-1 / -2, P. polypeptide, MC1R, Prostate-specific antigen, p-catenin, BRCA1 / 2, CML66, Fibronectin, MART-2, TGF-0RII, or VEGF receptors (e.g., VEGFR2), for example. The CAR may be a first, second, third, or more generation CAR. The CAR may be bispecific for any two nonidentical antigens, or it may be specific for more than two non-identical antigens.L. Kits

[0126] Certain aspects of the present disclosure further provide kits containing compositions of the disclosure or compositions to implement methods of the invention. In some aspects, kits can be used to evaluate one or more biomarkers. In certain aspects, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1 ,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein.

[0127] Kits may comprise, in certain embodiments, components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0128] Individual components, in particular embodiments, may also be provided in a kit in concentrated amounts. In one embodiment, a component is provided individually in the same concentration as it would be in a solution with other components. In certain embodiments, concentrations of components may be provided as lx, 2x, 5x, 1 Ox, or 20x or more.

[0129] In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit aspects. In some embodiments, a kit may include a sample that is a negative or positive control for methylation of one or more biomarkers.

[0130] The term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted otherwise. The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that “comprises,” “has,” or “includes” one or more components is not limited to possessing only those components and covers other unlisted components. As used herein, the term “consists essentially of,” when used in reference to a nucleotide or amino acid sequence of the present disclosure, means that the nucleotide sequence or amino acid sequence, may contain additional nucleotides or amino acids so long as the additional nucleotides or amino acids do not materially alter the function of the recited sequences. The term “materially alter,” as applied to a nucleotide sequence or amino acid sequence of the present disclosure, refers to a decrease in the antigen binding activity of the encoded polypeptide or polypeptide sequence of at least 25%. For example, additional nucleotides or amino acids added to a nucleotide or an amino acid sequence of the present disclosure may be deemed to “materially alter” the encoded polypeptide or polypeptide sequence, if such additions decrease the antigen-specific binding activity by at least 25%.

[0131] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0132] All references herein are incorporated herein by reference in their entirety.EXAMPLES

[0133] The following examples are included to illustrate embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.Example 1: Production and in vitro Testing of PD-1 Specific Antibodies

[0134] PD-1 deficient mice were immunized with both full-length recombinant extracellular domain and a recombinant subdomain most involved in PD-Ligand binding derived from mouse PD-1. Following immunization mice were bled and antibody titer determined by ELISA. Mouse B cells were then collected and fused with myeloma cells to produce hybridoma clones, each expressing a different antibody. Culture medium supernatant from selected clones was tested to analyze antibody binding to recombinant mouse PD-1 by ELISA (FIG. 1). As shown in FIG. 1 a number of antibody clones, including clones 384-4, 384-18, 384-30, 384-35, and 384-39, demonstrated specific binding to mouse PD-1. To further analyze antibody binding to mouse PD-1, monoclonal antibodies from selected clones were purified and antigen-specific binding was measured by ELISA over a wide range of concentrations (FIG. 2). As shown in FIG. 2 antibody 384-35 demonstrates superior binding affinity to mouse PD-1 compared to the other antibody clones tested. To determine whether selected antibody clones were able to block binding of mouse PD-1 to mouse PD-L1-FC, Alexa_594-labeled mouse PD-L1-FC was used to stain DC2.4-cells expressing mouse PD-1 that had been pre-blocked with 1 pg / mL of the generated anti-PD-1 antibodies, with RMP1- 14, or with PBS (negative control). The fold reduction in PD-Ll-Fc-Alexa_594 staining relative to staining of the unblocked cells is shown as a measure of how effective each clone is in blocking PD-L1 binding to PD-1 (i.e., higher fold reduction equates with better PD-L1 to PD-1 blockade) (FIG. 3). The antibody RMP1-14 was used as a positive control. RMP1-14 is a rat anti-mouse PD-1 blocking antibody, which is well-known in the art. Importantly, however, because RMP1-14 is a rat antibody, it cannot be administered long term to an immunocompetent mouse without eliciting a host anti-drug antibody response. As demonstrated in FIG. 3, antibody clone 384-35 reduced PD-L1-FC binding to PD-1 by approximately 2.5-fold, whereas the commonly used blocking antibody RMP1-14 reduced PD-L1-FC binding to PD-1 by only about 2-fold.Example 2: In vivo Testing of PD-1 Specific Antibodies

[0135] To determine whether selected antibody clones could reduce tumor burden or improve survival in an immunocompetent mouse model of colorectal cancer, 1 x 105MC38 colorectal cancer cells were injected into the flank of immunocompetent mice. Tumors were allowed to establish for one week prior to the beginning of treatment with intra-peritoneal injections of the selected antibodies at 250 pg on days 7, 10, and 13. Negative control mice were not treated, and the antibody RMP1-14 was used as a positive control. Tumor volume was measured with calipers (FIG. 4A) and the tumor-free survival calculated (FIG. 4B) with the survival endpoint set at a tumor size of 1000 mm3and the experiment concluding when only tumor flee mice remained alive. As shown in FIG. 4A, treatment with antibody 384-35 significantly reduced tumor growth rate compared to the negative control and compared to treatment with antibody RMP1-14. Furthermore, as shown in FIG. 4B, treatment with antibody 384-35 significantly increased tumor-free survival (30% versus 0%) compared to PBS control treatment, as well as showing a trend of increased survival (30% versus 20%) relative to antibody RMP1-14. The nucleotide and amino acid sequences of the heavy chain variable region of antibody 384-35 are shown in FIG. 5, and the nucleotide and amino acid sequences of the light chain variable region of antibody 384-35 are shown n FIG. 6.* * *All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments or aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described hereinwhile the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

CLAIMS1. An isolated antigen binding protein that specifically binds programmed death cell protein 1 (PD-1) comprising: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC- CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC- CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein said variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof.

2. The isolated antigen binding protein of claim 1 , wherein: a) the HC-CDR1 region comprises the amino acid sequence of SEQ ID NO:1, the HC-CDR2 region comprises the amino acid sequence of SEQ ID NO:2, and the HC-CDR3 region comprises the amino acid sequence of SEQ ID NO:3; and b) the LC-CDR1 region comprises the amino acid sequence of SEQ ID NO:5, the LC-CDR2 region comprises the amino acid sequence of SEQ ID NO:6, and a LC-CDR3 region comprises the amino acid sequence of SEQ ID NO:7.

3. The isolated antigen binding protein of claim 1, wherein said antigen binding protein is selected from the group consisting of a monoclonal antibody, an antigen binding fragment, a chimeric antibody, a bispecific antibody, and a chimeric antigen receptor.

4. The isolated antigen binding protein of claim 1, wherein said antigen binding protein specifically binds mouse PD-1.

5. The isolated antigen binding protein of claim 1, wherein said heavy chain variable region further comprises an amino acid sequence having at least about 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4.

6. The isolated antigen binding protein of claim 1 , wherein said light chain variable region further comprises an amino acid sequence having at least about 80% amino acid sequence identity to SEQ ID NO:8.

7. The isolated antigen binding protein of claim 1, wherein the antigen binding protein is a monoclonal antibody, and wherein the monoclonal antibody is a murine, a rodent, a rabbit, a chimeric, a humanized, or a human antibody.

8. The isolated antigen binding protein of claim 7, wherein the monoclonal antibody is a murine antibody.

9. The isolated antigen binding protein of claim 7, wherein the monoclonal antibody is a humanized antibody.

10. The isolated antigen binding protein of claim 1, wherein the antigen binding protein comprises a ScFv fragment, a Fab fragment, Fab' fragment, a F(ab’)2 fragment, or a Fv fragment.

11. An antibody-drug conjugate comprising the isolated antigen binding protein of claim 1 and an anticancer drug or a detectable label.

12. The antibody-drug conjugate of claim 11, wherein the anticancer drug or detectable label is conjugated to the isolated antigen binding protein through a cleavable linker.

13. The antibody-drug conjugate of claim 11, wherein the anticancer drug is a chemotherapeutic agent, an immunotherapeutic agent, or a radiotherapeutic agent, or wherein the detectable label is a paramagnetic ion, a radioactive isotope, a fluorochrome, an NMR-detectable agent, or an X-ray imaging agent.

14. A composition comprising the isolated antigen binding protein of claim 1.

15. A recombinant polynucleotide molecule comprising a nucleotide sequence that encodes an antigen binding protein that specifically binds programmed death cell protein 1 (PD-1), wherein the antigen binding protein comprises: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof, a HC-CDR2 regioncomprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC- CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC- CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein said variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof.

16. A cell comprising the recombinant polynucleotide molecule of claim 15.

17. A method of producing an engineered cell, the method comprising introducing the recombinant polynucleotide molecule of claim 15 into a cell.

18. A hybridoma or an engineered cell that produces an antigen binding protein that specifically binds programmed death cell protein 1 (PD-1), wherein the antigen binding protein comprises: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC- CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof, and a LC- CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein said variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof.

19. A method of producing an antigen binding protein, the method comprising culturing the hybridoma or engineered cell of claim 18.

20. A recombinant antigen binding protein comprising: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC- CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof, and a LC- CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein said variant of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof.

21. The recombinant antigen binding protein of claim 20, wherein the recombinant antigen binding protein is selected from the group consisting of a recombinant antibody, a bispecific antibody, a chimeric antibody, a chimeric antigen receptor, and antigen binding fragments thereof.

22. A method of treating cancer, the method comprising administering the isolated antigen binding protein of claim 1 to a subject in need thereof.

23. The method of claim 22, wherein the cancer comprises a cancer cell that expresses a PD-1 ligand.

24. The method of claim 23, wherein the cancer cell is selected from the group consisting of a breast cancer cell, a lung cancer cell, a brain cancer cell, a skin cancer cell, a gastric cancer cell, a colorectal cancer cell, a esophageal cancer cell, a pancreatic cancer cell, a thymic cancer cell, a cervical cancer cell, a bladder cancer cell, a thyroid cancer cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a thymoma cell, an adrenocortical carcinoma cell, a head and neck squamous cancer cell, an oropharyngeal squamous cell carcinoma cell, a glioma cell, a mesothelioma cell, a merkel cell carcinoma cell, a hepatocellular carcinoma cell, anintrahepatic cholangiocarcinoma cell, a renal cell carcinoma cell, and a nasopharyngeal carcinoma cell.

25. The method of claim 22, wherein the subject is a rodent subject, a murine subject, or a human subject.

26. The method of claim 22, the method further comprising administering a second therapy to said subject.

27. The method of claim 26, wherein said second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, or surgery.

28. A method of treating cancer, the method comprising administering the composition of claim 14 to a subject in need thereof.

29. The method of claim 28, wherein the cancer comprises a cancer cell that expresses a PD-1 ligand.

30. The method of claim 29, wherein the cancer cell is selected from the group consisting of a breast cancer cell, a lung cancer cell, a brain cancer cell, a skin cancer cell, a gastric cancer cell, a colorectal cancer cell, a esophageal cancer cell, a pancreatic cancer cell, a thymic cancer cell, a cervical cancer cell, a bladder cancer cell, a thyroid cancer cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a thymoma cell, an adrenocortical carcinoma cell, a head and neck squamous cancer cell, an oropharyngeal squamous cell carcinoma cell, a glioma cell, a mesothelioma cell, a merkel cell carcinoma cell, a hepatocellular carcinoma cell, an intrahepatic cholangiocarcinoma cell, a renal cell carcinoma cell, and a nasopharyngeal carcinoma cell.

31. The method of claim 28, wherein the subject is a rodent subject, a murine subject, or a human subject.

32. The method of claim 28, the method further comprising administering a second therapy to said subject.

33. The method of claim 32, wherein said second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, or surgery.

34. A method of treating cancer, the method comprising administering the hybridoma or engineered cell of claim 18 to a subject in need thereof.

35. The method of claim 34, wherein the cancer comprises a cancer cell that expresses a PD-1 ligand.

36. The method of claim 35, wherein the cancer cell is selected from the group consisting of a breast cancer cell, a lung cancer cell, a brain cancer cell, a skin cancer cell, a gastric cancer cell, a colorectal cancer cell, a esophageal cancer cell, a pancreatic cancer cell, a thymic cancer cell, a cervical cancer cell, a bladder cancer cell, a thyroid cancer cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a thymoma cell, an adrenocortical carcinoma cell, a head and neck squamous cancer cell, an oropharyngeal squamous cell carcinoma cell, a glioma cell, a mesothelioma cell, a merkel cell carcinoma cell, a hepatocellular carcinoma cell, an intrahepatic cholangiocarcinoma cell, a renal cell carcinoma cell, and a nasopharyngeal carcinoma cell.

37. The method of claim 34, wherein the subject is a rodent subject, a murine subject, or a human subject.

38. The method of claim 34, the method further comprising administering a second therapy to said subject.

39. The method of claim 38, wherein said second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, or surgery.

40. A method of treating cancer, the method comprising administering the recombinant antigen binding protein of claim 20 to a subject in need thereof.

41. The method of claim 40, wherein the cancer comprises a cancer cell that expresses a PD-1 ligand.

42. The method of claim 41, wherein the cancer cell is selected from the group consisting of a breast cancer cell, a lung cancer cell, a brain cancer cell, a skin cancer cell, a gastric cancer cell, a colorectal cancer cell, a esophageal cancer cell, a pancreatic cancer cell, a thymic cancer cell, a cervical cancer cell, a bladder cancer cell, a thyroid cancer cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a thymoma cell, an adrenocortical carcinoma cell, a head and neck squamous cancer cell, an oropharyngeal squamous cell carcinoma cell, a glioma cell, a mesothelioma cell, a merkel cell carcinoma cell, a hepatocellular carcinoma cell, an intrahepatic cholangiocarcinoma cell, a renal cell carcinoma cell, and a nasopharyngeal carcinoma cell.

43. The method of claim 40, wherein the subject is a rodent subject, a murine subject, or a human subject.

44. The method of claim 40, the method further comprising administering a second therapy to said subject.

45. The method of claim 44, wherein said second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, or surgery.

46. A composition comprising an antigen binding protein comprising: a) a heavy chain variable region comprising a HC-CDR1 region comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof, a HC-CDR2 region comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HC- CDR3 region comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof; and b) a light chain variable region comprising a LC-CDR1 region comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof, a LC-CDR2 region comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, and a LC- CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, wherein said variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises one or two amino acid substitutions, deletions, or additions, or combinations thereof.

47. The composition of claim 46, wherein said composition is serum-free, endotoxin-free, or sterile.

48. A method of treating cancer, the method comprising administering the composition of claim 46 to a subject in need thereof.

49. The method of claim 48, wherein the cancer comprises a cancer cell that expresses a PD-1 ligand.

50. The method of claim 49, wherein the cancer cell is selected from the group consisting of a breast cancer cell, a lung cancer cell, a brain cancer cell, a skin cancer cell, a gastric cancer cell, a colorectal cancer cell, a esophageal cancer cell, a pancreatic cancer cell, a thymic cancer cell, a cervical cancer cell, a bladder cancer cell, a thyroid cancer cell, an ovarian cancer cell, a leukemia cell, a melanoma cell, a thymoma cell, an adrenocortical carcinoma cell, a head andneck squamous cancer cell, an oropharyngeal squamous cell carcinoma cell, a glioma cell, a mesothelioma cell, a merkel cell carcinoma cell, a hepatocellular carcinoma cell, an intrahepatic cholangiocarcinoma cell, a renal cell carcinoma cell, and a nasopharyngeal carcinoma cell.

51. The method of claim 48, wherein the subject is a rodent subject, a murine subject, or a human subject.

52. The method of claim 48, the method further comprising administering a second therapy to said subject.

53. The method of claim 52, wherein said second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, or surgery.

54. A method of detecting PD-1 in a sample, the method comprising: a) contacting the sample with the isolated monoclonal antibody or antigen binding protein of claim 1 ; and b) detecting binding of said isolated monoclonal antibody or antigen binding protein to said sample.

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