ISOLATED ANTAGONIST ANTIBODY THAT SPECIFICALLY BINDS TO PD-1, NUCLEIC ACID THAT ENCODES SAID ANTIBODY, AND EXPRESSION VECTOR

Abstract

Antibodies that bind to programmed cell death protein 1 (PD-1) and methods for their use. Anti-PD-1 antibodies can be used therapeutically alone or in combination with other treatments to treat cancer and other diseases. Host cell, DNA, vector, method for producing the antibody, pharmaceutical composition.

Description

The present invention relates to antibodies, eg, full length antibodies that bind to PD-1. The invention also relates to compositions comprising antibodies against PD-1 and methods of using anti-PD-1 antibodies as a medicament. Some embodiments relate to methods of using anti-PD-1 antibodies for the treatment, prevention, and / or diagnosis of various diseases, including hyperproliferative diseases, such as cancer. BACKGROUND PD-1 is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line that undergoes activation-induced apoptosis. PD-1 is expressed on T cells, B cells, and macrophages. The ligands for PD-1 are the PD-L1 (B7-H1) and PD-L2 (B7-DC) members of the B7 family. PD-1 is a member of the immunoglobulin (Ig) superfamily that contains a single Ig V-like domain in its extracellular region. The cytoplasmic domain of PD1 contains two tyrosines, with the tyrosine closest to the membrane (VAYEEL in mouse PD-1) located within an ITIM (immunoreceptor tyrosine-based inhibitory motif). The presence of an ITIM in PD-1 indicates that this molecule functions to attenuate antigen receptor signaling by recruiting cytoplasmic phosphatases. Human and murine PD-1 proteins share about 60% amino acid identity with the conservation of four possible N-glycosylation sites, and residues defining the Ig-V domain. The ITIM in the cytoplasmic region and the ITIM-like motif surrounding the carboxy-terminal tyrosine are also conserved between the human and murine orthologs. Traditionally, cancer immunotherapy has involved complicated and time-consuming methods using individualized cells and preparations. Recently, cancer immunotherapy based on monoclonal antibodies based on the interruption of suppressive signals that are delivered to the adaptive immune system has shown promise in the clinic within the framework of available systemic immunotherapy. However, there is a continuing need in the art for safer and more effective cancer treatments. SYNTHESIS Antibodies that selectively interact with PD-1 are provided. Certain anti-PD-1 antibodies have been shown to be effective in vivo in preventing and / or treating cancer. Advantageously, the anti-PD-1 antibodies provided herein bind to human, Macaca fascicularis, and mouse PD-1. Also advantageously, the anti-PD-1 antibodies provided herein are also effective in vivo to stimulate T cell proliferation. Isolated antagonist antibodies that specifically bind to PD-1 and prevent or reduce the biological effect of PD-1 are provided herein. In some embodiments, the antagonist antibody can be, for example, a human, humanized, or chimeric antibody. The invention described herein relates to antibodies that bind to PD1. In one aspect, the invention provides an isolated antagonist antibody that specifically binds to PD-1, wherein the antibody comprises a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR1), VH CDR2 and VH CDR3 of VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6; and a light chain (VL) variable region comprising VL CDR1, VL CDR2 and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO:7; SEQ ID NO: 8; and SEQ ID NO: 9. In some embodiments, the VH region comprises the amino acid sequence shown in SEQ ID NO: 3, 4, 5, or 6, or a variant with one or more conservative amino acid substitutions at residues not found in a CDR. , and / or the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 7, 8 or 9, or a variant thereof with one or more amino acid substitutions in amino acids not found in a CDR. In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO: 39 and / or a heavy chain comprising the sequence shown in SEQ ID NO: 29 or 38. In some forms of embodiment, the antibody comprises a VH region produced by the expression vector ATCC Accession No. PTA-121183. In some embodiments, the antibody comprises a VL region produced by the expression vector ATCC Accession No. PTA-121182. In another aspect, the invention provides an isolated antibody that specifically binds to PD-1, wherein the antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, 24, 25, 27, 28, 35 or 36, a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, 23, 26 or 37, a CDR1 VL comprising the amino acid sequence shown in SEQ ID NO: 10, 22, 30 or 32, a CDR2 VL comprising the amino acid sequence shown in SEQ ID NO: 11, 20 or 33, and a CDR3 VL comprising the amino acid sequence shown in SEQ ID NO: 12, 21, 31 or 34. In some embodiments, the antibody can be a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody comprises a constant region. In some embodiments, the antibody is of the human IgGi, IgG2, IgG2Aa>Ιθθ3, IgG4, IgGíib, IgG4¿Cj, IgG4 S228P, IgG4ib S228P, and IgG4¿c S228P subclass. In some embodiments, the antibody is of the IgG4 isotype and comprises a stabilized hinge, eg, S228P. In another aspect, the invention provides an isolated antibody that specifically binds to PD-1 and competes with and / or binds the same PD-1 epitope as the antibodies described herein. In some embodiments, an anti-PD-1 antibody provided herein promotes IFNγ and / or TNF secretion from T cells. In some embodiments, an anti-PD-1 antibody provided herein promotes T cell proliferation. In some embodiments, an anti-PD-1 antibody provided herein inhibits tumor growth. In some embodiments, an anti-PD-1 antibody provided herein binds to human PD-1 and mouse PD-1. In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody against PD-1 described herein, and a pharmaceutically acceptable carrier. In another aspect, the invention provides an isolated polynucleotide comprising a nucleotide sequence encoding an antibody against PD-1 described herein. In another aspect, the invention provides a vector comprising the polynucleotide. In another aspect, the invention provides an isolated host cell that recombinantly produces an antibody against PD-1 described herein. In another aspect, the invention provides a method of producing an antagonistic anti-PD-1 antibody, wherein the method comprises: culturing a cell line that recombinantly produces the antibody described herein under conditions where the antibody; and recovering the antibody. In another aspect, the invention provides a method of producing an anti-PD-1 antagonist antibody, wherein the method comprises: culturing a cell line comprising a nucleic acid encoding an antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 29 or 38, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 39 under conditions where the antibody is produced; and recovering the antibody. In some embodiments, the light and heavy chains are encoded on separate vectors. In other embodiments, the light and heavy chains are encoded on the same vector. In another aspect, the invention provides a method of treating a condition in a subject, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described herein. In some embodiments, the condition is cancer. In some embodiments, the cancer is selected from the group consisting of gastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma , colon cancer, oral cancer, skin cancer and melanoma. In some embodiments, the subject is a previously treated adult patient who has locally advanced or metastatic melanoma, squamous cell head and neck cancer (NCHSC), ovarian carcinoma, sarcoma, or refractory classical Hodgkin's lymphoma or relapsing (cHL). In some embodiments, the cancer may be platinum-resistant and / or platinum-refractory cancer, for example, platinum-resistant and / or refractory ovarian cancer, platinum-resistant and / or refractory breast cancer, or platinum-refractory cancer. platinum resistant and / or refractory lung. In some embodiments, an anti-PD-1 antibody is administered at a dose of about 0.5 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, or about 10 mg. / kg. In some embodiments, the anti-PD-1 antibody is administered once every 7, 14, 21, or 28 days. In some embodiments, the anti-PD-1 antibody is administered intravenously or subcutaneously. In another aspect, the invention provides a method of inhibiting tumor growth or progression in a subject having a tumor comprising administering to the subject an effective amount of the pharmaceutical composition described herein. In another aspect, the invention provides a method of inhibiting or preventing cancer cell metastasis in a subject, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described herein. In another aspect, the invention provides a method of inducing tumor regression in a subject having a tumor that expresses PD-1, comprising administering to the subject an effective amount of the pharmaceutical composition described herein. In some embodiments, the antibody herein can be administered parenterally to a subject. In some embodiments, the subject is a human. In some embodiments, the method may also comprise administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is, for example, crizotinib, paibociclib, an anti-CTLA4 antibody, an anti-4-1BB antibody, or a PD-1 second antibody. Also provided is the use of any of the anti-PD-1 antagonist antibodies provided herein in the manufacture of a medicament for the treatment of cancer or for inhibiting tumor growth or progression in a subject in need thereof. In some embodiments, the anti-PD-1 antagonist antibody reduces weight gain in the subject. Also provided are anti-PD-1 antagonist antibodies for use in treating cancer or for inhibiting tumor growth or progression in a subject in need thereof. In some embodiments, the cancer is, for example, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, stomach, thyroid cancer, lung cancer (including, for example, non-small cell lung carcinoma), ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, myeloma multiple, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer and melanoma, among others. In another aspect, the present description provides a method for improving the immunogenicity or therapeutic effect of a vaccine for the treatment of cancer in a mammal, in particular, a human being, wherein the method comprises administering to the mammal that receives the vaccine a effective amount of antagonistic anti-PD-1 antibody provided by the present disclosure. In another aspect, the present disclosure provides a method of treating cancer in a mammal, in particular, a human, wherein the method comprises administering to the mammal (1) an effective amount of a vaccine capable of eliciting an immune response against cells cancer and (2) an effective amount of an anti-PD-1 antagonist antibody provided by the present disclosure. BRIEF DESCRIPTION OF THE FIGURES AND THE DRAWINGS Figure 1A shows a graph summarizing the body weight of mice treated with anti-PD-1 antagonist antibody. Figure 1B shows a graph summarizing the body weight of mice treated with anti-PD-1 antagonist antibody. Figure 1C shows a graph summarizing the body weight of mice treated with anti-PD-1 antagonist antibody. Figure 1D shows a graph summarizing the body weight of mice treated with anti-PD-1 antagonist antibody. Figure 1E shows a graph summarizing the body weight of mice treated with anti-PD-1 antagonist antibody. Figure 2A shows a graph summarizing EC50 for anti-PD1 antibody binding to primary activated human T cells. Figure 2B shows a graph summarizing EC50 for anti-PD1 antibody binding to primary activated Macaca fascicularis T cells. Figure 3 shows a bar graph summarizing the proliferation of cultured activated CD4 T cells treated as follows (a) no antibody; (b) isotype control; (c) EH12.1; (d) C1; (e) C2; (f) C3; (g) mAb1; (h) mAbX; (i) mAb4; u) mAb5; (k) mAb6; (I) mAb7; (m) mAb9; (n) mAb10; (o) mAb11; (p) mAb14; (q) mAb15; (r) mAb16. Figure 4 shows a bar graph summarizing the proliferation of cultured activated CD8 T cells treated as follows (a) no antibody; (b) isotype control; (c) EH12.1; (d) C1; (e) C2; (f) C3; (g) mAb1; (h) mAbX; (i) mAb4; (j) rnAb5; (k) mAb6; (I) mAb7; (m) mAb9; (n) mAb10; (o) mAb11; (p) mAb14; (q) mAb15; (r) mAb16. DETAILED DESCRIPTION Described herein are antibodies that specifically bind to PD-1. Methods of obtaining anti-PD-1 antibodies, compositions comprising these antibodies, and methods of using them as medicaments are provided. Anti-PD-1 antibodies can be used to inhibit tumor progression, and can be used in the prevention and / or treatment of cancer and / or other diseases. General techniques In the practice of the present invention, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the purview of the people of the world, will be used, unless otherwise indicated. mid-level job. These techniques are explained in detail in the literature, eg, Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.l. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B, Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunoology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullís et al., eds., 1994); Current Protocols in Immunoology (J.E, Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies; a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995). Definitions Unless otherwise indicated, the following terms shall have the meanings set forth below: the term "isolated molecule" refers to a molecule (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) that, due to its origin or source of derivation, (1) it is not related to naturally associated components that accompany it in its native state, (2) it is practically free of other molecules from the same source, eg, species, cell from from which it is expressed, library, etc., (3) is expressed by a cell of a different species, or (4) does not occur naturally. Thus, a molecule that is chemically synthesized or expressed in a different cellular system than the system from which it naturally occurs will be considered isolated from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques known in the art. Molecular homogeneity or purity can be analyzed by various methods known in the state of the art. For example, the purity of a polypeptide sample can be analyzed by polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide, using techniques known in the art. For certain purposes, higher resolution can be provided using HPLC or other purification methods known in the art. An "antibody" is an immunoglobulin molecule capable of specifically binding a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the molecule. of immunoglobulin. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding portion thereof that competes with the intact antibody for specific binding, protein fusion compounds comprising an antigen binding portion and any other modified configuration of the immunoglobulin molecule comprising an antigen recognition site. Antigen-binding moieties include, for example, Fab, Fab', F(abj2, Fd, Fv, domain antibodies (dAb, eg, shark and camelid antibodies), fragments including complementarity determining regions ( CDR), single-chain variable fragment (scFv) antibodies, maxibodies, minibodies, intrabodies, diabodies, tribodies, tetrabodies, v-NARs, and bis-scFvs, and polypeptides containing at least a portion of an immunoglobulin that is sufficient to confer antigen-specific binding to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or a subclass of these), and the antibody need not be of any particular class. Based on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of Immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), for example IgGi, IgG2, IgG3, IgG4, IgAq and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known. A "variable region" of an antibody refers to the light chain variable region of the antibody or the heavy chain variable region of the antibody, either alone or in combination. As is known in the art, heavy chain and light chain variable regions consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions, and they contribute to the formation of the antigen-binding sites of antibodies. If variants of a subject variable region are desired, in particular with substitution at amino acid residues outside a CDR region (i.e., in the framework region), the appropriate amino acid substitution, preferably conservative amino acid substitution, can be used. identify by comparing the subject variable region with the variable regions of other antibodies that contain CDR1 and CDR2 sequences in the same canonical class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4): 901-917, 1987 ). In some embodiments, definitive delineation of a CDR and identification of residues that comprise the binding site of an antibody are achieved by dissolving the antibody structure and / or the structure of the antibody-ligand complex. In some embodiments, this may be accomplished by various techniques known to those of ordinary skill in the art, such as X-ray crystallography. In some embodiments, various methods of analysis may be used to identify or approximate regions CDR. In some embodiments, different analysis methods can be used to identify or approach the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, and the conformational definition. The Kabat definition is a standard for numbering residues in an antibody and is generally used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. Chothia's definition is similar to Kabat's, but Chothia's definition takes into account the positions of certain regions of the structural loop. See, eg, Chothia et al., 1986, J. Mol. BioL, 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group, which model the structure of the antibody. See, eg, Martin et al., 1989, Proc Nati Acad Sci (USA), 86:9268-9272; “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The definition of AbM models the tertiary structure of an antibody from a primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The definition of contact is based on an analysis of the available complex crystal structures. See, eg, MacCallum et al., 1996, J. Mol. BioL, 5:732-45. In another approach, referred to herein as "conformational definition" of CDRs, the positions of the CDRs can be identified as the residues that make enthalpy contributions to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Still, other CDR boundary definitions may not necessarily follow one of the above approaches, but will still overlap with at least a portion of the Kabat CDRs; however, they may be shortened or lengthened by virtue of experimental or predictive findings that particular residues or groups of residues do not have a significant impact on antigen binding. As used herein, a CDR can refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods herein may use CDRs defined according to any of these approaches. For any embodiment that contains more than one CDR, the CDRs can be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformational definitions. As is known in the art, a "variable region" of an antibody refers to the antibody light chain constant region or the antibody heavy chain constant region, either individually or in combination. As used herein, monoclonal antibody refers to an antibody derived from a population of substantially homogeneous antibodies, that is, the individual antibodies comprised in the population are identical except for possible natural mutations which may occur in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, unlike polyclonal antibody preparations, which generally include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The monoclonal modifier '' indicates the character of the antibody obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be obtained by the hybridoma method first obtained by Kohler and Milstein, 1975, Nature 256:495, or they can be obtained by recombinant DNA methods, such as as described in US Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated by techniques described, for example, in McCafferty et al., 1990, Nature 348:552-554. As used herein, "humanized antibody" refers to non-human (eg, murine) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding antibody subsequences) that contain a minimal sequence derived from non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibody), wherein residues of a recipient CDR are replaced by residues of a CDR from a non-human species (donor antibody), such as mouse, rat or rabbit, which it has the desired specificity, affinity and capacity. . The humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize the performance of the antibody. A human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or obtained by any of the techniques for obtaining human antibodies, as described herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species, and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from an antibody. mouse and the constant region sequences are derived from a human antibody. The term "epitope" refers to that portion of a molecule capable of being recognized and bound by an antibody at one or more of the antibody's antigen-binding regions. Epitopes often consist of a surface cluster of molecules, such as sugar or amino acid side chains, and have specific three-dimensional characteristics and specific charge characteristics. In some embodiments, the epitope can be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. Non-linear epitope or conformational epitope comprise non-contiguous polypeptides (or amino acids) within the antigenic protein to which an epitope-specific antibody binds. As used herein, the term "antigenic epitope" is defined as a portion of an antigen to which an antibody can specifically bind, as determined by any method known in the art, eg, conventional immunoassays. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, for example, using the techniques described herein. Alternatively, during the discovery process, the generation and characterization of antibodies can shed light on desirable epitopes. Based on this information, antibodies can be competitively screened for binding to the same epitope. One approach to achieving this is to perform competition and cross-competition studies to find antibodies that compete or cross-compete with each other for binding to PD-1, eg, antibodies compete for binding to antigen. As used herein, the term "PD-1" refers to any form of PD-1 and its variants that retain at least some of the activity of PD-1. Unless otherwise indicated, eg, by specific reference to human PD-1, PD-1 includes all mammalian species of native sequence PD-1, eg, human, canine, feline, equine and cattle. An exemplary human PD-1 is found as Uniprot Accession Number Q15116 (SEQ ID NO: 1). The term "agonist" refers to a substance that promotes (ie, induces, causes, enhances, or augments) the biological effect or activity of another molecule. The term agonist encompasses substances that bind to the receptor, such as an antibody, and substances that promote function without binding to the receptor (eg, by activating an associated protein). The terms "antagonist" or "inhibitor" refer to a substance that prevents, blocks, inhibits, neutralizes, or reduces a biological effect or activity of another molecule, such as a receptor. The term "antagonistic antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of that target. In some embodiments, the expression may indicate an antibody that prevents the target, eg, PD-1, to which it binds from performing a biological function. As used herein, an "anti-PD-1 antagonist antibody" refers to an antibody capable of inhibiting the biological activity of PD-1 and / or the downstream events mediated by PD-1. Antagonistic anti-PD-1 antibodies include antibodies that block, antagonize, suppress, or reduce (to any degree, even significantly) the biological activity of PD-1, including downstream events mediated by PD-1, such as binding to PD -L1 and downstream signaling, PD-L2 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, IL-10 induction, and inhibition of antitumor immune responses. For purposes of the present invention, the term "anti-PD-1 antagonist antibody" (referred to interchangeably as "anti-PD-1 antibody," "anti-PD-1 antagonist antibody, or PD-1 antagonist antibody") is expressly understood to mean encompasses all of the above-identified terms, titles, and functional states and features by which PD-1, a biological activity of PD-1, or the consequences of biological activity are substantially abolished, diminished, or neutralized in any considerable degree. In some embodiments, an antagonistic anti-PD-1 antibody binds to PD-1 and up-regulates an anti-tumor immune response. Examples of antagonistic anti-PD-1 antibodies are provided herein. The terms polypeptide", "oligopeptide", "peptide" and protein" are used herein interchangeably to refer to amino acid chains of any length. The chain may be straight or branched, may comprise modified amino acids, and / or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling partner. Also included in the definition are, for example, polypeptides that contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can be produced as single chains or as associated chains. As is known in the art, "polynucleotide" or "nucleic acid", used interchangeably herein, refer to nucleotide chains of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, bases or modified nucleotides and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications can be made to the nucleotide structure before or after chain assembly. The nucleotide sequence may be interrupted by components that are not nucleotides. A polynucleotide can also be modified after polymerization, for example, by conjugation with a marker component. Other types of modifications include, for example, "caps," substitution of one or more of the natural nucleotides with an analog, internucleotide modifications, eg, those with uncharged bonds (eg, methyl phosphonates, phosphotriesters , phosphoamidates, carbamates, etc.) and with charged linkages (eg, phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, eg, proteins (eg, nucleases, toxins, antibodies, signal peptides, poly -L-lysine, etc.), those with intercalators (eg, acridine, psoralen, etc.), those containing chelators (eg, metals, metals radioactive, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (eg, alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotides. In addition, any of the hydroxyl groups generally present in sugars may be replaced, for example, by phosphonate groups or phosphate groups, protected by standard protecting groups or activated to make additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic terminating group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized at standard protecting groups. The polynucleotides may also contain analogous forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-, 2'-0-allyl, 2'-fluoro- or 2 '-azido-ribose, carboxylic acid analogs, alpha or beta anomeric sugars, epimeric sugars, such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and raw nucleoside analogs, such as riboside methyl. One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments where the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate") , P(O)R, P(O)OR', CO, or CH2 ("formacetal), wherein each of R or R' is independently H or substituted or unsubstituted (1-20C) alkyl optionally containing a ether (Ό-), aryl, aichenyl, cycloalkenyl, cycloalkenyl, or araldyl bond. It is not necessary that all bonds in a polynucleotide be identical. The above description applies to all polynucleotides indicated herein, including RNA and DNA. As used herein, an antibody "interacts with PD-1 when the equilibrium dissociation constant is equal to or less than 20 nM, preferably less than about 6 nM, more preferably less than about 1 nM, most preferably, less than about 0.2 nM, as measured by the methods described herein in Example 7. An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term within the prior art, and methods for determining specific binding or preference! are also known in the state of the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for a longer duration, and / or with greater affinity to a molecule. particular cell or substance compared to alternative cells or substances. An antibody "binds specifically" or binds preferentially to a target if it binds with greater affinity, avidity, ease, and / or duration than it would bind to other substances. For example, an antibody that binds specifically or preferentially to a PD-1 epitope is an antibody that binds to this epitope with greater affinity, avidity, ease, and / or duration than it would bind to other PD-1 epitopes or epitopes. that are not PD-1. Reading this definition it is also understood that, for example, an antibody (or moiety or epitope) that binds specifically or preferentially to a first target may or may not bind specifically or preferentially to a second target. As such, "specific fixation" or "preferred fixation!" does not necessarily require (but may include) exclusive fixation. In general, but not necessarily, reference to binding means preferential binding. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e. free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure. pure, even more preferably at least 98% pure, and most preferably at least 99% pure. A "host cell" includes an individual cell or cell culture that may be or was a recipient of the vectors for incorporation of polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny need not be completely identical (in terms of morphology or genomic DNA complement) to the cell of origin due to natural, accidental, or deliberate mutations. A host cell includes cells transfected in vivo with a polynucleotide of the invention. As is known in the art, the term Fc region is used to define a C-terminal region of an immunoglobulin heavy chain. The Fc region may be a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain may vary, in general, the Fc region of human IgG heavy chain is defined to span from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus thereof. The numbering of the residues in the Fe region is that of the Kabat EU index. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. In general, the Fc region of an immunoglobulin comprises two constant domains: CH2 and CH3. As is known in the art, an Fe region can be present in dimeric or monomeric form. As used in the prior art, Fc receptor and FcR describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRIl, and FcγRIII subclasses, including S allelic variants and, alternatively, spliced ​​forms of these receptors. FcγRIl receptors include FcγRIIA (an activating receptor) and FcγRIIB (an inhibitory receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and from Haas et al., 1995, J. Lab. Clin. Med., 126:330-41, "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249). The term "competitive", as used herein with respect to an antibody, means that a first antibody, or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antigen. or an antigen-binding portion thereof, such that the result of binding of the first antibody to its cognate epitope is detectably decreased in the presence of the second antibody, compared to binding of the first antibody in the absence of the second antibody. Alternatively, it is possible that the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, but this is not necessarily the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, when each antibody detectably inhibits the binding of the other antibody to its ligand or cognate epitope to a lesser, equal, or greater degree, the antibodies are said to "cross-compete" with each other for the binding of their respective epitopes. The present invention encompasses both competing and cross-competing antibodies. Regardless of the mechanism by which competition or cross-competition occurs (e.g., spherical hindrance, conformational change, or binding to a common epitope or portion thereof), those of the mid-level trade will understand, based on the explanations provided herein, which include competing and / or cross-competing antibodies and may be useful for the methods described in Present. A "functional Fc region" has at least one effector function of a Fc region of a native sequence. Examples of effector functions include binding to C1q; complement dependent cytotoxicity; Fe receptor binding; antibody-dependent cellular cytotoxicity; phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor), etc. In general, effector functions require that the Fe region be combined with a binding domain (for example, an antibody variable domain), and can be analyzed by different assays known in the state of the art to evaluate the effector functions of the antibody. . A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a natural Fc region. An Fc region variant comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, but still retains at least one effector function of the native sequence Fc region. Preferably, the Fc region variant has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, eg, from about 1 to about 10 amino acid substitutions. and, preferably, from about 1 to about 5 amino acid substitutions in an Fc region of the native sequence or in the Fc region of the parent polypeptide. The Fc region variant herein preferably has at least about 80% sequence identity with a native sequence Fc region and / or with a Fc region of a parent polypeptide, and most preferably at least about 90% sequence identity therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% of sequence identity with that. As used herein, "treatment" is an approach to obtain desired or beneficial clinical results. For purposes of the present invention, beneficial or desirable results include, but are not limited to, one or more of the following: reduction in the proliferation (or destruction) of neoplastic or cancer cells, inhibition of metastasis of neoplastic cells, reduction in cell size of a tumour, remission of cancer, decrease in symptoms resulting from cancer, increase in quality of life for those who have cancer, reduction in the dose of other medicines needed to treat cancer, slow down the progression of cancer cancer, cancer cure and / or prolongation of survival of cancer patients. "Ameliorate" means a decrease or improvement in one or more symptoms, as compared to no administration of an anti-PD-1 antagonist antibody. "Improve" also includes shortening or reducing the duration of a symptom. As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve one or more desired or beneficial results. In more specific aspects, an effective amount prevents, alleviates or ameliorates the symptoms of a disease and / or prolongs the survival of the treated subject. For prophylactic use, desired or beneficial outcomes include elimination or reduction of risk, decrease in severity, or delay in onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and phenotypes. pathological intermediates that occur during the development of the disease. For therapeutic use, desired or beneficial results include clinical results, such as reduction of one or more symptoms of a disease, such as cancer, including but not limited to gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, head and neck squamous cell cancer, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer and melanoma, decreased the dose of other drugs needed to treat the disease, enhancing the effect of another drug and / or slowing the progression of cancer in patients. An effective dose can be provided in one or more administrations. For purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment, either directly or indirectly. As is known in the clinical setting, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Therefore, an "effective dose" can be considered in the context of the administration of one or more therapeutic agents, and a single agent can be considered administered in an effective amount if, together with one or more additional agents, it can be achieved or a desirable result is achieved. An individual or subject is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals (eg, cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice, and rats. As used herein, vectori' means a construct, which is capable of delivering and, preferably, expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. As used herein, "expression control sequence" means a nucleic acid sequence that directs the transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive promoter or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" include any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions, such as oil / water emulsion, and various types of wetting agents. The preferred diluents for parenteral or aerosol administration are phosphate buffered saline (PBS) or normal saline (0.9%). Compositions comprising such carriers are formulated by known conventional methods (see, eg, Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th edition, Mack Publishing, 2000). As used herein, the term Κ,η refers to the rate of association constant of an antibody to antigen. Specifically, rate constants (kqn and koff) and equilibrium dissociation constants are measured using full-length antibodies and / or Fab (ie, univalent) and PD-1 antibody fragments. As used herein, the term "koff" refers to the rate of dissociation constant of an antibody from the antibody / antigen complex. As used herein, the term "kD" refers to the equilibrium dissociation constant of an antibody-antigen interaction. Reference to "about" a value or parameter herein includes (and describes) embodiments referring to that value or parameter itself. For example, the description referring to "around X" includes the description of "X. Numeric ranges include the numbers that define the range. The terms immune effector cell enhancer or "IEC enhancer" refer to a substance capable of increasing or enhancing the quantity, quality, or function of one or more types of immune effector cells in a mammal. Examples of immune effector cells include CD8 cytolytic T cells, CD4 T cells, NK cells, and B cells. The term "immune modulator" refers to a substance capable of altering (eg, inhibiting, reducing, increasing, enhancing, or stimulating) the immune response (as defined herein) or the function of any component of the innate, humoral, or immune system. cell of a mammalian host. Thus, the term "immune modulator" encompasses "immune effector cell enhancer" defined herein and "immune suppressor cell inhibitor" defined herein, as well as substance that affects other components of the immune system of a mammal. The term "immune response" refers to any detectable response to a particular substance (such as an antigen or immunogen) by the immune system of a mammalian host, such as innate immune responses (eg, activation of the Toll receptor signaling cascade). ), cellular immune responses (eg, responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (eg, responses mediated by B cells, such as generation and secretion of antibodies in the plasma, ganglia and / or interstitial fluids). The term "immunogenic" refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response, or to enhance, potentiate, augment, or prolong a pre-existing immune response, against a particular antigen, either alone or when present. bound to a carrier, in the presence or absence of an adjuvant. The terms "immune suppressor cell inhibitor" or "ISC inhibitor" refer to a substance capable of reducing or suppressing the number of immune suppressor cell functions of a mammal. Examples of immune suppressor cells include T-regulatory cells ("T reg"), myeloid-derived suppressor cells, and tumor-associated macrophages. The term "intradermal administration" or "intradermally administered" in the context of administering a substance to a mammal, including a human, refers to the administration of a substance into the dermis layer of a mammal. Mammalian skin is composed of an epidermis layer, a dermis layer, and a subcutaneous layer. The epidermis is the outer layer of the skin. The dermis, which is the middle part of the skin, contains nerve endings, sweat glands, sebaceous glands, hair follicles, and blood vessels. The subcutaneous layer is made of fat and connective tissue that houses larger blood vessels and nerves. Unlike intradermal administration, "subcutaneous administration" refers to the administration of a substance to the subcutaneous layer, and "topical administration" refers to the administration of a substance to the surface. The term "neoplastic disorder" refers to a condition in which cells proliferate at an abnormally high and uncontrolled rate, where the rate exceeds and is uncoordinated with that of surrounding normal tissues. Generally, it results in a solid lesion or lump, known as a "tumor." This term encompasses benign and malignant neoplastic disorders. The term "malignant neoplastic disorder", which is used in the same way as the term "cancer" in the present description, refers to a neoplastic disorder characterized by the ability of tumor cells to spread to other places in the body (this is known as as metastases). The term "benign neoplastic disorder" refers to a neoplastic disorder in which tumor cells do not have the ability to metastasize. The term "prevent" refers to (a) preventing a disorder from occurring or (b) delaying the onset of a disorder or the onset of symptoms of a disorder. The term "tumor associated antigen" or "TAA" refers to an antigen that is specifically expressed by tumor cells or that is expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type. Tumor-associated antigens may be antigens that are not normally expressed by the host; can be mutated, truncated, incorrectly folded, or they may be abnormal manifestations of molecules normally expressed by the host; they may be identical to normally expressed molecules but expressed at abnormally high levels; or they may be expressed in an abnormal context or environment. Tumor-associated antigens can be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biological molecules. The term "vaccine" refers to an immunogenic composition for administration to a mammal to elicit an immune response against a particular antigen in the mammal. Generally, a vaccine contains an agent (known as an "antigen" or "immunogen") that resembles or is derived from the target of the immune response, such as a disease-causing microorganism or tumor cells. A vaccine for the treatment of a tumor, such as cancer, generally contains an antigen that is derived from a TAA found in the target tumor and can elicit immunogenicity against the TAA in the target tumor. The term "vaccine immunotherapy regimen" refers to a therapeutic regimen in which a vaccine is administered together with one or more immune modulators. The vaccine and immune modulators can be administered together in a single formulation, or separately. It is understood that wherever embodiments are described herein with the term "comprising", other analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided. When aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group indicated in its entirety, but also each member of the group separately and all possible subgroups of the main group, and also the main group in the absence of one or more of the members of the group. The present invention also provides for the explicit exclusion of one or more of any of the group members in the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning ordinarily given to them by a person in the mid-level trade to which the invention pertains. In the event of a conflict, this specification, including definitions, shall prevail. Throughout the specification and claims, the term "comprising" or variations such as "comprises" or "comprising" shall imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless the context indicates otherwise, singular terms shall include the plural, and plural terms shall include the singular. All examples after the expressions “p. ex.'' or for example are not intended to be exhaustive or limiting. Examples of methods and materials are described herein, although methods and materials similar or equivalent to those described herein may also be used to practice or evaluate the present invention. The materials, methods and examples are illustrative only and not limiting. Anti-PD-1 antagonist antibodies Provided herein are anti-PD-1 antagonist antibodies that block, suppress, or reduce (even significantly reduce) the biological activity of PD-1, including downstream events mediated by PD-1. An antagonistic anti-PD-1 antibody should exhibit one or more of the following characteristics: (a) binding to PD-1 and blocking of downstream signaling events; (b) blockade of PD-L1 binding to PD-1; (c) upregulation of the T cell-mediated immune response; (d) stimulation of IFN[gamma] secretion; (e) stimulation of TNF secretion; (f) increased proliferation of T lymphocytes; and (g) reduction of inhibitory signal transduction via PD-1. For purposes of the present invention, the antibody preferably reacts with PD-1 in a manner that inhibits PD-1 signaling function. In some embodiments, the antagonistic anti-PD-1 antibody specifically binds to primate PD-1. Antibodies useful in the present invention may encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Fab, Fab', F(ab')2, Fv, Fe, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies. , single-chain (ScFv), mutants of these, fusion proteins comprising an antibody portion (for example, an antibody domain), humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. The antibodies can be murine, rat, human, or of any other origin (including chimeric or humanized antibodies). In some embodiments, the antagonistic anti-PD-1 antibody is a monoclonal antibody. In some embodiments, the antibody is a human or humanized antibody. Antagonist anti-PD-1 antibodies can be obtained by any method known in the state of the art. General techniques for the production of human and mouse antibodies are known in the art and / or are described herein. Antagonistic anti-PD-1 antibodies can be identified or characterized using methods known in the art, whereby reduction, enhancement or neutralization of PD-1 biological activity is detected and / or measured. In some embodiments, an antagonistic anti-PD-1 antibody is identified by incubating a candidate agent with PD-1 and monitoring for concomitant binding and / or reduction or neutralization of PD-1 biological activity. Binding assay can be performed, for example, with purified PD-1 polypeptides or with cells naturally expressing (eg, different strains), or transfected to express, PD-1 polypeptides. In one embodiment, the binding assay is a competition binding assay, wherein the ability of a candidate antibody to compete with a known anti-PD-1 antagonist antibody for binding to PD-1 is assessed. it can be performed in various formats, including the ELISA format. In some embodiments, an antagonistic anti-PD-1 antibody is identified by incubating a candidate antibody with PD-1 and monitoring for binding. After initial identification, the activity of a candidate anti-PD-1 antagonist antibody can be confirmed and further refined by bioassays, which are known to assess desired biological activities. In some embodiments, an in vitro cellular assay is used to further characterize a candidate anti-PD-1 antibody. For example, a candidate antibody is incubated with primary human T cells, PD-L1 is added, and IFN[gamma] secretion is monitored. Alternatively, bioassays can be used to test candidates directly. Antagonistic anti-PD-1 antibodies of the invention exhibit one or more of the following characteristics: (a) binding to PD-1 and blocking of downstream signaling events; (b) blockade of PD-L1 binding to PD-1; (c) upregulation of the T cell-mediated immune response; (d) stimulation of IFN[gamma] secretion; (e) stimulation of TNF secretion; (f) increased proliferation of T lymphocytes; (g) reduction of inhibitory signal transduction via PD-1; and (h) blocking the binding of PD-L2 to PD-1. Preferably, the anti-PD-1 antibodies have two or more of these characteristics. More preferably, the antibodies have three or more of the characteristics. More preferably, the antibodies have four or more of the characteristics. More preferably, the antibodies have five or more of the characteristics. More preferably, the antibodies have six or more of the features. More preferably, the antibodies have seven or more of the characteristics. Most preferably, the antibodies have all eight characteristics. Antagonist anti-PD-1 antibodies can be characterized using methods known in the art. For example, one approach is to identify the epitope to which it binds or "epitope mapping." There are many methods known in the state of the art to map and characterize the location of epitopes on proteins, including dissolving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and peptide assays. synthetics, as described, for example, in Harlow and Lane, Chapter 11, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In a further example, mapping can be used of epitopes to determine the sequence to which an anti-PD-1 antagonist antibody binds. Epitope mapping is commercially available from various sources, eg, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, the Netherlands). The epitope can be linear, that is, it can be contained in a single region of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that are not necessarily contained in a single portion. Peptides of various lengths (eg, at least 4-6 amino acids in length) can be isolated or synthesized (eg, recombinantly) and used for binding assays with an antagonist anti-PD-1 antibody. In another example, the epitope to which the anti-PD-1 antagonist antibody binds can be determined in a systematic assay using overlapping peptides derived from the PD-1 sequence and determining the binding of the anti-PD-1 antagonist antibody. According to gene fragment expression assays, the open reading frame encoding PD-1 is fragmented randomly or by specific gene constructs, and the reactivity of the expressed PD-1 fragments with the antibody to be tested is determined. . Gene fragments can be produced, for example, by PCR and then transcribed and translated into protein in vitro, in the presence of amino acids. radioactive. Antibody binding to radioactively labeled PD-1 fragments is determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified using large collections of random peptide sequences displayed on the surface of phage (phage collections) or yeast (yeast display) particles. Alternatively, a defined collection of overlapping peptide fragments can be assessed for binding to test antibody in simple binding assays. In another example, antigen mutagenesis, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, alanine scanning mutagenesis experiments can be performed using a mutant PD1, in which different residues of the PD-1 polypeptide have been replaced by alanine. By evaluating antibody binding to mutant PD-1, the importance of particular PD-1 residues in antibody binding can be assessed. Another method that can be used to characterize an antagonistic anti-PD-1 antibody is to use competition assays with other antibodies known to bind to the same antigen, i.e., different PD-1 fragments, to determine if the antagonistic anti-PD-1 antibody binds to the same epitope as other antibodies. Proficiency assays are known to those in the mid-level trade, which are included in an ELISA format. The binding affinity (KD) of an anti-PD-1 antagonist antibody to PD-1 can be from about 0.001 to about 200 nM. In some embodiments, the binding affinity is any of about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM or about 1 pM. In some embodiments, the binding affinity is less than any of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 pM, about 1 pM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM or about 2 pM. Accordingly, the invention provides any of the following, or compositions (including pharmaceutical compositions) comprising an antibody with a partial light chain sequence and a partial heavy chain sequence, as indicated in Table 1, or variants thereof . In Table 1, the underlined sequences are CDR sequences. In Table 1, the KD indicates the affinity to human PD-1 as measured using surface plasmon resonance at 25°C, unless otherwise indicated. Table 1: Variable Region Sequences of Antagonist Anti-PD-1 mAb Light Chain mAb1 DIVMTQSPDSLAVSLGERATINCK Antibodies SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 2) mAb2 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 7) mAb3 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPHTFGGGTKVEIK (SEQ ID NO: 8) mAb4 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSTNQKNFLTWYQQKP GQPPKLLIYWTSTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAVY YCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 9) mAb5 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 2) mAb6 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 7) mAb7 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPHTFGGGTKVEIK (SEQ ID NO: 8) mAb8 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSTNQKNFLTWYQQKP GQPPKLLIYWT STRESGVPDRFS GSGSGTDFTLTISSLQAEDVAVY YCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 9) Heavy chain KD (nM) QVQLVQSGAEVKKPGASVKVSCKAS 64.24 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLLT GTFAYWGQGTLVTVSS (SEQ ID NO: 3) QVQLVQSGAEVKKPGASVKVSCKAS 2.22 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLLT GTFAYWGQGTLVTVSS (SEQ ID NO: 3) QVQLVQSGAEVKKPGASVKVSCKAS 1.43 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLLT GTFAYWGQGTLVTVSS (SEQ ID NO: 3) QVQLVQSGAEVKKPGASVKVSCKAS 89 (at GYTFTSYWINWVRQAPGQGLEWMG 37° C) NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLLT GTFAYWGQGTLVTVSS (SEQ ID NO: 3) QVQLVQSGAEVKKPGASVKVSCKAS 12.82 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLST GTFAYWGQGTLVTVSS (SEQ ID NO: 4) QVQLVQSGAEVKKPGASVKVSCKAS 1.16 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLST GTFAYWGQGTLVTVSS (SEQ ID NO: 4) QVQLVQSGAEVKKPGASVKVSCKAS 0.73 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLST GTFAYWGQGTLVTVSS (SEQ ID NO: 4) QVQLVQSGAEVKKPGASVKVSCKAS 17.35 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSLTNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLST GTFAYWGQGTLVTVSS (SEQ ID NO: 4) mAb Light chain mAb9 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 2) mAb10 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQN D YFYPLTFGGGTKVEIK (SEQ ID NO: 7) mAb11 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQN DYFYPHTFGGGTKVEIK (SEQ ID NO: 8) mAb12 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSTNQKNFLTWYQQKP GQPPKLLIYWTSTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAVY YCQNDYFYPLTFGGGTKVEIK (SEQ ID NO : 9) mAb13 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 2) mAb14 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 7) mAb15 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSGNQKNFLTWYQQK PGQPPKLLIYWTSYRESGVPDRF SGSGSGTDFTLTISSLQAEDVAV YYCQNDYFYPHTFGGGTKVEIK (SEQ ID NO: 8) mAb16 DIVMTQSPDSLAVSLGERATINCK SSQSLWDSTNQKNFLTWYQQKP G QPPKLLIYWSTRESSGVPDRFS GSGSGTDFTLTISSLQAEDVAVY YCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 9) Heavy chain KD (nM) QVQLVQSGAEVKKPGASVKVSCKAS 13.54 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSITNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLTT GTFAYWGQGTLVTVSS (SEQ ID NO: 5) QVQLVQSGAEVKKPGASVKVSCKAS 0.98 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSITNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLTT GTFAYWGQGTLVTVSS (SEQ ID NO: 5) QVQLVQSGAEVKKPGASVKVSCKAS 0.93 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSITNYNEKFKNRVTMTRDTS TSTVYM ELSS LRS EDTAVYYCARLTT GTFAYWGQGTLVTVSS (SEQ ID NO: 5) QVQLVQSGAEVKKPGASVKVSCKAS 17.27 GYTFTSYWINWVRQAPGQGLEWMG NIYPGSSITNYNEKFKNRVTMTRDTS TSTVYMELSSLRSEDTAVYYCARLTT GTFAYWGQGTLVTVSS (SEQ ID NO: 5) QVQLVQSGAEVKKPGASVKVSCKAS 5.87 GYTFTSYWINWVRQAPGQGLEWMG NIWPGSSLTNYNEKFKNRVTMTRDT STSTVYMELSSLRSEDTAVYYCARLL TGTFAYWGQGTLVTVSS (SEQ ID NO: 6) QVQLVQSGAEVKKPGASVKVSCKAS 0.6 GYTFTSYWINWVRQAPGQGLEWMG NIWPGSSLTNYNEKFKNRVTMTRDT STSTVYMELSSLRSEDTAVYYCARLL TGTFAYWGQGTLVTVSS (SEQ ID NO: 6) QVQLVQSGAEVKKPGASVKVSCKAS 0.49 GYTFTSYWINWVRQAPGQGLEWMG NIWPGSSLTNYNEKFKNRVTMTRDT STSTVYMELSSLRSEDTAVYYCARLL TGTFAYWGQGTLVTVSS (SEQ ID NO: 6) QVQLVQSGAEVKKPGASVKVSCKAS 7.51 GYTFTSYWINWVRQAPGQGLEWMG NIWPGSSLTNYNEKFKNRVTMTRDT STSTVYMELSSLRSEDTAVYYCARLL TGTFAYWGQGTLVTVSS (SEQ ID NO: 6) The invention also provides CDR portions of antibodies against PD1. The determination of CDR rules is known to those in the mid-level trade. It should be noted that, in some embodiments, the CDRs may be a combination of the Kabat and Chothia CDRs (also called combined CDRs or extended CDRs). In another approach, referred to herein as "conformational definition" of CDRs, the positions of the CDRs can be identified as the residues that make enthalpic contributions to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. In general, "conformational CDRs" include the positions of residues in the Kabat CDRs and Vernier regions that are constrained, so as to maintain a proper loop structure for the antibody to bind to a specific antigen. The determination of conformational CDRs is known to those in the mid-level trade. In some embodiments, the CDRs are the Kabat CDRs. In other embodiments, the CDRs are the Chothia CDRs. In other embodiments, the CDRs are the extended, AbM, conformational, or contact CDRs. In other words, in embodiments having more than one CDR, the CDRs can be any of Kabat, Chothia, extended, AbM, conformational, contact, or combinations of these. In some embodiments, the antibody comprises three CDRs from any of the heavy chain variable regions listed in Table 1. In some embodiments, the antibody comprises three CDRs from any of the light chain variable regions listed in Table 1. In some embodiments, the antibody comprises three CDRs from any one of the heavy chain variable regions shown in Table 1, and three CDRs from any one of the light chain variable regions shown in Table 1. Table 2 provides examples of CDR sequences of antagonist anti-PD-1 antibodies provided herein. Table 2. Antagonist anti-PD-1 antibodies (mAb) and their antigen-binding CDR sequences according to Kabat (underlined) and Chothia (bold) mAb1 ID NO: 1 H NO: 13 14 mAb2 ID NO: 1 NO: 13 14 mAb3 L ID . 1 H-ID NO: 13 14 mAb4 ID NO: 11 ID . 1 ID NO: 16 ID . 1 D ID NO: 1 ID NO: 16 ID NO: 1 ID .21 ID NO: 16 ID . 1 mAb5 mAb6 mAb7 mAb8 mAb10 mAb1 mAb16 L H L H L H H L H H H L H ID NO: NO: 13 14 ID , 1 14 ID NO: 1 14 ID . 1 14 ID NO: 14 ID NO: 1 14 ID . 1 14 ID NO: 1 14 Yo ID 14 ID . 1 14 ID NO: 1 14 ID NO: 1 14 ID NO: 14 ID NO: 16 NOT: 16 ID NO: 16 NOT: 16 ID NO: 16 ID NO: 24 ID ID NO: 24 ID NO: 24 ID NO: 24 ID NO: 27 ID ID NO: 27 ID NO: 27 D ID NO: 27 ID . 1 . 1 ID . 12 ID NO: ID NO: 1 ID NO: D NOT: 21 ID . 1 ID NO: ID NO: 1 ( NO: ID No. 1 ID ID NO: 21 ID NO: ID NO: 1 ID NO: ID NO: 1 ( ID . 1 ID . 1 ID NO: 1 ID .21 ID NO: ID . 1 ( ID NO: 1 In some embodiments, the antibody comprises three light chain CDRs and three heavy chain CDRs from Table 2. An alignment of the light chain CDRs of anti-PD-1 antibodies is provided in Table 3. Variable residues are shown in bold. The light chain CDR consensus sequences are provided in the last row of Table 3. Table 3. Alignment of anti-PD-1 light chain CDRs. SEQ SEQ SEQ mAb VL CDR1 ID NO: VL CDR2 ID NO: VL CDR3 ID NO: 1,5, 9, 13 KSSQSLWDSGNQK NFLT 10 WTSTRES '11 QNDYFYPLT 12 2, 6, 10, 14 KSSQSLWDSGNQK NFLT 10 WTSYRES 20 QNDYFYPLT 12 3, 7, 11, 15 kssqslwdsgnqk nflt 10 wtsyres 20 qndyfypht 21 4 8, 12, 16 kssqslwdstnqkn flt 22 wtstres 11 qndyfyplt 12 17 kssqslldsgnqkn flt 30 wtstres 11 qndysyplt 31 kssqslx, dsx2nqk nflt or T 32 WTSXiRES, where Χί isToY 33 QNDY^YPXjT, where X·! is F or S, and X2 is L or H 34 An alignment of the heavy chain CDRs of anti-PD-1 antibodies is provided in Table 4. Variable residues are shown in bold. The heavy chain CDR consensus sequences are provided in the last row of Table 4. Table 4. Anti-PD-1 heavy chain CDR alignment. mAb VH CDR1 SEQ ID NO: VH CDR2 SEQ ID NO: VH CDR3 SEQ ID NO: 1-4 GYTFTSYWIN 13 NIYPGSSLTNYNEKFK N 17 LLTGTFA Y 18 5-8 GYTFTSYWIN 13 NIYPGSSLTNYNEKFK N 17 LSTGTFA Y 23 9-12 GYTFTSYWIN 13 NIYPGSSITNYNEKFKFTTN V25 LGTKFTTN 26 13-16 GYTFTSYWIN 13 NIWPGSSLTNYNEKFK N 28 1 LLTGTFA Y 18 17 GYTFTSYWIN 13 NIYPGSSSTNYNEKFK N 35 LLTGTFA Y 18 GYTFTSYWIN 13 NlX^GSSXjTNYNEKF KN, where X) is Y or W, and X2 is LAY, I or S 36 LXJGTF AY , where X1 is L or S 37 In some embodiments, the antibody comprises three light chain CDRs from Table 3 and three heavy chain CDRs from Table 4. In some embodiments, the antibody comprises the full-length heavy chain, with or without the C-terminal lysine, and / or the full-length light chain of the antagonistic anti-PD-1 antibodies mAb7 or mAb15. The amino acid sequence of the full length heavy chain of mAb7 (SEQ ID NO: 29) is shown below: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGS SLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPPPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 29) La secuencia de aminoácidos de la cadena pesada de longitud completa de mAb7 sin la lisina del terminal C (SEQ ID NO: 38) se muestra a continuación: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGS SLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTL V7VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:38) The amino acid sequence of the full-length light chain of mAb7 (SEQ ID NO: 39) is shown below: DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWT SYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIKR GTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSGLSTLSKADYPVGESSVYACEVTHQ: NOCDSTYSLSGLSTLSTKEKHKSSVYACEVTHQ: NOCDSTYSLSGLSTLSTKEKHKSSVYACEVT9 The invention also provides methods for generating, selecting and preparing antagonistic anti-PD-1 antibodies. The antibodies of the present invention can be prepared by methods known in the state of the art. In some embodiments, the antibodies can be recombinantly obtained and expressed by any method known in the art. In some embodiments, antibodies can be prepared and selected by phage display technology. See, for example, US Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150; and Winter et al., Annu. Rev. Immunoi. 12:433-455, 1994. Alternatively, phage display technology (McCafferty et al., Nature 348:552-553, 1990) can be used to produce human antibodies and antibody fragments in vitro, from gene repertoires. of immunoglobulin variable domains (V) from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and expressed as functional antibody fragments on the phage particle surface. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selections made for the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Therefore, the phage mimics some of the properties of the B cell. Phage display can be carried out in a variety of formats; for a review, see, eg, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571, 1993. Numerous sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628, 1991, isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial collection of V genes derived from the spleens of immunized mice. A repertoire of human donor V genes can be constructed, and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially according to the techniques described by Mark et al., J. Mol. Biol. 222:581-597, 1991, or Griffith et al., EMBO J. 12:725-734, 1993. In a natural immune response, antibody genes accumulate mutations at a rapid rate (somatic hypermutation). Some of the changes introduced will confer higher affinity, and preferentially, B cells displaying surface immunoglobulin with higher affinity replicate and differentiate upon subsequent exposure to antigen. This natural process can be mimicked using a technique called chain suffling. (Marks et al., Bio / Technol. 10:779-783, 1992). In this method, the affinity of "primary" human antibodies obtained by phage display can be enhanced by sequential replacement of the light chain and heavy chain V region genes by naturally occurring variant repertoires (repertoires) of V domain genes obtained from unimmunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for obtaining very large phage antibody repertoires (also called "the mother of all libraries") was described in Waterhouse et al., Nucí. Acids Res. 21:2265-2266, 1993. Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibody has similar specificities and affinities as the starting rodent antibody. According to this method, which is also called “epitope printing”, the light chain or heavy chain V domain gene of rodent antibodies obtained by the phage display technique is replaced by a repertoire of human genes. domain V, which allows the creation of rodent-human chimeras. Antigen selection results in the isolation of human variable regions capable of restoring a functional antigen-binding site, ie, the epitope controls (imprints) mate choice. When the process is repeated so that the remaining rodent V domain can be replaced, a human antibody is obtained (see PCT Publication No. WO 93 / 06213). Unlike the traditional humanization of rodent antibodies by CDR grafting, this technique yields fully human antibodies, which do not have rodent framework or CDR residues. In some embodiments, antibodies can be obtained using hybridoma technology. It is contemplated that any mammalian subject, including humans, or the antibody-producing cells thereof, can be engineered to function as a basis for the production of mammalian, including human, hybridoma cell lines. Generally, the route and schedule of immunization of the host animal follows established and conventional techniques for the stimulation and production of antibodies, as also described herein. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of immunogen, as described herein. Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. 1975, Nature 256:495-497 or the modification of Buck, D. W., et al., In Vitro, 18:377-381, 1982. In hybridization, available myeloma lines can be used including, but not limited to, X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA. The technique generally involves the fusion of myeloma cells and lymphoid cells using a fusogen, such as polyethylene glycol, or by electrical means known to those of ordinary skill in the art. After fusion, the cells are separated from the fusion medium and cultured in a selective growth medium, such as hypoxanthine-aminopterinthymidine (HAT) medium, to remove unhybridized cells of origin. Any of the media described herein, supplemented with or without serum, can be used for the cultivation of hybridomas secreting monoclonal antibodies. As another alternative to the cell fusion technique, EBV-immortalized B cells can be used to produce the PD-1 monoclonal antibodies of the invention. The hybridomas or other immortalized B cells are expanded and subcloned, if desired, and the supernatants assayed for anti-immunogenic activity by standard immunoassay procedures (eg, radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay). Hybridomas that can be used as a source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies specific for PD-1, or a portion thereof. Hybridomas producing such antibodies can be cultured in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture medium or body fluids by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration, if desired. Unwanted activity, if present, can be removed, for example, by pouring the preparation onto adsorbents prepared with the immunogen bound to a solid phase and eluting or releasing the desired antibodies out of the immunogen. Immunization of a host animal with a PD-1 polypeptide, or a fragment containing the target amino acid sequence conjugated to an immunogenic protein in the species to be immunized, for example, keyhole limpet hemocyanin, serum, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N =C=NR, where R and R1 are different alkyl groups, may allow a population of antibodies (eg monoclonal antibodies) to be obtained. If desired, the anti-PD-1 antagonist antibody (monoclonal or polyclonal) of interest can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained on a vector in the host cell, and the host cell can then be expanded and frozen for later use. The production of recombinant monoclonal antibodies in cell culture can be carried out through the cloning of B-lymphocyte antibody genes through methods known in the state of the art. See, for example, Tiller et al., 2008, J. Immunol. Methods 329, 112; US Patent No. 7,314,622. In some embodiments, the polynucleotide sequence can be used for genetic manipulation, in order to "humanize" the antibody or improve the affinity or other characteristics of the antibody. The antibodies can be customized for use in, for example, dogs, cats, primates, equines, and bovines. In some embodiments, fully human antibodies can be obtained using commercially available mice genetically engineered to express specific human immunoglobulin proteins. Transgenic animals that are engineered to produce a more desirable (eg, fully human antibodies) or stronger immune response can also be used for the generation of humanized or humanized antibodies. Examples of such technology are Xenomouse™ from Abgenlx, Inc. (Fremont, CA) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Prlnceton, NJ). Antibodies can be obtained recombinantly by first isolating the antibodies and antibody-producing cells from host animals, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in host cells (e.g. , CHO cells). Another method that can be used is to express the antibody sequence in transgenic plants (eg tobacco) or milk. Methods for recombinantly expressing antibodies in plants or milk were also described. See, eg, Peeters, et al, Vaccine 19:2756 2001; Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65 1995; and Pollock, et al., J Immunol Methods 231:147, 1999. Methods for making antibody derivatives, eg, domain, single chain, etc., are known in the art. Immunoassays and flow cytometric separation techniques, such as fluorescence activated cell sorting (FACS) can also be used to isolate antibodies that are specific for PD-1. DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells function as the preferred source of DNA. Once isolated, the DNA can be placed into expression vectors (such as the expression vectors described in PCT Publication No. WO 87 / 04462), which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to elicit monoclonal antibody synthesis in the recombinant host cells. See, for example, PCT Publication No. WO 87 / 04462. The DNA can also be modified, for example, by substituting the human heavy and light chain constant domains for the coding sequence in place of the homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci. 81:6851,1984, or by covalent attachment to the immunoglobulin coding sequence of all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of a PD-1 monoclonal antibody hereof. Antibody fragments can be produced by proteolytic or other degradation of the antibodies, by recombinant methods (ie, single or fusion polypeptides) as described above, or by chemical synthesis. Antibody polypeptides, especially short polypeptides of up to 50 amino acids, are conveniently obtained by chemical synthesis. Chemical synthesis methods are known in the state of the art and are commercially available. For example, an antibody could be produced by an automated polypeptide synthesizer using the solid phase method. See also US Patent Nos. 5,807,715; 4,816,567 and 6,331,415. In some embodiments, a polynucleotide comprises a sequence encoding the heavy chain and / or light chain variable regions of antibodies mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11 , mAb12, mAb13, mAb14, mAb15 or mAb16. The sequence encoding the antibody of interest can be maintained on a vector in the host cell, and the host cell can then be expanded and frozen for later use. Vectors (including expression vectors) and host cells are also described herein. The invention includes affinity mature embodiments. For example, affinity matured antibodies can be produced by methods known in the art (Marks et al., 1992, Bio / Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813;Schier et al., 1995, Gene, 169:147-155;Yelton et al., 1995, J. Immunol,, 155:1994-2004;Jackson et al., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and PCT Publication No. W02004 / 058184). The following methods can be used to adjust the affinity of an antibody and to characterize a CDR. One way to characterize a CDR of an antibody and / or to alter (eg, improve) the binding affinity of a polypeptide, such as an antibody, is called library scanning mutagenesis. Generally, library scanning mutagenesis works as follows. One or more amino acid positions in the CDR are replaced by two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids by methods known in the state of the art. This generates small collections of clones (in some embodiments, one for each amino acid position being analyzed), each with a complexity of two or more members (if two or more amino acids are substituted at each position). Typically, the library also includes a clone comprising the native (unsubstituted) amino acid. A small number of clones, eg, about 20-80 clones (depending on library complexity), from each library are screened for binding affinity to the target polypeptide (or other binding agent), and candidates are identified. with greater, equal or lesser affinity, or no affinity. Methods for determining binding affinity are known in the art. Binding affinity can be determined using, for example, Biacore™ surface plasmon resonance assay, which detects differences in binding affinity of about 2-fold or more, Biosensor Kinexa® and scintillation proximity assays, ELISA , the ORIGEN® immunoassay, fluorescence quenching, fluorescence transfer, and / or yeast expression. Binding affinity can also be tested using a suitable bioassay. Biacore™ is particularly useful when the starting antibody binds with a relatively high affinity, eg, a KD of about 10 nM or less. In some embodiments, each amino acid position in a CDR is replaced (in some embodiments, one at a time) with the 20 natural amino acids by mutagenesis methods known in the art (some of which are described below). at the moment). This generates small collections of clones (in some embodiments, one for each amino acid position being analyzed) each with a complexity of 20 members (if all 20 amino acids at each position are substituted). In some embodiments, the library to be analyzed comprises substitutions at two or more positions, which may be at the same CDR or at two or more CDRs. Thus, the library may comprise substitutions at two or more positions in a CDR. The library may comprise substitutions at two or more positions, at two or more CDRs. The library may comprise substitutions at 3, 4, 5 or more positions; these positions are found at 2, 3, 4, 5 or 6 CDRs. The substitution can be made using low redundancy codons. See, for example, Table 2 of Balintetal., 1993, Gene 137(1):109-18, The CDR may be the heavy chain CDR3 variable region (VH) and / or the light chain CDR3 variable region (VL). The CDR may be one or more of CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and / or CDR3 VL. The CDR can be a Kabat CDR, a Chothia CDR, an extended CDR, an AbM CDR, a contact CDR or a conformational CDR. Candidates with better binding can be sequenced, thereby identifying a CDR substitution that results in better affinity (also called an improved substitution). Binding candidates can also be sequenced, thereby identifying a CDR substitution that retains binding. Multiple rounds of analysis can be performed. For example, candidates (comprising an amino acid substitution at one or more positions in one or more CDRs) with improved binding are also useful for the design of a second library containing at least the original and substituted amino acid at each CDR position. improved (ie, amino acid position in the CDR where a substitution mutant showed improved binding). The preparation and analysis or selection of this library is discussed in greater detail below. Library scanning mutagenesis also provides a means of characterizing a CDR, provided that the frequency of clones with better, equal or lesser affinity, or no affinity also provides information on the importance of each amino acid position for the stability of the complex. antibody-antigen For example, if a position in the CDR retains binding when changed to the 20 amino acids, that position is identified as a position that is unlikely to be required for antigen binding. In contrast, if a CDR position retains binding for only a small percentage of substitutions, that position is identified as a position that is important for CDR function. Therefore, library-scanning mutagenesis methods generate information about the positions in the CDRs that can be changed to several different amino acids (even all 20 amino acids) and about the positions in the CDRs that cannot be changed or can only be changed. change to a few amino acids. Candidates with improved affinity can be combined into a second library, which includes the improved amino acid, the original amino acid at that position, and may also include additional substitutions at that position, depending on the complexity of the library desired or permitted by the method of analysis. or planned selection. Furthermore, if desired, the adjacent amino acid position may be random for at least two or more amino acids. The randomness of adjacent amino acids may allow additional conformational flexibility in the mutant CDR which, in turn, may allow or facilitate the introduction of a large number of improved mutations. The library may also comprise substitutions at positions that did not show improved affinity in the first round of analysis. The second library is screened or screened for members with enhanced and / or altered binding affinity using any method known in the art, including screening by Kinexa™ biosensor analysis and screening by any method known in the state of the art. technique for selection, including phage expression, yeast expression and ribosome expression. For the purpose of expressing the anti-PD-1 antibodies of the present invention, the DNA fragments encoding the VH and VL regions can first be obtained using any of the methods described above. Various modifications, eg, mutations, deletions, and / or additions, can be introduced into the DNA sequences using standard methods known in the art. For example, mutagenesis can be carried out using standard methods, such as PCR-mediated mutagenesis, where mutated nucleotides are incorporated into PCR primers such that the PCR product contains the desired mutations, or site-directed mutagenesis. The invention encompasses modifications of the variable regions shown in Table 1 and the CDRs shown in Tables 2, 3 or 4. For example, the invention includes antibodies comprising functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants that have improved or decreased affinity and / or activity. For example, the amino acid sequence can be mutated, in order to obtain an antibody with the desired binding affinity for PD-1. Modification of polypeptides is common practice in the state of the art and need not be described in detail here. Examples of modified polypeptides include polypeptides with conservative amino acid residue substitutions, one or more amino acid deletions or additions that do not substantially or detrimentally change functional activity, or mature (enhance) the affinity of the polypeptides for its ligand, or use of chemical analogs. Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal inserts include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or polypeptide that increases the half-life of the antibody in the bloodstream. Substitutional variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but framework alterations are also contemplated. Conservative substitutions are shown in Table 5 under the title conservative substitutions. If the substitutions result in a change in biological activity, more substantial changes, referred to as exemplary substitutions in Table 5 or as described below in relation to amino acid classes, can be introduced and the products analyzed. Table 5: Amino Acid Substitutions Residue Conservative substitutions Substitutions of Val The N asn asn Residue gln asn glu I have (I) Leu Met; Ph; Leu (L)lie norleucine Norleucine; lie; To; Ph Leu Pro Val (V) Leu To Thr Ph be lie; Leu; Met; Ph; To; norleucine Substantial modifications of the biological properties of the antibody are achieved by selection of substitutions that differ considerably in their effect of maintaining (a) the polypeptide backbone structure in the area of ​​the substitution, for example, as a helical or helical conformation. lamellar □, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Natural residues are divided into groups based on common side chain properties: (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, lie; (2) Polar uncharged: Cys, Ser, Thr, Asn, Gln; (3) Acid (negative charge): Asp, Glu; (4) Basic (positive charge): Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His. Non-conservative substitutions are obtained by exchanging a member of one of these classes for another class. For example, one type of substitution that can be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as alanine or serine. For example, there may be a non-canonical cysteine ​​substitution. The substitution can be made in a CDR or framework region of a variable domain or in the constant region of an antibody. In some embodiments, the cysteine ​​is canonical. Any cysteine ​​residue not involved in maintaining the correct conformation of the antibody can also be substituted, thus usually with serine, in order to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Rather, cysteine ​​linkages can be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment, such as an Fv fragment. Antibodies may also be modified, eg, in the variable domains of the heavy and / or light chains, eg, to alter a binding property of the antibody. Changes in the variable region may alter binding specificity and / or affinity. In some embodiments, within a CDR domain, no more than 1 to 5 conservative amino acid substitutions are made. In other embodiments, within a CDR domain, no more than 1 to 3 conservative amino acid substitutions are made. For example, a mutation in one or more CDR regions can be obtained, in order to increase or decrease the KD of the PD-1 antibody, to increase or decrease the koff, or to alter the binding specificity of the antibody. Site-directed mutagenesis techniques are known in the state of the art. See, for example, Sambrook et al. and Ausubel et al., supra. A modification or mutation can also be made in a framework region or constant region, in order to increase the half-life of an anti-PD-1 antibody. See, for example, PCT Publication No. WO 00 / 09560. A mutation in a framework region or constant region may also be obtained in order to alter the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, or to alter such properties as complement fixation, binding FcR and antibody-dependent cellular cytotoxicity. In some embodiments, within a framework region or constant region, no more than 1 to 5 conservative amino acid substitutions are made. In other embodiments, within a framework region or constant region, no more than 1 to 3 conservative amino acid substitutions are made. In accordance with the invention, a single antibody may have mutations in one or more of the CDRs or framework regions of the variable domain or in the constant region. Modifications also include glycosylated and non-glycosylated polypeptides, polypeptides with other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conservative positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunoi. 65:111-128; Wright and Morrison, 1997, TibTECH 15:2632). The oligosaccharide side chains of immunoglobulins affect the protein function (Boyd et al·, 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and intramolecular interaction between portions of the glycoprotein, which may affect the conformation and three-dimensional surface of the glycoprotein (Jefferls and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides can also serve to target a particular glycoprotein to particular molecules based on specific recognition structures. Antibody glycosylation has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, antibodies produced by CHO cells with tetracycline-regulated expression of p(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes bisector GIcNAc formation, were reported to have enhanced ADCC activity ( Umana et al., 1999, Nature Biotech.17:176-180). Glycosylation of antibodies is generally N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-Xthreonine, and asparagine-X-cysteine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a possible glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Conveniently, the addition of glycosylation sites to the antibody is achieved by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The alteration can also be obtained by the addition of one or more serine or threonine residues to the original antibody sequence or by substitution therewith (for O-linked glycosylation sites). The glycosylation pattern of antibodies can also be altered without altering the underlying nucleotide sequence. Glycosylation is highly dependent on the host cell used to express the antibody. Since the cell type that is used for the expression of recombinant glycoproteins, e.g., antibodies, as a potential therapeutic, is rarely the native cell, variations in the glycosylation pattern of antibodies can be anticipated (see, for example, example, Hse et al. 1997, J. Biol. Chem. 272:9062-9070). In addition to the choice of host cells, factors that affect glycosylation during recombinant antibody production include growth mode, media formulation, culture density, oxygenation, pH, purification schemes, and the like. Various methods have been proposed to alter the glycosylation pattern obtained in a particular host organism, including introducing or overexpressing certain enzymes involved in oligosaccharide production (US Patent Nos. 5,047,335, 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be removed enzymatically from the glycoprotein, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, the recombinant host cell can be genetically engineered to be defective in processing certain types of polysaccharides. These and other similar techniques are known in the state of the art. Other methods of modification include the use of coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, for the attachment of labels for immunoassay. The modified polypeptides are obtained using procedures established in the state of the art and are analyzed by standard assays known in the state of the art, some of which are described below and in the Examples. In some embodiments, the Fe can be human IgG2 or human IgG4. In some embodiments, the antibody comprises an IgG4 constant region comprising the following mutations (Armour et al., 2003, Molecular Immunology 40 585-593): E233F234L235 to P233V234A235 (lgG4¿c), where the numbering is with reference to wild-type lgG4. In yet another embodiment, the Fe is human IgG4 E233F234L235 to P233V234A235 with the G236 deletion (IgG4ib). In some embodiments, the Fe is any human IgG4 Fe (IgG4, IgG4¿b, or IgG4ic) that contains the stabilizing hinge mutation S228 to P228 (Aalberse et al., 2002, Immunology 105, 9-19). In other embodiments, the Fe can be human IgG2, containing the mutation A330P331 to S330S331 (IgG^a), where the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur. J. Immunol., 1999, 29:2613-2624. In some embodiments, the antibody comprises a modified constant region that has an increased or decreased binding affinity to the human Fe gamma receptor, is immunologically or partially inert, that is, does not trigger complement-mediated lysis, does not stimulates antibody-dependent cellular cytotoxicity (ADCC) or activates microglia; or has decreased activity (compared to unmodified antibody) of one or more of the following: triggering of complement-mediated lysis, stimulation of ADCC, or activation of microglia. Different modifications of the constant region can be used to achieve an optimal level and / or combination of effector functions. See, eg, Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Publication No. WO99 / 058572. In some embodiments, an antibody constant region can be modified, so as to prevent interaction with the Fe gamma receptor, complement, and immune systems. Techniques for the preparation of such antibodies are described in WO 99 / 58572. For example, the constant region can be engineered to be more similar to human constant regions, in order to avoid immune response if the antibody is used in human clinical trials and treatments. See, for example, US Patent Nos. 5,997,867 and 5,866,692. In still other embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutation of the oligosaccharide attachment residue and / or side residues. that are part of the N-glycosylation recognition sequence in the constant region. For example, the N N297 glycosylation site can be mutated to, eg, A, Q, K, or H. See, Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically (for example, by removing carbohydrates by the enzyme PNGase) or by expression in a glycosylation-deficient host cell. Other antibody modifications include antibodies that were modified as described in PCT Publication No. WO 99 / 58572. These antibodies comprise, in addition to a binding domain directed at the target molecule, a effector domain having an amino acid sequence substantially homologous to all or part of a human immunoglobulin heavy chain constant region. These antibodies can bind to the target molecule without triggering, to a large extent, complement-dependent lysis or cell destruction of the target. In some embodiments, the effector domain is capable of specifically binding FcRn and / or FcyRIlb. These are generally based on chimeric domains derived from two or more CH2 domains of the human immunoglobulin heavy chain. Antibodies modified in this way are particularly suitable for use in chronic antibody treatment, in order to avoid inflammatory and other adverse reactions to conventional antibody treatment. In some embodiments, the antibody comprises a modified constant region that has a higher binding affinity for FcRn and / or a higher serum half-life compared to the unmodified antibody. In a process that crosses the germ line, certain amino acids in the VH and VL sequences can mutate to match those naturally found in the germline VH and VL sequences. In particular, the amino acid sequences of the framework regions in the VH and VL sequences can be mutated to match the germline sequences, in order to reduce the risk of immunogenicity when the antibody is administered. Germline DNA sequences for human VH and VL genes are known in the art (see, e.g., Vbase Human Germline Sequence Database; see also Kabat, E. A., et al ., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Tomlinson et al., 1992, J. Mol. Biol. 227:776798, and Cox et al., 1994, Eur. J. Immunol. 24:827-836). Another type of amino acid substitution that can be obtained involves removing possible proteolytic sites on the antibody. Such sites can be obtained in a CDR or framework region of a variable domain or in the constant region of an antibody. Substitution of cysteine ​​residues and removal of proteolytic sites can decrease the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution involves removing asparagine-glycine pairs, which form potential deamination sites by altering one or both residues. In another aspect, the C-terminal lysine of the heavy chain of an anti-PD-1 antibody can be cleaved from the invention. In various embodiments of the invention, the heavy chain and light chain of the anti-PD-1 antibodies may optionally include a signal sequence. Once the DNA fragments encoding the VH and VL segments of the present invention are obtained, these DNA fragments can also be manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into the full length antibody chain, on Fab fragment genes or on a scFv gene. In these manipulations, the VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" means that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in frame. Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably ligating the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., Kabat, E.A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments spanning these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGi, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is more preferably an IgGi or IgG2 constant region. The IgG constant region sequence can be any of several alleles or allotypes that occur in different subjects, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes represent natural amino acid substitutions in the IgG1 constant regions. For a Fab fragment heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only the constant region CH1 of the heavy chain. The CH1 heavy chain constant region can be derived from any of the heavy chain genes. Isolated DNA encoding the VL region can be converted into a full-length light chain gene (such as a Fab light chain gene) (operably linking the VL-encoding DNA to another DNA molecule encoding the constant region). light chain, CL Human light chain constant region gene sequences are known in the art (see, e.g., Kabat, E.A., et al., 1991, Sequences of Proteins of Immunological Interest , Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments spanning these regions can be obtained by standard PCR amplification.The light chain constant region can be a region kappa or lambda constant. The kappa constant region can be any of several alleles that occur in different subjects, such as lnv(1), lnv(2), and lnv(3). The lambda constant region can be derived from any of the three lambda genes . In order to create an scFv gene, the VH and VL encoding DNA fragments are operably linked to another fragment encoding a flexible linker such that the VH and VL sequences can be expressed as a contiguous single chain protein, with the VL and VH regions linked by the flexible linker (see, eg, Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Nati. Acad. Sci. USA 85:5879-5883 McCafferty et al., 1990, Nature 348:552-554 An example of a linker peptide is (GGGGS)3 (SEQ ID NO: 19), which establishes an approximately 3.5 nm bridge between the carboxy terminus of a variable region and the amino terminus of the other variable region. Linkers from other sequences were designed and used (Bird et al., 1988, supra). In turn, linkers can be modified to allow additional functions, such as attachment of drugs or fixation to solid supports The single-chain antibody can be monovalent if a single VH is used and VL, bivalent if two VH and VL are used, or polyvalent if more than two VH and VL are used. Bispecific or polyvalent antibodies can be generated that specifically bind to PD-1 and one other molecule. Single chain variants can be produced recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid-containing polynucleotide encoding the scFv can be introduced into a suitable host cell, whether eukaryotic, such as yeast, plant, insect or mammalian cells; or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be obtained by routine manipulations, such as polynucleotide ligation. The resulting scFv can be isolated using standard purification techniques known in the art. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to to pair with complementary domains of another chain and allows the creation of two antigen binding sites (see, eg, Holliger, P., et al., 1993, Proc. Nati. Acad Sci. USA 90:6444-6448; Poljak, R.J., et al., 1994, Structure 2:1121-1123). Heteroconjugate antibodies, comprising two covalently linked antibodies, are also within the scope of the invention. Such antibodies have been used to target cells of the immune system to unwanted cells (US Patent No. 4,676,980) and for the treatment of HIV infection (PCT Publication Nos. WO 91 / 00360 and WO 92 / 200373; EP 03089 ). Heteroconjugate antibodies can be obtained using any convenient cross-linking method. Suitable crosslinking agents and techniques are known in the art and are described in US Patent No. 4,676,980. Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate. The invention also encompasses fusion proteins comprising one or more fragments or regions of the antibodies described herein. In some embodiments, a fusion antibody comprising all or part of an anti-PD-1 antibody of the invention linked to another polypeptide can be obtained. In another embodiment, only the variable domains of the anti-PD-1 antibody bind to the polypeptide. In another embodiment, the VH domain of an anti-PD-1 antibody binds to a first polypeptide, while the VL domain of an anti-PD-1 antibody binds to a second polypeptide that associates with the first polypeptide, such that the VH and VL domains can interact with each other to form an antigen binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker, so that the VH and VL domains can interact with each other. The VH-linker-VL antibody then binds to the polypeptide of interest. Also I know they can create fusion antibodies where two (or more) single-chain antibodies are linked together. This is useful if it is desired to create a divalent or polyvalent antibody on a single polypeptide chain, or if it is desired to create a bispecific antibody. In some embodiments, a fusion polypeptide is provided that comprises at least 10 contiguous amino acids from the variable light chain region shown in SEQ ID NO: 2, 7, 8 or 9 and / or at least 10 heavy chain variable region amino acids shown in SEQ ID NO: 3, 4, 5 or 6. In other embodiments, a fusion polypeptide is provided comprising at least about 10, at least about 15 , at least about 20, at least about 25, or at least about 30 contiguous light chain variable region amino acids and / or at least about 10, at least about 15, at least about 20, at least about 25 or at least about 30 contiguous amino acids of the heavy chain variable region. In another embodiment, the fusion polypeptide comprises a light chain variable region and / or a heavy chain variable region, as shown in any of the selected sequence pairs of SEQ ID NOs: 2 and 3, 7 and 3, 8 and 3, 8 9 and 3, 2 and 4, 7 and 4, 8 and 4, 9 and 4, 2 and 5, 7 and 5, 8 and 5, 9 and 5, 2 and 6, 7 and 6, 8 and 6, and 9 and 6. In another embodiment, the fusion polypeptide comprises one or more CDRs. In yet other embodiments, the fusion polypeptide comprises CDR3 VH and / or CDR3 VL. For the purposes of the present invention, a fusion protein contains one or more antibodies and another amino acid sequence to which it does not bind in the natural molecule, for example, a heterologous sequence or a homologous sequence from another region. Examples of heterologous sequences include, but are not limited to, a tag, such as a FLAG or 6His tag. Labels are known in the state of the art. A fusion polypeptide can be created by methods known in the art, for example, by synthesis or recombinantly. The fusion proteins of this invention are often made by making and expressing a polynucleotide encoding them using the recombinant methods described herein, although they can also be made by other means known in the art, including, for example, chemical synthesis. In other embodiments, other modified antibodies can be prepared using anti-PD-1 antibody encoding nucleic acid molecules. For example, "Kappa bodies" (III et al., 1997, Protein Eng. 10:949-57), "Minibodies" (Martin et al., 1994, EMBO J. 13:5303-9), "Diabodies" (Holliger et al., supra) or "Janusins" (Trauneckeret al., 1991, EMBOJ. 10:3655-3659 and Traunecker et al., 1992, Int. J. Cancer (Suppl.) 7:51-52) can be prepared by standard molecular biological techniques in accordance with the teachings of the specification. For example, bispecific antibodies, monoclonal antibodies that have binding specificities for at least two different antigens, can be prepared using the antibodies described herein. Methods for obtaining bispecific antibodies are known in the art (see, eg, Suresh et al., 1986, Methods in Enzymology 121:210). For example, bispecific antibodies or antigen-binding fragments can be produced by fusion of hybridomas or binding of Fab fragments. See, for example, Songsvilai & Lachmann, 1990, Clin. Exp, Immunol. 79:315-321, Kostelny et al., 1992, J. Immunol. 148:1547-1553. Traditionally, the recombinant production of bispecific antibodies was based on the coexpression of two immunoglobulin heavy chain-light chain pairs; the two heavy chains have different specificities (Millstein and Cuello, 1983, Nature 305, 537-539). Furthermore, bispecific antibodies can be formed as “diabodies” or “Janusins. In some embodiments, the specific antibody binds to two different epitopes of PD-1. In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions of the anti-PD-1 antibody provided herein. According to one approach to obtain bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused with immunoglobulin constant region sequences. Preferably, the fusion is with an immunoglobulin heavy chain constant region, comprising at least part of the CH2 and CH3 hinge regions. It is preferred that the first heavy chain constant region (CH1), which contains the necessary site for light chain attachment, is present in at least one of the fusions. DNAs encoding fusions of the immunoglobulin heavy chain and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where the unequal proportions of the three polypeptide chains used in construction provide optimal performance. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in equal ratios results in high yield or when the ratios are not particularly high. transcendence. In one approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. . This asymmetric structure, with an immunoglobulin light chain in only one half of the bispecific molecule, facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This approach is described in PCT Publication No. WO 94 / 04690. This invention also provides compositions comprising antibodies conjugated (eg, linked) with an agent that facilitates coupling to a solid support (such as biotin or avidin). For the sake of simplicity, reference will generally be made to antibodies; however, it should be noted that these methods apply to any of the PD-1 antagonist and / or PD-1 binding embodiments described herein. In general, conjugation refers to the ligation of these components, as described herein. Ligation (which generally involves preparing these components in close association for administration) can be accomplished in a variety of ways. For example, a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydric group, in one may react with a carbonium-containing group, such as an anhydride or acid halide, or with an alkyl group containing a suitable leaving group (for example, one halide) in the other. Antibodies can be attached to various carriers. Carriers can be active and / or inert. Examples of known carriers include the following: polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. For the purposes of the invention, the nature of the carrier may be soluble or insoluble. Those in the mid-level trade will know of other suitable carriers for binding antibodies, or will be able to determine which ones are, using routine experiments. In some embodiments, the carrier comprises a portion that addresses the lung, heart, or heart valve. An antibody or polypeptide of the present invention may be linked to a labeling agent, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art. The labels that are known in the state of the art generally provide (directly or indirectly) a signal. Polynucleotides, Vectors, and Host Cells The invention also provides polynucleotides encoding any of the antibodies, including antibody fragments and modified antibodies, described herein, such as, for example, antibodies having impaired effector function. In another aspect, the invention provides a method of obtaining any of the polynucleotides described herein. The polynucleotides can be obtained and expressed by procedures known in the state of the art. Accordingly, the invention provides polynucleotides or compositions, including pharmaceutical compositions, encoding any of the following: antibodies mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, mAb14, mAb15, mAb16 and mAb17 or any fragment or part of these that have the ability to antagonize PD-1. The present invention also encompasses polynucleotides complementary to any such sequence. Polynucleotides can be single-stranded (sense or antisense) or double-stranded, and can be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to an individual DNA molecule, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but need not, be present in a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and / or carrier materials. The polynucleotides may comprise a native sequence (ie, an endogenous sequence encoding an antibody or fragment thereof) or may comprise a variant of such sequence. Variant polynucleotides contain one or more substitutions, additions, deletions, and / or insertions, in a manner that does not decrease the immunoreactivity of the encoded polypeptide, relative to a naturally occurring immunoreactive molecule. In general, the effect on the immunoreactivity of the encoded polypeptide can be assessed as described herein. Preferably, the variants exhibit at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 90% identity. 95% identity to a polynucleotide sequence encoding a native antibody or fragment thereof. Two polynucleotide or polypeptide sequences are considered to be identical if the nucleotide or amino acid sequence in the two sequences is the same when aligned for closest correspondence, as described below. Typically, comparisons between two sequences are made by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a comparison window refers to a segment having at least about 20 contiguous positions, typically 30 to about 75 or 40 to about 50, in which to compare. a sequence with a reference sequence having the same number of contiguous positions after optimally aligning the two sequences. Optimal alignment of sequences for comparison can be performed using the MegAlign® program in the Lasergene® suite of bioinformatics programs (DNASTAR®, Inc., Madison, Wl), using default parameters. This program encompasses several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC vol. 5, Suppl. 3, p. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol.Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ. and Lipman, D.J., 1983, Proc, Nati. Acad. Sci. USA 80:726-730. Preferably, percent sequence identity is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) of 20% or less, typically 5-15% or 10-12%, compared to the reference sequences (comprising neither additions nor deletions) for optimal alignment of the two sequences. To calculate the percentage, the number of positions in which identical amino acid residues or nucleic acid bases are obtained in both sequences must be determined to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the reference sequence (ie, window size) and multiply the results by 100 to obtain percent sequence identity. Additionally or alternatively, the variants may be substantially homologous to a native gene or a portion or complement thereof. Polynucleotide variants can hybridize under moderately stringent conditions to a natural DNA sequence encoding a native antibody (or a complementary sequence). The term "suitable moderately stringent conditions" includes prior washing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C-65°C, 5X SSC, overnight; then washed twice at 65°C for 20 min, each 2x, 0.5x, and 0.2x SSC containing 0.1% SDS. As used herein, the terms "high stringency" or "high stringency" are those that: (1) use low ionic strength and high temperature for washing, eg, 0.015 M sodium chloride / 0.0015 sodium citrate M / 0.1% sodium dodecyl sulfate at 50'C; (2) use during hybridization a denaturing agent, such as formamide, eg 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42'C; or (3) use 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 pg / mL), 0.1% SDS and 10% dextran sulfate at 42'C, with washes at 42'C in 0.2x SSC (sodium chloride / sodium citrate) and 50% formamide at 55'C, followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55'C. Those in the mid-level trade will know how to adjust temperature, ion strength, etc., as necessary to accommodate factors such as probe length, and the like. It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. However, the present invention specifically contemplates polynucleotides that vary due to differences in codon usage. Furthermore, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified by standard techniques (such as hybridization, amplification and / or database sequence comparison). The polynucleotides of the present invention can be obtained by chemical synthesis, recombinant methods or PCR. Methods of chemical polynucleotide synthesis are known in the art, and need not be described in detail herein. A person of ordinary skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence. To prepare polynucleotides by recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector, in turn, can be introduced into a suitable host cell for replication and amplification, as discussed herein. Polynucleotides can be inserted into host cells by methods known in the art. Cells are transformed by introduction of an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-pairing, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the genome of the host cell. The polynucleotide amplified in this way can be isolated from the host cell by methods known in the art. See, for example, Sambrook et al., 1989. Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in US Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, as well as in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994. RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated by methods known to those of ordinary skill in the art, as outlined in Sambrook et al., 1989, supra, for example. Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the state of the art. While the cloning vector selected may vary depending on the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and / or may have genes for a marker that can be used to select clones containing the vector. Suitable examples include bacterial plasmids and viruses, eg pUC18, pUC19, Bluescript (eg pBS SK+) and their derivatives mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and carrier vectors, such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors, such as BioRad, Strategene, and Invitrogen. Expression vectors are also provided. Expression vectors are generally replicable polynucleotide constructs containing a polynucleotide according to the invention. It goes without saying that an expression vector must be able to replicate in host cells as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vectors described in PCT Publication No. WO 87 / 04462. Components of vectors generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers and terminators). For expression (ie, translation), one or more translational control elements, such as ribosome binding sites, translation initiation sites, and stop codons, are also generally required. Vectors containing the polynucleotides of interest can be introduced into the host cell by any number of suitable means, including electroporation, transfection by calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; bombardment with microprojectiles; lipofection and infection (eg, when the vector is an infectious agent, such as a vaccinia virus). Often the choice regarding the introduction of vectors or polynucleotides will depend on the characteristics of the host cell. The invention also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding the antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisiae, S. pombe; or K. lactis). Preferably, the host cells express the cDNAs at a level about 5-fold, more preferably 10-fold, even more preferably 20-fold higher than that of the corresponding endogenous protein or antibody of interest, if present, in the host cells. Analysis of host cells for specific binding to PD-1 or a PD-1 domain is performed by immunoassay or FACS. A cell that overexpresses the antibody or protein of interest can be identified. An expression vector can be used to direct the expression of an antagonist anti-PD-1 antibody. A person of average level is familiar with the administration of expression vectors to obtain the expression of an exogenous protein in vivo. See, for example, US Patent Nos. 6,436,908; 6,413,942 and 6,376,471. Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or gavage administration, and topical administration. In another embodiment, the expression vector is administered directly into the sympathetic trunk or ganglion, or into a coronary artery, atrium, ventricle, or pericardium. Targeted delivery of therapeutic compositions containing an expression vector or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J.A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al., J, Biol. Chem., 1994, 269:542; Zenke et al., Proc. Nati. Acad. Sci. USA, 1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338, Therapeutic compositions containing a polynucleotide are delivered in a range from about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges from about 500 ng to about 50 mg, about 1 pg to about 2 mg, about 5 pg to about 500 pg, and about 20 pg to about 100 pg of DNA can also be used during a gene treatment protocol. Therapeutic polypeptides and polynucleotides can be delivered using gene delivery vehicles. The gene delivery vehicle may be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy, 1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy, 1995, 1:185 and Kaplitt, Nature Genetics, 1994, 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. The expression of the coding sequence can be constitutive or regulated. Virus-based vectors for delivery of a desired polynucleotide and expression in a desired cell are known in the art. Exemplary virus-based carriers include, but are not limited to, recombinant retroviruses (see, for example, PCT Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; US Patent Nos. 5,219,740 and 4,777,127; UK Patent No. 2,200,651; and EP Patent No. 0 345 242), vectors a alphavirus-based (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)) and adeno-associated virus (AAV) vectors (see, for example, PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Administration of DNA linked to inactivated adenovirus, as described in Curiel, Hum. Gene Ther., 1992, 3:147. Non-viral delivery vehicles and methods may also be employed, including, but not limited to, polycationic condensed DNA bound or unbound to inactivated adenovirus alone (see, eg, Curiel, Hum. Gene Ther., 1992, 3:147); ligand-bound DNA (see, eg, Wu, J. Biol. Chem., 1989, 264:16985); eukaryotic cell manager carrier cells (see, eg, US Patent No. 5,814,482; PCT Publication Nos. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763 and WO 97 / 42338) and Nucleic fusion or charge neutralization with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90 / 11092 and US Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in US Patent No. 5,422,120; in PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445 and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol., 1994, 14:2411, and in Woffendin, Proc. Nati. Acad. Sci., 1994, 91:1581. compositions The invention also provides pharmaceutical compositions comprising an effective amount of an anti-PD-1 antibody described herein. Examples of such compositions, as well as how to formulate them, are described herein. In some embodiments, the composition comprises one or more antibodies against PD-1. In other embodiments, the anti-PD-1 antibody recognizes PD-1. In other embodiments, the anti-PD-1 antibody is a human antibody. In other embodiments, the anti-PD-1 antibody is a humanized antibody. In some embodiments, the anti-PD-1 antibody comprises a constant region that is capable of eliciting a desired immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the anti-PD-1 antibody comprises a constant region that does not trigger an unwanted or inappropriate immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the anti-PD-1 antibody comprises one or more antibody CDRs (such as one, two, three, four, five, or, in some embodiments, all six CDRs). It should be noted that the compositions may comprise more than one anti-PD-1 antibody (eg, a mixture of anti-PD-1 antibodies that recognize different epitopes of PD-1). Other exemplary compositions comprise more than one anti-PD-1 antibody that recognizes the same epitopes, or different species of anti-PD-1 antibody that bind to different epitopes of PD-1. In some embodiments, the compositions comprise a mixture of anti-PD-1 antibodies that recognize various PD-1 variants. The compositions used in the present invention may also comprise pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and practice of Pharmacy 20th ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form of lyophilized formulations or aqueous solutions. Suitable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and may comprise buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrans; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg Zn protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are also described herein. The anti-PD-1 antibody and compositions thereof may also be used in conjunction with other agents that enhance and / or complement the efficacy of the agents, or may be used separately, simultaneously, or sequentially. The invention also provides compositions, including pharmaceutical compositions, comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding the antibody as described herein. In another embodiment, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies described herein. Methods for Preventing or Treating Conditions Mediated by PD-1 The antibodies and antibody conjugates of the present invention are useful in a variety of applications including, but not limited to, therapeutic methods of treatment and diagnostic methods of treatment. In one aspect, the invention provides a method of treating cancer. In some embodiments, the method of treating cancer in a subject comprises administering to the subject in need thereof an effective amount of a composition (eg, pharmaceutical composition) comprising any of the PD-1 antibodies described herein. As used herein, cancer types include, but are not limited to, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, gastric cancer, glioblastoma, glioma, brain tumor, cancer head and neck cancer, kidney cancer, lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, liver cancer, uterine cancer, bone cancer, leukemia, lymphoma, sarcoma, blood cancer, thyroid cancer, thymus cancer, eye cancer and skin cancer. In some embodiments, a method of inhibiting tumor growth or progression in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising the PD-1 antibodies or antibody conjugates. against PD-1 described herein. In some embodiments, the tumor is a PD-L1 expressing tumor. In other embodiments, the tumor does not express PD-L1. In other embodiments, a method of inhibiting cancer cell metastasis in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising any of the PD-1 antibodies described herein. In other embodiments, a method of inducing tumor regression in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising any of the PD-1 antibodies described herein. In another aspect, a method of detecting, diagnosing and / or monitoring cancer is provided. For example, the PD-1 antibodies described herein can be labeled with a detectable moiety, such as an imaging agent and an enzyme substrate label. The antibodies described herein can also be used for in vivo diagnostic assays, such as in vivo imaging (eg, PET or SPECT), or a staining reagent. In some embodiments, the methods described herein further comprise a step of treating a subject with an additional form of treatment. In some embodiments, the additional form of treatment is additional cancer treatment including, but not limited to, chemotherapy, radiation, surgery, hormonal treatment, and / or additional immunotherapy. With respect to all of the methods described herein, reference to antagonistic anti-PD-1 antibodies also includes compositions comprising one or more additional agents. These compositions may also comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, known in the art. The present invention can be used alone or in combination with other methods of treatment. The anti-PD-1 antagonist antibody can be administered to a subject by any suitable route. Those of ordinary skill in the art will understand that the examples described herein are not intended to be limiting, but rather illustrative of the available techniques. Accordingly, in some embodiments, the anti-PD-1 antagonist antibody is administered to a subject according to known methods, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, via the intravenous route. intramuscular, intraperitoneal, intracerebrospinal, transdermal, subcutaneous, intraarticular, sublingual, intrasynovial, insufflation, intrathecal, oral, topical, or inhalation. Administration may be systemic, eg, intravenous, or localized. Commercially available nebulizers for liquid formulations, including jet-type nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be used directly for nebulization, and the lyophilized powder can be used for nebulization after reconstitution. Alternatively, the anti-PD-1 antagonist antibody can be employed as an aerosol using a fluorocarbon formulation and metered dose inhaler, or it can be inhaled as a ground, lyophilized powder. In some embodiments, an anti-PD-1 antagonist antibody is administered by site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various sources of implantable anti-PD-1 antagonist antibody depots or local delivery catheters, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, tunica adventitia , shunts and stents or other implantable devices, site-specific carriers, direct injection or direct application. See, for example, PCT Publication No. WO 00 / 53211 and US Patent No. 5,981,568. For administration, different formulations of an anti-PD-1 antagonist antibody can be used. In some embodiments, the anti-PD-1 antagonist antibody can be administered neat. In some embodiments, the anti-PD-1 antagonist antibody and a pharmaceutically acceptable carrier may be present in different formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of a pharmacologically effective substance. For example, an excipient can give shape or consistency, or can act as a diluent. Suitable excipients include, but are not limited to, stabilizing, moisturizing and emulsifying agents, salts of various osmolarities, encapsulating agents, buffers and skin penetration enhancers. Carriers and formulations for parenteral and non-parenteral drug delivery are listed in Remington, The Science and Practice of Pharmacy, 20th ed. Wlack Publishing, 2000. In some embodiments, these agents are formulated for administration by injection (eg, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents can be combined with pharmaceutically acceptable carriers, such as saline, Ringer's solution, dextrose solution, and the like. The particular dosage regimen, ie dose, time and repetition, will depend on the particular subject and his or her medical history. An anti-PD-1 antagonist antibody can be administered by any suitable method, including injection (eg, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Anti-PD-1 antibodies can also be administered topically or by Inhalation, as described herein. In general, for the administration of anti-PD-1 antibodies, a possible starting dose may be around 2 mg / kg. For purposes of the present invention, a typical daily dose may range from about 3 pg / kg to 30 pg / kg, to 300 pg / kg, to 3 mg / kg, to 30 mg / kg, to 100 mg / kg or more, depending on the factors mentioned above. For example, a dose of about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 10 mg / kg and about 25 mg / kg can be used. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired suppression of symptoms occurs or until therapeutic levels sufficient, eg, to reduce cancer-associated symptoms, are achieved. The evolution of this treatment is easily controlled by means of conventional techniques and assays. The dosage regimen (including the anti-PD-1 antagonist antibody used) may vary over time. For purposes of the present invention, the appropriate dose of an anti-PD-1 antagonist antibody will depend on the anti-PD-1 antagonist antibody (or compositions thereof) used, the type and severity of the symptoms to be treated. , whether the agent is administered for prophylactic or therapeutic purposes, prior treatment, the patient's medical history and response to the agent, the rate of clearance of the patient from the administered agent, and the judgment of the treating physician. In general, the clinician will administer an anti-PD-1 antagonist antibody until a dose is reached to achieve the desired result. Dosage and / or frequency may vary during treatment. Empirical considerations, such as half-life, generally help determine dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the half-life of the antibody and to prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted during treatment and is generally, but not necessarily, based on treatment and / or suppression and / or improvement and / or delay of symptoms. Alternatively, sustained continuous release formulations of anti-PD-1 antagonist antibodies may be suitable. Various formulations and devices to achieve sustained release are known in the state of the art. In one embodiment, doses for an antagonist antibody can be determined empirically in subjects who received one or more administrations of an antagonist antibody. Subjects receive increasing doses of an anti-PD-1 antagonist antibody. To assess the effectiveness, an indicator of the disease can be followed. Administration of an anti-PD-1 antagonist antibody according to the method of the present invention may be continuous or intermittent, for example, depending on the physiological condition of the recipient, whether the administration is therapeutic or prophylactic, and other factors known to clinicians. experts. Administration of an anti-PD-1 antagonist antibody may be substantially continuous for a preselected period or may be in a series of spaced doses. In some embodiments, there may be more than one antagonist anti-PD-1 antibody. There may be at least 1, at least 2, at least 3, at least 4, at least 5 or more different antagonist antibodies. In general, antagonistic anti-PD-1 antibodies may have complementary activities that do not harm each other. An anti-PD-1 antagonist antibody can also be used in conjunction with other antibodies and / or other treatments. An anti-PD-1 antagonist antibody can also be used in conjunction with other agents that enhance and / or complement the efficacy of the agents. In some embodiments, the anti-PD-1 antagonist antibody can be administered in combination with one or more additional therapeutic agents. These include, but are not limited to, administration of a chemotherapeutic agent, a vaccine, a CAR-T cell treatment, radiation therapy, a cytokine treatment, a vaccine, a bispecific anti-PD-1 antibody, an inhibitor of other immunosuppressive pathways , angiogenesis inhibitors, a T cell activator, a metabolic pathway inhibitor, an mTOR inhibitor, an adenosine pathway inhibitor, a tyrosine kinase inhibitor including but not limited to inlyta, ALK inhibitors, and sunitinib , a BRAF inhibitor, an epigenetic modifier, a Treg cell and / or myeloid-derived suppressor cell inhibitor or depressant, a JAK inhibitor, a STAT inhibitor, a cyclin-dependent kinase inhibitor, a biotherapeutic agent ( including, but not limited to, antibodies against VEGF, VEGFR, EGFR, Her2 / neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1 BB, and ICOS), a immunogenic agent (for example, cancer cells attenuated antigens, tumor antigens, cell-presenting antigens such as nucleic acid-driven dendritic cells or tumor-derived antigens, immunostimulatory cytokines (eg, IL-2, IFNa2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines , such as GMCSF). Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizeiesin); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogs, KW2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, novembiquine, fenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranímustine; antibiotics, such as the enodiyne antibiotics (eg, calicheamicin, especially calicheamicin gammall and calicheamicin Phill, see, eg, Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemycin, including dynemycin A; bisphosphonates, such as clodronate; anesperamycin; in addition to the chromophore neocarzinostatin and related enodiyne chromoprotein antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carabycin, camlnomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo-L- norleucin, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin , potphyromycin, puromycin, chelamicin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); photic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxurldine; androgens, such as calusterone, dromostanolone proplonate, epithiostanol, mepitiostane, testolactone; antiadrenals, such as aminoglutethimide, mitotane, trilostane; folic acid regenerator, such as frolinic acid; Aceglatone; aldophosphamide glucoside; aminolevulinic acid; eniluracil; ansacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptlnium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitoclines; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (especially T2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine; dacarbazine; manomustine; mitobronitoi; mitoiactol; pipobroman; gacytosin; arabinoside ("Ara-C); cyclophosphamide; thiotepa; taxoids, eg, paclitaxei and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;. mitoxantrone; vincristine; vinorelbine; Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; Ibandronate; CPT-11; RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifen, 4-hydroxytamoxifen , trlooxfene, keoxfene, LY117018, onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. In some embodiments, an antagonist anti-PD-1 antibody is used in conjunction with one or more other therapeutic agents that target an immune system checkpoint modulator, such as an agent that targets PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, B7-DC (PD-L2), B7-H5, B7-H6, B7-H8, B7-H2, B7-1, B7 -2, ICOS, ICOS-L, TIGIT, CD2, CD47, CD80, CD86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA (PD-H1 ), 0X40, OX40L, GITR, GITRL , CD70, CD27 , 4-1 BB, 4-BBL, DR3, TL1A, CD40, CD40L, CD30, CD30L, LIGHT, HVEM, SLAM (SLAMF1, CD150), SLAMF2 (CD48) , SLAMF3 (CD229), SLAMF4 (2B4, CD244), SLAMF5 (CD84), SLAMF6 (NTB-A), SLAMCF7 (CS1), SLAMF8 (BLAME), SLAMF9 (CD2F), CD28, CEACAM1(CD66a ), CEACAM3, CEACAM4 , CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM13AS CEACAM3C2, CEACAM1-15, PSG1-11, CEACAM1-4C1, CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, CD39-CD73-adenosine pathway (A2AR), BTK , TIK, CXCR2, CCR4, CCR8, CCR5, VEGF pathway, CSF-1, or a mod ulator of the innate immune response. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with, for example, an anti-PD-L1 antagonist antibody, such as BMS936559 (MDX-1105) and MPDL3280A; an anti-PD-1 antagonist antibody, such as nivolumab, pembrolizumab, and pidilizumab; an anti-CTLA-4 antagonist antibody, such as ipilimumab; an anti-LAG-3 antagonist antibody, such as BMS-986016 and IMP701; an anti-TIM-3 antagonist antibody; an anti-B7-H3 antagonist antibody, such as MGA271; an anti-VISTA antagonist antibody; an anti-TIGlT antagonist antibody; an antagonist anti-CD28 antibody; an anti-CD80 antibody; an anti-CD86 antibody; an anti-B7-H4 antagonist antibody; an agonist anti-ICOS antibody; an agonist anti-CD28 antibody; a modulator of the innate immune response (eg TLR, KIR, NKG2A) and an IDO inhibitor. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with a 4-1BB (CD137) agonist, such as PF-05082566 or BMS-663513. In some embodiments, an antagonistic anti-PD-1 antibody is used in conjunction with an 0X40 agonist, such as an agonistic anti-OX-40 antibody. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with a GITR agonist, such as an anti-GITR agonist antibody, eg, TRX518. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with an IDO inhibitor. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with cytokine treatment, eg, IL-15, CSF-1, MCSF-1, etc. In some embodiments, an antagonist anti-PD-1 antibody is used in conjunction with one or more therapeutic antibodies, such as an antibody that targets CD19, CD22, CD40, CD52, or CCR4. In some embodiments, anti-PD-1 antibody treatment can be performed before or after treatment with another agent at intervals ranging from minutes to weeks. In embodiments where the other agents and / or proteins or polynucleotides are administered separately, it should be ensured that no considerable period elapses between each administration, so that the agent and the composition of the present invention can still exert a combined effect. advantageous in the subject. In those cases, it is contemplated that both modalities can be administered within about 12-24 hours of each other, and more preferably within about 6-12 hours of each other. However, in some situations, it may be desirable to extend the administration period considerably, in which case several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) between the respective administrations. In some embodiments, an anti-PD-1 antagonist antibody composition comprises a second agent selected from crizotinib, palbociclib, gemcitabine, cyiophosphamide, fluorouracil, FOLFOX, folinic acid, oxaliplatin, axitinib, sunitinib malate, totacitinib, bevacizumab, rituximab, and traztuzumab. In some embodiments, an anti-PD-1 antibody composition is combined with a treatment regimen that also comprises traditional treatment selected from the group consisting of: surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal treatment, inhibition of angiogenesis and palliative care. Use of PD-1 Antibodies in Cancer Vaccine Immunotherapy Regimens In some particular embodiments, the present description provides a method for improving the immunogenicity or therapeutic effect of a vaccine for the treatment of cancer in a mammal, in particular, a human being, wherein the method comprises administering to the receiving mammal the vaccine an effective amount of anti-PD-1 antagonist antibody provided by the present description. In some particular embodiments, the present description provides a method for treating cancer in a mammal, in particular, a human being, wherein the method comprises administering to the mammal (1) an effective amount of a vaccine capable of eliciting an immune response against cancer cells and (2) an effective amount of an anti-PD-1 antagonist antibody provided by the present description. The method of treating a neoplastic disorder in a mammal and the method of enhancing the immunogenicity or therapeutic effect of a vaccine for the treatment of a neoplastic disorder in a mammal described herein are collectively referred to as vaccine immunotherapy regimens. against cancer” (or VBIR against cancer). In VBIR against cancer, the vaccine can be presented in any form or formulation, for example, (i) cell vaccines, (ii) subunit vaccines, (iii) protein vaccines, (iv) peptide vaccines or (v) vaccines. nucleic acids (such as DNA vaccines, RNA vaccines, plasmid vaccines or viral vector vaccines). The anticancer VBIRs provided by the present disclosure may be applicable to any type of cancer. Examples of specific cancer types include: small cell lung cancer, non-small cell lung cancer, glioma, gastric cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, bladder cancer, breast cancer, and head and neck cancer. Vaccines to treat cancer usually contain an antigen (in the form of a peptide, protein, cellular component, whole cell, or nucleic acid molecule encoding a peptide antigen) that is capable of eliciting an immune response against a particular TAA expressed on target tumor cells or via these. In the state of the art, many TAAs are known. Some examples of known TAAs include: PSA, PSCA and PSMA against prostate cancer; CEA, MUC-1, Ep-CAM, 5T4, hCG-b, K-ras and TERT against colorectal cancer; CEA, Muc-1, p53, mesothelin, survivin and NY-ESO-1 against ovarian cancer; Muc-1, 5T4, WT-1, TERT, CEA, EGF-R, and MAGE-A3 against non-small cell lung cancer; 5T4 against renal cell carcinoma; and Muc-1, mesothelin, K-Ras, annexin A2, TERT, and CEA against pancreatic cancer. In some particular embodiments, the vaccine used in VBIRs against cancer provided by the present disclosure is selected from the group consisting of: (1) a vaccine capable of eliciting an immune response against a TAA selected from PSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF-R, or MAGE-A3; (2) a vaccine containing a peptide antigen derived from a TAA selected from PSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF-R or MAGE-A3; and (3) a vaccine containing a nucleic acid molecule encoding a peptide antigen, wherein the peptide antigen is derived from a TAA selected from PSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF-R or MAGE-A3. In yet other particular embodiments, the vaccine contains a nucleic acid molecule that encodes one or more PSA-derived immunogenic polypeptides, one or more PSCA-derived immunogenic polypeptides, or one or more PSMA-derived immunogenic polypeptides. In a specific embodiment, the nucleic acid molecule is selected from the group consisting of: (1) a nucleic acid molecule encoding an immunogenic polypeptide derived from human PSMA of SEQ ID NO:42; (2) a nucleic acid molecule encoding an immunogenic polypeptide comprising amino acids 15-750 of SEQ ID NO:42; (3) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 43, or a degenerate variant thereof; (4) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44, or a degenerate variant thereof; (5) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 45, or a degenerate variant thereof; (6) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46, or a degenerate variant thereof; (7) a nucleic acid molecule encoding an immunogenic polypeptide derived from human PSA of SEQ ,D NO:47; (8) a nucleic acid molecule encoding an immunogenic polypeptide comprising amino acids 25-261 of SEQ ID NO:47; (9) a nucleic acid molecule encoding an immunogenic polypeptide derived from human PSCA of SEQ ID NO:48; (10) a nucleic acid molecule encoding (i) an immunogenic polypeptide derived from human PSMA of SEQ ID NO:42, (ii)) an immunogenic polypeptide derived from human PSA of SEQ ID NO:47 and (iii) a polypeptide immunogen derived from human PSCA of SEQ ID NO:48; and (11) a nucleic acid molecule encoding (i) an immunogenic polypeptide comprising amino acids 15-750 of SEQ ID NO:42, (ii) an immunogenic polypeptide comprising amino acids 25-261 of SEQ ID NO:47 and (iii) an immunogenic polypeptide of SEQ ID NO:48. Nucleic acid molecules encoding one or more immunogenic polypeptides derived from prostate-associated antigens may be in the form of plasmids or vectors. An example of such a plasmid is the nucleic acid construct of SEQ ID NO.46 (also referred to as plasmid 458). The nucleotide sequence of a vector expressing an immunogenic human PSMA-derived polypeptide is set forth in SEQ ID NO:44 (also referred to as AdC68W vector), The nucleotide sequence of a vector expressing an immunogenic human PSMA-derived polypeptide, a polypeptide human PSA-derived immunogen and a human PSCA-derived immunogenic polypeptide is set forth in SEQ ID NO:45 and a human PSMA vector (AdC68W-734 vector). Various immunogenic polypeptides derived from human PSMA, PSA, and PSCA, nucleic acid constructs (including plasmids and vectors) encoding such immunogenic polypeptides, and methods for preparing the immunogenic polypeptides and nucleic acid constructs, including the 458 plasmid, and the AdC68W and AdC68W-734 vectors are described in international application publications WO2013 / 164754 and WO 2015 / 063647, each of which is incorporated herein in its entirety by reference. In one aspect, the invention provides an isolated antagonist antibody that specifically binds to PD-1, wherein the antibody comprises a heavy chain variable region (VH) comprising a complementarity determining region (CDR1) VH, CDR2 VH and VH CDR3 of VH having an amino acid sequence selected from the group consisting of SEQ ID NO; 3, SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6; and a light chain (VL) variable region comprising VL CDR1, VL CDR2 and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO:7; SEQ ID NO: 8; and SEQ ID NO: 9. Any anti-PD-1 antagonist antibody described herein can be used in VBIRs against cancer. In some embodiments, the anti-PD-1 antagonist antibody comprises a VH region and / or a VL region, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 3, 4, 5 or 6 , or a variant with one or more conservative amino acid substitutions at residues not found in a CDR, and wherein the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 7, 8 or 9, or a variant thereof with one or more amino acid substitutions in amino acids not found in a CDR. In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO: 39 and / or a heavy chain comprising the sequence shown in SEQ ID NO: 29 or 38. In some forms of particular embodiments, the antibody comprises a VH region produced by the expression vector ATCC Accession No. PTA-121183. In some embodiments, the antibody comprises a VL region produced by the expression vector ATCC Accession No. PTA-121182. The anticancer VBIR provided by the present disclosure may also comprise one or more immune modulators (in addition to the PD-1 antagonist antibody provided by the present disclosure). The other immune modulators may be an enhancer of immune effector cells ("IEC enhancer") or an inhibitor of immune suppressor cells (ISC Inhibitor). The additional IEC enhancer or the additional ISC inhibitor can be used alone or in combination with the anticancer VBIR. Additional IEC Enhancer and Additional ISC Inhibitor can also be used in combination with the anti-cancer VBIR. Examples of classes of ISC inhibitors include protein kinase inhibitors, cyclooxygenase-2 (COX-2) inhibitors, phosphodiesterase type 5 (PDE5) inhibitors, and DNA crosslinkers. Examples of COX-2 inhibitors include celecoxib and rofecoxib. Examples of PDE5 inhibitors include avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, and zaprinast. An example of DNA crosslinkers is cyclophosphamide. The term "protein kinase inhibitor" refers to any substance that acts as a selective or non-selective inhibitor of a protein kinase. Examples of protein kinase inhibitors suitable for use in VBIR against cancer include Lapatinib, AZD 2171, ET18OCH 3, Indirubin-3'-oxime, NSC-154020, PD 169316, quercetin, roscovitine, triciribin, ZD 1839, 5 -iodotubercidin, adafostin, aloisin, alsterpaullone, aminogenlstein, API-2, aplgenin, arctigenin, ARRY-334543, axitinib (AG-013736), AY-22989, AZD 2171, bisindolylmaleimide IX, CCl-779, chelerythrine, DMPQ, DRB, edelfosine, ENMD-981693, erbstatin analog, erlotinib, fasudil, gefitinib (ZD1839), H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudll, kenpaullone , KN-62, KY12420, LFM-A13, luteolin, LY294002, LY-294002, malotoxin, ML-9, MLN608, NSC-226080, NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucin, oxindole I, PD 153035, PD 98059, Ploridzin, Piceathanol, Plcropodophyllin, PKI, PP1, PP2, PTK787 / ZK222584, PTK787 / ZK-222584, Purvalanol A, Rapamune, Rapamycin, Ro 31-8220, Rottlerin, SB202, SB202190, SL3,2 Sirolimus SP600125, and staurosporine, STI-571, SU 1498, SU4312, SU5416, SU5416 (Semaxanib), SU6656, SU6668, Syk inhibitor, TBB, TCN, tyrphostin AG 1024, tyrphostin AG 490, tyrphostin AG 825, tyrphostin AG 957, U0126, W- 7, Wortmannin, Y27632, Zactima (ZD6474), ZM 252868. gefitinib (Iressa.RTM.), sunitinib malate (SUTENT; SU11248), erlotinib (TARCEVA; OSI-1774), lapatinib (GW572016; GW2016), canertinib (Cl 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec.RTM .; STI571), dasatinib (BMS-354825), leflunomide (SU 101), vandetanib (ZACTIMA; ZD6474), and nilotinib. In some particular embodiments, the tyrosine kinase inhibitor is sunitinib malate, sorafenib tosylate, or axitinib. Sunitinib malate, which is marketed by Pfizer Inc. under the brand name SUTENT, is described chemically as butandioic acid, hydroxy-, (2S)-, compound with W-[2-(diethylamino)ethylj5-[(Z)-( 5-Fluoro-1,2-dihydro-2-oxo-3H-indole-3-ylidin)methyl]-2,4-dimethyl-'H-pyrrole-3carboxamide (1:1). The compound, its synthesis, and particular polymorphs are described in US Patent No. 6,573,293. Sunitinib malate was approved in the US for the treatment of gastrointestinal stromal tumor, advanced renal cell carcinoma, and progressive differentiated pancreatic neuroendocrine tumors in patients with unresectable locally advanced or metastatic disease. The recommended dose of sunitinib malate for gastrointestinal stromal tumor (GIST) and advanced renal cell carcinoma (RCC) in humans is 50 mg, administered orally once daily, on a 4-week treatment schedule. followed by 2 weeks off (schedule 4 / 2). The recommended dose of sunitinib malate for pancreatic neuroendocrine tumors is 37.5 mg administered orally once daily. In VBIR for cancer, sunitinib malate can be administered orally in a single dose or in multiple doses. Generally, sunitinib malate is given for 2, 3, 4 or more consecutive weeks, followed by a “off” period of about 1 or 2 weeks or longer when sunitinib malate is not given. In one embodiment, the doses are administered for about 4 weeks, with a 2-week break. Generally, the effective amount of sunitinib malate administered orally to a human is less than 40 mg per person per day, such as 37.5, 31.25, 25, 18.75, 12.5, or 6.25 mg. mg per person per day. In some embodiments, sunitinib malate is administered orally in the range of 1-25 mg per person per day. In some different embodiments, sunitinib malate is administered orally in the range of 6.25, 12.5, or 18.75 mg per person per dose. Other dosage regimens and variations may be envisioned and will be determined with the assistance of a physician. Sorafenib tosylate, which is marketed under the brand name NEXAVAR, has the chemical name 4-(4-{3-[4-chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N-methylpyrid-in-2-carboxamide. It is approved in the US for the treatment of primary kidney cancer (advanced renal cell carcinoma) and advanced primary liver cancer (hepatocellular carcinoma). The recommended daily dose is 400 mg, administered orally twice daily. In the anticancer VBIR provided by the present disclosure, the effective amount of orally administered sorafenib tosylate is generally less than 400 mg per person per day. In some embodiments, the effective amount of orally administered sorafenib tosylate is in the range of 10-300 mg per person per day. In some different embodiments, the effective amount of orally administered sorafenib tosylate is between 10-200 mg per person per day, such as 10, 20, 60, 80, 100, 120, 140, 160, 180, or 200 mg. mg per person per day. Axitinib, which is marketed under the brand name INLYTA, has the chemical name (W-methyl-2-(3-((E)-2-pyridin-2-yl-vin¡l)-1 / - / -¡ ndazol-6-ylsulfanyl]-benzamide Is approved for the treatment of advanced renal cell carcinoma after failure of prior systemic therapy The starting dose is 5 mg orally twice daily Dose adjustments can be made based on the safety and tolerability of the subject.In the anticancer VBIRs provided herein, the effective amount of axitinib administered orally is less than 5 mg twice daily.In some different embodiments , the effective amount of axitinib administered orally is 1-5 mg twice daily In some different embodiments, the effective amount of axitinib administered orally is between 1, 2, 3, 4 and 5 mg twice daily. Examples of IEC enhancers that can be used in the anticancer VBIRs provided by the present disclosure include TNFR agonists, CTLA-4 antagonists, TLR agonists, other PD-1 antagonists (such as BMS-936558 and antibody anti-PD-1 CT-011), programmed cell death protein ligand 1 (PD-L1) antagonists (such as BMS-936559), lymphocyte activating gene 3 (LAG3) antagonists, and molecule containing T-lymphocyte immunoglobulin and mucin domain -3 (TIM-3). Examples of TNFR agonists include agonists of 0X40, 4-1BB (such as BMS-663513), GITR (such as TRX518), and CD40. Examples of specific CD40 agonists are described in detail below. In some different embodiments, the additional immune modulator is an antagonist anti-CD40 antibody. The antibody may be a human, humanized or chimeric partially human anti-CD40 antibody. Examples of specific anti-CD40 monoclonal antibodies include G28-5, mAb89, EA-5, or the monoclonal antibody S2C6, and CP870893. In a particular embodiment, the agonist anti-CD40 antibody is CP870893 or dacetuzumab (SGN-40). CP-870,893 is a fully human agonist CD40 monoclonal antibody that was clinically investigated as an antitumor treatment. The structure and preparation of CP870.893 are described in W02003040170, where the CP870.893 antibody is identified as antibody “21.4.1. The heavy chain and light chain amino acid sequences of CP-870,893 are set forth in SEQ ID NO: 46 and SEQ ID NO: 48, respectively, and in Table 7, in / W02003040170. In clinical trials, CP870.893 was administered by intravenous infusion at doses generally in the range of 0.05 - 0.25 mg / kg per infusion. In the anticancer VBIRs provided by the present disclosure, CP78 870,893 can be administered intradermally, subcutaneously, or topically. In general, the effective amount of CP870893 to be administered in the regimen is less than 0.2 mg / kg, generally in the range of 0.01 mg - 0.15 mg / kg, or 0.05 - 0. 1mg / kg. Dacetuzumab (also known as SGN-40 or huS2C6; CAS number 88486-59-9) is another agonist anti-CD40 antibody that has been investigated in clinical trials for indolent lymphomas, diffuse large B-cell lymphomas, and multiple myeloma. In the anticancer VBIRs provided by the present disclosure, acetuzumab can be administered intradermally, subcutaneously, or topically. In general, the effective amount of dacetuzumab to be administered is less than 16 mg / kg, generally in the range of 0.2 mg - 14 mg / kg, 0.5 - 8 mg / kg, or 1 - 5 mg / kg. kg. In yet other embodiments, the additional immune modulator is an anti-CTLA-4 antagonist. Examples of suitable anti-CTLA-4 antagonists include anti-CTLA-4 antibodies (such as human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, anti-CTLA-4 antibodies). -4 humanized, monotional anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, anti-CTLA-4 domain antibodies) and CTLA-4 inhibitors that agonize the co-stimulatory pathway. In some embodiments, the CTLA-4 inhibitor is Ipilimumab or Tremelimumab. Ipilimumab (marketed as YERVOY; also known as MEX-010, MDX-101, or by its CAS Registry No. 477202-00-9) is described as antibody 10DI in PCT Publication No. WO 01 / 14424, incorporated herein by reference in its entirety and for all purposes. Examples of pharmaceutical compositions comprising pilimumab are provided in PCT Publication No. WO 2007 / 67959. Ipilimumab is approved in the US for the treatment of unresectable or metastatic melanoma. In the methods provided by the present invention, Ipilimumab can be administered intradermally or subcutaneously. The effective amount of ipilimumab administered locally is generally in the range of 5 - 200 mg / dose per person. In some embodiments, the effective amount of Ipilimumab is in the range of 10-150 mg / dose per person per dose. In some particular embodiments, the effective amount of Ipilimumab is about 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg / dose per person. Tremelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has the CAS number 745013-59-6; Tremelimumab is described as antibody 11.2.1 in US Patent No.': 6,682,736, incorporated herein by reference in its entirety and for all purposes. In the anticancer VBIRs provided by the present invention, Tremelimumab can be administered intravenously, intradermally, or subcutaneously. The effective amount of Tremelimumab administered intradermally or subcutaneously is generally in the range of 5 - 200 mg / dose per person. In some embodiments, the effective amount of Tremelimumab is in the range of 10-150 mg / dose per person per dose. In some particular embodiments, the effective amount of Tremelimumab is about 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg / dose per person. In yet other embodiments, the additional immune modulator is a Toll-like receptor (TLR) agonist. The term "toll-like receptor agonist" or "TLR agonist" refers to a compound that acts as an agonist of a toll-like receptor (TLR). This includes agonists of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 and TLR11 or a combination of these. TLR agonists useful in the method of the present invention include small organic molecules and large biological molecules. Examples of small molecule TLR agonists include 4-amino-alpha, alpha,2- trimethyl-IHimidazo[4,5-c]qumol¡n-l-ethanol, N-(2-{2-[4-amino-2 -(2-methoxyethyl)-IH-imidazo[4,5c]quinolin-l-yl]ethoxy-}ethyl)-N-methylmorpholine-4-carboxamide, l~(2~amino-2-methylpropyl)-2(ethoxymethyl -)-IH-imidazo[4,5-c]quinolin-4-arnine, N-[4-(4-amino-2-ethyl-IH-imidazo[4,5c]quinolin-l-yl)b- ethyljmethanesulfonamide , N-[4-(4-amino-2-propyl-IH-imidazo[4.5c]quinolin-l-yl)butyl]methanesulfonamide and imiquimod. Some particularly useful TLR agonists in the methods or regimen provided by the present disclosure are discussed in the review article: Folkert Steinhagen, et al.: TLRbased immune adjuvants. Vaccine 29 (2011): 3341-3355. In some embodiments, the TLR agonists are TLR9 agonists, in particular CpG (or CpG.ODN) oligonucleotides. A CpG oligonucleotide is a short nucleic acid molecule containing a cytosine and then a guanine linked by a phosphate bond, where the pyrimidine ring of the cytosine is unmethylated. Some examples of particular CpG oligonucleotides useful in the methods provided by the present disclosure include: 5' TCGTCGTTTTGTCGTTTTGTCGTT3' (CpG 7909; SEQ ID NO:49); 5' TCGTCGTTTTTCGGTGCTTTT3' (CpG 24555; SEQ ID NO:50); Y 5' TCGTCGTTTTTCGGTCGTTTT3' (CpG 10103; SEQ ID NO:51). CpG7909, a synthetic single-stranded 24mer, has been extensively investigated for cancer treatment as monotherapy and in combination with chemotherapeutic agents, as well as an adjuvant for vaccines against cancer and infectious diseases. In the methods provided by the present disclosure, CpG7909 can be administered by injection into muscle or any other suitable method. For use with a nucleic acid vaccine, such as a DNA vaccine, a CpG can be co-formulated with the vaccine in a single formulation and administered by intramuscular injection coupled with electroporation. The effective amount of CpG7909 by intramuscular, intradermal or subcutaneous administration is generally in the range of 10 pg / dose -10 mg / dose. In some embodiments, the effective amount of CpG7909 is in the range of 0.05mg - 14mg / dose. In some particular embodiments, the effective amount of CpG7909 is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 05 1 mg / dose. Other CpG oligonucleotides, including CpG 24555 and CpG 10103, can be administered in a similar manner and at similar dose levels. In VBIR for cancer, the anti-PD-1 antagonist, the vaccine, and additional immune modulators can be administered simultaneously or sequentially. In some embodiments, a vaccine is administered sequentially with respect to the anti-PD-1 antagonist antibody, but simultaneously (eg, in admixture) with respect to one or more additional immune modulators. In cases where a nucleic acid vaccine is administered in combination with a CpG, the vaccine and the CpG may be contained in a single formulation and may be administered together by any suitable method. In some embodiments, the nucleic acid vaccine and CpG in a coformulation (mixture) are administered by intramuscular injection in combination with electroporation. formulations Therapeutic anti-PD-1 antagonist antibody formulations used in accordance with the present invention are prepared for storage by admixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers ( Remington, The Science and Practice of Pharmacy, 20th ed., Mack Publishing, 2000), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and may comprise buffers such as phosphate, citrate and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (such as octadeclldlmethylbenzylammonium chloride; hexametonl chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m -cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, hstldlnine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg Zn protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Liposomes containing the anti-PD-1 antagonist antibody are prepared by methods known in the art, such as described in Epsteln, et al., Proc. Nati. Acad. sel. USA 82:3688 (1985); Hwang, et al., Proc. Nati Acad. sel. US 77:4030 (1980); and US Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are described in US Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and phosphatidylethanolamine derivatized from PEG (PEG-PE). The liposomes are extruded through filters having a defined pore size to obtain liposomes with the desired diameter. Active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., gelatin or hydroxymethylcellulose microcapsules and poly(methylmethacrylate) microcapsules, respectively, in drug delivery systems. colloidal (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are described in Remington, The Science and Practice of Pharmacy, 20th ed. Mack Publishing (2000). Sustained release preparations can be made. Suitable examples of sustained release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody; the matrices have the appearance of shaped articles, eg, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), L-glutamic acid copolymers, and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ™ (injectable microsyres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate and acid poly-D-(-)-3-hydroxybutyric. Formulations to be used for in vivo administration must be sterile. This is easily achieved, for example, by filtration through sterile filter membranes. In general, therapeutic anti-PD-1 antagonist antibody compositions are placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable with an injection needle. hypodermic Compositions according to the present invention may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions, suspensions or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation. . To prepare solid compositions, such as tablets, the main active ingredient is mixed with a pharmaceutical carrier, for example, conventional tabletting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, eg, water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention or a pharmaceutically acceptable non-toxic salt thereof. When these preformulation compositions are indicated as homogeneous, the active ingredient is uniformly dispersed throughout the composition so that it can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel composition can be coated or otherwise combined to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may comprise an inner dosage component and an outer dosage component; the latter wraps the former. The two components can be separated by an enteric coating that serves to resist disintegration in the stomach and to allow the inner component to pass intact into the duodenum or to have its release delayed. Various materials can be used for the enteric coatings or layers, including various polymeric acids and mixtures of polymeric acids with those materials, such as shellac, cetyl alcohol, and cellulose acetate. Suitable surfactants include, in particular, non-ionic agents, such as polyoxyethylene sorbitans (eg Tween™ 20, 40, 60, 80 or 85) and other sorbitans (eg Span™ 20, 40, 60, 80 or 85). . Compositions with a surfactant conveniently comprise from 0.05 to 5% surfactant, and may be from 0.1 to 2.5%. It should be noted that other ingredients, eg, mannitol or other pharmaceutically acceptable carriers, may be added if necessary. Suitable emulsions can be prepared using commercially available lipid emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition or alternatively may be dissolved in an oil (for example, soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and an emulsion formed by mixing it with a phospholipid (for example, egg phospholipids, soy phospholipids, or soy lecithin) and water. It should be noted that other ingredients, for example glycerol or glucose, can be added to adjust the tonicity of the emulsion. In general, suitable emulsions contain up to 20% oil, for example between 5 and 20%. The lipid emulsion may comprise fat droplets of 0.1 to 1.0 pm, in particular 0.1 to 0.5 pm, and may have a pH in the range of 5.5 to 8.0. Emulsion compositions may be those prepared by mixing an antagonistic anti-PD-1 antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol, and water). Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients, as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably sterile pharmaceutically acceptable solvents may be employed for nebulization by the use of gases. Nebulized solutions can be aspirated directly from the nebulizer, or the nebulizer can be attached to a face mask, tent, or intermittent positive pressure respirator. The compositions in solution, suspension or powder can be administered, preferably orally or nasally, from devices that deliver the formulation in a suitable manner. kits The invention also provides kits comprising any or all of the antibodies described herein. Kits of the invention include one or more containers comprising an antagonistic anti-PD-1 antibody described herein and instructions for use in accordance with any of the methods of the invention described herein. These instructions generally comprise a description of the administration of antagonistic anti-PD-1 antibodies for the therapeutic treatments described above. In some embodiments, kits for producing a single dose administration unit are provided. In some embodiments, the kit may contain both a first container having a dehydrated protein and a second container having an aqueous formulation. In some embodiments, kits containing prefilled single-chamber or multi-chamber syringes (eg, liquid syringes and lyosyringes) are included. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. Instructions regarding the use of an anti-PD-1 antibody generally include information regarding the dosage, dosage schedule, and route of administration for the intended treatment. Containers may be unit doses, batch packs (eg, multi-dose packs), or subunit doses. In general, the instructions provided in the kits of the invention are instructions written on a label or package insert (for example, a sheet of paper included in the kit), but instructions in machine-readable format (for example, instructions included in an optical or magnetic storage disk). The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, polyethylene terephthalate or plastic bags), and the like. Containers for use in combination with a specific device, such as an inhaler, a device for nasal administration (eg, an atomizer), or an infusion device, such as a minipump, are also contemplated. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced with a hypodermic injection needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable with a hypodermic injection needle). At least one active agent in the composition is an anti-PD-1 antibody. The container may also comprise a second pharmaceutically active agent. Kits can optionally provide additional components such as shock absorbers and interpretive information. Typically, the kit comprises a container and a label or insert on the container, or associated with the container. biological deposit Materials representative of the present invention are deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 201102209, USA, on Apr 29, 2014. The vector msb7-LC having ATCC Accession No. PTA-121182 is a polynucleotide encoding the light chain variable region of mAb7, and the vector mab7-HC having ATCC Accession No. PTA121183 is a polynucleotide that encodes the heavy chain variable region of mAb7. The deposits were obtained in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Related Regulations (Budapest Treaty). This guarantees the maintenance of a viable culture of the deposit for 30 years from the date of deposit. The repository will be made available through the ATCC under the terms of the Budapest Treaty, and will be subject to an agreement between Pfizer, Inc. and ATCC, which ensures the continued and unrestricted availability of the repository culture's progeny to the public at the time of the issuance of the applicable United States patent, or of disclosing to the public any United States or foreign patent application, whichever comes first, and guarantees the availability of progeny to whom the Commissioner of Patents and Trademarks of the United States determines to be entitled to do so in accordance with § 122 of Title 35 of the United States Code and the Commissioner's regulations thereon (including § 1.14 of Title 37 of the Code of Federal Regulations, with particular reference to 886 OG 638). The assignee of the present application has agreed that if a culture of the materials on deposit dies, is lost, or is destroyed when grown under proper conditions, the materials will be promptly replaced upon notice. The availability of the deposited material shall not be construed as a license to practice the invention contrary to the rights granted by the authority of any government in accordance with its patent laws. The following examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will be apparent to those of ordinary skill in the art and will fall within the scope of the appended claims. examples Example 1: Effect of the anti-PD-1 antibody on the secretion of IFN-v and TNF This example illustrates the effect of anti-PD-1 antibody on IFN-γ and TNF secretion in a mixed lymphocyte reaction (MLR) assay. Primary human T cells isolated from blood (Stanford University blood bank) were activated with allogeneic dendritic cells (DCs) expressing high levels of PD-L1 and PD-L2, which were previously differentiated using IL-4 and GMCSF from myeloid cells. CD14+. In this study, the following antibodies were used: isotype control (IgG4 kappa hinge-stabilized), anti-PD-1 C1 antagonist antibody, anti-PD-1 C2 antagonist antibody, anti-PD-1 C3 antagonist antibody, EH12 .1 (BD Biosciences mouse anti-human anti-PD-1 antibody, mouse IgG1 Kappa isotype), mAb7-G4, mAb15-G4, mAb-AAA, mAb15-AAA (G4 = hinge-stabilized IgG4; AAA = mutant lgG1 that does not bind to FcγR). The antibodies were evaluated at the following concentrations: 0, 0.1, 1 or 10 pg / ml). For the MLR assay, cultures were incubated with control or test antibody in 96-well plates in triplicates at ratios of 1:10 DC:T cells and incubated in a humidified 37°C incubator with 5% HCl. CO2. Supernatants were harvested on day 5, and cytokines were measured using cytometric bead array (CBA) using the Flex Set Soluble Human Protein Kit. System (BD Biosciences, Cat #558265) according to the manufacturer's protocol with the following human analytes: IFNγ (BD Biosciences, Cat #558269), TNF (BD Biosciences, Cat #558273). Briefly, 96-well filter plates (Millipore, Cat. No. MSBVN1250) were washed with wash buffer (proprietary formula from BD Biosciences) and aspirated through a vacuum manifold. Kit-supplied standards and samples were diluted in assay diluent (BD Biosciences proprietary formula) and added to capture bead plates (capture beads are beads coated with antibodies to a specific soluble protein coated with a different fluorescence). Plates were mixed for 5 minutes at 500 rpm using a plate shaker and incubated for 1 hour at room temperature. Detection reagent (phycoerythrin (PE)-conjugated antibodies, provided by k) was added to the plates, and the plates were mixed for 5 minutes. The plates are incubated for 2 hours at room temperature. They were then [omitted in source text] with wash buffer for 5 minutes, and samples were acquired on BD Fortessa platforms. Data was analyzed using FCAP Array v3 (BD), MLR assay results are shown in Tables 6A and B below. Table 6A shows IFNγ levels (in pg / ml), and Table 6B shows TNF levels (in pg / ml). Data are presented as an average ± S.E.M of biological triplicates. Samples are representative of an MLR experiment. Table 6A: IFNy secretion Antibody 0 1 1 2 7 EH12.1 7 2 1 mAb15-G4 5 1 1 mAb7-G4 11 11 1 mAb7-AAA 1 1 5 1 09.45 AAA 1 5 C1 1 C2 C3 11 1 4735 Table 6B: TNF secretion Antibody 371 17 19 .4 17 9 .123 60, EH12.1 7 3 17 7 116 56, mAb15-G4 3 7 371 7.011 730.47 mAb7-G4 3 7 8 371.323 17.01 9 113 mAb7-AAA 1 1 mAb15- AAA 3 37 17 19 1 C1 3 7 17 9 C2 3 7 6 7 1 39, C3 7 7 14, 1501.17 35, 5 .1 .623 16.0493 7,3774 49, .44 1 7 Treatment of activated T cells with antagonistic anti-PD-1 antibodies produced an increase in IFN[gamma] levels compared to isotype control (Table 6A). For example, treatment with 0.1 pg / ml of mAb15-G4 and mAb7-G4 produced an IFNγ level of 4728.295 + 2.035 pg / ml and 8567.203 + 2085.826 pg / ml, respectively. Treatment with 1 pg / ml of mAb15-G4 and mAb7-G4 produced an IFNγ level of 8893.595 + 365.125 pg / ml and 11876.86 + 1259.788 pg / ml, respectively. Treatment with 10 pg / ml of mAb15-G4 and mAb7-G4 produced an IFNy level of 7790.95 + 1700.012 pg / ml and 11794.82 + 1827.243 pg / ml, respectively. In contrast, treatment with 0.1, 1, or 10 pg / mL of isotype control produced IFNγ levels of 3760 + 367.4262 pg / mL, 3693.972 + 1033.879 pg / mL, and 3525, 655 + 744.676 pg / ml, respectively. Treatment of activated T cells with antagonistic anti-PD-1 antibodies produced an increase in TNF levels compared to isotype control (Table 6B). For example, treatment with 0.1 pg / ml of mAb15-G4 or mAb7-G4 produced a TNF level of 743.1167 + 56.75547 pg / ml and 730.47 ± 33.35488 pg / ml, respectively. Treatment with 1 pg / ml of mAb15-G4 and mAb7-G4 produced a TNF level of 686.32 + 45.63348 pg / m, and 793.05 + 21.19019 pg / ml, respectively. Treatment with 10 pg / ml of mAb15-G4 or mAb7-G4 produced a TNF level of 798.853 + 9.14366 pg / ml and 930.623 + 16.0494 pg / ml, respectively. In contrast, treatment with 0.1, 1, or 10 pg / mL of isotype control produced TNF levels of 407.4133 + 49.58195 pg / mL, 486.7167 + 4.4241 pg / mL, and 501 0.17 + 5.033334 pg / ml, respectively. These results demonstrate that anti-PD-1 antibodies mAb7 and mAb15 stimulate IFNγ and TNF secretion from T cells at least as much as anti-PD-1 antibodies C1, C2 and C3 or more. A second MLR study was performed to assess the effect of lower concentrations of antibody on T cell activation. Primary human T cells isolated from blood were activated as described above. In the second study, the following antibodies were evaluated: mAb7 (G4), mAb15 (G4), C1 (G4), EH12.1, and control isotype G4. The antibodies were evaluated at the following concentrations: 0.0001, 0.001, 0.01, 0.1, 1 and 10 pg / ml. The MLR assay was carried out as described above. The results are summarized in Tables 7A and 7B below. Table 7A: IFNy secretion Antibody + + 1 4.37 + 1.96 ± 1.96 + + de 1 1 + 2284, + 4384, + + 4 + 1 + EH12.1 417 5 1 176 1 4 + .01 + 41 1 + 13 + 4 + 1 + C1 290 77 194 168 61 ± + + + 1 ± 1 + 1 1 1 + 1410, + .49 + 5416 + + 1 3 + 1 1 1 Table 7B: TNF secretion Antibody 452, 33 + + 31 1 1 1107 + 1.008 + 44 + + de + 687 + + + + EH12.1 + 444, + + + + + C1 1.6± + + + 1 + 14 9, 88 + 4 1 711 . ± + + , + + + Yo Treatment of activated T lymphocytes with antagonistic anti-PD-1 antibodies produced an increase in IFNγ levels compared to isotype control (Table 7A). In cultures without antibody, the IFNy level was 901,453 + 216,472 pg / ml. In cultures given 0.0001, 0.001, 0.01, 0.1, 1, or 10 pg / ml of isotype control, IFNγ levels were 558.9629 +, 489.4828 pg / ml, 753.3767 + 291.6092 pg / ml, 1074.37 + 324.2031 pg / ml, 1667.96 + 144.7286 pg / ml, 1867.96 + 282.7461 pg / ml, 2501.293 + 220, 1829 pg / ml, respectively. In contrast, in cultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10 pg / ml of mAb7 (G4), IFNγ levels were 2082.07 + 720.9931 pg / ml , 3062.09 + 370.2791 pg / ml, 5067.823 + 111.4903 pg / ml, 7082.667 + 1336.082 pg / ml, 11928.81 + 1457.723 pg / ml, 11862.13 + 800.586 pg / ml, respectively. In cultures treated with 0.0001, 0.001, 0.01,0.1, 1, or 10 pg / ml mAb15 (G4), IFNγ levels were 1678.27 + 233.82 pg / ml, 1410.758 + 439.9474 pg / ml, 4734.49 + 322.2087 pg / ml, 5416 + 1054.075 pg / ml, 9140.337 + 1320.499 pg / ml and 10992.13 + 1008.533 pg / ml, respectively. Treatment of activated T cells with antagonistic anti-PD-1 antibodies produced an increase in TNF levels compared to isotype control (Table 7B). In cultures without antibody, the level of TNF was 365.523 + 84.6607 pg / ml. In cultures treated with 0.0001, 0.001, 0.01, 0.1.1, or 10 pg / ml isotype control antibody, TNF levels were 452.7133 + 62.85 pg / ml, 282, 9287 + 56.77266 pg / ml, 310.9144 + 21.7811 pg / ml, 358.948 + 81.09122 pg / ml, 338.1107 + 46.88385 pg / ml and 331.008 + 31.35559 pg / ml, respectively . In contrast, in cultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10 pg / ml of mAb7 (G4), TNF levels were 227.6 + 50.63436 pg / ml , 394.4233 + 30.47005, 452.65 + 30.64335 pg / ml, 1089.377 + 174.7824 pg / ml, 1583.52 + 267.2131 pg / ml and 1419.88 ± 108.711 pg / ml , respectively. In cultures given 0.0001, 0.001, 0.01, 0.1, 1, or 10 pg / ml mAb15(G4), TNF levels were 494.7967 + 48.1810 pg / ml, 489.2333 + 30.63302 pg / ml, 593.34 + 65.87622 pg / ml, 811.16 + 89.50238 pg / ml, 1143.54 + 136.3954 pg / ml and 1109.063 + 57, 70232 pg / ml, respectively. These results demonstrate that the anti-PD-1 antibodies mAb7 and mAb15 block PD-1 signaling and promote IFN[gamma] and TNF secretion from primary human T cells. Example 2: Effect of Anti-PD-1 Antibodies on T Lymphocyte Proliferation This example illustrates the effect of anti-PD-1 antibodies on T cell proliferation, In this study, T cell proliferation was measured in an MLR assay, wherein T lymphocytes were cultured in the presence of antagonist anti-PD-1 or isotype control antibodies. For the MLR assay, primary human T cells isolated from blood (obtained from the Stanford University blood bank) were activated with allogeneic dendritic cells (DCs) expressing high levels of PD-L1 and PD-L2, which had been previously differentiated using IL-4 and GM-CSF from CD14+ myeloid cells. Two experiments were carried out. In the first experiment, mAb7 (IgG4 kappa hinge-stabilized), mAb15 (IgG4 kappa hinge-stabilized), C1, EH12.1, and isotype control were compared. In the second experiment, mAb7, mAb15, C2, EH12.1 and isotype control clones were compared. In both experiments, antibodies were added at the following concentrations: 0, 0.0001; 0.001; 0.01; 0.1; 1 and 10 pg / ml. For both experiments, cultures were incubated with antibody in 96-well plates in triplicates at 1:10 ratios of DCJT-lymphocytes and incubated in a humidified 37°C incubator with 5% CO2. On day 5, cultures were boosted for 18 h with 1 pCiper well of [ 3 H]-thymidine before harvesting. Plates were then harvested onto DNA-specific filter papers (Perkin Elmer) using Harvester96 (Tomtec Life Sciences). Radiolabeled filters were covered with beta scintillation fluid (Perkin Elmer) and read on Microbeta® counting plates (Perkin Elmer). Thymidine incorporation was analyzed as counts per minute (CPM). Results are shown as the mean of triplicates ± SEM. Table 8A Control antibody concentration of + 205 90 + 1 , + , ± 1 EH12.1 mAb7 1 ± 1 + C1 1 + 1 + 41 + 226145 + 21 7 + 1 .3 + 4 + .3 + + + 1 + + 67 + + 7 + .7 + 1 + + .7 + + + + 1 77 + + 7 + + 1 41 1 Table 8B EH12.1 mAb7 control antibody concentration 5 + + + + 1 1 + + 11 ± 31 4101 97 + + + + 1 1 + + + + + 31 + .3± + 1 1 1 4 1 24 + , + 7 + + 1 1.7 ± + .7 ± 1.3 + 1 10 Treatment of activated T cells with antagonistic anti-PD-1 antibodies at a concentration of 0.01 pg / ml or more produced significantly greater T cell proliferation compared to isotype control (Tables 8A and 8B). For example, treatment with 0.01, 0.1, 1, or 10 pg / mL mAb7 produced thymidine incorporation rates of 365625.3 + 30171.07 CPM, 380054.3 + 9774.328 CPM, 392256.7 + 15341.19 CPM and 372889.7 + 14826.49 CPM, respectively (Table 8B). Treatment with 0.01, 0.1, 1, or 10 pg / mL of mAb15-G4 and mAb7 produced thymidine incorporation rates of 317377 + 31915.29 CPM, 360226.3 + 1802.69 CPM, 421229 + 27865, 13 CPM and 323441.3 + 64476.55 CPM, respectively (Table 8B). In contrast, treatment with 0.01, 0.1, 1, or 10 pg / mL of isotype control produced thymidine incorporation rates of 278072.3 + 32671.62 CPM, 268939.7 + 12332.06 CPM, 241164 + 13776.81 CPM and 231897.7 + 25865.95 CPM, respectively (Table 8B). These results demonstrate that the anti-PD-1 antibodies mAb7 and mAb15 block PD-1 signaling and promote the proliferation of primary human T cells. Example 3: Effect of anti-PD-1 antibodies in a mouse model of GvHD This example illustrates the effect of anti-PD-1 antibodies on T cell proliferation and body weight loss in a mouse model of graft versus host disease (GvHD). In this study, IL-2 receptor gamma chain (NSG) NOD-scid-null mice were used to assess the effects of antagonistic anti-PD-1 antibodies. in the proliferation of T lymphocytes in vivo. Because NSG mice lack T cells and B cells, and have defective NK cells, human cell engraftment can be easily achieved. When human PBMCs are grafted into these mice, human T cell proliferation occurs and GvHD is induced. GvHD affects the myeloid compartment of the host and human cells. Large-scale proliferation of human lymphocytes can be observed in the blood at early stages, followed by high infiltration of the cells into mouse organs, for example, liver, spleen, kidney, intestines, etc., which results in a loss in mouse body weight and skin lesions, hyperkyphosis, and death. The severity of the patterns depends on the donor PBMC and may differ between donors. In this study, the following antagonistic anti-PD-1 antibodies were used: mAb7 (hinge-stabilized human IgG4 or AAA), mAb15 (hinge-stabilized human IgG4), C1, C2 and C3. For the negative control, an isotype control human IgG4 hinge-stabilized antibody was used. Primary human PBMC were isolated from blood (Stanford University blood bank), using Ficoll gradient. 107 human PBMC were injected into NSG mice (8-week-old female, Jackson Laboratories). On day 0, mice were randomized based on body weight, and PBMC were injected intravenously. For experiments 1-4, on day 2 and day 8, antibodies were administered intraperitoneally at 10 mg / kg. For experiment 5, on day 2 and day 8, antibodies were administered intraperitoneally at 1 mg / kg or 10 mg / kg. Table 9 summarizes the antibodies used in each experiment. Table 9: Antibodies used in experiments 1-5 Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 control control control control control control isotype isotype isotype isotype isotype mAb7 mAb15 mAb15 mAb15 mAb7 mAb15 mAb7 mAb7 mAb7 mAb7-AAA C1 C3 C2 C1 Body weight was measured periodically. The results are summarized in Figures 1A-1E. Mice were bled periodically to assess T cell proliferation. Anti-PD-1 antibody treatment accelerated the course of the disease as measured by the rate of body weight loss. Compared to control mice, antibody-treated mice anti-PD-1 antagonist lost body weight more rapidly (Figures 1A-1E). Human T cell proliferation was measured by flow cytometry using CD45 as a marker (clone HI30; BD Biosciences). The results of flow cytometry are summarized below in Table 10. T cell proliferation was greater in mice treated with anti-PD-1 antagonist antibody than in mice treated with isotype control. A higher percentage of CD45 indicates a higher level of proliferation of CD45-expressing cells and thus more severe GvHD. In experiment 1, the percentage of blood cells expressing CD45 was 63.86% in control mice (Table 10). In contrast, the percentage of blood cells expressing CD45 in mice treated with anti-PD-1 antibody mAb7, mAb15, C1, or mAb7-AA was 80.34%, 77.62%, 77.26%, and 76. 9%, respectively (Table 10). Table 10: T-lymphocyte proliferation measured as a function of the presence of CD45, animals treated in two: in SEM in Average SEM 4: in mAb7 mAb7 In summary, anti-PD-1 antibody-treated mice suffered more rapid body weight loss and had greater T cell proliferation compared to isotype control-treated mice. These results demonstrate that anti-PD-1 antibody treatment stimulates human T cell proliferation in vivo. Example 4: Binding of anti-PD-1 antibodies This example illustrates the binding of anti-PD-1 antibodies to activated human T cells and Macaca fascicularis T cells. Primary human T cells were isolated from PBMC (Stanford University blood bank) using a PAN human T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130-096-353). Macaca fascicularis PBMC were purchased from (BioreclamationlVT), and PAN T cells were isolated using a non-human primate PAN T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130-091-993). Human T cells were activated for 3 days with DYNABEADS™ CD3 / CD28 Human T Cell Activators for Cell Expansion and Activation (Life Technologies; 11131D). The microsphere to cell ratio used was 1:1 microsphere:T cell, respectively. Macaca fascicularis T cells were activated for 3 days using the non-human primate T cell activation / expansion kit according to the manufacturer's protocol (Miltenyi Biotec; 130-092-919). The microsphere-to-cell ratio used was 1:1 microsphere:T cell; respectively. After 3 days, the cultures were harvested, the microstrands were separated from the activated T lymphocytes using magnetic force. Cells were washed and incubated with FACS buffer (including 2% FBS) and human Fe receptor binding inhibitor (Affymetrix eBioscience Cat. No. 16-9161-73). For Macaca fascicularis cells, Fe blocking reagent (BD Biosciences Cat. No. 564765) was used. Cells were incubated for 10 minutes at room temperature and then stained with Live / Dead color to exclude dead cells (LIVE / DEAD® Fixation Blue Dead Cell Staining Kit, for UV excitation, Cat. A10346) for a further 5 minutes. Anti-PD-1 antibodies were added (concentrations of anti-PD-1 clones were incubated on cells at serial dilution ratios of 1:3 starting at 10 pg / ml - 0 pg / ml). 1x106 cells were used in each reaction in a total of 100 μΙ, and the cells were incubated on ice for 30 minutes. Cells were then washed in FACS buffer to remove access of primary antibodies and incubated with secondary [source text omitted] anti-human (donkey anti-human IgG (H+L) F(ab')2 fragment AffiniPure allophycocyanin-conjugated (APC; Cat. No. 709-136-149). Cells were stained for 30 min on ice. Cells were washed and kept on ice until read using a BD LSRFortessa Cell Analyzer (BD Biosciences, Cat# 647465). Data was analyzed using FlowJo™ software. The results are summarized in Figures 2A and 2B. Figure 2A shows the EC50 measured for anti-PD-1 antibody bound to activated human cells, and Figure 2B shows the EC50 measured for anti-PD-1 antibody bound to activated Macaca fascicularis cells. Anti-PD-1 antibodies mAb7 and C1 bind to activated T cells with similar EC50s (Figures 2A and 2C). Example 5: Inhibition of Binding to PD-L1 by Anti-PD-1 Antibody This example illustrates the inhibition of PD-1 ligand (PD-L1) binding by anti-PD-1 antibody. Primary human T cells were isolated from PBMC (Stanford University blood bank) using a human PAN T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130-096-353). PBMC were purchased from Macaca fascicularis from (BioreclamationlVT), and PAN T cells were isolated using a non-human primate PAN T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130-091-993). Human T cells were activated for 3 days with DYNABEADS™ CD3 / CD28 human T cell activators (for cell expansion and activation, Life Technologies; 11131D). The ratio of microspheres to cells used was 1:1, respectively. lymphocytes Macaca fascicularis T cells were activated for 3 days using the non-human primate T cell activation / expansion kit according to the manufacturer's protocol (Miltenyi Biotec; 130-092-919). The ratio of microspheres to cells used was 1:1, respectively. After 3 days, the cultures were harvested, the microspheres separated from the activated T cells using magnetic force. Cells were washed and incubated with FACS buffer (including 2% FBS) and human Fe receptor binding inhibitor (Affymetrlx eBioscience; cat# 16-9161-73). For Macaca fascicularis cells, Fe blocking reagent (BD Biosclences; Cat# 564765) was used. Cells were incubated for 10 minutes at room temperature and stained with Live / Dead color to exclude dead cells (LIVE / DEAD® Fixation Blue Dead Cell Staining Kit, for UV excitation; Cat. No. A10346 ) for 5 more minutes. Recombinant human PD-L1 Fe (R&D Systems, cat# 156-B7) or buffer alone were incubated with cells at 10 ng / ml. Each ligand was incubated separately and incubated on ice for 30 minutes. Cells were then washed and incubated with anti-PD-1 antibodies (concentrations of anti-PD-1 clones were incubated on cells at serial dilution ratios of 1:3 starting at 1 pg / ml - 0 pg / ml ). 1x106 cells were used in each reaction for a total of 100 pl, and the cells were incubated on ice for 30 minutes. Cells were then washed with FACS buffer to remove access of primary antibodies and incubated with allophycocyanin-conjugated anti-human kappa (APC) (Life Technologies; Cat# MH10515). Cells were stained for 30 minutes on ice, then washed and kept on ice until read using a BD LSRFortessa Cell Analyzer (BD Blosciences, Cat# 647465). Data was analyzed using FlowJo™ software, and mean fluorescence intensity (MFI) and geometric means (Geo.M) of APC staining in living cells were calculated in FlowJo™ software. After calculation of the geometric mean, the 1C50 was calculated using GraphPD Prism software. The results are summarized in Tables 11 and 12 below. Table 11: Antl-PD-1 Blockade of PD-L1 Binding to PD-1 in Human T Cells Geo Mean Antibody Concentration (pg / mL) mAb7 C1 0.0083375 107 143 0.00416875 129 190 IC50 (μM) 0.002084375 162 245 0.001042188 205 327 0.000521094 482 415 0.000260547 358 469 0.000130273 445 484 0.000065137 503 458 0.000032568 450 420 L 0.001117 | 0.00224 Table 12: Anti-PD-1 blockade of PD-L1 binding to PD-1 in Macaca fascicularis T cells Average Concentration Geo of Antibody (PG / ML) MAB7 C1 0.0083375 114 108 0.00416875 135 144 0.002084375 174 183 0.001042188 240 264 0.000521094 322 325 0.000260547 440 404 0.000130273 494 494 494 494 491 472 0.000032568 410 406 i 0.00092 | 0.00108 These results demonstrate that the anti-PD-1 antibodies mAb7 and C1 inhibit the binding of PD-L1 to human and Macaca fascicularis T lymphocytes with a similar IC50. Example 6: Effect of Anti-PD-1 Antibodies on T Lymphocyte Proliferation This example illustrates the effect of anti-PD-1 antibodies on T cell proliferation. CD4 and CD8 (AlICells, LLC) were activated for 2 days with DYNABEADS™ CD3 / CD28 human T cell activators (for cell expansion and activation, Life Technologies; 11131D). The ratio of microspheres to cells used was 1:1, respectively, to induce PD-1. On day 2, the cultures were harvested, and the microspheres were separated from the activated T cells using magnetic force. The cells were then activated into dendritic cells expressing PD-L1 and the cells were incubated with different anti-PD-1 clones at 1 pg / ml in 96-well plates in triplicates at ratios of 1:10 DC:T cells and they were incubated in a humidified 99 incubator at 37 °C with 5% CO2. On day 3, cultures were boosted for 18 h with 1 pCi per well of [ 3 H]-thymidine before harvesting. Plates were then harvested onto DNA-specific filter papers (PerkinElmer) using Harvester96 (Tomtec Life Sciences). Radiolabeled filters were covered with beta scintillation fluid (Perkin Elmer) and read on Microbeta® counting plates (Perkin Elmer). Thymidine incorporation was analyzed as counts per minute (CPM). The results are shown as the mean of triplicates + SEM in Figures 3 and 4, Example 7: Determination of the Kinetics and Affinity of Human, Macaca fascicularis and Mouse PD-1 in Interaction with Humanized Anti-PD-1 Antibodies This example illustrates the binding of anti-PD-1 antibodies to human, Macaca fascicularis, or mouse PD-1. Interaction analysis was performed on label-free biosensors at 25 °C, unless otherwise noted. Surface plasmon resonance biosensors (ProteOn-XPR™ from BíoRad™, and Biacore 2000™ and Biacore T200™ from GE Life Sciences) were used to study human and Macaca fascicularis PD-1, and a biofilm interferometry biosensor was used (Octet-Red384, Fortebio / Pall Life Sciences) to study mouse PD-1. ProteOn experiments were performed in PBS, pH 7.4 + 0.01% Tween-20 running buffer (PBST). Biacore experiments were performed in 10 mM Hepes, pH 7.4, 150 mM NaCl, 0.05% Tween-20 (HBST+), and Octet experiments were performed in HBST+ with 1 g / L BSA. . ProteOn data was processed in ProteOn Manager software, Biacore data was processed in Biaevaluation, and Octet data was simply zeroed in control software. The SPR data were double checked (Myszka, 1999, J Mol Recognita2(5):279-284) and globally fitted to a simple Langmuir model to determine the equilibrium dissociation constant, KD, from the ratio of the kinetic rate constants (KD = kd / ka). Calibration Free Concentration Analysis (CFCA) The active concentration of human PD-1 (hPD-1) monomer (Sino Biologicals, Cat. No. 10377-H08H) for use as analyte in immobilized IgG kinetic experiments was determined empirically using a CFCA assay in a Biacore T200™ equipped with a CM5 sensor chip. To prepare the 100 For these experiments, a high capacity (approximately 12,000 RU) of mAb15 hlgG4 (or, in some experiments, competitor antibody C2-hlgG1) was amine-coupled into flow cell 2, leaving flow cell 1 empty (only “ activated and blocked”, without any IgG) to provide a reference surface. The hPD-1 samples were injected at nominal concentrations of 0.1, 1, and 10 pg / ml for 36 seconds at low (5 μΙ / min) and high (100 μΙ / min) flow rates. Surfaces were regenerated with a 2:1 v / v cocktail of Pierce IgG Elution Buffer (pH 2.8):4M NaCl. Data was analyzed in the CFCA tool in T200 software to derive a value of apparent activity for analyte hPD-1, which was used to correct its "nominal" protein concentration, as determined by absorbance at 280 nm with appropriate extinction coefficient, to obtain an "active" protein concentration. Some batches were found to be 32% active while others were 100% active. Kinetic analysis of human PD-1 (hPD-1) bound to amine-coupled mAb mAb7, mAb15, C1, C2, C3 and C4 A ProteOn-XPR36 equipped with GLC sensor chips (BioRad™, Hercules, CA) was used to determine the kinetics and affinity of hPD-1 monomer bound to a panel of amine-coupled anti-hPD-1 mAbs (mAb7, mAb15 , C1, C2, C3 and C4) in PBST running buffer. The surfaces for these experiments were prepared in three stages; (1) ligand channels were minimally activated for two minutes using a fresh mix of the final 0.8 mM EDC and 0.2 mM sulfo-NHS activating reagents in water, (2) IgGs were coupled for three minutes at 15 pg / ml in 10 mM sodium acetate, pH 4.5, and (3) excess reactive esters were blocked for three minutes with 1M ethanolamine, HCl, pH 8.5. Final levels of coupled IgG ranged from 400 RU to 1157 RU. The hPD-1 monomer was injected in a one-shot kinetic mode (Bravman et al., 2006, Anal Biochem 358(2):281-288) along with the "analyte" channels as a three-fold dilution series. with maximum "active" concentrations of 30, 44 or 36 nM, depending on the experiment. Association and dissociation times were 3 min and 20 min, respectively, and all analytes were injected in duplicate fixation cycles. Surfaces were regenerated with a 2:1 v / v cocktail of Pierce IgG Elution Buffer (pH 2.8): 4M NaCl. Table 13: Kinetic analysis of hPD-1 monomer bound to IgG coupled by 101 amine xx a 4 7 = = Interactions of hPD-1 monomer with mAb7 and mAb15 showed no visible decline in the binding response within the allowed dissociation phase, thus an upper limit was placed on the kd and KD values, applied according to the “5% rule” (Katsamba et al, 2008, Anal Biochem 352(2):208-221) to place an upper limit on your kd and KD values. N = 2 refers to two independent experiments on different chips. Cross-reactivity of mAb7 and mAb15 with Macaca fascicularis PD-1 Binding kinetics of recombinant purified Fab fragments (mAb7 and mAb15) to hPD-1-hFd (R&D systems cat. no. 1086PD) and cynoPD-1-hFd (prepared in-house) was determined using a Biacore 2000™ equipped with a CM4 sensor chip and HBST+ stroke damper. A polyclonal anti-hFc antibody was amine-coupled to the chip and used to capture approximately 90 RU of hPD-1-hFc1 and 125 RU of cynoPD-1-hFd in flow cells 2 and 3, leaving flow cell 1 empty. (bare anti-hFc capture surface) to provide a reference channel. Recombinant purified Fabs were injected for two minutes as anate at 0, 10 and 100 nM onto freshly captured PD-1-hFc1 fusion proteins, allowing 15 min dissociation time. Capture surfaces were regenerated using 75 mM phosphoric acid and mAb7 Fab samples injected in duplicate fixation cycles. All Fab / PD-1 complexes were very stable such that none of the interactions showed a visible decline in their binding responses within the allowed dissociation time, so the 5% rule was applied” (Katsamba et al , 2008) to place an upper bound on the kd and KD values. Table 14: Fab affinity determination of mAb7 and mAb15 with hPD-1-hFc1 and cynoPD-1-hFd fusion proteins 102 Analyte mAb7 Fab mAb7 Fab mAb15Fab mAb15Fab on chip hPD-1-hFc1 cynoPD-1-hFd hPD-1-hFd cynoPD-1-hFd ka (1 / Ms) X105 *kd (1 / s) x 10.5 KD (pNl) at 25 °C 5.67 <5.7 <101 5.26 <5.7 <108 9.16 <5, 7 <62 8.24 <5.7 <69 Temperature dependence of hPD-1 binding affinity to mAb15, mAb7 and C3 A Biacore T200™ equipped with a CM4 sensor chip was used to determine the kinetics and binding affinity of hPD-1 monomer to a panel of hlgG4 molecules (mAb15, mAb7 and C3) that were captured at low levels by amine-coupled anti-hFc polyclonal antibody. hlgG4 mAbs were captured at 10 pg / ml in individual flow cells, with flow cell 1 left empty to serve as a reference surface (bare capture surface). hPD-1 was injected at active concentrations of 0, 10, and 100 nM for three minutes, allowing for a dissociation phase of 18 min. Capture surfaces were regenerated with 75 mM phosphoric acid after each fixation cycle. Table 15: Kinetic analysis of hPD-1 monomer binding as analyte to hlgG4 molecules captured by anti-hFc hlgG4 on chip Temp (25 °C) ka (1 / Ms) kd (1 / s) Ko (nM) mAb15 25 3.49 x 105 1.71 x 10 0.49 mAb 15 37 6.94x105 3.73x10 0 .54 mAb7 25 2.37 x 105 1.73x10 0.73 mAb7 37 4.28 x 105 3.63 x 10 0.85 C3 25 2.14 x 105 2.25 x 10'3 10.5 C3 37 9 .70 x 105 1.56 x 10'2 16.1 Cross-reactivity of mAb7 and mouse PD-1 An Octet-Red384 equipped with streptavidin sensor tips was used to determine if mouse PD-1 binds to mAb7. An avidity propensity assay format was chosen to increase the detection sensitivity of the assay. Sensors were coated with biotinylated anti-human kappa polyclonal and used to capture a panel of anti-hPD-1 hlgG4 mAbs (mAb7, C1, C2 and C3) at 10 pg / ml; each mAb was captured on eight sensors. As a positive control, eight streptavidin sensors were coated with biotinylated J43 (eBioSciences), an anti-mouse PD-1 antibody. Each mAb-coated sensor was exposed to the following analytes; buffer, 1 µM mouse-PD-1-hFc binding sites, 1 µM hPD-1hFc binding sites (positive control), or 1 pM hGHR-hFc binding sites (negative control). All recombinant Fe fusion proteins were from R&D systems. Therefore, 103 each analyte / mAb interaction was evaluated on duplicate sensors. Anti-PD-1 antibodies C1, C2 and C3 did not bind to mouse-PD-1-hFd (data not shown). Anti-PD-1 mAb7 antibody bound weakly to mouse-PD-1-hFd (data not shown). All anti-PD-1 antibodies tested bound to hPD-1hFc. These results demonstrate that mAb7 is weakly cross-reactive with mouse PD-1, while C1, C2 and C3 are not cross-reactive with mouse PD-1. Example 8: Treatment of cancer with anti-PD-1 antibodies This is a predictive example illustrating the use of anti-PD-1 antibodies of the present invention to treat cancer. Patients with histologically confirmed, previously untreated, mild metastatic rectal cancer are selected for treatment with an anti-PD-1 antibody. Patients are assigned to one of two treatment groups: chemotherapy and placebo or chemotherapy and mAb7. A dynamic randomization algorithm is used to achieve an overall balance and within each of the following categories: study center, baseline ECOG activity grade (0 vs. §1), site of primary disease (colon vs. §1). rectum) and number of metastatic sites (1 vs. >1). Chemotherapy treatment is given weekly for the first 6 weeks of each 8-week cycle. Chemotherapy continues until the end of the study (96 weeks) or until disease progression. 5 mg / kg of mAb7 or placebo are given every 2 weeks. Patients in the mAb7 arm who have a confirmed complete response or experience unacceptable toxicity as a result of chemotherapy treatment may discontinue chemotherapy and continue to receive mAb7 alone as first-line treatment. Only patients who are randomized to the mAb7 arm can receive mAb7 as a component of second-line treatment. After completion of the study, patients are followed for further treatment and survival every 4 months until death, loss to follow-up, or study completion. Patients will be evaluated for tumor status at the start and end of each 8-week cycle using appropriate radiographic techniques, typically helical computed tomography. The investigator and the independent radiology center (IRF) will determine tumor response or progression using the Solid Tumor Response Evaluation Criteria. Therasse et al. (2000). The evaluation in an IRF will be carried out without knowledge of the allocation of the 104 treatment or evaluation of the investigator. In addition, patients will complete the Functional Assessment of Cancer Treatment—Colorectal (FACT-C), version 4, a validated instrument to assess quality of life (QOL) in patients with colorectal cancer, at baseline and before each cycle of treatment. until disease progression. Ward et al. (1999) Qual. Life Res. 8:181-195. Safety is assessed from reports of adverse events, laboratory test results, and measurement of vital signs. Adverse events and abnormal laboratory results are categorized using the National Cancer Institute Common Toxicity Criteria (NCI-CTC), version 2. Prespecified safety measures include four adverse events of special interest (hypertension, proteinuria, thrombosis and bleeding). The primary endpoint is the duration of overall survival. Secondary endpoints include: progression-free survival, objective response rate (complete and partial), duration of response, and change in FACT-C QOL index. Survival duration is defined as the time from randomization to death. For patients alive at the time of analysis, duration of survival will be assessed at the date of last contact. Progression-free survival is defined as the time from randomization to the onset of disease progression or death during the study, which is defined as death from any cause within 30 days after the last dose of the drug. study or chemotherapy. For patients alive without disease progression at the time of analysis, progression-free survival will be assessed at the last tumor assessment, or on Day 1 (the first day of study treatment) if no assessment was performed after baseline. In objective response analysis, patients without tumor assessments are categorized as non-responders. Disease progression analysis and response analysis are based on IRF assessments. The change in quality of life is analyzed as the time of deterioration in the QOL (TDQ), defined as the period of time from randomization to the start of a 3-point decrease in the initial value of the index of the FACT-C subscale. colon cancer-specific (CCS), disease progression or death during the study. The TDQ will also be determined for TOlC (sum of CCS, physical and functional well-being) and total FACT-C to determine changes from baseline. Example 9: Treatment of cancer with anti-PD-1 antibodies 105 This example illustrates the use of the anti-PD-1 antibodies of the present invention to treat cancer. The study in this example is a Phase 1, open-label, multi-center, multiple-dose and dose-escalation, safety, pharmacokinetic (PK), and pharmacodynamic (PD) study of the monotional anti-PD-1 antibody mAb7 administered intravenously. in previously treated adult patients with locally advanced or metastatic melanoma, squamous cell head and neck cancer (SCHNC), ovarian carcinoma, sarcoma, or refractory or relapsed classical Hodgkin's lymphoma (cHL). The study protocol is summarized below in Table 16. Table 16 Arms Group 1: mAb7 0.5 mg / kg every 21 days Group 1: mAb7 1.0 mg / kg every 21 days Group 1: mAb7 3.0 mg / kg every 21 days Group 1; mAb7 10 mg / kg every 21 days Assigned interventions Drug mAb7 IV every 21 days Drug mAb7 IV every 21 days Drug mAb7 IV every 21 days Drug mAb7 IV every 21 days Inclusion criteria: - Histological or orthological diagnosis of locally advanced or metastatic melanoma, SCCHN, ovarian cancer, sarcoma, or refractory or recurrent cHL: - Patient should receive at least 1 and no more than 5 prior lines of treatment for recurrent or metastatic disease, including standards of care and experimental treatments. - At least one measurable lesion defined by RECIST version 1.1, or (for cHL) at least 1 measurable lesion by positron emission tomography (FDG PET) fluorodeoxyglucose uptake (Deauville 4 / 5) >1.5 cm as defined using the response criteria for malignant lymphoma that was not previously irradiated. - For the Part 1B expansion and all Part 2 cohorts: Patient consented to prior treatment and treatment biopsy. - Exclusion criteria for adequate kidney, liver, and bone marrow function Active leptomeningeal or brain metastasis. -Ocular melanoma - Known or apparent active autoimmune disease. Patients with vitiligo, type I diabetes mellitus, residual hypothyroidism due to an autoimmune condition requiring hormone replacement only, psoriasis not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger may be enrolled. Diagnosis of prior immunodeficiency or organ transplant that required immunosuppressive treatment, - For Part 2: prior treatment with an antibody against PD 1 or PD L1. - History of grade £3 immune-mediated AE (including AST / ALT elevations considered drug-related, and 106 cytokine release syndrome), which was considered related to immunomodulatory therapy (eg, checkpoint inhibitors). of the immune system, co-stimulatory agents, etc.) and who required immunosuppressive treatment. The number of patients with ORR (objective response rate) is measured at baseline and every six weeks until disease progression or unacceptable toxicity, up to 24 months. Example 9: Antagonistic activity of anti-PD-1 antibodies in primary human and Macaca fascicularis T lymphocytes This example illustrates the activity of anti-PD-1 antibodies in primary human and Macaca fascicularis T lymphocytes, In this study, the antagonistic activities of the anti-PD-1 monoclonal antibody mAb7 were examined in vitro using mixed lymphocyte reaction (MLR). Primary T lymphocytes were isolated from human and Macaca fascicularis peripheral blood mononuclear cells (PBMC). After exposure to Ab7, cell proliferation and cytokine secretion were assessed in vitro under different activation conditions using an MLR for human and Macaca fascicularis and a cytokine release assay using Macaca fascicularis blood activated with the enterotoxin superantigen. Staphylococcal B (SEB). Methods Human T lymphocytes Human buffy coat was obtained from the Stanford Blood Center (Stanford, CA), diluted with phosphate buffered saline (PBS) and plated on Ficoll for PBMC isolation. The huPBMCs were washed 4 times with PBS, and the T cells were isolated using a human Pan-specific T cell isolation kit with negative selection, as described in the manufacturer's protocol (Mlltenyi Biotec, San Diego, CA). Macaca fascicularis T lymphocytes Fresh Macaca fascicularis PBMC were obtained from Bioreclamation IVT (New York, NY) and washed twice with PBS. T cells were isolated using a non-human primate specific Pan T cell isolation kit with negative selection, as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA). Generation of Human Dendritic Cells Expressing High Levels of PD-L1 Human buffy coat was obtained from Stanford Blood Center (Stanford, CA), 107 diluted with PBS and plated on Ficoll to isolate hu-PBMCs. hu-PBMCs were washed 4 times with PBS, and differentiation cluster 14 (CD14+) monocyts were isolated using a positive selection human CD14-specific lymphocyte isolation kit, as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA). Cells were then seeded at 5 x 10 5 cells / mL in complete Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) for 7 days. Cultures were spiked with recombinant human IL-4 (rh-) (1000 U / mL) (R&D Systems, Minneapolis, MN) and recombinant human granulocyte-macrophage colony-stimulating factor (GMCSF) (rh-GMCSF) (500 U / mL). U / ml) (R&D Systems, Minneapolis, MN) on Days 0, 2, and 5. Immature DC were harvested, washed, and counted on Day 7. A sample from each preparation was evaluated for PD- expression. L1 using r-phycoerythrin (RPE)-tagged anti-huPD-L1 (eBioscience / Affymatrix, San Diego, CA) by flow cytometry using an LSRFortessa™ analyzer (BD Biosciences, San Jose, CA). Generation of Macaca fascicularis dendritic cells expressing high levels of PDL1 Macaca fascicularis PBMCs were obtained from Bioreclamation IVT (New York, NY) and washed twice with PBS. CD14+ monocytes were isolated using a nonhuman primate-specific CD14 lymphocyte isolation kit with positive selection, as described in the manufacturer's protocol (Miltenyi Biotech, San Diego, CA). Cells were then seeded as 5 x 105 cells / ml in complete RPMI 1640 medium supplemented with 10% FBS for 7 days. Cultures were spiked with rhIL-4 (1000 U / ml) (R&D Systems, Minneapolis) and rhGMCSF (500 U / ml) (R&D Systems, Minneapolis, MN) on Days 0, 2, and 5. Immature DC were harvested, were washed and counted on Day 7. A sample from each preparation was assessed for PDL1 expression using RPE-labeled anti-hu-PD-L1 (eBioscience / Affymatrix, San Diego, CA) by flow cytometry using an analyzer. LSRFortessa™ (BD Biosciences, San Jose, CA). Generation of JeKo-1-Luc-Green Fluorescent Protein Cell Clones Expressing High Levels of Human PD-L1 The JeKo-1 cell line (a mantle cell lymphoma) was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The JeKo-1 -luc2A-GFP cell line was produced at Pfizer (South San Francisco, CA) by a transduction process using lentiviral particles individually expressing luciferase. Firefly 108 (luc2A) and green fluorescent protein (GFP) via a bicistronic system with a blasticidin label (AMSBIO, LVP323, 1*10E7 particles per 200 μ!) according to the manufacturer's protocol. JeKol cells were pelleted and diluted to 1 x 10 6 cells / ml in RPMI with 20% FBS medium. Lentiviral particles were added to the cells diluted in a ratio of 50 μΙ of virus per 0.5 ml of cells. To generate a JeKo-1 cell line expressing huPD-L1, hu-PD-L1 cDNA was custom synthesized and cloned into a generic expression vector (pcDNA3.1) by Life Technologies (San Diego, CA). A JeKol Luc-GFP cell line stably expressing hu-PDL-1 was produced at Pfizer (South San Francisco, CA) by electroporation using the Amaxa® Nucleofector system (Lonza, Walkersville, MD) and Kit V, according to with the manufacturer's protocol (Lonza, Walkersville, MD). Cells were then cultured in the presence of 250 pg / ml hygromycin for 2 weeks and then selected by cell sorting using the BD FACSAria™ II Cell Sorter (BD Biosciences, San Jose, CA). Cell sorting was performed with the anti-hu-PD-L1 antibody clone MIH-1 (Affymetrix / eBioscience, San Diego, CA) directly labeled with the allophycocyanin (APC) tag. Positive clones were expanded and evaluated for elevated PDL1 expression using flow cytometry (LSRFortessa™ analyzer, BD Biosciences, San Jose, CA). Clones generated from single detached cells and containing high levels of PD-L1 expression were selected. Antibody generation mAb7s were generated in CHO cells (Pharmaceutical Sciences, Pfizer Inc, Saint Louis, MO) using Good Laboratory Practice (GLP) material (Lot #STL0005717) and supplied in 20 mM de-histidine, 85 mg / ml sucrose, 0.2 mg / ml polysorbate-80, 0.05 mg / ml disodium EDTA, pH 5.5 buffer. Endotoxin was measured as <0.01 EU / mg. The control antibody used in all in vitro assays was an anti-bovine herpes virus cloned in frame IgG4-HG (same frame as MAB7). Control antibody was generated from Pfizer (South San Francisco, CA), Lot #4945, with <0.3 EU / mg endotoxin. Two anti-hu-PD-1 antibodies were generated from the sequences published in earlier patents and expressed in the framework of IgG4-HG, so as to resemble that used for MAB7. Antibodies were generated at Pfizer (South San Francisco, CA) and designated Positive Control 1 and Positive Control 2 (Lots 109 Nos. 5053 and 4255, respectively). Endotoxin was measured as <0.13 EU / mg and <0.056 EU / mg, respectively. Human trial using dendritic cells expressing high levels of PD-L1 The protocol was adapted from Kruisbeek et al, 2004, with some modifications. Differentiated primary huDCs were harvested on Day 7 and checked by flow cytometry for high levels of PD-L1 expression and by costimulatory signals for T cell activation; markers included CD80 and CD86 (antibodies obtained from BD Bioscience, San Jose, CA). Cells were counted and irradiated at 3000 radiation units (rad) using an RS2000 X-ray machine (Radsource, Brentwood, TN) to prevent DC from secreting cytokines, but functioning only as Ag presenting support for T lymphocytes. Therefore, the assay result was induced by T cells only. On Day 7, freshly isolated human T cells were harvested from allogeneic donors. T cells were plated with irradiated DC in a ratio of 10.Ί (optimal assay conditions determined to be 2 x 105 T cells incubated with 2 x 104 DC in 200 μΙ cultures) in the presence of different concentrations of mAb7, positive and negative control antibodies, or medium alone (to assess reaction at baseline). All conditions were plated in tissue culture treated 96-well flat bottom plates (Fisher Scientific Pittsburgh, PA). Cells were cultured using serum-free X-vivo15 medium (Lonza, Walkersville, MD) to avoid variability of human serum between experiments. Cultures were incubated at 37°C with 5% CO2 for 5 days. On Day 5, supernatants were harvested, and cytokine concentrations were measured by cytometric bead array (CBA) (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Data were acquired by flow cytometry (LSRFortessa™ Analyzer, BD Biosceinces, San Jose, CA), and data analysis was performed using BD FCAP Array software, version 3.0 (BD Biosceinces, San Jose, CA). Proliferation was measured in parallel cultures by adding 1 pCi of 3 H methyltitrated thymidine (Perkin Elmer, Waltham, MA) to each well and also incubating for 16-18 hours. Cultures were then harvested onto deoxyribonucleic acid (DNA) incorporation filters (Perkin Elmer, Waltham, MA), and tritiated thymidine incorporation provided a cell proliferation index measured as counts per minute (cpm) using the MicroBeta2 machine (Perkin Elmer, Waltham, Mass.). 110 Assay in humans using the JeKo1-PDL1 expressing cell line This assay was performed using a 5:1 ratio of T cells to the JeKo-1-PD-L1 cell line, as this ratio provided a more ideal approach to capture cytokine secretion on Day 5. Therefore , each well was incubated with 2 x 105 T cells with 4 x 104 JeKo-1-PD-L1. On Day 5, supernatants were collected, and cytokine concentrations were measured using CBA (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. This cell line expresses a very modest amount of costimulatory molecules (CD80 and CD86), and thus cell proliferation is very slight after antibody treatment. In contrast, cytokine secretion, including IL-2, is considerable, allowing measurement of cytokine secretion after! mAb7 treatment and not the proliferation of T lymphocytes. Assay in Macaca fascicularis using dendritic cells expressing high levels of PDL1 Differentiated DCs were harvested on Day 7 and verified by flow cytometry (using the LSRFortessa™ Analyzer, BD Biosceinces, San Jose, CA) for elevated expression of PDL1 and costimulatory signals required for T cell activation; markers included CD80 and CD86 (antibodies from BD Bloscience, San Jose, CA). Cells were counted and irradiated at 3000 rad using an RS2000 X-ray machine (Radsource, Brentwood, TN). Freshly isolated Macaca fascicularis T lymphocytes were collected from allogeneic donors. T cells were plated with irradiated DC using a 10:1 ratio of T cells to DC (assay was optimal when 2 x 105 T cells were incubated with 2 x 104 DC in 200 μΙ culture) in presence of different concentrations of mAb7, positive and negative control antibodies, or medium alone to assess the reaction at baseline. All conditions were plated in tissue culture-treated 96-well flat bottom plates (Fisher Scientific, Pittsburgh, PA). Cells were cultured using serum-free X-vivo15 medium (Lonza, Walkersville, MD) to avoid serum variability between experiments. Cultures were incubated at 37°C with 5% CO2 for 5 days. On Day 5, supernatants were collected, and cytokine concentrations were measured using CBA (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Data was acquired by flow cytometry (LSRFortessa™ Analyzer, BD Biosceinces, San Jose, CA) and data analysis was performed using BD FCAP Array software, 111 version 3.0 (BD Biosciences, San Jose, CA). At the same time and in similar cultures, 1 pCI of 3 H methyltritiated thymidin (Perkin Elmer, Waltham, MA) was added to each well; cells were also cultured for 16-18 hours to measure proliferation. Cultures were harvested on Printed glass filtermate A DNA Incorporation filters (Perkin Elmer, Waltham, MA), and Incorporation of tritiated thymidine, which provides an index of cell proliferation, was measured as cpm using the MicroBeta2 machine (Perkin Elmer , Waltham, Mass.). Macaca fascicularis blood cytokine release by stimulation with superantigen (staphylococcal enterotoxin B) Blood was collected from Macaca fascicularis, 225 μΙ of blood was aliquoted into tissue culture-treated 96-well plates (Flsher Scientific, Plttsburg, PA). Samples were incubated in duplicate at 37°C in 5% CO 2 in the presence of mAb7 or an isotope-matched negative control antibody at concentrations ranging from 0.1 to 100 pg / ml. One hour after antibody addition, samples were stimulated with 0.1 pg / ml SEB (Toxlc Technologies, Sarasota, FL), and cultures were incubated for 3 days. On Day 3, plasma was collected, pooled and frozen at 80°C. Concentrations of IFN-γ, IL-2, and TNF-α in thawed serum samples were measured in duplicate according to the manufacturer's protocol using MSD immunoassay plates (Meso Scale Diagnostics, Rockville, MD) and an MSD reader (Model 1200) with MSD Discovery Workbench software (version 4.0.12). Means of duplicates were reported. Results Antagonist activity of mAb7 in primary human T cells When primary human T cells were activated in MLRs with allogeneic human DC expressing PD L1, mAb7 increased T cell proliferation (measured by tritiated thymidine incorporation) and T cell activation (measured by proinflammatory cytokine secretion) of a dose-dependent manner. Treatment of T cells with mAb7 (10 pg / ml) resulted in an increase in T cell proliferation of up to 2.5-fold compared to treatment with a negative control antibody (10 pg / ml). IFN-γ and TNF-α levels increased up to 8-fold and 5-fold, respectively, when compared to the negative control antibody. The increase in IFN-γ was greater than the increase in TNFa. IL-2 expression was not detected in these cultures. When primary human T cells were activated in MLRs, using the JeKo 1 PD-L1 tumor cell line as allogeneic antigen-presenting cells, mAb7 induced an increase 112 dose-dependent increase in secretion of IFN-γ (up to 2.5-fold), TNF-α (up to 2-fold) and IL-2 (up to 5-fold) compared to the negative control antibody. The effect of mAb7 in this assay was similar to the data obtained with both positive control antibodies. No increased proliferation of T lymphocytes was observed under these conditions. Cell proliferation was minimal with a weak signal provided by CD80 and CD86, compared to MLR mediated by primary DCs. iL-10, IL-4, IL-17A, and IL-6 levels were minimal to undetected in all assays described above. Antagonistic activity of mAb7 in primary Macaca fascicularis T lymphocytes The binding affinity of mAb7, in solution, to human PD-1 and Macaca fascicularis PD-1 was very similar in the kinetic exclusion assay (KinExA) (KD = 23 and 28 pM for human and Macaca fascicularis PD-1). , respectively). The EC50 of mAb7 in cells expressing human PD-1 and Macaca fascicularis PD-1 was also similar. In a functional MLR assay using T cells and DC isolated from different Macaca fascicularis monkeys, mAb7 induced T cell proliferation and activation in a dose-dependent manner (measured by incorporation of tritiated thymidine). This effect was also seen with the positive control (antibody 1 and antibody 2), but not with the negative control antibody. mAb7 also enhanced cytokine secretion (ie, IFN-γ and TNF-α, by up to 5-fold and 3-fold, compared with negative control antibody treatment. IL2 expression was not detected in these cultures. In another cytokine release assay using Macaca fascicularis blood stimulated with 0.1 pg / ml SEB superantigen for 3 days, mAb7 (0.1-100 pg / ml) induced increased secretion of IFN-γ, IL- 2 and TNF-α compared to the negative control antibody. MLR studies were used to create an in vitro environment resembling the tumor environment, where T cells are activated and express high levels of PD-1 in the presence of allogeneic DC or PDL1-expressing tumor cells. The PD-1 / PD-L1 interaction inhibits further T cell proliferation and cytokine release. Addition of anti-PD-1 antibodies to these MLRs restored activation and increased T cell proliferation and cytokine secretion, especially IFN-γ, due to blockade of the PD-1 / PD-L1 axis. mAb7 accelerated the proliferation and secretion of iFN-γ, TNF-α, and IL-2 by human T cells when cultured with allogeneic cells expressing high levels of PD-L1 (JeKo 1 PD-L1 or DC cell line). In contrast, the negative control antibody, which was shown to be similar to medium alone, did not enhance these effects. Of Similarly, mAb7 enhanced T cell proliferation, IFN-γ and TNFα secretion in the Macaca fascicularis MLR system, and also enhanced Macaca fascicularis blood cytokine release using the SEB superantigen. These results demonstrate that the anti-PD-1 mAb7 antibody enhanced T cell proliferation and proinflammatory cytokine secretion, including interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL )two. These activities were observed in primary human and Macaca fascicularis T lymphocytes. Example 10: Effect of Anti-PD-1 Antibodies in Graft Versus Host Disease This example illustrates the effect of anti-PD-1 antibodies on in vivo activation and expansion of T cells using a xeno-aGvHD model in NSG mice transferred to hu-PBMC. In this study, the effect of anti-PD-1 antibody mAb7 on in vivo T cell activation and expansion was studied in an acute xenograft-versus-host disease (aGvHD) model in non-obese diabetic (NOD) mice, with Severe combined immunodeficiency (SCID), interleukin 2 gamma (IL2rYnul') receptor null (NSG) using human peripheral blood mononuclear cells (PBMC). Immunocompromised female NSG mice (5 per group), 8 to 10 weeks old (formal name, NOD, CgPrkdcscldH2rgtmlW|l / SzJ) were obtained from Jackson Laboratory (Bar Harbor, ME). All animals were housed in a pathogen-free facility at Pfizer (South San Francisco, CA) in accordance with the Institutional Animal Care and Use Committee (IACUC). Human buffy coat was obtained from the Stanford Blood Center (Standard, CA), diluted with phosphate buffered saline (PBS) and plated on Ficoll for PBMC isolation. PBMCs were washed 2 times with PBS. Red blood cells (RBCs) were lysed using ammonium chloride-potassium (ACK) lysis buffer as indicated in the manufacturer's protocol (Life Technologies, San Diego, CA). After RBC lysis, cells were washed once more and diluted in PBS to 5 x 10 7 cells per ml. For induction of xeno-aGvHD, NSG mice received hu-PBMC via intravenous injection in the tail vein. Each mouse received 1 x 10 7 cells in 200 pl PBS. In all experiments, mice were weighed 3 times per week and 114 were monitored for xenoaGvHD-like symptoms, including weight loss, stooped posture, wavy fur, reduced mobility, and, in some cases, diarrhoea. Mice were euthanized after losing 20% ​​body weight or losing 1 g / day within 2 days; this time point was recorded as the survival time. mAb7, negative control antibody, and positive control antibodies were generated as described above in Example 9. NSG mice were treated with negative control antibodies, positive control antibodies, or mAb7, at doses ranging from 0.1-10 milligrams per kilogram (mg / kg). Antibodies were administered to mice using the appropriate vehicle on Day 2 and Day 8 after hu-PBMC transfer. Antibodies were administered intraperitoneally (ip). Blood, spleens, and livers were collected from mice transplanted with hu-PBMC. Single cell suspensions from each organ were prepared as follows: Peripheral blood was collected, and RBCs were lysed using ACK lysis buffer and washed with PBS. Spleens and livers were mechanically homogenized using the back of a syringe plunger to macerate cells through a 70 μM filter and washed once with PBS. The RBCs were lysed, and the cells were washed an additional 2 times and macerated again through a 70 μM filter. At this stage, the spleen cells were ready. To isolate liver leukocytes, single cell suspensions were layered using a 30% to 80% Percoll gradient (Percoll® Plus, GE Healthcare Bio-Sciences, Pittsburgh, PA) and subjected to high speed centrifugation. Leukocytes were collected from the middle layer and washed twice with PBS. All single cell suspensions were counted and 1x10 ® cells from each sample were used for fluorescence activated cell sorting (FACS). For human cytokine secretion assays, mouse CD45+ lymphocytes were depleted using a mouse CD45-specific lymphocyte isolation kit by positive selection, as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA), to ensure that cytokine secretion was from human immune cells only. Cells were counted, and 1 x 10 ® cells from each sample were used in the assay. Single cell suspensions from peripheral blood, spleens and livers were obtained from xenoaGvHD mice. A total of 1 x 10® cells per sample was incubated for 30 minutes at 4°C protected from light with a mixture of antibodies 115 fluorescently labeled monocells in FACS buffer including 1 x PBS containing 2% fetal bovine serum (FBS), then cells were washed with FACS buffer. For intracellular protein Kiel 67 (Ki67, to measure cell proliferation) staining, cells were fixed after surface staining using the Intracellular Permeabilization and Fixation Buffer Set (Affymetrix / eBioscience, San Diego, CA) according to with the manufacturer's protocol. Intracellular staining was performed for 30 minutes at 4°C in the dark. At the end of staining, samples were washed and subjected to multicolor flow cytometry using the BD LSR Fortessa™ Cellular Analyzer (BD Biosciences, San Jose, CA). Data analyzes were performed using FlowJo software (FLOWJO LLC, Ashland, OR). The antibodies used in different combinations for the recognition of cell surface molecules were: anti-hu-CD45 Pacific Blue (PB) or Amcyan, anti-mouse CD45-Brilliant Violet (BV)-711, anti-hu-CD3-PerCPCy5. 5, anti-hu-CD8 phycoerythrin-Cy7 (PE-Cy7) or BV-786, anti-hu-CD4-fluorescein isothiocyanate (FITC) or BV-650, anti-hu-PD-1 (clone EH12.1) BV -786 or PE, anti-hu-PD-1 (clone MIH-4) PE or FITC, anti-hu-K¡67 allophycocyanin (APC) (all antibodies from BD Biosciences, San Jose, CA), anti-hu -PD-L1 (PEAffymatrix-eBioscience, San Diego, CA) and Fluorescent Reactive Dye for Live / Dead Staining (Life Technologies, Grand Island, NY). Single cell suspensions of human CD45-enriched population from spleens and livers were obtained from xeno-aGvHD mice (after mouse CD45 knockdown). A total of 1 x 106 cells per sample was incubated in 96-well tissue culture-treated plates (Fisher Scientific; Pittsburg, PA) in 200 μΙ of X-vivo 15 medium. Cells were then left unstimulated or stimulated with acetate. of phorbol myristate (PMA) 10 ng / ml and Ionomycin (iono) 125 ng / ml as the low stimulation condition, or PMA 50 ng / ml and Ionomycin 1 pg / ml as the high stimulation condition (both obtained from Sigma-Aldrich, Saint Louis, MO). Cultures were incubated for 8 hours at 37°C in 5% CO2 to ensure maximal cytokine secretion. Supernatants were collected, and human cytokine concentrations were measured using the human cytokine-specific cytometric bead array (CBA) (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Data was obtained using flow cytometry (LSRFortessa™ analyzer, BD Biosciences, San Jose, CA), and data analysis was performed using FCAP array™ version 3.0 software (BD Biosciences, San Jose, CA). The hu-IFN-γ, huTNF-ct, and hu-IL-2 microsphere array kits were obtained from BD Biosciences (San Jose, CA), and it was confirmed that 116 they are not cross-active with mouse cytokines. All analyzes involved a comparison of means using independent samples t-test or two-way ANOVA using Graphpad Prism (Graphpad Software, San Diego, CA). P values ​​<0.05 were considered statistically significant. Graft data are reported in the figures as concentration means, including standard error of the mean (sem). Treatment of immunosuppressed NSG mice with anti-PD-1 mAb7 antibody accelerated body weight loss (Table 17) and induced other signs of disease commonly observed in this model, such as hunched posture, wavy fur, reduced mobility, and, in some cases, diarrhea. In this model, body weight loss is predicted to accelerate between Day 20 and Day 30 after transfer (see, eg, Schroeder and DiPersio, 2011). Because treatment with mAb7 and positive controls 1 and 2 accelerated xeno-aGvHD symptoms, mice had to be euthanized at earlier time points, and a survival curve could not be obtained; therefore, body weight loss was e! primary endpoint. In the first experiment, mice were treated with 0.1, 1, and 10 mg / kg of mAb7 or negative control antibody at 10 mg / kg. In Table 17, body weight loss was calculated by normalizing body weight differences between treated groups on Day 23 relative to Day 0. Values ​​indicate an average of 5 mice per group ± sem. Table 17 Body Weight Groups Weight Weight # per Loss of treatment Day 0 body Day 14 body Day 23 group body weight Negative control mAb; 10 mg / kg 20.68 ± 0.36 22.00 ± 0.36 20.88 ± 1.11 5 0% mAb7: 10 m / kg 20.78 ± 0.36 17.76 ±0.57 17, 74 ± 0.86 5 15% mAb7:1 mg / kg 21.52 ±0.36 19.26 ±0.35 17.7 ±0.58 5 15% mAb7: 0.1 mg / kg 20.5 ± 0.78 19.78 ± 1.38 19.22 ± 1.95 5 7.95% Mice were treated with the indicated antibodies on Day 2 and Day 8 after hu-PBMC transfer. While body weight loss became evident in the control group on Day 23, body weight loss and disease progression were detected in individual mice from all treated groups. 117 with anti-PD-1 mAb7 antibody from Day 10-11 after transfer of human PBMC. In both the 1 mg / kg and 10 mg / kg treated groups, body weight loss reached significance between Days 14 and 23, compared to the negative control treated group (p<0.0001). On Day 23, a 15% weight loss was detected in the 1 mg / kg and 10 mg / kg treated groups, and a 7.9% weight loss was detected in the 0.1 mg / kg treated group. kg (Table 17). FACS analyzes of peripheral blood, liver, and spleen demonstrated an increased percentage of huCD45-expressing cells and huCD3-expressing T cells in the mAb7-treated group compared to the control group. Representative data from spleens also showed an increase in hu-CD8-expressing T-cell counts and hu-CD4-expressing T-cell counts (but not less) in mAb7, compared with the control antibody treatment group. negative. A similar increase in human T cell counts was also observed in liver and blood. To assess whether the increase in T cells was due to blockade of PD-1 by binding of mAb7 to human T cells, T cells were stained with a commercial antibody to detect PD-1 (clone EH12.1) that competed with mAb7 binding. mAb7-treated lymphocytes from all organs (analyzed by FACS) did not show any binding to EH12.1. To confirm that mAb7-treated T cells still expressed PD-1, T cells were stained with a different anti-hu-PD-1 clone (clone MIH4) that partially competed with mAb7 for binding to T cells. The results showed that mAb7-treated T cells partially bound MIH4; and this indicates that PD-1 was expressed in the treated T cells and that blockade of PD-1 by mAb7 was evident (data not shown; maintained on internal Pfizer registries). No detectable changes were observed for hu-PDL1 expression in the spleen, in T cells (expressing hu-CD3), or in non-human T cells (not expressing huCD3). Ki67, a marker of lymphocyte proliferation, was elevated in hu-CD3-expressing cells from the PF0681591-treated groups compared to the negative control-treated group in the spleen (middle panel). Similar results were seen in blood and liver. To examine the effects of mAb7 on cytokine release during xenoaGvHD, human CD45-expressing cells (from livers and spleens) were further isolated from mouse CD45-expressing cells. These lymphocytes were then treated with a mixture of PMA and ionomycin (in 2 concentrations: low vs. high) or left untreated for 8 hours at 37°C. The secretion of 118 cytokine was measured in the supernatants by CBA (Table 18). No detectable toxins were obtained without stimulation (Table 18). After mAb7 treatment, hu-IFN-γ, hu-IL2, and hu-TNF-α were elevated in human lymphocytes isolated from spleen and liver compartments compared to control groups. Under weak ex vivo stimulation conditions, mAb7-treated T cells increased secretion of hu-IFN-γ to levels that were considerably higher than those of T cells isolated from the negative control group (p<0.05). Under stringent ex vivo stimulation conditions, all cytokines were induced in all groups, but were increased more in mAb7-treated T cells compared to T cells isolated from negative controls. Table 18 shows the data collected from 5 different mice in each group. In Table 18, *p<0.05, **p<0.01 in unpaired t-test comparing mAb7 group and negative control group; aGvHD = acute graft-versus-host disease; Hu = human; - IFN-γ = interferon gamma; IL-2 = interleukin-2; lno = ionomycin; N = number; ns = Not significant; P = P-value; PMA = phorbol myristate acetate; TNFa = tumor necrosis factor alpha; Xeno = xenogeneic. Table 18 Lymphocytes Human Human Organ Cytokine Stimulation Conditions pg / ml isolated ex vivo (PMA / lono) (ng / ml) Liver Weak Spleen (10 / 125 ng / ml) IFN-γ Liver Spleen IL-2 Liver Spleen TNF-a Liver Spleen Liver Rigorous (50 / 1000 ng / ml) IFN-γ Spleen IL-2 In vivo treatment N Unpaired t-test statistic mAb7 10 mg / kg Negative control mAb 10 mg / kg 414.1 ± 163.9 ± 5 **P<0.05 59.4 43.41 2599.96 ± 503, 9 ± 5 *P<0.01 863.45 111.9 783.1 ± 19.41 ± 5 ns 776.26 5.29 1813.98 ± 14.26 ± 5 ns 840.26 1.40 80.75 ± 9.92 ± 5 ns 60.45 2.55 368.81 ± 14.1 ± 5 ns 209.73 2.51 3838.65 ± 2931.26 ± 5 ns 178.84 556.98 3084.1 ± 2523 .9 ± 5 ns 204.1 141.2 7759.44 ± 4565.26 ± 5 ns 809.24 1240.8 16735.57 13204.5 ± 5 ns 119 ± 1417 1834.3 Liver 1611.9 ± 758.1 ± 5 **P<0.01 TMC ~ 150.82 188.2 Spleen 1 Nr-a 2842.6 ± 2311.7 ± 5 ns 224.2 281, 36 Liver None 3.1 ± 1.25 4.6 ± 0.96 5 ns Spleen (0 / 0 ng / ml) IFN-y 3.94 ± 4.86 ± 5 ns 0.97 1.16 Liver 3, 9 ±0.14 4.66 ± 5 ns Spleen IL-2 3.23 ± 0.41 4.39 ± 5 ns 0.77 0.55 Liver TNF-a 0.1+0.09 0.3 ±0 .2 5 ns Spleen 0.4 ±0.15 0.27 ±0.3 5 ns These results showed that treatment of xeno-aGvHD with anti-PD-1 mAb7 antibody accelerated the development of xeno-aGvHD in NSG mice, as measured by body weight loss, T cell proliferation, and increased cytokine secretion, including interferongamma (IFN-γ) and interleukin-2 (IL-2). Example 11: Characterization of anti-PD-1 antibodies This example illustrates the binding affinity, specificity, and ligand blocking activity of anti-PD-1 antibody mAb7 on cells expressing the PD-1 surface receptor. mAb7, negative control antibody, and positive control antibodies were generated as described above in Example 9. Human anti-PD-1 antibody clone EH12.1, initially labeled with phycoerythrin (PE) or Brilliant Violet (BV) -786, and isotype control antibodies labeled with the same dyes were purchased from BD Biosciences (San Jose, CA). PE-labeled mouse anti-PD-1 clone J43 and hamster anti-lgG isotype control antibody were purchased from Affymetrix / eBiosciense (San Diego, CA). Biotinylated hu-PD-L1 and biotinylated hu-PDL2 (CD273) were obtained from AGRO Biosystems (Newark, DE), Macaca fascicularis PD-L1 and PDL2 recombinant proteins were purchased from Creative BioMart® (Shirley, NY), and both Macaca fascicularis PD-L1 and PD-L2 were internally labeled with Alexa Fluor® 647 stain using an Alexa Fluor® 647 protein labeling kit (LifeTechnologies, San Diego, CA) as directed by the manufacturer's protocol. were evaluated to determine their binding to the Macaca fascicularis PD-1 expressing cell line. The Fe tags of PD-L1 from 120 rat and mouse (Fe region of human IgG1 at the C-terminus) recombinant proteins were purchased from Creative BioMart® (Shirley, NY). Detection of biotinylated PDL1 and PDL2 was achieved using streptavidin-PE or aiophycocyanin (APC) (Affymetrix / eBiosciense, San Diego, CA). Detection of mouse or rat PD-L1 was achieved using F(ab')z donkey anti-human IgG, specific for gamma Fe (Fcy) fragment, labeled with APC AffiniPure (Jackson ImmunoResearch Laboratories Inc, West Grave, PA). ). Vectors for transient transfection The hu-PD-1 expression plasmid (in the pCMV6 entry vector) was purchased from OriGene Technologies, Inc (Rockville, MD), Catalog No. RC210364 / Accession No. NM_005018. The mouse PD-1 expression plasmid (in the pCMV6 entry vector) was purchased from OriGene Technologies, Inc (Rockville, MD), Catalog No. MR227347 / Accession No. NM_008798. Life Technologies (San Diego, CA) codon optimized and synthesized Macaca fascicularis PD-1, Lot No. 1482149 / Accession No. EF443145. It was cloned into a proprietary cytomegalovirus (CMV)-based expression plasmid by Pfizer (San Francisco, CA), in frame with a mouse kappa secretion signal sequence that added a C-terminal FLAG tag to the C-terminal. Rat PD-1 deoxyribonucleic acid (DNA) was custom synthesized according to the sequence Accession No. NM_001106927 and cloned into BamHI-Notl sites of the expression vector pEF1V5-His from LifeTechnologies (San Diego, CA ), Lot No. 1598305. PD-1 expression vectors for stable transfection Human PD-1 DNA was custom synthesized according to the sequence Accession No. NM_005018 and cloned into BamHI-Notl sites of the pEF1V5-His B expression vector from InVitroGen, Lot No. 513478. Macaca fascicularis PD-1 DNA was custom synthesized according to Accession No. EF443145 and cloned into BamHI-Notl sites of pEF1V5-His B expression vector from InVitroGen, Lot No. 1482149. Mouse PD-1 DNA was custom synthesized according to Accession No. NM_008798 and cloned into BamHI-NotI sites of pEF1V5-His B expression vector from InVitroGen, Lot No. 1513476. Life Technologies synthesized and cloned all vectors into the pEF1V5-His B expression vector from InVitroGen (San Diego, CA). All vectors contain the complete PD-1 sequence including the extracellular domain, the 121 membrane and cytosolic domain. All vectors encoded a V5-6His epitope tag at the C-terminus of PD-1. The neomycin resistance gene was included in each vector for selection using G418 sulfate antibiotics. Transient transfection using the HEK-293T cell line HEK-293T cells were purchased from the American Type Culture Collection (ATCC®, Manassas, VA), and cells were maintained in Duibecco's Modified Eagle's Medium (DMEM) (Corning CellGro, Manassas, VA) supplemented with 10% serum. fetal bovine (FBS) and 1 x penicillin / streptomycin (Pen / Strep) and cultured in 6% carbon dioxide (CO2) at 37°C. One day prior to transfection, cells were trypsinized and plated at 4x10 ® cells per T75 flask (Fisher Scientific, Pittsburgh, PA). On the day of transfection, the old medium was replaced with prewarmed growth medium free of antibiotics, and cells were further incubated for 2 hours at 37°C in 6% CO2. The expression vector or the empty vector (10 pg) was added to 1.5 ml of OptiMEM medium (Life Technologies, San Diego, CA). Lipofectamine 2000 reagent (20 pl) (Life Technologies, San Diego, CA) was then added to an additional 1.5 ml of OptiMEM. Plasmid OptiMEM and Lipofectamine OptiMEM tubes were then mixed and incubated at room temperature for 25 minutes. The OptiMEM mixture was then added dropwise to the appropriate culture flask, and the flask was kept overnight at 37°C in 6% CO2. The next day, the medium was removed from the flask and replaced with complete growth medium. Forty-eight (48) hours after transfection, cells were harvested using StemProAccutase (Life Technologies, San Diego, CA), allowing cells to be carefully removed from the culture surface without affecting surface expression of PD-1. The cells were then subjected to antibody binding and fluorescence-activated cell sorting (FACS) analysis. Stable transfection using the Jurkat cell line The Jurkat cell line (clone E6-1-TIB-152™, ATCC®, Manassas, VA) was used to stably express human and Macaca fascicularis PD-1. Cell lines were generated using electroporation using the Amaxa nucleotransfector system (Lonza, Walkersville, MD). Transfections were performed at Pfizer (San Francisco, CA) using a V kit as directed by the manufacturer's protocol (Lonza, Walkersville, MD). Cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 culture medium supplemented with 10% FBS and 1 x L-glutamine (LifeTechnologies, San Diego, CA) at 37 °C in 5% CO2 at a density 0.3 to 1.0*10® cells / ml. For each vector, 2 pg / ml was used to transfect 2 x 10® 122 cells. After transfection, cells were cultured in the presence of G418 sulfate (600 pg / ml) for 2 weeks for selection and maintenance of stable transfected cells. In order to prevent long-term contamination, 1 x Pen / Strep was added to the medium. Single cell clones were selected by cell culture at a dilution of 1:200 (1 cell in 200 μl complete medium supplemented with G418 sulfate antibiotics). In order to select clones expressing high human or Macaca fascicularis PD-1, the PE-labeled anti-human PD-1 clone EH12.1 was used in flow cytometry assays. Samples were collected using the BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA). Data analyzes and mean fluorescence intensity (MFI) were calculated using Flowjo software (FLOWJO LLC, Ashland, OR). Cell lines with high MFI were chosen for assay development. HEK-293T cells transfected with human, Macaca fascicularis, mouse or rat PD-1 vectors, or with an empty vector were harvested 48 hours after transfection. Prior to mAb7 fixation, cells from each specific transfection were screened with commercial antibodies or ligands to ensure that the appropriate PD-1 receptor was highly expressed on the cell surface. For human and Macaca fascicularis, a cross-reactive commercial anti-PD1 antibody (clone EH12.1) was used. For mouse, a commercial mouse anti-PD-1 antibody (clone J43) was used. For rat, a rat PD-L1 ligand was used (since no cross-reactive rat anti-PD-1 antibody was commercially available). After adequate PD-1 expression was confirmed, cells were counted, and 2 x 105 cells were incubated with a functional grade mixture of human Fe receptor binding inhibitor (receptor blockade and Fe at 1 pg / 1 x 106 cells, Affymetrix / eBiosclense, San Diego, CA) for 20 min and blue fluorescent reactive dye, used for Live / Dead staining, was added to the mixture (Life Technologies, San Diego, CA). Serial dilutions of mAb7 or of! negative control antibody (1-0.00001 pg / ml) to the cell mixtures and then incubated on ice for 1 hour. Then, the cells were washed and AffiniPure Fc-specific APC-tagged F(ab')2 fragment donkey anti-human IgG (1:100 dilution) (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) was added to the mixture. , and then incubated on ice for an additional 30 min. Data were obtained on the BD LSRFortessa™ Cellular Analyzer (BD Biosciences, San Jose, CA), and analyzed using Flowjo software (FLOWJO LLC, Ashland, OR). Stable Jurkat cell clones were generated and transfected with 123 human, Macaca fascicularis or mouse PD-1 receptor. The PD1 cell clones chosen for these assays from each species expressed similar amounts of PD-1 receptors (~400,000 receptors / cell; receptors were previously quantified during the production of these cell lines). After adequate PD-1 expression was confirmed, cells were counted, and 2 x 10 5 cells were incubated with a functional grade of human Fe receptor binding inhibitor (Fe receptor blockade at 1 pg / 1 x 10 6 cells). , Affymetrix / eBlosciense, San Diego, CA) for 20 min and blue fluorescent reactive dye, used for Light / Dead staining, was added to the mixture (Life Technologies, San Diego, CA). Serial dilutions (1:3) of mAb7, positive control 1, positive control 2, or negative control antibodies (using an IgG4 framework) were added to the cell mixture and then incubated on ice for 1 hour. The cells were then washed, and APC-tagged, Fcy-specific, APC-tagged donkey anti-human IgG (1:100 dilution) (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) was added to the mixture, and the cells were then incubated for an additional 30 minutes. Data were obtained on the BD LSRFortessa™ Cellular Analyzer (BD Biosciences, San Jose, CA). Analyzes were performed using Flowjo software (FLOWJO LLC, Ashland, OR), and geometric means were calculated. ECSo values ​​were calculated using Graph Pad Prism (Log agonist versus response [binding]). Activated T cells expressing high levels of PD-1 receptors were generated. Proper PD-1 expression was confirmed in T lymphocytes, obtained from each species, using commercial reagents (human anti-PD-1 clone EH12.1 for human and Macaca fascicularis, anti-PD-1 clone J43 for Macaca fascicularis). mouse for mouse PD-1 and rat PD-L1 for rat PD-1). Cells were counted, and 1 x 10 ® cells were incubated with a functional grade of human Fe receptor binding inhibitor (Fe receptor blockade at 1 pg / 1 x 10 6 cells, Affymetrix / eBiosciense, San Diego, CA) for 20 minutes and blue fluorescent reactive dye, used for Live / Dead staining, was added to the mixture (Life Technologies, San Diego, CA). Serial dilutions of mAb7 (1:3 dilution) were added to the cell mixture and then incubated on ice for 1 hour. Cells were then washed before Fc-specific APC-tagged F(ab')2 fragment anti-human donkey IgG secondary antibody was added to AffiniPure (1:100 dilution) (Jackson ImmunoResearch Laboratories Inc, West Grove, PA), and the cells were incubated for 30 minutes. Data were obtained on the BD LSRFortessa™ Cellular Analyzer (BD Biosciences, San Jose, CA). Data analyzes were performed using Flowjo software (FLOWJO LLC, 124 Ashland, OR). Geometric means were counted, and ECS0 values ​​were calculated using Pad Graph Prism (Log agonist versus response [fixation]). Stable Jurkat cell clones expressing high levels of hu-PD-1 were chosen for testing. Twenty (20) pg / mL of biotinylated hu-PD-L1 or biotinylated hu-PD-L2 saturated all PD-1 receptors in this cell line (after planned binding titration assays). Therefore, this concentration was chosen as the optimal concentration in our studies. Jurkat cells expressing HuPD-1 (2 x 108) were incubated with a functional grade of human Fe receptor binding inhibitor (receptor blockade / Fe at 1 pg / 1 x 106 cells, Affymetrix / eBiosciense, San Diego, CA ) for 20 min, and blue fluorescent reactive dye, used for Live / Dead staining, was also added to the mixture (Life Technologies, San Diego, CA). Biotinylated hu-PD-L1 or biotinylated hu-PD-L2 were added to cells at 20 pg / mL, and then serial dilutions of mAb7, positive control 1, positive control 2, or negative control antibodies ( all on the IgG4 backbone and in 1:3 serial dilutions). Cells were incubated on ice for 1 hour. Cells were then washed, and streptavidin-PE (1:100 dilution) (Affymetrix / eBiosciense, San Diego, CA) was added, and cells were incubated on ice for 30 minutes. Data were then collected on the BD LSRFortessa™ Cellular Analyzer (BD Biosciences, San Jose, CA), and analyzed using Flowjo software (FLOWJO LLC, Ashland, OR), and geometric means calculated. EC50 values ​​were calculated using Graph Pad Prism (Log inhibitory to response [binding]). Jurkat cells were stably transfected with high levels of human or Macaca fascicularis PD-1 receptors. Cells expressing Macaca fascicularis PD-1 receptors (-400,000 receptors / cell) were chosen for this assay. Binding to Macaca fascicularis PD-L1 and PDL2 was assessed using these cells, and the results showed that 20 pg / ml of Macaca fascicularis PD-L1 or PD-L2 was sufficient to saturate all PD-1 receptors based on the geometric mean analyzed for different concentrations (50 ng / ml-50 pg / ml) of these ligands when bound to this cell line. Macaca fascicularis PD-1-expressing Jurkat cells (2 x 105) were incubated with a functional grade of human Fe receptor binding inhibitor (Fcy receptor blockade at 1 pg / 1 x 106 cells, Affymetrix / eBiosciense, San Diego). , CA) for 20 min, and blue fluorescent reactive dye, used for Live / Dead staining, was added to the mixture (Life Technologies, San Diego, CA). Added PDL1 from Macaca fascicularis Alexa125 Fluor®-647 or Macaca fascicularis Alexa-Fiuor®-647 PD-L2 to the cells at 20 pg / mL, and then different concentrations of mAb7, Positive Control 1, Positive Control 2, or Negative Control antibodies were added immediately. (using IgG4 backbone, in 1:3 serial dilution). Cells were then incubated on ice for 1 hour. After washing, cells were obtained on the BD LSRFortessa™ Cellular Analyzer (BD Biosciences, San Jose, CA). Data analyzes were performed using Flowjo software (FLOWJO LLC, Ashland, OR), and geometric means were calculated. EC50 values ​​were calculated with Graph Pad Prism (Log inhibitory to response [binding]). Human buffy coat, purchased from Stanford Blood Center (Stanford, CA), was diluted with PBS and plated on Ficoll to isolate PBMC. PBMCs were washed 4 times with PBS. RBCs were lysed using ACK lysis buffer as indicated in the manufacturer's protocol (Life Technologies, San Diego, CA). After RBC lysis, cells were washed once more and diluted in PBS to 5 x 10 7 cells per ml. Half of the PBMCs were counted and frozen in freezing medium (90% FBS with 10% dimethylsulfoxide [DMSO]), and the remaining cells were subjected to T cell purification. From the remaining PBMC, T cells were isolated using negative selection human Pan T cell isolation kit as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA). For the ADCC assay, freshly isolated T cells (target cells) were counted, and 1 x 106 T cells per mL were cultured in serum-free X-vivo medium (Lonza, Walkersville, MD) and stimulated using coated microspheres. with anti-CD3 and anti-CD28 (CD3 / CD28 T-cell activators Dynabeads® for T-cell activation and expansion (Life Technologies, San Diego, CA) and 100 U / mL human (rh)-IL-2 (R&D Systems, Minneapolis, MN). Cultures were incubated at 37°C in 5% CO2 for 72 hours. When T cell activation reached the 48-hour time point, PBMCs (effector cells derived from the same donor ) were thawed, counted, and then stimulated with rh-IL-2 (50 U / ml culture at 5 x 106 cells / ml) for a total of 18 to 24 hours in complete RPM 1-1640 medium with 10% FBS On the day of the assay, both cell types were counted and evaluated by flow cytometry to ensure adequate activation. For T cells, high PD-1 expression was examined, and for PBMC, the expression level of CD16, CD32, and CD64 receptors (Fcγ receptor gamma [FcγR] III [a and b], FcγRlIa, FcγRI, respectively) that mediate 126 ADCC. Cells were plated at a 5:1 effector to target ratio (as this was an optimal ratio) in tissue culture-treated 96-well flat-bottom plates (Fisher Scientific, Pittsburg, PA), and then antibodies were added: mAb7 (IgG4 and IgG1 frameworks) and positive control antibodies. All antibodies were tested at concentrations from 0.01 to 100 pg / mL (in 1:10 serial dilutions). Assay controls, including only target cells, were added to assess maximal lysis, effector cells only, and one effector to achieve a 5:1 ratio without adding antibodies. Assay plates were incubated at 37°C in 5% CO2 for 4 hours. Data were analyzed using the CytoTox 96® non-radioactive cytotoxicity assay (Promega US, Madison, Wl) as directed by the manufacturer's protocol. The assay quantitatively measured lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released following cell lysis. Percent (%) cytotoxicity was measured as = 100 x (experimental LDH release [OD490] / maximal LDH release [OD490]). Maximal LDH release was calculated when only target cells (T cells) were chemically lysed to obtain maximal clearance. The ability of mAb7 to bind to human, Macaca fascicularis, mouse, and rat PD-1 was assessed using flow cytometric cell binding assays (FACS) that included the transiently transfected HEK-293T cell line, primary activated T cells. and Jurkat cells stably transfected with hu-PD-1 or Macaca fascicularis and mouse PD-1. mAb7 bound to Macaca fascicularis hu-PD-1 and PD-1 with high affinity and showed similar EC50 values ​​for the 2 species (the amino acid sequences for these isoforms are the most similar of the species tested). The data is summarized in Tables 19, 20 and 21. Binding to mouse PD-1 was only achieved at a high concentration of mAb7 which is biologically irrelevant and could be due to the affinity maturation process. Binding of mAb7 to rat PD-1 was not detected when transfected cells or primary activated T cells were used (Table 19). Table 19: Binding of mAb7 to primary naive and activated T cells by FACS mAb7 (% binding) Negative control (% binding) Species Human T lymphocytes No Activated No prior treatment prior treatment 9.68 ± 4.82 87.95 + 1.65 3.49 ± 0.59 Activated 4.75 + 0.59 127 Macaca fascicularis T lymphocytes Mouse T lymphocytes rat T cells 9.21 ± 0.89 4.63 ± 0.62 3.82 ±1.72 84.35 ± 0.75 4.14 ± 1.87 0.74 ± 0.05 5.385 + 0.38 0.93 ± 0.07 0.82 ±0.16 4.67 ± 0.47 0.68 ±0.10 4.36 ± 0.34 Table 20. Binding of mAb7 to primary activated T lymphocytes obtained by expressing human and Macaca fascicularis PD-1 (EC50) Subject Subject 1 Subject 2 Mean ± sem Human T lymphocytes (pM) 45.9 56.11 51.04 ±5.07 Macaca fascicularis T lymphocytes (pM) 73.61 97.07 85.34 ± 11.73 In Table 20, each number represents ECS0 values ​​for mAb7 binding to a different donor. The last row represents the mean ± sem. sem = standard error of the mean. Table 21: Binding of mAb7 to Jurkat cell lines stably transfected with human PD-1 or Macaca fascicularis PD-1 (EC50) mAb (igG4) Macaca Jurkat / PD-1 Jurkat / PD-1 number human repeat (pM) fascicularis (pM) mAb7 1 62.84 181.3 2 66.61 263.8 *Mean± sem 64.725 ± 1, 89 222.55 ±41.3 Control mAb 1 54.52 265.2 positive 1 2 57.60 267.2 'Mean ± sem 56.01 ± 1.55 266.2 ± 1.0 Control mAb 1 181 .6 380.4 positive 2 2 179.6 375.9 'Mean ± 180.6 ± 1.0 378.15 ±2.5 week In Table 21, each number represents EC50 values ​​for mAb7 binding in a single experiment, ' indicates mean ± sem. mAb(lgG4) = IgG4-framed monoclonal antibody; PD1= programmed death-1; sem= standard error of the mean. Binding of mAb7 to human and Macaca fascicularis PD-1 was examined using different cellular assay systems. In all systems, binding was found to have high affinity and specificity in both species. Using transiently transfected HEK-293T cell lines expressing human or Macaca fascicularis PD-1, mAb7 showed similar binding patterns as indicated by MFI. Minimal or no binding was observed in the cell line 128 parent transfected with an empty vector (vehicle). ECS0 values ​​of mAb7 binding to activated primary human and Macaca fascicularis T cells were determined 72 hours after activation, when PD-1 cell surface expression and cell viability were optimal (Table 19). ECS0 values ​​were calculated for 2 different donors of each species (Table 20). In activated human and Macaca fascicularis T cells, low EC50 values ​​were obtained, and EC50 values ​​were found to be close between the 2 species, 51.04 ± 5.07 pM and 85.34 ± 11.73 pM, respectively { + standard error of the mean [wk]). No binding was observed with the negative control antibody above baseline. The Jurkat T cell line, which minimally expresses hu-PD-1 receptors, was used to generate stably transfected hu-PD-1, Macaca fascicularis PD-1, and mouse PD-1 cell lines. Cell lines were subcloned, and clones expressing high levels of human PD-1 and Macaca fascicularis PD-1 receptors were selected (~400,000 PD-1 receptors per cell was the highest expression obtained). In this system, mAb7 showed high affinity to human PD-1 and Macaca fascicularis PD-1 receptors. EC5D values ​​for the two species were similar to primary activated T cell data (Table 21); EC50= 64.725 ± 1.89 for human PD-1 and 222.55 ± 41.3 for Macaca fascicularis PD1. The EC50 values ​​for cells expressing Macaca fascicularis PD-1 were more variable than the human EC50 values ​​between the 2 experimental runs. The data for the two species were similar to the data obtained with the positive control antibodies used in any given replicate (Table 21). The negative control antibody did not exhibit binding above baseline in any of the experimental replicates. The ability of mAb7 to block the interaction of PD-L1 and PD-L2 ligands with the PD1 receptor was examined in a PD-1 transfected Jurkat cell line (expressing hu-PD-1 or Macaca fascicularis PD-1). . In this assay, cells were incubated with labeled ligands at saturating concentrations and then incubated with unlabeled mAb7 at different concentrations. As shown in Table 22, mAb7 inhibited the binding of PD-L1 and PD-L2 ligands to the PD-1 receptor in a dose-dependent manner. Positive control antibodies to PD-1 showed comparable inhibition The IC50 of mAb7 could be compared between human and Macaca fascicularis PD-1 (880.15 ± 289.85 and 1058 ± 355.4 for hu-PD-L1 and hu - PDL2, respectively, and 942.9 ± 110.1 and 839 ± 89.5 for Macaca fascicularís PD-L1 and Macaca fascicularís PD-L2, 129 respectively). A summary of IC50 values ​​is provided in Table 22. In Table 22, each number represents IC50 values ​​for mAb7 in a single experiment; ‘indicates mean ± sem; íindicates the repetition number in parentheses; mAb IgG4=IgG4 framed monoclonal antibody; Pd1=programmed death-1; PDL1=programmed death ligand 1; PD-L2=programmed death ligand 2; sem = standard error of the mean. Table 22: Inhibitory Concentrations (IC50; pM) for Blocking the Binding of Human PD-1 or Macaca fascicularis PD-1 to PD-L1 and PD-L2 Using a Stably Transfected Jurkat Cell Line System Human PD-1 / Jurkat Macaca PD-1 mAb (lgG4) fascicularis! Jurkat PD-1 / PD-L1 Blockade PD-1 / PD-L1 Blockade PD-1 / PD-L2 Blockade PD-1 / PD-L2 Blockade mAb7(1)t 590.3 703.2 832.8 749.5 (2)t 1170 1414 1053 928.5 'Mean ± 880.15 ± 1058±355.4 942.9±110.1 839±89.5 wk 289.85 C1 (1)í 1022 1226 1286 1316 (2)í 629 811.1 861.8 1289 'Mean ± 825.5 ± 196.5 1081.55 ± 1073.9 ±212.1 1302.5 ± 13.5 wk 207.45 C2 (1)t 1715 1972 1809 1961 (2)í 1597 1560 1881 2111 'Mean ± 1656 ±59 1766 ±206 1845 ±36 2036 ± 75 wk mAb7 showed weak binding to complement component 1, subcomponent q (C1q) and CD64. C1q and CD64 (for the IgG4 framework) are considered possible surrogates for complement dependent cytotoxicity (CDC) and ADCC, respectively. To further investigate the lack of ability of mAb7 to induce T cell clearance in vitro, an ADCC assay was performed with activated T cells (target cells) expressing high levels of PD-1 receptors and PBMCs (effector cells) that express high levels of Fcy receptors. Cells were selected from two 2 healthy donors. mAb7 (in its original IgG4 frame) showed minimal ADCC activity in both donors. The lack of ADCC activity was in agreement with the activity exhibited by the positive control anti-PD-1 antibody in the IgG4 frame, and both were similar to that of the positive control IgG4 antibody. When anti-PD-1 (mAb7 or positive control anti-PD-1 antibodies) were tested in the setting of human IgG1, which is known to induce stronger ADCC, the 130 anti-PD-1 induced ADCC up to 4-fold higher than when the antibody is in frame with IgG4. Maximal LDH release was calculated when only target cells (T cells) were chemically lysed to obtain maximal clearance (kill was calculated as 100% lysis for each donor according to assay calculation). Lysis of T cells using the IgG1 framework corresponds to the level of PD-1 in activated T cells (PD-1 expression of donor 1 and lysis were higher than that of donor 2), which confirmed the accuracy of the assay. trial. These results demonstrate that the anti-PD-1 mAb7 binds with high affinity to human and Macaca fascicularis PD-1 receptors expressed on cells with EC50 values ​​ranging from 46 to 270 pM, depending on the evaluation system. mAb7 did not bind to cells expressing mouse PD-1 or rat PD-1 at physiological concentrations. mAb7 blocked the interaction of PD-L1 and PD-L2 ligands with cell surface PD-1 receptors; IC50 values ​​ranged from 500 to 1000 pM, indicating its high antagonistic role in blocking PD1 function induced by ligand binding. mAb7, in its IgG4 framework, produced minimal or no antibody-induced toxicity, which is consistent with the properties of the IgG4 antibody. Example 12: Characterization of Anti-PD-1 mAb7 Antibody Binding to PD-1, FcRn, FcvR and C1Q using label-free biosensors v ELISA. This example illustrates the in vitro binding affinities of mAb7 to recombinant purified PD1 from different species with respect to toxicology studies using SPR biosensors. The ability of the Fc region of mAb7 to bind FCGR and FcRn was also evaluated by SPR to confirm that it exhibited properties consistent with those of an isotype-matched control. By ELISA, mAb7 and an isotype-matched control were analyzed for binding to human C1q. The ability of mAb7 to block the binding interaction of PD1 with its ligands, PDL1 and PDL2, was also assessed using label-free biosensors to support its mechanism of action. All affinity and kinetic experiments were performed at 25 °C in running buffer of phosphate buffered saline (PBS) + 0.01% Tween 20, unless otherwise noted. Kinetic studies were performed on a ProteOn XPR36 SPR biosensor equipped with NLC (neutravidin-coated) chips (BioRad, Hercules, CA). The ProteOn was also used to determine the 131 active concentrations of human PD1 and Macaca fascicularis PD1 analytes by titration against mAb7 as the reference standard. Concentrations of protein analytes used herein refer to "active" or "nominal" values. The active concentration of human gamma Fe receptor (hu-FCGR) I, human neonatal Fe receptor (hu-FcRn) (Lot No. R3091), and Macaca fascicularis neonatal Fe receptor (Macaca fascicularis FcRn) (Lot No. lot JCR) was determined using calibration free concentration analysis (CFCA) experiments on a Biacore T200 equipped with CMS sensor chip (GE Life Sciences, Marlborough, MA). All other analytes were used at their nominal concentrations as determined by light absorbance at A280nm with a suitable extinction coefficient. Solution affinities were determined at room temperature (approximately 23°C) using a KinExA 3000 or 3200 instrument equipped with an autosampler (Sapidyne). Secondary detection antibodies were labeled with DyLight 650 (Pierce Biotechnology, Grand Island, NY.) according to the manufacturer's instructions. Immunoglobulin G (IgG) biotinylations were performed in an equimolar ratio of linker:IgG using EZ-Link™ SulfoNHS-LCLC-Biotin (Pierce Biotechnology Grand Island, NY) according to the manufacturer's instructions. Unless otherwise stated, immobilized IgGs were regenerated with a 'Pierce / salt cocktail comprising a 2:1 v / v mixture of Pierce IgG Elution Buffer (pH 2.8) / Pierce chloride. 4M sodium (NaCl). The binding interactions of human PD1 and Macaca fascicularis PD1 (both His-tagged monomers) to mAb7 were determined in solution using the KinExA method in a running buffer of PBS + 0.01% Tween 20 and a sample buffer of PBS + 0.01% Tween 20. PBS + 0.01% Tween 20 + 1 g / l bovine serum albumin (BSA). Two different assay formats were used. In the first assay format, mAb7 was titrated in a constant concentration of human PD-1 (nominal 200, 400 or 4000 pM), and the samples reached equilibrium. Free human PD1 was captured on polymethylmethacrylate (PMMA) microspheres that had been coated (by absorption) with anti-hu-PD1 monoclonal antibody mAb7 (which was prepared in-house under non-GLP conditions [N. lot no. R5432]). Bead-captured hu-PD1 was then detected with 0.5 pg / mL Dylight-tagged anti-His mAb (R&D Systems, Minneapolis, MN). In the second assay format, human PD1 or Macaca fascicufaris PD-1 were titrated at a constant concentration of mAb7 (20, 50, 100 or 500 pM), and these mixtures were equilibrated. Free mAb7 was captured on PMMA microspheres which were 132 had been adsorbed with a mouse blocker anti-idiotypic anti-mAb7 mAb 1699.1H6 that specifically binds to free mAb7 but not to PD-1 saturated mAb7. mAb7 captured on microspheres was then detected with Dylight (H+L)-labeled goat anti-human IgG (Jackson ImmunoResearch Inc, West Grove, PA). All titrations were prepared as a 12-membered two-fold dilution series varying the upper nominal binding site concentration to be within the range of 1 nM to 10 nM, depending on the experiment....

Claims

1. An isolated antagonistic antibody that binds specifically to PD-1 and characterized in that it comprises: a heavy chain variable region (HV) comprising the amino acid sequence shown in SEQ ID NO: 4 or 6 and a light chain variable region (LV) comprising the amino acid sequence shown in SEQ ID NO:

8. 11 Claims follow