Anti-PD-1 antibodies and compositions

Abstract

This invention relates to anti-PD-1 antibodies and methods for using them in the treatment of diseases and conditions related to PD-1 activity, for example, cancer.

Description

Anti-PD-1 antibodies and compositions Background of the invention PD-1, also known as Programmed Cell Death Protein 1 and CD279, is a 268-amino-acid cell surface receptor belonging to the immunoglobulin superfamily. PD-1 is a member of the CD28 family of T cell regulators and is expressed on T cells, B cells, and macrophages. It binds to the ligands PD-L1 (also known as B7 homolog) and PD-L2 (also known as B7-DC). PD-1 is a type I membrane protein whose structure includes an extracellular IgV domain, a transmembrane region, and an intracellular tail containing two phosphorylation sites. Known as an immune checkpoint protein, PD-1 functions as an inducible immunomodulatory receptor, playing a role, for example, in the negative regulation of T cell responses to antigen stimulation. PD-L1 is the predominant ligand for PD-1. The binding of PD-L1 to PD-1 inhibits T cell activity by reducing cytokine production and suppressing T cell proliferation. Cancer cells that express PD-L1 can exploit this mechanism to inactivate the antitumor activity of T cells by binding PD-L1 to the PD-1 receptor. Given its regulatory properties on the immune response, PD-1 has been investigated as a potential target for immunotherapy, including the treatment of cancer and autoimmune diseases. Two anti-PD-1 antibodies, pembrolizumab and nivolumab, have been approved in the United States and Europe to treat certain cancers. In view of PD-1's critical role as an immunomodulator, there is a need for new and improved immunotherapies that target PD-1 to treat cancers and certain disorders of the immune system. Compendium of the invention The present invention relates to novel recombinant antibodies targeting PD-1 as defined in the claims, as well as to pharmaceutical compositions comprising one or more of these antibodies, and to the use of antibodies and pharmaceutical compositions to enhance immunity in a patient and for the treatment of cancers originating in tissues such as skin, lung, intestine, ovary, brain, prostate, kidney, soft tissues, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus, and pancreas. Compared to currently available treatments for such cancers, including antibody therapies, it is envisaged that the antibodies of the invention may provide a superior clinical response, either alone or in combination with another anticancer therapeutic agent, such as an antibody targeting another immune checkpoint protein. In one embodiment, the present invention provides an anti-PD-1 antibody or an antigen-binding portion thereof, wherein the antibody binds to the same human PD-1 epitope as 12819.15384. In some embodiments, the anti-PD-1 antibody comprises H-CDR1-3 comprising the H-CDR1-3 sequences, respectively, of antibody 12819.15384, Wang et al., (Cancer Immunology Research, vol. 2, no. 9, pp. 846-856) disclose an in vivo characterization of the anti-PD-1 antibody nivolumab and in vivo toxicology in non-human primates. In some embodiments, the anti-PD-1 antibody has a variable heavy chain (VH) domain whose amino acid sequence is at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) identical to the Vh domain of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody has a Vh comprising the antibody Vh amino acid sequence 12819.15384. In some embodiments, the anti-PD-1 antibody has a heavy chain (HC) comprising the amino acid sequence of antibody Vh 12819.15384 and the amino acid sequence of the heavy chain constant region of SEQ ID NO: 67. In some embodiments, the anti-PD-1 antibody comprises L-CDR1-3 comprising the L-CDR1-3 sequences, respectively, of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody has a light chain variable domain (Vl) whose amino acid sequence is at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) identical to the Vl domain of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody has a Vl comprising the amino acid sequence of antibody Vl 12819.15384. In some embodiments, the anti-PD-1 antibody has a light chain (LC) comprising the amino acid sequence VL of antibody 12819.15384 and the amino acid sequence of the light chain constant region of SEQ ID NO: 68. In some embodiments, the anti-PD-1 antibody comprises any of the heavy chain sequences described above and any of the light chain sequences described above. In some embodiments, the anti-PD-1 antibody comprises the H-CDR3 and L-CDR3 amino acid sequences of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody comprises the amino acid sequences H-CDR1-3 and L-CDR1-3 of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody has a Vh and a Vl whose amino acid sequence is at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) identical to Vh and Vl, respectively, of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody has a Vh and a Vl comprising or consisting of the amino acid sequences of Vh and Vl, respectively, of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody has an HC and an LC comprising or consisting of the amino acid sequences HC and LC, respectively, of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody has (1) an HC comprising the amino acid sequence of Vh from antibody 12819.15384, and the amino acid sequence of the heavy chain constant region of SEQ ID NO: 67; and (2) an LC comprising the amino acid sequence of Vl from that antibody and the amino acid sequence of the light chain constant region of SEQ ID NO: 68. This document discloses an anti-PD-1 antibody or antigen-binding portion comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of: O: 24, 25, 26, 27, 28 and 29, respectively; NO: 30, 31, 32, 33, 34 and 35, respectively; O: 36, 37, 38, 39, 40 and 41, respectively; O: 42, 43, 44, 45, 46 and 47, respectively; O: 48, 49, 50, 51, 52 and 53, respectively; f) SEQ ID NO: 54, 55, 56, 57, 58 and 59, respectively; or g) SEQ ID NO: 60, 61, 62, 63, 64 and 65, respectively. This document discloses an anti-PD-1 antibody or antigen-binding portion comprising a heavy chain variable domain and a light chain variable domain having the amino acid sequences of: a) SEQ ID NO: 4 and 5, respectively; b) SEQ ID NO: 4 and 66, respectively; c) SEQ ID NO: 6 and 7, respectively; d) SEQ ID NO: 8 and 9, respectively; e) SEQ ID NO: 10 and 11, respectively; f) SEQ ID NO: 12 and 13, respectively; g) SEQ ID NO: 14 and 15, respectively; or h) SEQ ID NO: 16 and 17, respectively. This document discloses an anti-PD-1 antibody comprising: a) an HC comprising the amino acid sequences of SEQ ID NO: 4 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 5 and 68; b) an HC comprising the amino acid sequences of SEQ ID NO: 4 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 66 and 68; c) an HC comprising the amino acid sequences of SEQ ID NO: 6 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 7 and 68; d) an HC comprising the amino acid sequences of SEQ ID NO: 8 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 9 and 68; e) an HC comprising the amino acid sequences of SEQ ID NO: 10 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 11 and 68; f) an HC comprising the amino acid sequences of SEQ ID NO: 12 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 13 and 68; (g) an HC comprising the amino acid sequences of SEQ ID NO: 14 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 15 and 68; or h) an HC comprising the amino acid sequences of SEQ ID NO: 16 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 17 and 68. In some embodiments, the antibody or antigen-binding portion of the invention comprises H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 18-20 and SEQ ID NO: 21-23, respectively. In certain embodiments, the anti-PD-1 antibody comprises a Vh comprising the amino acid sequence of SEQ ID NO: 2 and a Vl comprising the amino acid sequence of SEQ ID NO: 3. In specific embodiments, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO: 2 and 67 and a light chain comprising the amino acid sequences of SEQ ID NO: 3 and 68. The invention also provides an anti-PD-1 antibody or an antigen-binding portion thereof that binds to a PD-1 epitope comprising amino acid residues K131, P130, A132, V64, and L128 (e.g., antibody 12819). An anti-PD-1 antibody or antigen-binding portion thereof that binds to a PD-1 epitope comprising amino acid residues K131 and E136 (e.g., antibody 12865) is disclosed herein. This document discloses an anti-PD-1 antibody or an antigen-binding portion thereof that binds to a PD-1 epitope comprising amino acid residues V44 and T145 of SEQ ID NO: 1 (e.g., a 13112 antibody such as those listed in Tables 1, 4-7, 9 and 11-14). In specific embodiments, the antibody or portion binds to a PD-1 epitope comprising amino acid residues V64, L128, P130, K131 and A132 of SEQ ID NO: 1 (e.g., an antibody 12819). In the context of the present invention, a monoclonal antibody or an antigen-binding portion thereof is disclosed that binds to a PD-1 epitope comprising amino acid residues 69-90 and 122-140 of SEQ ID NO: 1 (e.g., antibody 12819 or 12865). In certain embodiments, the monoclonal antibody or antigen-binding portion binds to a PD-1 epitope comprising amino acid residues 56-64, 69-90, and 122-140 of SEQ ID NO: 1 (e.g., antibody 12819). An antibody or portion thereof that binds to residues 69-75 (or a fragment thereof) of SEQ ID NO: 1 (e.g., antibody 12819 or 12865) is disclosed herein. This document discloses an antibody or portion thereof that binds to residues 136-140 (or a fragment thereof) of SEQ ID NO: 1 (e.g., an antibody 12819 or 12865).This document discloses an antibody or portion thereof that binds to residues 69-75 (or a fragment thereof) and residues 136-140 (or a fragment thereof) of SEQ ID NO: 1 (e.g., an antibody 12819 or 12865). In some embodiments, the anti-PD-1 antibody or antigen-binding portion of the invention has at least one of the following properties: a) bind to human PD-1 with a Kd of 750 pM or less; b) They bind to cynomolgo PD-1 with a Kd of 7 nM or less; c) They bind to mouse PD-1 with a Kd of 1 nM or less; d) do not bind to rat PD-1; e) increase IL-2 secretion in a SEB whole blood assay; f) increase IFN-γ secretion in a unidirectional lymphocyte mixed reaction assay; g) inhibit the interaction of PD-1 with PD-L1 by at least 60% at a concentration of 10 g / ml in a competitive assay by flow cytometry; (h) block the binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 pg / ml as determined by Bio-Layer interferometry analysis; and i) inhibit tumor growth in vivo. An example of such an antibody is, without limitation, antibody 12819 (which has properties ai). The antibodies disclosed herein that have some of the properties are antibodies 12748, 12892, and 12777 (which have at least properties a, b, and eh); antibodies 12865 and 12796 (which have at least properties a, b, e, f, and h); and antibodies 12760 and 13112 (which have at least properties a, b, e, and f). In some embodiments, the anti-PD-1 antibody or the antigen-binding portion of the invention has all the aforementioned properties. In some embodiments, the anti-PD-1 antibody or the antigen-binding portion has at least properties a, b, and eh. In some embodiments, the anti-PD-1 antibody or the antigen-binding portion has at least properties a, b, e, f, and h. In some embodiments, the anti-PD-1 antibody or the antigen-binding portion has at least properties a, b, e, and f. Unless otherwise noted, 12819, 12748, 12865, 12892, 12796, 12777, 12760, and 13112 refer to a group of antibodies that have the same six CDRs and share the first five digits in their ten-digit numerical designations. For example, 12748 includes antibody variants 12748.15381 and 12748.16124, which have the same six CDRs (as shown in Table 2). Each antibody group is expected to share the same or substantially the same biological properties. In some embodiments, the anti-PD-1 antibody or the antigen-binding portion of the invention does not compete for PD-1 binding with pembrolizumab or nivolumab. In some embodiments, the anti-PD-1 antibody or the antigen-binding portion of the invention does not bind to the same epitope as pembrolizumab or nivolumab; for example, the antibody or the portion of the invention binds to one or more moieties on PD-1 that do not bind to pembrolizumab or nivolumab. In another aspect, the present invention provides pharmaceutical compositions comprising at least one anti-PD-1 antibody or an antigen-binding portion thereof as described herein and a pharmaceutically acceptable excipient. The present invention further provides isolated nucleic acid molecules comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, or both, of an anti-PD-1 antibody as described herein. The present invention also provides vectors comprising an isolated nucleic acid molecule of this type, wherein said vector further comprises an expression control sequence. The present invention also provides host cells comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, or both, of a PD-1 antibody as described herein. The present invention also provides a method for producing an antibody or antigen-binding portion thereof as described herein, comprising providing a host cell comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof and a nucleotide sequence encoding the light chain or an antigen-binding portion thereof of an anti-PD-1 antibody as described herein, culturing said host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion. The present invention also provides a bispecific binding molecule having the binding specificity of an anti-PD-1 antibody described herein and the binding specificity of another anti-PD-1 antibody (e.g., another anti-PD-1 antibody described herein) or an antibody that targets a different protein, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease such as cancer. The present invention also provides a method for improving immunity in a patient (e.g., a human patient) who needs it, comprising administering to said patient an anti-PD-1 antibody or an antigen-binding portion thereof, a pharmaceutical composition, or a bispecific binding molecule as described herein. The present invention further provides a method for treating cancer in a patient (e.g., a human patient), comprising administering to said patient an anti-PD-1 antibody or an antigen-binding portion thereof, a pharmaceutical composition, or a bispecific binding molecule as described herein. In some embodiments, the cancer originates in a tissue selected from the group consisting of skin, lung, intestine, ovary, brain, prostate, kidney, soft tissues, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus, and pancreas. The cancer may be, for example, advanced or metastatic melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, renal cell carcinoma, or Hodgkin lymphoma.In some embodiments, the method additionally comprises administering a chemotherapeutic agent, an antineoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, or a PD-1 pathway inhibitor. The present invention further provides antibodies or antigen-binding portions of the present invention for use in the treatments mentioned above, and the use of the antibodies or antigen-binding portions of the present invention for the manufacture of medicaments for the treatments mentioned above, i.e., the treatment of a human being in need of it to improve his immune system, and the treatment of a human being with cancer, such as one of the cancers mentioned above. Brief description of the drawings Figure 1 shows a PCR product containing the Vh and Vl regions of the anti-PD-1 antibody AAS-12819 (shown in black) frame-cloned with the corresponding human heavy chain IgG1-CH1-CH2-CH3 and human light chain constant lambda fragments, respectively. The restriction sites for this cloning are Apal and Avrll. The AscI and Nhel restriction sites are shown between the 5' ends of Vh and Vl. The plasmid origin of replication is represented as pUCo, and the gene conferring ampicillin resistance is represented as AmpR. Figure 2 shows an expression construct with a double CMV promoter inserted between the 5' ends of V and Vl using AscI and NheI restriction sites. The Vh and Vl sequences are shown in black, and other annotated genetic elements are shown in white. Figures 3A-3C show representative flow cytometry dot plots for (A) an antibody clone that binds specifically to cells transfected with human PD-1, (B) a clone that does not bind specifically to CHO-S cells, and (C) a clone that does not bind to any of the cell populations used in the screening. Figure 4 shows the frequency of PD-1 expressing lymphocytes in six donors (D1-D6) before and after stimulation with SEB (Staphylococcus Enterotoxin B). Figures 5 A-1 show the titration of candidate anti-PD-1 antibodies in an SEB assay. Figures 6A-H show the titration of candidate anti-PD-1 antibodies in a one-way MLR assay. Figures 7A-B show the binding of PD-L1 to cells expressing PD-1 in the presence of anti-PD-1 antibodies. Figure 8 shows an overview of the identified epitope clusters (epitope pools) for the assayed anti-PD-1 antibodies 12866.13188, 12807.13177, 12819.17149, 12865.17150, 12892.13195, 12777.15382, 12760.13169, 13112.15380, and nivolumab and pembrolizumab analogues. Antibodies connected by black lines indicate cross-blocking activity. Antibodies are grouped according to competition patterns with other anti-PD-1 antibodies. Nivo: nivolumab analogue; Pembro: pembrolizumab analogue. Figure 9 (AG panels) shows the location of antibody epitopes on the human PD-1 structure (PDB 4ZQK and 2M2D). A) Vignette of the extracellular domain (ECD) of human PD-1 (residues 33-150). The location of GFCC, the ABED p-sheet, and the C'-D loop are illustrated. B) Vignette of human PD-1:human PD-L1 complex at the same viewing angles as in (A). C) Molecular model of the pembrolizumab epitope shown as a density map with darker areas representing regions that mediate stronger binding. Black areas represent contact residues found by alanine scanning. D) Molecular model of the nivolumab epitope represented as in (C). E) Molecular model of the antibody 12819 epitope represented as in (C). F) Molecular model of antibody epitope 12865 represented as in (C). Figure 10 (AD panels) shows the effect of treatment with the anti-PD-1 antibody 12819, 17149 or a vehicle on tumor growth in four syngeneic tumor models. A) CT26 (colon cancer). B) C38 (colon cancer). C) ASB-XIV (lung cancer). D) Sa1N (fibrosarcoma). The gray area indicates the treatment period. Data are presented as means ± SEM. *P < 0.001. Figure 11 shows the effect of treatment with the anti-PD-1 antibody 12819.17149, pembrolizumab (Keytruda®), or vehicle on tumor growth in a semi-humanized xenograft tumor model, where the human melanoma cell line A375 was mixed with purified human CD8+ and CD4+ T cells prior to inoculation. The gray area indicates the treatment period. Data are presented as means ± SEM. *P < 0.001. Detailed description of the invention The present invention provides novel anti-human PD-1 antibodies as defined in the claims that can be used to enhance the immune system in a human patient, such as a cancer patient. Unless otherwise stated, as used herein, "PD-1" refers to human PD-1. A polypeptide sequence of human PD-1 is available under Uniprot Accession Number Q15116 (PDCD1_HUMAN), shown herein as SEQ ID NO: 1. The term "antibody" (Ab) or "immunoglobulin" (Ig), as used herein, refers to a tetramer comprising two heavy chains (H) (approximately 50–70 kDa) and two light chains (L) (approximately 25 kDa) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable domain (Vh) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (Vl) and a light chain constant region (CL). The Vh and Vl domains may be further subdivided into hypervariable regions, called "complementarity-determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FRs).Each Vh and Vl is composed of three CDRs (H-CDR here designates a heavy chain CDR, and L-CDR designates a light chain CDR) and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid numbers in the heavy or light chain may be in accordance with the definitions of the IMGT® (Lefranc et al, Dev Comp Immunol 27 (1):55-77 (2003)); or the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989). The term "recombinant antibody" refers to an antibody that is expressed from a cell or cell line comprising the nucleotide sequence or sequences encoding the antibody, where said nucleotide sequence or sequences are not naturally associated with the cell. The terms "isolated protein," "isolated polypeptide," or "isolated antibody" refer to a protein, polypeptide, or antibody that, by virtue of its origin or source, (1) is not associated with the naturally associated components that accompany it in its native state, (2) is free of other proteins of the same species, (3) is expressed by a cell of a different species, and / or (4) does not occur in nature. Therefore, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein can also be made substantially free of naturally associated components through isolation, using well-established protein purification methods. As used in this dissertation, the term "germline" refers to the nucleotide and amino acid sequences of antibody genes and gene segments as they are passed from parent to offspring through germ cells. Germline sequences are distinct from the antibody-coding nucleotide sequences in mature B cells, which have been altered by recombination and hypermutation events during B cell maturation. An antibody that "uses" a particular germline sequence has a nucleotide or amino acid sequence that aligns more closely with that germline nucleotide sequence or the amino acid sequence it specifies than with any other germline nucleotide or amino acid sequence. The term "affinity" refers to a measure of the attraction between an antigen and an antibody. The intrinsic attraction of an antibody for an antigen is typically expressed as the binding affinity equilibrium constant (Kd) of a specific antibody-antigen interaction. An antibody is said to bind specifically to an antigen when the Kd is <1 mM, preferably <100 nM. A binding affinity constant (Kd) can be measured, for example, by surface plasmon resonance (BIAcore™) or biolayer interferometry, using, for example, the Bio-Rad ProteOn™ XPR36 SpR system or the Octet™ system. The term "koff" refers to the dissociation rate constant of a specific antibody-antigen interaction. A dissociation rate constant koff can be measured by Bio-Layer Interferometry, for example, using the Octet™ system. The term "epitope," as used herein, refers to a portion (determinant) of an antigen that binds specifically to an antibody or a related molecule, such as a bispecific binding molecule. Epitope determinants generally consist of chemically active surface clusters of molecules such as amino acids, carbohydrates, or sugar side chains and typically have specific three-dimensional structural features as well as specific charge characteristics. An epitope can be either "linear" or "conformational." In a linear epitope, all interaction points between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) occur linearly along the protein's primary amino acid sequence.In a conformational epitope, interaction points occur through amino acid residues in the protein that are separated from each other in the primary amino acid sequence. Once a desired epitope on an antigen is identified, antibodies can be generated for that epitope using well-established techniques. For example, an antibody for a linear epitope can be generated by immunizing an animal with a peptide containing the amino acid residues of the linear epitope. An antibody for a conformational epitope can be generated by immunizing an animal with a minidomain containing the relevant amino acid residues of the conformational epitope. An antibody for a specific epitope can also be generated by immunizing an animal with the target molecule of interest or a relevant portion thereof (e.g., the ECD of p D-1), then selecting for epitope binding. It can be determined whether an antibody binds to the same epitope or competes for binding with an anti-PD-1 antibody of the invention using methods known in the art, including, without limitation, competitive assays, epitope clustering, and alanine sweeping. The test antibody and an anti-PD-1 antibody of the invention can bind to at least one common residue (e.g., at least two, three, four, or five common residues) on PD-1. The contact residues on PD-1 can be completely identical between the test antibody and the anti-PD-1 antibody of the invention. The anti-PD-1 antibody of the invention can be allowed to bind to PD-1 under saturation conditions, and then the ability of the test antibody to bind to PD-1 is measured. If the test antibody can bind to PD-1 at the same time as the reference anti-PD-1 antibody, then the test antibody binds to a different epitope than the reference anti-PD-1 antibody.However, if the test antibody is unable to bind to PD-1 simultaneously, it binds to the same epitope, an overlapping epitope, or an epitope very close to the epitope bound to the anti-PD-1 antibody of the invention. This experiment can be performed using ELISA, RIA, BlAcORE™, Bio-Layer Interferometry, or flow cytometry. To test whether one anti-PD-1 antibody cross-competes with another anti-PD-1 antibody, the competition method described above can be used in two directions, i.e., to determine whether the known antibody blocks the test antibody and vice versa. Such cross-competition experiments can be performed, for example, using an IBIS MX96 SPR apparatus or the Octet™ system. The term "chimeric antibody" refers, in its broadest sense, to an antibody containing one or more regions of one antibody and one or more regions of one or more different antibodies. Typically, it is an antibody that is partly of human origin and partly of non-human origin, meaning it is obtained in part from a non-human animal, such as a mouse, rat, or other rodent, or a bird such as a chicken. Chimeric antibodies are preferred over non-human antibodies to reduce the risk of an anti-human antibody response, such as a human anti-mouse antibody response in the case of a murine antibody. A typical example of a chimeric antibody is one in which the variable domain sequences are murine, while the constant region sequences are human. In the case of a chimeric antibody, the non-human parts can undergo further alteration to humanize the antibody.The chimeric antibodies described in this dissertation have chicken variable domain sequences and human constant region sequences. The term "humanize" refers to the fact that when an antibody is wholly or partially of non-human origin (e.g., a murine or chicken antibody obtained from the immunization of mice or chickens, respectively, with an antigen of interest, or a chimeric antibody based on such a murine or chicken antibody), it is possible to replace certain amino acids, particularly in the framework and constant regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. Although it is not possible to predict exactly the immunogenicity and therefore the human anti-antibody response of a particular antibody, non-human antibodies tend to be more immunogenic in humans than human antibodies. Chimeric antibodies, in which the foreign constant regions have been replaced (e.g.,Chimeric antibodies (from rodents or birds) derived from human sequences are generally less immunogenic than antibodies of entirely foreign origin, and the trend in therapeutic antibodies is toward humanized or fully human antibodies. Chimeric antibodies or other antibodies of non-human origin can therefore be humanized to reduce the risk of an anti-human antibody response. For chimeric antibodies, humanization typically involves modifying the framework regions of the variable domain sequences. Amino acid residues that are part of the complementarity-determining regions (CDRs) are most often unaltered in relation to humanization, although in certain cases it may be desirable to alter the amino acid residues of the individual CDR, for example, to remove a glycosylation site, a deamidation site, an aspartate isomerization site, or an unwanted cysteine ​​or methionine residue. N-linked glycosylation occurs by the attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except proline. Removal of an N-glycosylation site can be achieved by mutating the Asn or Ser / Thr residue to a different residue, preferably by means of a conservative substitution.Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, particularly Asn-Gly, is present in a CDR sequence, it may be desirable to remove the site, typically by conservative substitution to eliminate one of the residues involved. Numerous methods for humanizing an antibody sequence are known in the field; see, for example, the review by Almagro and Fransson, Front Biosci. 13:1619-1633 (2008). One commonly used method is CDR grafting, which, for example, for a murine-derived chimeric antibody, involves identifying human germline counterparts of the murine variable domain genes and grafting the murine CDR sequences onto this framework. The specificity of an antibody's interaction with a target antigen resides primarily in the amino acid residues located in the six heavy- and light-chain CDRs. The amino acid sequences within the CDRs are therefore much more variable among individual antibodies than the sequences outside the CDRs.Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody, or more generally any specific antibody with a given amino acid sequence, for example, by constructing expression vectors that express CDR sequences from the specific antibody grafted into framework sequences of a different antibody. As a result, it is possible to "humanize" a non-human antibody and still substantially maintain the binding specificity and affinity of the original antibody. CDR grafting can be based on Kabat's CDR definitions, although a more recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225 (2007)) has suggested using the IMGT® definition (the international ImMunoGeneTics information system®, www.imgt.org).org) can improve the outcome of humanization (see Lefranc et al., Dev. Comp Immunol., 27:55-77 (2003) ). In some cases, CDR grafting can reduce the binding specificity and affinity, and therefore the biological activity, of a non-human antibody grafted with CDRs compared to the parental antibody from which the CDRs are derived. Back mutations (sometimes called "framework repair") can be introduced at selected positions in the CDR-grafted antibody, typically in the frame regions, to restore the binding specificity and affinity of the parental antibody. Identifying positions for potential back mutations can be done using information available in the literature and antibody databases. Amino acid residues that are candidates for back mutations are typically those found on the surface of an antibody molecule, while residues that are hidden or have a low degree of surface exposure will not normally be altered. An alternative humanization technique to CDR grafting and retromutation is remodeling, in which unexposed surface remains of non-human origin are retained, while surface remains are altered to human remains. In certain cases, it may also be desirable to alter one or more amino acid residues of the CDR to improve binding affinity to the target epitope. This is known as "affinity maturation" and can be optionally performed in conjunction with humanization, for example, in situations where humanization of an antibody leads to reduced specificity or binding affinity and it is not possible to sufficiently improve specificity or binding affinity solely through back mutations. Several affinity maturation methods are known in the art, for example, the in vitro sweep saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method of Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998). The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more portions or fragments of an antibody that retain the ability to bind specifically to an antigen (e.g., human pD-1, or a portion thereof). Certain fragments of a complete antibody have been shown to perform the antigen-binding function of the antibody. Examples of binding fragments included within the term "antigen-binding portion" include (i) a Fab fragment: a monovalent fragment consisting of the V1, Vh, Cl, and Ch1 domains (e.g., Fab fragments 12819.17149 and 12865).17150 described below); (ii) an F(ab')2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the Vh and Ch1 domains; (iv) an Fv fragment consisting of the Vl and Vh domains of a single antibody arm; (v) a dAb fragment, consisting of a Vh domain; and (vi) an isolated complementarity-determining region (CDR) capable of specifically binding to an antigen. Furthermore, although the two domains of the Fv fragment, V and Vh, are encoded by separate genes, they can be joined, using recombinant methods, by means of a synthetic connector that allows them to form as a single protein chain in which the V and Vh domains are joined to form monovalent molecules (known as single-chain Fv (scFv)). Also within the invention are antigen-binding molecules comprising a Vh and / or a Vl.In the case of a Vh antibody, the molecule may also comprise one or more CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bispecific bivalent antibodies in which the Vh and Vl domains 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. This forces the domains to pair with complementary domains on another chain, creating two antigen-binding sites. Antibody fragments, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. In addition, antibodies, antibody fragments, and immunoadherence molecules can be obtained using conventional recombinant DNA techniques, for example, as described herein. The class (isotype) and subclass of anti-PD-1 antibodies can be determined using any known method. In general, the class and subclass of an antibody can be determined using antibodies that are specific to a particular class and subclass. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blot, and other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant regions of the antibody's heavy and / or light chains, comparing their amino acid sequences with the known amino acid sequences of various immunoglobulin classes and subclasses, and determining the antibody's class and subclass. When referring to specific amino acid residues at a particular position in an antibody sequence, an indication of, for example, "35S" refers to both the position and the residue; that is, in this case, it indicates that a serine (S) residue is present at position 35 of the sequence. Similarly, an indication of, for example, "13Q+35S" refers to both residues at their respective positions. Unless otherwise stated, all antibody amino acid residue numbers referenced in this description are those under the IMGT® numbering scheme. Anti-PD-1 antibodies The present invention provides antibodies directed against PD-1 as defined in the claims and antigen-binding portions thereof. The antibodies may be chimeric, with variable domains derived from chickens and human constant regions, or they may be humanized. The antibodies described herein are, in particular, humanized antibodies. The amino acid sequences of Vh and Vl (SEQ ID NO: 2 to 17) of eight selected humanized anti-PD-1 antibodies are shown below in Table 4 (Example 4). For reference, the SEQ ID NO. are provided below in Table 1. The anti-PD-1 antibodies described herein may be referred to by a 5-digit number, e.g., "12819", or by a 10-digit number, e.g., "12819.15384". As used herein, the 5-digit number refers to all antibodies having the heavy and light chain CDR1-3 sequences shown for that number in Table 2, whereas the use of a 10-digit number refers to a specific humanized variant. For example, 12819.15384 is a specific humanized variant that has the CDR sequences of an antibody 12819 as shown in Table 2. The 5-digit number encompasses, for example, antibodies that are identical to the 10-digit variants shown below in Table 1, except for some changes in the FRs (e.g., they lack the sY residues at the N-terminus of the mature light chain, or they have SS residues instead of SY). These modifications do not change the functional properties (e.g.,e.g., antigen binding) of antibodies. Table 1 SEQ ID NO for the amino acid sequences of the heavy and light chain variable domains of humanized anti-PD-1 antibodies Table 2 below provides the SEQ ID NO for the heavy and light chain CDR amino acid sequences of the antibodies. Table 2 SEQ ID NO for anti-PD-1 antibody CDR amino acid sequences In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: a) an antibody whose H-CDR1-3 comprises the amino acid sequences of SEQ ID NO: 18-20, respectively; b) an antibody whose heavy chain variable domain (Vh) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 2; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 2 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 21-23, respectively; f) an antibody whose variable light chain (VL) domain has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3; g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 3; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 3 and 68; i) an antibody having H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 18-23, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3; (k) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 2 and whose Vl comprises the amino acid sequence of SEQ ID NO: 3; and l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO: 2 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 3 and 68. An anti-PD-1 antibody disclosed herein may be selected from the group consisting of: a) an antibody whose H-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 24-26, respectively; b) an antibody whose heavy chain variable domain (Vh) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 4; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 4 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 27-29, respectively; f) an antibody whose light chain variable domain (Vl) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5 or 66; g) an antibody whose Vl comprises the amino acid sequence of SEQ ID NO: 5 or 66; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 5 or 66 and the amino acid sequence of SEQ ID NO: 68; i) an antibody whose H-CDR1-3 and L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 24-29, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5 or 66; (k) an antibody whose Vh comprises the amino acid sequences of SEQ ID NO: 4 and whose Vl comprises the amino acid sequences of SEQ ID NO: 5 or 66; and l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO: 4 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 5 or 66 and SEQ ID NO: 68. An anti-PD-1 antibody disclosed herein may be selected from the group consisting of: a) an antibody whose H-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 30-32, respectively; b) an antibody whose variable heavy chain (VH) domain has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 6; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 6 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 33-35, respectively; f) an antibody whose variable light chain (VL) domain has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7; g) an antibody whose Vl comprises the amino acid sequence of SEQ ID NO: 7; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 7 and 68; i) an antibody having H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 30-35, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7; (k) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 6 and whose Vl comprises the amino acid sequence of SEQ ID NO: 7; and l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO:6 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 7 and 68. An anti-PD-1 antibody disclosed herein may be selected from the group consisting of: a) an antibody whose H-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 36-38, respectively; b) an antibody whose variable heavy chain (VH) domain has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 8; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 8 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 39-41, respectively; f) an antibody whose light chain variable domain (Vl) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9; g) an antibody whose Vl comprises the amino acid sequence of SEQ ID NO: 9; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 9 and 68; i) an antibody having H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 36-41, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9; (k) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 8 and whose Vl comprises the amino acid sequence of SEQ ID NO: 9; and l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO: 8 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 9 and 68. An anti-PD-1 antibody disclosed herein may be selected from the group consisting of: ) an antibody whose H-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 42-44, respectively; b) an antibody whose heavy chain variable domain (Vh) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 10; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 10 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 45-47, respectively; f) an antibody whose variable light chain (VL) domain has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11; g) an antibody whose Vl comprises the amino acid sequence of SEQ ID NO: 11; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 11 and 68; i) an antibody having H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 42-47, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11; (k) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 10 and whose Vl comprises the amino acid sequence of SEQ ID NO: 11; and l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO: 10 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 11 and 68. An anti-PD-1 antibody disclosed herein may be selected from the group consisting of: a) an antibody whose H-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 48-50, respectively; b) an antibody whose variable heavy chain (VH) domain has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 12; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 12 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 51-53, respectively; f) an antibody whose light chain variable domain (Vl) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13; g) an antibody whose Vl comprises the amino acid sequence of SEQ ID NO: 13; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 13 and 68; i) an antibody having H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 48-53, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13; (k) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 12 and whose Vl comprises the amino acid sequence of SEQ ID NO: 13; and l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO: 12 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 13 and 68. An anti-PD-1 antibody disclosed herein may be selected from the group consisting of: a) an antibody whose H-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 54-56, respectively; b) an antibody whose heavy chain variable domain (Vh) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 14; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 14 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 57-59, respectively; f) an antibody whose light chain variable domain (Vl) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; g) an antibody whose Vl comprises the amino acid sequence of SEQ ID NO: 15; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 15 and 68; i) an antibody having H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 54-59, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; (k) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 14 and whose Vl comprises the amino acid sequence of SEQ ID NO: 15; and l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO: 14 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 15 and 68. An anti-PD-1 antibody disclosed herein may be selected from the group consisting of: a) an antibody whose H-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 60-62, respectively; b) an antibody whose heavy chain variable domain (Vh) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16; c) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 16; d) an antibody whose heavy chain (HC) comprises the amino acid sequences of SEQ ID NO: 16 and 67; e) an antibody whose L-CDR1-3 comprise the amino acid sequences of SEQ ID NO: 63-65, respectively; f) an antibody whose light chain variable domain (Vl) has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17; g) an antibody whose Vl comprises the amino acid sequence of SEQ ID NO: 17; h) an antibody having a light chain (LC) comprising the amino acid sequences of SEQ ID NO: 17 and 68; i) an antibody having H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 60-65, respectively; j) an antibody whose Vh has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and whose Vl has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17; k) an antibody whose Vh comprises the amino acid sequence of SEQ ID NO: 16 and whose Vl comprises the amino acid sequence of SEQ ID NO: 17; l) an antibody whose HC comprises the amino acid sequences of SEQ ID NO: 16 and 67 and whose LC comprises the amino acid sequences of SEQ ID NO: 17 and 68. In some embodiments, the anti-PD-1 antibody or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 12819 (e.g., antibody 12819.15384). An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 12748 (e.g., antibody 12748.15381 or antibody 12748.16124). An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 12865 (e.g., antibody 12865.15377). An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 12892 (e.g., antibody 12892.15378). An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 12796 (e.g., antibody 12796.15376). An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of an antibody 12777 (e.g., antibody 12777.15382).An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 12760 (e.g., antibody 12760.15375). An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 13112 (e.g., antibody 13112.15380). In another embodiment, the anti-PD-1 antibody or an antigen-binding portion thereof has a Vh and a Vl having an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to that of Vh and Vl, respectively, of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody or an antigen-binding portion thereof has a Vh and a Vl comprising the amino acid sequences of Vh and Vl, respectively, of antibody 12819.15384. In some embodiments, the anti-PD-1 antibody or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of Se Q ID NO: 18, 19, 20, 21, 22, and 23, respectively. An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of: a) SEQ ID NO: 24, 25, 26, 27, 28 and 29, respectively; b) SEQ ID NO: 30, 31, 32, 33, 34 and 35, respectively; c) SEQ ID NO: 36, 37, 38, 39, 40 and 41, respectively; d) SEQ ID NO: 42, 43, 44, 45, 46 and 47, respectively; e) SEQ ID NO: 48, 49, 50, 51, 52 and 53, respectively; f) SEQ ID NO: 54, 55, 56, 57, 58 and 59, respectively; or g) SEQ ID NO: 60, 61, 62, 63, 64 and 65, respectively. In some embodiments, the anti-PD-1 antibody or an antigen-binding portion thereof comprises a Vh that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and a V l that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 2 and 3, respectively. An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises a Vh that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and a Vl that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of: a) SEQ ID NO: 4 and 5, respectively; b) SEQ ID NO: 4 and 66, respectively; c) SEQ ID NO: 6 and 7, respectively; d) SEQ ID NO: 8 and 9, respectively; e) SEQ ID NO: 10 and 11, respectively; f) SEQ ID NO: 12 and 13, respectively; g) SEQ ID NO: 14 and 15, respectively; or h) SEQ ID NO: 16 and 17, respectively. In some embodiments, the anti-PD-1 antibody or an antigen-binding portion thereof comprises a V and a Vl which are the amino acid sequences of SEQ ID NO: 2 and 3, respectively. An anti-PD-1 antibody disclosed herein or an antigen-binding portion thereof comprises a VH and a VL which are the amino acid sequences of: a) SEQ ID NO: 4 and 5, respectively; b) SEQ ID NO: 4 and 66, respectively; c) SEQ ID NO: 6 and 7, respectively; d) SEQ ID NO: 8 and 9, respectively; e) SEQ ID NO: 10 and 11, respectively; f) SEQ ID NO: 12 and 13, respectively; g) SEQ ID NO: 14 and 15, respectively; or h) SEQ ID NO: 16 and 17, respectively. In some embodiments, the anti-PD-1 antibody comprises an HC comprising the amino acid sequences of SEQ ID NO: 2 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 3 and 68. An anti-PD-1 antibody disclosed herein comprises: a) an HC comprising the amino acid sequences of SEQ ID NO: 4 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 5 and 68; b) an HC comprising the amino acid sequences of SEQ ID NO: 4 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 66 and 68; c) an HC comprising the amino acid sequences of SEQ ID NO: 6 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 7 and 68; d) an HC comprising the amino acid sequences of SEQ ID NO: 8 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 9 and 68; e) an HC comprising the amino acid sequences of SEQ ID NO: 10 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 11 and 68; f) an HC comprising the amino acid sequences of SEQ ID NO: 12 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 13 and 68; (g) an HC comprising the amino acid sequences of SEQ ID NO: 14 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 15 and 68; or h) an HC comprising the amino acid sequences of SEQ ID NO: 16 and 67 and an LC comprising the amino acid sequences of SEQ ID NO: 17 and 68. In some embodiments, the anti-PD-1 antibody comprises a HC consisting of the amino acid sequences of SEQ ID NO: 2 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 3 and 68. An anti-PD-1 antibody disclosed herein comprises: a) an HC consisting of the amino acid sequences of SEQ ID NO: 4 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 5 and 68; b) an HC consisting of the amino acid sequences of SEQ ID NO: 4 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 66 and 68; c) an HC consisting of the amino acid sequences of SEQ ID NO: 6 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 7 and 68; d) an HC consisting of the amino acid sequences of SEQ ID NO: 8 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 9 and 68; e) an HC consisting of the amino acid sequences of SEQ ID NO: 10 and 67 and an LC consisting of the amino acid sequences of SEQ ID NOs: 11 and 68; f) an HC consisting of the amino acid sequences of SEQ ID NO: 12 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 13 and 68; (g) an HC consisting of the amino acid sequences of SEQ ID NO: 14 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 15 and 68; or h) an HC consisting of the amino acid sequences of SEQ ID NO: 16 and 67 and an LC consisting of the amino acid sequences of SEQ ID NO: 17 and 68. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions of the invention may have at least one of the following properties: a) binds to human PD-1 with a Kd of 750 pM or less; b) binds to cynomolgo PD-1 with a Kd of 7 nM or less; c) binds to mouse PD-1 with a KD of 1 nM or less; d) does not bind to rat PD-1; e) increases IL-2 secretion in a SEB whole blood assay; f) increases IFN-γ secretion in a unidirectional lymphocyte mixed reaction assay; g) inhibits the interaction of PD-1 with PD-L1 by at least 60% at a concentration of 10 µg / ml in a competitive flow cytometry assay; h) blocks the binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 jg / ml as determined by Bio-Layer interferometry analysis; e i) inhibits tumor growth in vivo. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions of the invention can bind to human PD-1 with a Kd of at least 900, at least 850, at least 800, at least 750, at least 700, at least 650, at least 600, at least 550, at least 500, at least 450, at least 400, at least 350, at least 300, at least 250, at least 200, at least 150, at least 100, at least 50, at least 40, at least 30, or at least 20 pM. In certain embodiments, the Kd is determined using surface plasmon resonance. In specific realizations, anti-PD-1 antibodies or antigen-binding portions bind to human PD-1 with a higher affinity than nivolumab, pembrolizumab, or both. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions of the invention can be bound to PD-1 of cinomolgo (SEQ ID NO: 89) with a Kd of at least 9000, at least 8000, at least 7000, at least 6000, at least 5000, at least 4000, at least 3000, at least 2500, at least 2000, at least 1500, at least 1000, at least 900, at least 800, at least 700, at least 600, at least 500, at least 400, at least 300, at least 200, at least 100, at least 75, at least 50, at least 25, at least 20, at least 15, at least 10 or at least 5 pM. In certain embodiments, Kd is determined using surface plasmon resonance. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions of the invention can be bound to mouse PD-1 (SEQ ID NO: 91) with a Kd of at least 1000, at least 950, at least 900, or at least 850 pM. In certain embodiments, the Kd is determined using surface plasmon resonance. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions of the invention can inhibit the interaction of PD-1 with PD-L1 by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% at a concentration of 10 jg / ml in a competitive flow cytometry assay. In certain embodiments, anti-PD-1 antibodies or antigen-binding portions can inhibit the interaction of PD-1 with PD-L1 by at least 83%. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions of the invention can block the binding of PD-L1 and PD-L2 to PD-1 by at least 20%, at least 30%, to at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, to at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% at a concentration of 10 jg / ml as determined by Bio-Layer Interferometry analysis. In certain embodiments, anti-PD-1 antibodies or antigen-binding portions block the binding of PD-L1 and PD-L2 to PD-1 by at least 90%. Any of the anti-PD-1 antibodies or antigen-binding portions described herein may compete or cross-compete for PD-1 binding with antibodies 12865, 12892, and 12777 (e.g., antibodies 12865.15377, 12892.15378, and 12777.15382). Any of the anti-PD-1 antibodies or antigen-binding portions described herein may compete or cross-compete for PD-1 binding with antibody 12819 (e.g., antibody 12819.15384). Any of the anti-PD-1 antibodies or antigen-binding portions described herein may compete or cross-compete for PD-1 binding with antibodies 12760 and 13112 (e.g., antibodies 12760.15375 and 13112.15380). An anti-PD-1 antibody disclosed herein, or an antigen-binding portion thereof, binds to a PD-1 epitope that includes at least one (e.g., at least one, at least two, at least three, at least four, or at least five) of the following SEQ iD NO: 1 residues: V44, V64, L128, P130, K131, A132, E136, and T145. The antibody of the invention or antigen-binding portion binds to a PD-1 epitope that includes SEQ ID NO: 1 residues V64, L128, P130, K131, and A132 (such as a 12819 antibody, for example, antibody 12819.15384). An antibody disclosed in the present memory or antigen-binding portion binds to a PD-1 epitope that includes the K131 and E136 residues of SEQ ID NO: 1 (such as an antibody 12865, for example, antibody 12865.15377).An antibody disclosed in the present memory or antigen-binding portion binds to a PD-1 epitope that includes the V44 and T145 residues of Se Q ID NO: 1 (such as an antibody 13112, for example, antibody 13112.15380). In some embodiments, an anti-PD-1 antibody of the invention, or an antigen-binding portion thereof, binds to a PD-1 epitope comprising residues 56-64, 69-90, and 122-140 of SEQ ID NO: 1. An antibody disclosed herein or an antigen-binding portion binds to a PD-1 epitope comprising residues 69-90 and 122-140 of SEQ ID NO: 1 (such as antibodies 12865, e.g., antibody 12865.15377). In certain embodiments, the antibody or antigen-binding portion binds to a PD-1 epitope comprising residues 56-64, 69-90, and 122-140 of SEQ ID NO: 1 (e.g., antibody 12819). An antibody disclosed in the present memory or antigen-binding portion binds to a PD-1 epitope comprising residues 69-90 and 122-140 of SEQ ID NO: 1 (e.g., an antibody 12865).An antibody disclosed in this memory or portion binds to residues 69-75 (or a fragment thereof, such as a fragment of one, two, three, four, five, or six residues) of SEQ ID NO: 1 (such as antibodies 12865, e.g., antibody 12865.15377). An antibody disclosed in this memory or portion binds to residues 136-140 (or a fragment thereof, such as a fragment of one, two, three, or four residues) of SEQ ID NO: 1 (such as antibodies 12865, e.g., antibody 12865.15377). An antibody disclosed in this memory or portion binds to residues 69-75 (or a fragment thereof) and residues 136-140 (or a fragment thereof) of SEQ ID NO: 1 (such as antibodies 12865, e.g., antibody 12865.15377). An epitope with any combination of the above residues is also contemplated. An amino acid sequence comprising a PD-1 epitope as described herein (e.g., administered to an animal or in the form of an antigen to screen antibody libraries) can be used as an immunogen to generate or identify anti-PD-1 antibodies or antigen-binding portions thereof that bind to said epitope. The class of an anti-PD-1 antibody obtained by the methods described herein can be replaced or changed to another class or subclass. In one aspect of the invention, a nucleic acid molecule encoding Vl or Vh is isolated using procedures well known in the art such that it does not include nucleic acid sequences encoding Cl or Ch. The nucleic acid molecules encoding Vl or Vh are then operatively connected to a nucleic acid sequence encoding a Cl or Ch, respectively, of a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule comprising a Cl or Ch chain, as described above. For example, an anti-PD-1 antibody that was originally IgM can be reclassified to IgG. Furthermore, the reclassification can be used to convert one IgG subclass into another, e.g., from IgG1 to IgG2.A light chain constant region k can be replaced by a light chain constant region A. A preferred method for producing an antibody of the invention with a desired Ig isotype comprises the steps of isolating a nucleic acid molecule encoding the heavy chain of an anti-PD-1 antibody and a nucleic acid molecule encoding the light chain of an anti-PD-1 antibody, obtaining the variable domain of the heavy chain, ligating the variable domain of the heavy chain with the constant region of a heavy chain of the desired isotype, expressing the linked light chain and heavy chain in a cell, and collecting the anti-PD-1 antibody with the desired isotype. The anti-PD-1 antibody of the invention can be an IgG, an IgM, an IgE, an IgA, or an IgD, but is typically of the IgG isotype, e.g., of the IgG subclass IgG1, IgG2a, or IgG2b, IgG3, or IgG4. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG4. In one embodiment, the anti-PD-1 antibody may comprise at least one mutation in the Fc region. Several different Fc mutations are known, and these mutations alter the effector function. For example, in many cases it will be desirable to reduce or eliminate the effector function, e.g., when ligand / receptor interactions are undesirable or in the case of antibody-drug conjugates. In one embodiment, the anti-PD-1 antibody comprises at least one mutation in the Fc region that reduces effector function. The amino acid positions in the Fc region that may be advantageously mutated to reduce effector function include one or more of positions 228, 233, 234, and 235, where the amino acid positions are numbered according to the IMGT® numbering scheme. In one embodiment, one or both amino acid residues at positions 234 and 235 may be mutated, e.g., from Leu to Ala (L234A / L235A). These mutations reduce the effector function of the Fc region of IgG1 antibodies. Additionally or alternatively, the amino acid residue at position 228 may be mutated, e.g., to Pro. In another embodiment, the amino acid residue at position 233 may be mutated, e.g., to Pro, the amino acid residue at position 234 may be mutated, e.g., to Val, and / or the amino acid residue at position 235 may be mutated, e.g., to Ala. The amino acid positions are numbered according to the IMGT® numbering scheme. In another embodiment, when the antibody is of the lgG4 subclass, it may contain the S228P mutation, meaning it has a proline at position 228, where the amino acid positions are numbered according to the IMGT® numbering scheme. This mutation is known to reduce unwanted Fab arm exchange. In certain embodiments, an antibody or antigen-binding portion thereof of the invention may be part of a larger immunoadherence molecule, formed by covalent or non-covalent association of the antibody or antibody portion with one or more proteins or peptides. Examples of such immunoadherence molecules include the use of the streptavidin core region to form a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)) and the use of a cysteine ​​moiety, a marker peptide, and a C-terminal polyhistidine tag to prepare bivalent, biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol., 31:1047-1058 (1994)). Other examples include when one or more c Dr of an antibody are incorporated into a molecule, either covalently or non-covalently, to convert it into an immunoadhesin that binds specifically to an antigen of interest.CDRs can be incorporated as part of a larger polypeptide chain, can be covalently connected to another polypeptide chain, or can be incorporated non-covalently. In another embodiment, a fusion antibody or immunoadhesin can be prepared comprising all or part of an anti-PD-1 antibody of the invention connected to another polypeptide. In certain embodiments, only the variable domains of the anti-PD-1 antibody are connected to the polypeptide. In certain embodiments, the VH domain of an anti-PD-1 antibody is connected to a first polypeptide, while the VL domain of an anti-PD-1 antibody is connected 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 (e.g., single-chain antibodies). The Vh-linker-Vl antibody is then attached to the polypeptide of interest. Furthermore, fusion antibodies can be created by linking two (or more) single-chain antibodies together. This is useful for creating a divalent or polyvalent antibody on a single polypeptide chain, or for creating a bispecific antibody. To create a single-stranded antibody (scFv), the DNA fragments encoding Vh and Vl are operatively connected to another fragment encoding a flexible connector, e.g., one encoding the amino acid sequence (Gly4-Ser)3, so that the Vh and Vl sequences can be expressed as a contiguous single-stranded protein, with the Vl and Vh domains linked by the flexible connector. See, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). The single-stranded antibody can be monovalent, if only one Vh and Vl are used; 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 bind specifically to human PD-1 and another molecule, for example. In other embodiments, other modified antibodies can be prepared using nucleic acid molecules that encode anti-PD-1 antibody. For example, "kappa antibodies" (III et al., Protein Eng. 10:949-57 (1997)), "minibodies" (Martin et al., EMBO J. 13:5303-9 (1994)), "bivalent fragments (diabodies)" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or "Janusins" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) can be prepared using conventional molecular biology techniques following the teachings of the descriptive report. An anti-PD-1 antibody or antigen-binding portion of the invention may be derivatized or linked to another molecule (e.g., another peptide or protein). In general, antibodies or portions thereof are derivatized in such a way that binding to PD-1 is not adversely affected by derivatization or labeling. Accordingly, the antibodies and antibody portions of the invention are intended to include both intact and modified forms of human anti-PD-1 antibodies. For example, an antibody or antibody portion of the invention may be functionally linked (by chemical docking, genetic fusion, non-covalent association, or otherwise) to one or more different molecular entities, such as another antibody (e.g.,, a bispecific antibody or a bivalent fragment), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag). A derivatized antibody type is produced by crosslinking two or more antibodies (of the same or different types, e.g., to create bispecific antibodies). Suitable crosslinking agents include those that are heterobifunctional, having two distinctly reactive groups separated by a proper spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available, e.g., from Pierce Chemical Company, Rockford, IL. An anti-PD-1 antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups can be useful for improving the antibody's biological characteristics, e.g., for increasing its half-life in serum. An antibody can also be labeled according to the present invention. As used herein, the terms "label" or "labeled" refer to the incorporation of another molecule into the antibody. In one embodiment, the label is a detectable marker, e.g., the incorporation of a radiolabeled amino acid or the anchoring to a polypeptide of biotinyl radicals that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker) or enzymatic activity that can be detected by optical or colorimetric methods. In another embodiment, the label or marker can be a therapeutic agent, e.g., a drug or toxin conjugate. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,, 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide-based phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper-pair sequences, secondary antibody binding sites, metal-binding domains, epitopic tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogues or homologues thereof.In some embodiments, the markers are joined by spacer arms of varying lengths to reduce potential stereo impedance. In certain embodiments, the antibodies of the invention may be present in a neutral form (including zwitterionic forms) or as a positively or negatively charged species. In some embodiments, the antibodies may be complexed with a counterion to form a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to a complex comprising one or more antibodies and one or more counterions, wherein the counterions are obtained from pharmaceutically acceptable inorganic and organic acids and bases. Bispecific binding molecules In a further aspect, the invention provides a bispecific binding molecule that has the binding specificity for an anti-PD-1 antibody of the invention and the binding specificity for another anti-PD-1 antibody (e.g., another anti-PD-1 antibody described herein) or an antibody that targets a different protein, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity is involved in a disease state such as cancer. Such bispecific binding molecules are known in the art, and examples of different types of bispecific binding molecules are provided elsewhere herein. Nucleic acid molecules and vectors The present invention also provides nucleic acid molecules and sequences encoding anti-PD-1 antibodies or antigen-binding portions thereof. In some embodiments, different nucleic acid molecules encode the heavy-chain and light-chain amino acid sequences of the anti-PD-1 antibody or an antigen-binding portion thereof. In other embodiments, the same nucleic acid molecule encodes the heavy-chain and light-chain amino acid sequences of the anti-PD-1 antibody or an antigen-binding portion thereof. A reference to a nucleotide sequence includes its complement unless otherwise specified. Therefore, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand, with its complementary sequence. The term "polynucleotide" as used herein means a polymeric form of nucleotides at least 10 bases in length, whether ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes both single-stranded and double-stranded forms. Also disclosed herein are nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to one or more nucleotide sequences mentioned herein, for example, to a nucleotide sequence selected from the group consisting of SEQ ID NO: 69-88. The term "sequence identity percentage" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of the sequence identity comparison may be at least approximately nine nucleotides, usually at least approximately 18 nucleotides, more commonly at least approximately 24 nucleotides, typically at least approximately 28 nucleotides, more typically at least approximately 32 nucleotides, and preferably at least approximately 36, 48, or more nucleotides.Several different algorithms are known in the technique that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs from Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, for example, the FASTA2 and FASTA3 programs, provides alignments and percentage sequence identity of the regions of best overlap between the problem and search sequences (see, for example, Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); and Pearson, J. Mol. Biol. 276: 7-84 (1998)). Unless otherwise specified, the default parameters for a particular program or algorithm are used.For example, the percentage of sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters provided in GCG Version 6.1. In one aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 69 and 70. In any of the above embodiments, nucleic acid molecules can be isolated. In a further aspect, the present invention provides a vector suitable for expressing one of the chains of an antibody of the invention or the antigen-binding portion thereof. The term "vector," as used herein, means a nucleic acid molecule capable of carrying another nucleic acid to which it is bound. In some embodiments, the vector is a plasmid, i.e., a circular piece of double-stranded DNA to which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, in which additional DNA segments can be ligated to the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, the vectors (e.g.Non-episomal mammalian vectors can integrate into the genome of a host cell after introduction into the host cell, and thus replicate along with the host genome. Furthermore, certain vectors are capable of directing the expression of the genes to which they are operationally connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). The invention provides vectors comprising nucleic acid molecules encoding the heavy chain of an anti-PD-1 antibody of the invention or an antigen-binding portion thereof, the light chain of an anti-PD-1 antibody of the invention or an antigen-binding portion thereof, or both the heavy and light chains of an anti-PD-1 antibody of the invention or an antigen-binding portion thereof. The invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and antibody probes. A nucleic acid molecule encoding the heavy and / or light chain of an anti-PD-1 antibody or an antigen-binding portion thereof of the invention can be isolated from any source that produces such antibody or portion. The nucleic acid molecules can be isolated from B cells expressing an anti-PD-1 antibody isolated from an animal immunized with a human PD-1 antigen, or from an immortalized cell produced from such a B cell. Methods for isolating nucleic acids encoding an antibody are well known in the art. The mRNA can be isolated and used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In certain embodiments, a nucleic acid molecule of the invention can be synthesized instead of isolated. In some embodiments, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a Vh domain of an anti-PD-1 antibody or antigen-binding portion of the invention frame-linked to a nucleotide sequence encoding a heavy-chain constant region of any source. Similarly, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a Vl domain of an anti-PD-1 antibody or antigen-binding portion of the invention frame-linked to a nucleotide sequence encoding a light-chain constant region of any source. In a further aspect of the invention, nucleic acid molecules encoding the variable domain of the heavy chain (Vh) and / or light chain (Vl) can be "converted" into complete antibody genes. In one embodiment, nucleic acid molecules encoding the Vh or Vl domains are converted into complete antibody genes by insertion into an expression vector that already encodes heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the Vh segment is operatively connected to the CH segment(s) within the vector, and / or the Vl segment is operatively connected to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding the Vh and / or Vl domains are converted into complete antibody genes by connecting, e.g.The ligand is a nucleic acid molecule encoding a Vh and / or Vl domain to a nucleic acid molecule encoding a CH and / or CL domain using conventional molecular biology techniques. The nucleic acid molecules encoding the complete heavy and / or light chains can then be expressed from a cell into which they have been introduced, and the nti-PD-1 antibody isolated. Nucleic acid molecules can be used to recombinantly express large quantities of anti-PD-1 antibodies. Nucleic acid molecules can also be used to produce chimeric antibodies, bispecific antibodies, single-stranded antibodies, immunoadhesins, bivalent antibodies, mutated antibodies, and antibody derivatives, as described in this dissertation. In another embodiment, a nucleic acid molecule of the invention is used as a PCR probe or primer for a specific antibody sequence. For example, the nucleic acid may be used as a probe in diagnostic methods or as a PCR primer to amplify DNA regions that could be used, among other things, to isolate additional nucleic acid molecules encoding variable domains of anti-PD-1 antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. The oligonucleotides disclosed herein may be derived from highly variable domains of the heavy and light chains of the antibody of interest. The oligonucleotides disclosed herein may encode all or part of one or more of the CDRs of the anti-PD-1 antibodies or antigen-binding portions thereof of the invention as described herein. In another embodiment, nucleic acid molecules and vectors can be used to produce mutated anti-PD-1 antibodies. The antibodies can be mutated in the variable domains of the heavy and / or light chains, e.g., to alter a binding property of the antibody. For example, a mutation can be made in one or more of the CDRs to increase or decrease the Kd of the anti-PD-1 antibody, to increase or decrease Koff, or to alter the binding specificity of the antibody. In another embodiment, one or more mutations are made in an amino acid residue known to be changed compared to the germline in a monoclonal antibody of the invention. The mutations can be made in a CDR or frame region of a variable domain, or in a constant region. In a preferred embodiment, the mutations are made in a variable domain.In some embodiments, one or more mutations are made in an amino acid residue known to be changed compared to the germline in a CDR or frame region of a variable domain of an antibody or antigen-binding portion thereof of the invention. In another embodiment, the frame region or regions are mutated so that the resulting frame region or regions have the amino acid sequence of the corresponding germline gene. A mutation in a frame region or constant region can be made to increase the half-life of the anti-PD-1 antibody. See, e.g., PCT publication WO 00 / 09560. A mutation in a frame region or constant region can also be made to alter the immunogenicity of the antibody and / or to provide a site for covalent or non-covalent binding to another molecule. According to the invention, a single antibody can have mutations in any one or more of the CDRs or frame regions of the variable domain or in the constant region. In some embodiments, the anti-PD-1 antibodies of the invention, or antigen-binding portions thereof, are expressed by inserting partial or complete light and heavy chain DNA, obtained as described above, into expression vectors such that the genes are operatively connected to necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, and EBV-derived episomes. The antibody coding sequence can be ligated to a vector such that the transcriptional and translational control sequences within the vector fulfill their intended function of regulating the transcription and translation of the antibody coding sequence.The expression vector and expression control sequences can be selected to be compatible with the host cell used for expression. The antibody light chain coding sequence and the antibody heavy chain coding sequence can be inserted into separate vectors and can be operatively connected to the same or different expression control sequences (e.g., promoters). In one embodiment, both coding sequences are inserted into the same expression vector and can be operatively connected to the same expression control sequences (e.g., a common promoter), separate identical expression control sequences (e.g., promoters), or different expression control sequences (e.g., promoters). The antibody coding sequences can be inserted into the expression vector using conventional methods (e.g.,, ligation of complementary restriction sites on the gene fragment of the antibody and vector, or ligation of blunt ends if restriction sites are not present). A convenient vector is one that encodes a functionally complete human immunoglobulin CH or CL sequence, with appropriate restriction sites designed so that any V or Vl sequence can be easily inserted and expressed, as described above. The genes encoding HC and LC in such vectors may contain intron sequences that will result in increased overall antibody protein yields by stabilizing the associated mRNA. The intronic sequences are flanked by splice donor and splice acceptor sites, which determine where RNA splicing will occur. The location of the intronic sequences can be in variable or constant regions of the antibody chains, or in both variable and constant regions when multiple introns are used. Polyadenylation and transcriptional termination can occur at native chromosomal sites downstream of the coding regions.The recombinant expression vector can also encode a signal peptide that facilitates the secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector so that the signal peptide frames-links to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain genes, the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed and the desired level of protein expression. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), and adenoviruses (e.g.,The adenovirus major late promoter (AdMLP), polyoma, and strong mammalian promoters such as native immunoglobulin and actin promoters are known in the art. For a further description of viral regulatory elements and their sequences, see, e.g., U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as plant transformation, are known in the art. See, e.g., U.S. Patent No. 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification), the neo gene (for selection with G418), and the glutamate synthase gene. The term "expression control sequence" as used herein means polynucleotide sequences that are necessary for the expression and processing of the coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that enhance protein stability; and, when desired, sequences that enhance protein secretion.The nature of such control sequences differs depending on the host organism. In prokaryotes, these control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence. In eukaryotes, they generally include both promoters and transcription termination sequences. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Host cells and methods of antibody production and antibody compositions A further aspect of the invention relates to methods for producing the antibody compositions and the antibodies and antigen-binding portions thereof of the invention. One embodiment of this aspect of the invention relates to a method for producing an antibody of the invention, comprising providing a recombinant host cell capable of expressing the antibody, culturing such host cell under conditions suitable for antibody expression, and isolating the resulting antibody. The antibodies produced by such expression in such recombinant host cells are referred to herein as "recombinant antibodies." The invention also provides progeny cells of such host cells and antibodies produced by them. The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. The invention provides host cells comprising a vector according to the invention described above. The invention also provides host cells comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, or both, of an anti-PD-1 antibody or antigen-binding portion thereof of the invention. It is to be understood that "recombinant host cell" and "host cell" mean not only the particular subject cell but also the progeny of such cell.Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may, in fact, not be identical to the parent cell, but is still included within the scope of the term "host cell" as employed in this memory. Nucleic acid molecules encoding anti-PD-1 antibodies and vectors comprising these nucleic acid molecules can be used for transfection of a suitable mammalian, plant, bacterial, or yeast host cell. Transformation can be performed by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells using viral vectors. Methods of cell transformation are well known in the art. See, e.g., U.S. Patents 4,399,216 and 4,912.040, 4,740,461 and 4,959,455. Methods for transforming plant cells are well known in the art, including, e.g., Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art. Mammalian cell lines available as hosts for expression are well known in the technique and include many immortalized cell lines available in the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, Freestyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster pup kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and several other cell lines. Cell lines of particular preference are selected by determining which cell lines have high levels of expression. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells.When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow antibody expression within the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells were grown. The antibodies can then be recovered from the culture medium using conventional protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, maize, wheat, and potato. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris. Additionally, the expression of antibodies of the invention or antigen-binding portions thereof from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach to enhancing expression under certain conditions. The GS system is discussed in whole or in part in connection with patents EP 0216846, 0256055, 0323997, and 0338841. Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation patterns. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, are part of the present invention, regardless of the glycosylation status of the antibodies, and more generally, regardless of the presence or absence of post-translational modifications. Pharmaceutical compositions Another aspect of the invention is a pharmaceutical composition comprising, as an active ingredient (or as the sole active ingredient), an anti-PD-1 antibody or antigen-binding portion thereof, or any anti-PD-1 antibody composition of the invention. The pharmaceutical composition may comprise any anti-PD-1 antibody composition or antibody or antigen-binding portion thereof of the invention. In some embodiments, the compositions are intended for the enhancement, prevention, and / or treatment of a PD-1-related disorder (e.g., a disorder characterized by PD-1 overexpression or hyperactivity) and / or cancer. In some embodiments, the compositions are intended for the activation of the immune system.In certain embodiments, the compositions are intended for the improvement, prevention and / or treatment of cancer originating in tissues such as skin, lung, intestine, ovary, brain, prostate, kidney, soft tissues, the hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus and pancreas. Generally, the antibodies of the invention or antigen-binding portions thereof are suitable for administration as a formulation in association with one or more pharmaceutically acceptable excipients, e.g., as described below. The pharmaceutical compositions of the invention comprise one or more anti-PD-1 antibodies or binding portions of the invention, e.g., one or two anti-PD-1 antibodies or binding portions. In one embodiment, the composition comprises a single anti-PD-1 antibody or binding portion thereof of the invention. In another embodiment, the pharmaceutical composition may comprise at least one anti-PD-1 antibody or antigen-binding portion thereof, e.g., an anti-PD-1 antibody or portion thereof and one or more additional antibodies that target one or more relevant cell surface receptors, e.g., one or more cancer-relevant receptors. The term "excipient" is used herein to describe any ingredient other than the compound or compounds of the invention. The choice of the excipient or excipients will depend largely on factors such as the specific route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersions, coatings, antibacterial and antifungal agents, absorption retardants, and isotonic agents that are physiologically compatible. Examples of pharmaceutically acceptable excipients include water, saline solution, phosphate-buffered saline solution, dextrose, glycerol, ethanol, and combinations thereof. In many cases, it will be preferable to include isotonic agents, e.g.Sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be present in the composition. Additional examples of pharmaceutically acceptable substances include wetting agents or minimal amounts of excipients such as humectants or emulsifiers, preservatives, or buffers, which enhance the shelf life or efficacy of the antibody. The pharmaceutical compositions of the present invention and the methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably manufactured under GMP (Good Manufacturing Practice) conditions. A pharmaceutical composition of the invention may be prepared, packaged, or marketed in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete quantity of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction thereof, such as, e.g., half or one-third of such dosage. Any method for administering peptides, proteins, or antibodies accepted in the technique may be appropriately employed for the antibodies and antigen-binding portions of the invention. The pharmaceutical compositions of the invention are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical disruption of a subject's tissue and administration of the pharmaceutical composition through the disruption into the tissue, generally resulting in direct administration into the bloodstream, muscle, or an internal organ. Parenteral administration thus includes the administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, or by application of the composition through a non-surgical wound that penetrates the tissue.Specifically, parenteral administration is considered, including subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intra-arterial, intrathecal, intraventricular, intraurethral, ​​intracranial, and intrasynovial injections or infusions; and renal dialysis infusion techniques. Regional perfusion is also considered. The preferred routes include intravenous and subcutaneous administration. Pharmaceutical formulations suitable for parenteral administration typically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline solution. Such formulations may be prepared, packaged, or marketed in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or marketed in unit-dose form, such as in ampoules or multidose containers containing a preservative. Parenteral formulations include suspensions, solutions, emulsions in oily or aqueous vehicles, and pastes. Such formulations may further comprise one or more additional ingredients, including suspending agents, stabilizers, or dispersants.In a parenteral formulation, the active ingredient is provided in dry form (i.e., powder or granules) for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffering agents (preferably at a pH of 3 to 9), but for some applications, they may be more appropriately formulated as a sterile, non-aqueous solution or as a dry form for use with a suitable vehicle such as sterile, pyrogen-free water. Illustrative parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous solutions of propylene glycol or dextrose. Such dosage forms may be appropriately buffered, if desired.Other useful parenteral formulations include those containing the active ingredient in microcrystalline form or in a liposomal preparation. Parenteral formulations can be formulated for immediate and / or modified release. Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. For example, in one aspect, sterile injectable solutions can be prepared by incorporating the anti-PD-1 antibody or its antigen-binding portion, or the anti-PD-1 antibody composition, in the required amount, into a suitable solvent with one or a combination of the ingredients listed above, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which produce a powder of the active ingredient plus any additional desired ingredients from a solution previously filtered under sterile conditions. The appropriate flowability of a solution can be maintained, e.g.Prolonged absorption of injectable compositions can be achieved by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including an absorption-delaying agent in the composition, for example, monostearate salts and gelatin, and / or by using modified-release coatings (e.g., slow-release coatings). The antibodies of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (alone, as a mixture, or as a particle of mixed components, for example, mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, as an aerosol from a pressurized container, pump, sprayer, atomizer (preferably an atomizer that uses electrohydrodynamics to produce a fine mist) or nebulizer, with or without the use of a suitable propellant, or as nasal drops. The pressurized container, pump, sprayer, atomizer or nebulizer generally contains a solution or suspension of an antibody of the invention comprising, e.g., an agent suitable for dispersing, solubilizing or prolonging the release of the active agent, one or more propellants as a solvent. Before use in a dry powder or suspension formulation, the pharmaceutical product is generally micronized to a size suitable for inhalation administration (typically less than 5 micrometers). This can be achieved by any appropriate grinding method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high-pressure homogenization, or spray drying. Capsules, ampoules, and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound of the invention, a suitable powder base, and a performance modifier. A solution formulation suitable for use in an atomizer that uses electrohydrodynamics to produce a fine mist may contain a suitable dose of the antibody of the invention per actuation and the actuation volume may vary, e.g., from 1 pl to 100 pl. Suitable flavorings, such as menthol and levomenthol, or sweeteners, such as saccharin or sodium saccharin, may be added to those formulations of the invention intended for inhalation / intranasal administration. Inhaled / intranasal formulations can be formulated for immediate and / or modified release. Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. In the case of dry powder inhalers and aerosols, the dosage unit is determined by a valve that delivers a measured amount. The units according to the invention are typically arranged to deliver a measured dose or "inhalation" of an antibody of the invention. The total daily dose is typically administered as a single dose or, more commonly, as divided doses throughout the day. The antibodies and antibody portions of the invention may also be formulated for oral administration. Oral administration may involve swallowing, whereby the compound enters the gastrointestinal tract, and / or buccal, lingual, or sublingual administration, whereby the compound enters the bloodstream directly from the mouth. Suitable formulations for oral administration include solid, semi-solid, and liquid systems such as tablets; soft or hard capsules containing multi- or nanoparticulate products, liquids, or powders; lozenges (including liquid-filled lozenges); chewing gum; gels; rapid dispersion dosage forms; films; ovules; sprays; and oral / mucoadhesive patches. Liquid formulations include suspensions, solutions, syrups, and elixirs. Such formulations can be used as fillers in soft or hard capsules (made, for example, of gelatin or hydroxypropyl methylcellulose) and typically comprise a vehicle, such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying and / or suspending agents. Liquid formulations can also be prepared by reconstituting a solid, for example, from a sachet. Therapeutic uses of antibodies and compositions of the invention In one aspect, the anti-PD-1 antibodies and their antigen-binding portions, the anti-PD-1 compositions, and the bispecific binding molecules of the invention are used to enhance or activate the immune system in a human being in need. In some embodiments, the patient has a condition characterized by overexpression or hyperactivity of PD-1. In some embodiments, the patient is immunosuppressed. In certain embodiments, the antibody or antigen-binding portion thereof, composition, or pharmaceutical composition of bispecific binding molecule is for use in the treatment of cancer, e.g., cancers originating in tissues such as skin, lung, intestine, ovary, brain, prostate, kidney, soft tissues, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus, and pancreas, and cancers or other conditions that are dependent on PD-1 activity or in which the patient expresses or overexpresses PD-L1, PD-L2, or both.The cancers treated by the anti-PD-1 antibodies, their antigen-binding portions, the anti-PD-1 antibody compositions, and / or the bispecific binding molecules of the invention may include, for example, melanoma (such as advanced or unresectable melanoma or metastatic melanoma), non-small cell lung cancer, bladder cancer, squamous cell carcinoma of the head and neck, ovarian cancer, colorectal cancer, Hodgkin lymphoma, and renal cell carcinoma (RCC). In some embodiments, the cancers treated using the anti-PD-1 antibodies, antigen-binding portions, anti-PD-1 compositions, and / or bispecific binding molecules of the invention may include, for example, melanoma (e.g., advanced or metastatic melanoma), non-small cell lung cancer, squamous cell cancer of the head and neck, renal cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, glioblastoma, glioma, squamous cell lung cancer, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper urinary tract cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, liver cancer, colon cancer, colorectal carcinoma, multiple myeloma, sarcomas, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, nasopharyngeal cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, ovarian cancer, gastrointestinal cancer,primary peritoneal cancer, fallopian tube cancer, urothelial cancer, HTLV-associated T-cell leukemia / lymphoma, prostate cancer, genitourinary cancer, meningioma, adrenal cortex cancer, gliosarcoma, fibrosarcoma, kidney cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell carcinoma of the skin, appendix cancer, biliary tract cancer, salivary gland cancer, advanced Merkel cell carcinoma, diffuse large B-cell lymphoma, follicular lymphoma, mesothelioma, and solid tumors. "Treat," "treating," and "treatment" refer to a method for alleviating or eliminating a biological disorder and / or at least one of its accompanying symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Furthermore, references in this document to "treatment" include curative, palliative, and prophylactic treatments. "Therapeutically effective amount" refers to the quantity of a therapeutic agent administered that will alleviate, to some degree, one or more of the symptoms of the disorder being treated. A therapeutically effective amount of an anticancer agent may result in tumor reduction, improved survival, elimination of cancer cells, slowed disease progression, reversal of metastases, or other clinical parameters desired by healthcare professionals. The antibody compositions or antibodies or antigen-binding portions thereof of the invention may be administered alone or in combination with one or more other drugs or antibodies (or as any combination thereof). The pharmaceutical compositions, methods, and uses of the invention also encompass embodiments of combinations (co-administration) with other active agents, as detailed below. As used herein, the terms "co-administration", "co-administered" and "combined with", which refer to the antibody compositions and antibodies and antigen-binding portions thereof of the invention with one or more different therapeutic agents, are intended to mean, and refer to and include, the following: - simultaneous administration of said combination of antibody / antibody / antigen-binding portion composition of the invention and therapeutic agent or agents to a patient in need of treatment, when such components are formulated together in a single dosage form that releases said components substantially at the same time in said patient, - substantially simultaneous administration of such a combination of antibody / antibody / antigen-binding portion composition of the invention and therapeutic agent or agents to a patient in need of treatment, where such components are formulated separately from each other in separate dosage forms that are taken practically at the same time by said patient, whereupon said components are released substantially at the same time in said patient, - sequential administration of such a combination of antibody / antibody / antigen-binding portion composition of the invention and therapeutic agent or agents to a patient in need of treatment, where such components are formulated separately from each other in separate dosage forms that are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times in said patient; and - sequential administration of such a combination of antibody / antibody / antigen-binding portion composition of the invention and therapeutic agent(s) to a patient in need of treatment, where said components are formulated together in a single dosage form that releases said components in a controlled manner, whereupon they are released simultaneously, consecutively and / or overlappingly at the same and / or different times to said patient, where each part can be managed by the same route or by a different one. The antibody compositions and antibodies and their antigen-binding portions of the invention can be administered without additional therapeutic treatments, i.e., as a standalone therapy. Alternatively, treatment with the antibody compositions and antibodies and their antigen-binding portions of the invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the antibody composition or antibody or antigen-binding portion thereof may be administered or formulated in conjunction with another medication / drug for the treatment of cancer. The additional therapeutic treatment may comprise, e.g., a chemotherapeutic, antineoplastic, or antiangiogenic agent, a different anticancer antibody, and / or radiotherapy. By combining the antibody compositions, antibodies, or antigen-binding portions of the invention with agents known to induce terminal differentiation of cancer cells, the effect can be further enhanced. Such compounds can, for example, be selected from the group consisting of retinoic acid, trans-retinoic acids, cis-retinoic acids, phenylbutyrate, nerve growth factor, dimethyl sulfoxide, the active form of vitamin D3, peroxisome proliferator-activated receptor gamma, 12-O-tetradecanoylphorbol 13-acetate, hexamethylene bis-acetamide, transforming growth factor-beta, butyric acid, cyclic AMP, and vesnarinone. In some embodiments, the compound is selected from the group consisting of retinoic acid, phenylbutyrate, all-trans-retinoic acid, and the active form of vitamin D. Pharmaceutical articles comprising an anti-PD-1 antibody composition or anti-PD-1 antibody or antigen-binding portion thereof of the invention and at least one other agent (e.g., a chemotherapeutic, antineoplastic, or antiangiogenic agent) may be used as a combination therapy for simultaneous, separate, or successive administration in cancer therapy.The other agent may be any agent suitable for the treatment of the particular cancer in question, for example, an agent selected from the group consisting of alkylating agents, for example, platinum derivatives such as cisplatin, carboplatin and / or oxaliplatin; plant alkaloids, for example, paclitaxel, docetaxel and / or irinotecan; antitumor antibiotics, for example, doxorubicin (Adriamycin), daunorubicin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin and / or mitomycin; topoisomerase inhibitors such as topotecan; and / or antimetabolites, for example, fluorouracil and / or other fluoropyrimidines. An anti-PD-1 antibody or an antigen-binding portion thereof, or an anti-PD-1 antibody composition of the invention, can also be used in combination with other cancer therapies such as vaccines, cytokines, enzyme inhibitors, and T-cell therapies. In the case of a vaccine, this may be, for example, a protein, peptide, or DNA vaccine containing one or more antigens relevant to the cancer being treated, or a vaccine comprising dendritic cells together with an antigen. Suitable cytokines include, for example, IL-2, IFN-gamma, and GM-CSF. An example of an enzyme inhibitor with anticancer activity is an indolamine-2,3-dioxygenase (IDO) inhibitor, for example, 1-methyl-D-tryptophan (1-D-MT). Adoptive T-cell therapy refers to various immunotherapy techniques involving the expansion or genetic modification of a patient's own T cells to recognize and attack their tumors. It is also envisaged that an anti-PD-1 antibody, or an antigen-binding portion thereof, or an anti-PD-1 antibody composition of the invention, may be used as an adjunct to tyrosine kinase inhibitors. These are synthetic molecules, primarily quinazoline derivatives, of low molecular weight that interact with the intracellular tyrosine kinase receptor domain and inhibit ligand-induced receptor phosphorylation by competing for the intracellular Mg-ATP binding site. In some embodiments, the antibody composition or antibody or antigen-binding portion thereof may be used in combination with another drug / medicine that mediates activation of the immune system, including, but not limited to, an agent that mediates the expression or activity of A2AR, BLTA, B7-H3, B7-H4, CTLA-4, CD27, CD28, CD40, CD55, CD73, CD122, CD137, CD160, CGEN-15049, CHK1, CHK2, CTLA-3, CEACAM (e.g., CEACAM-1 and / or CEACAM-5), GAL9, GITR, HVEM, ICOS, IDO, KIR, LAIR1, LAG-3, OX40, TIGIT, TIM-3, TGFR-beta, VISTA and / or 2B4. In certain embodiments, the agent is an antibody or an antigen-binding fragment thereof that binds to one of the above molecules. In certain embodiments, the antibody composition or antibody or antigen-binding portion thereof of the invention can be administered in combination with a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody such as tremelimumab or ipilimumab).In one embodiment, the antibody composition or antibody or antigen-binding portion thereof of the invention can be administered in combination with ipilimumab. In certain respects, the antibodies and antigen-binding portions of the invention can be administered in combination with another inhibitor of the PD-1 pathway, which may target PD-1 or one or more of its ligands. Examples of such inhibitors include other anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies. In some embodiments, an antibody composition, antibody and / or antigen-binding portion of the invention may be administered in combination with pembrolizumab and / or nivolumab. It is understood that the antibody compositions and antibodies and antigen-binding portions thereof of the invention can be used in a treatment method as described herein, can be used in a treatment as described herein, and / or can be for use in the manufacture of a medicament for a treatment as described herein. Dosage and route of administration The antibody compositions of the invention shall be administered in an amount effective for the treatment of the condition in question, i.e., at doses and for periods of time necessary to achieve a desired result. A therapeutically effective amount may vary according to factors such as the specific condition being treated, the patient's age, sex, and weight, and whether the antibodies are administered as a standalone treatment or in combination with one or more additional cancer treatments. Dosage regimens can be adjusted to provide the desired optimal response. For example, a single bolus can be administered, several divided doses can be given over time, or the dose can be reduced or increased proportionally as indicated by the demands of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit-dose forms for ease of administration and dosage uniformity. The term unit-dose form, as used herein, refers to physically discrete units suitable as unit doses for the patients / subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required carrier drug.The requirements for the unit dosage forms of the invention are generally dictated by, and are directly dependent on, (a) the unique characteristics of the particular chemotherapeutic agent or prophylactic effect to be achieved, and (b) the limitations inherent in the technique of composing such active compound for the treatment of sensitivity in individuals. Therefore, a person skilled in the art would appreciate, based on the description provided herein, that the dosage and dosing regimen are adjusted according to well-established methods in therapeutic techniques. That is, the maximum tolerable dose can be readily established, and the effective amount that provides a detectable therapeutic benefit to a patient can also be determined, as can the timing requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Consequently, although certain dosages and administration regimens are illustrated herein, these examples in no way limit the dosage and administration regimen that may be provided to a patient in the practice of the present invention. It should be noted that dosage values ​​may vary with the type and severity of the condition to be treated and may include single or multiple doses. It should further be understood that, for any particular individual, specific dosage regimens must be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the compositions, and the dosage intervals stated herein are illustrative only and are not intended to limit the scope or practice of the incorporated composition. Moreover, the dosage regimen with the compositions of this invention may be based on a variety of factors, including the type of disease, age, weight, sex, the patient's medical condition, the severity of the condition, the route of administration, and the specific antibody employed.Therefore, the dosage regimen can vary widely, but it can be routinely determined using conventional methods. For example, doses can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Therefore, gradual increases or decreases in intrapatient doses are considered as determined by the practitioner. The determination of appropriate dosages and regimens is well established in the relevant practice. It is envisaged that a suitable dose of an antibody composition of the invention will be in the range of 0.1 to 100 mg / kg, such as approximately 0.5 to 50 mg / kg, for example, approximately 1 to 20 mg / kg. The antibody composition may be administered, for example, at a dosage of at least 0.25 mg / kg, for example, at least 0.5 mg / kg, such as at least 1 mg / kg, e.g., at least 1.5 mg / kg, such as at least 2 mg / kg, e.g., at least 3 mg / kg, such as at least 4 mg / kg, e.g., at least 5 mg / kg; and, e.g., up to a maximum of 50 mg / kg, such as up to a maximum of 30 mg / kg, e.g., up to a maximum of 20 mg / kg, such as up to a maximum of 15 mg / kg. The administration will normally be repeated at appropriate intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long as the responsible physician deems appropriate, who may optionally increase or decrease the dosage as needed. An effective quantity for tumor therapy can be measured by its ability to stabilize disease progression and / or improve symptoms in a patient, and preferably to reverse disease progression, e.g., by reducing tumor size. The ability of an antibody or the composition of the invention to inhibit cancer can be evaluated by in vitro analysis, e.g., as described in the examples, as well as in suitable animal models that are predictive of efficacy in human tumors. Suitable dosage regimens will be selected to provide an optimal therapeutic response in each specific situation, e.g., administered as a single bolus or as a continuous infusion, with possible dosage adjustment as indicated by the requirements of each case. Diagnostic uses and compositions The antibodies of the present invention are also useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies can be used to detect and / or measure the level of PD-1 in a patient sample (e.g., a tissue sample or a body fluid sample such as inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassay, and immunohistology. Kits (e.g., diagnostic kits) comprising the antibodies described herein are also disclosed herein. Unless otherwise defined herein, the scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Illustrative methods and materials are described below. In case of conflict, this specification, including the definitions, shall prevail. Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the field. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as is commonly done in the field, or as described herein. Furthermore, unless the context requires otherwise, singular terms shall include plurals and plural terms shall include singular. Throughout this descriptive memorandum and realizations, the words "has" and "includes," or variations such as "has," "that has," "includes," or "that includes," shall be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Although several documents are cited in this report, this mention does not constitute an admission that any of these documents form part of the common general knowledge of the art. To help the present invention be better understood, the following examples are set out. Examples Example 1: Cloning of anti-PD-1 antibodies from chicken B cells Cloning of chicken-derived antibody genes from antibody-secreting B cells (ASCs) was performed using the Symplex™ antibody discovery technology. Briefly, ASCs were isolated from lymphoid organs of chickens immunized with PD-1 antigen, either as a soluble protein antigen and / or in its native cell membrane-bound form presented on eukaryotic cells. Staining the ASCs with fluorescently labeled antibodies allowed for their differentiation from other cells (e.g., T cells, untreated B cells, monocytes, etc.) prior to sorting into PCR vessels. Individual ASC sorting was performed using flow cytometry. Subsequently, the Symplex™ procedure was carried out to generate PCR products containing V and V1 pairs for each B cell, ordered as described below. The joining of Vh and Vl coding sequences was performed on sorted ASCs, facilitating cognate sequence pairing. The process used a two-step PCR procedure based on two-step multiplex overlap-extension RT-PCR followed by nested PCR. The principle for linking cognate Vh and Vl sequences using Symplex™ technology is described in detail in WO 2005 / 042774; WO 2008 / 104184; WO 2010 / 022738, and Meijer et al., J Mol Biol 358 (3):764-72 (2006). Briefly, the amplified cognate Vh and Vl fragments are joined by overlap-extension PCR in a step called nested PCR. In the subsequent procedure, the PCR products are pooled before cloning into a plasmid vector.This is done so that the cloned DNA fragments encoding the variable domains of the chicken antibody can be expressed as a complete chimeric antibody from a single plasmid expression construct in transfected mammalian cells. Consequently, it is possible to screen the cell supernatants to identify chimeric antibodies that exhibit specific binding to the PD-1 antigen. Materials and methods The Symplex™ technology, as described in the publications mentioned above, was modified to amplify Vl and Vh from sorted chicken B cells. Cloning of a functional expression construct was performed in two stages, as described below. Step 1. Amplified PCR products containing the paired Vh and Vl fragments were amplified in a nested PCR reaction. This allowed the addition of flanking restriction enzyme recognition sites for Apal and Avrll at each end. Since the cognate V and Vl sequences were paired in a single PCR product from each sorted ASC, cloning of the PCR products was performed after pooling all the PCR fragments. The pML392 plasmid was constructed to receive the Symplex™ PCR products by digestion of the corresponding Apal and Avrll restriction sites. The resulting ligation of pooled PCR products and pML392 is shown in Figure 1. Here, insertion of the PCR product positioned the V and Vl sequences opposite human CH1-CH2-CH3 and lambda constant cDNA regions, respectively, so that complete heavy and light strand reading frames were obtained. Step 2. In the initial constructs, the two reading frames encoding the heavy and light chain sequences were placed head-to-head and separated by a DNA sequence containing restriction enzyme recognition sites for Ascl and Nhel. By inserting a double-digested CMV promoter DNA fragment containing the corresponding Ascl / Nhel, which included the 5' UTRs and signal peptides between the two 5' ends of the heavy and light chain genes, a full expression construct was obtained, as depicted in Figure 2. Example 2: Cloning of anti-PD-1 reference antibody analogues This example briefly explains how the reference analogues of the anti-PD-1 antibodies nivolumab and pembrolizumab were generated. The amino acid sequences encoding the variable heavy and light chain domains of the nivolumab and pembrolizumab antibody analogues were obtained from the IMGT® website imgt.org / mAb-DB / ; see Table 3 below. The protein sequences were reverse-translated into DNA sequences using human codons. The corresponding DNA sequences were then synthesized and cloned into expression vectors containing human IgG4 heavy or kappa light chain constant domains, resulting in the expression of complete antibodies. To avoid Fab arm exchange, the serine moiety at position 228 was replaced with proline (Angal et al., Mol. Immunol., 30:105-108 (1993)). CHO cells were transfected with the corresponding expression plasmids using a conventional protein expression system.The corresponding antibody supernatants were purified using conventional protein A purification column chromatography. Table 3 Genetically synthesized antibody analogues Example 3: Screening of antibody repertoires for their binding to PD-1 expressed on the cell surface Cloned antibodies from the anti-PD-1 repertoire were individually transfected and expressed in HEK293 cells using 293fectin™ Transfection Reagent (Invitrogen, Cat. No. 12347-019) in a 384-well format, and antibody-containing supernatants were collected on day 6 post-transfection. For cell-based antibody screening, CHO-S cells were transfected in a 384-well format to express full human PD-1 using Freestyle™ MAX reagent (Invitrogen, Cat. No. 16447-100), and untransfected cells were used as a negative control. To enable a multiplexed screening setup, untransfected cells were labeled using CFSE and mixed with unlabeled PD-1-transfected cells at a 1:1 ratio, with a density of 1E6 cells per ml each. In 384 well plates, 40 pl of this cell mixture were mixed with 10 pl of the antibody-containing supernatant, and the cell-bound antibody was revealed by the addition of goat anti-human IgG (H+L) secondary antibody AF647 (Molecular Probes, Cat. No. A21445) in a no-wash setting.Samples were acquired using high-throughput flow cytometry (iQue® Screener, Intellicyt) and data were analyzed using ForeCyt® software by plotting CFSE against human IgG binding (AF647). PD-1-specific primary hits were identified as antibody clones that bind only to cells transfected with human PD-1 (CSFE-negative) but not to control cells (CFSE-positive), and plaque numbers and plaque coordinates were collected for hit selection and subsequent sequence analysis. Figures 3A-3C show representative flow cytometry dot plots for (A) an antibody clone that binds specifically to cells transfected with human PD-1, (B) a clone that does not bind specifically to CHO-S cells, and (C) a clone that does not bind to any of the cell populations used in the screening. Example 4: Humanization of anti-PD-1 antibodies Humanization of the framework regions of chicken anti-PD-1 antibodies was performed in order to produce antibody molecules that have minimal immunogenicity when administered to humans, while substantially retaining the specificity and affinity of the parent chicken antibodies. Materials and methods Humanization of chicken-derived antibodies was performed using the "CDR grafting" approach, a method originally described by Jones et al., Nature 321:522-525 (1986). First, the BLAST tool was used with the variable heavy (Vh) and variable light (Vl) domains of the antibodies against human IgG databases to find the closest human germline genes. This identified the human genes IGHV3-23*01 (M99660) and IGLV3-19*01 (X56178) as the closest to the chicken Vh and Vl genes, respectively. Similarly, the human amino acid sequences selected for humanization of the J gene region were obtained from IGHJ1*01 (J00256) and IGLJ6*01 (M18338) for Vh and Vl, respectively.Furthermore, the antibody Vh and Vl genes were aligned against the chicken immunoglobulin germline genes to identify somatic mutations in framework regions that may play a role in antibody function and / or structure. Such fragments can be incorporated into the final humanized antibody genes as so-called "backmutation" fragments. Finally, certain amino acid positions, known as "Vernier fragments" (Foote and Winter, J Mol Biol. 224 (2):487-99 (1992)), which are known to play an important role in antibody structure, stability, and function, are thought to generate alternative humanized antibody variants that include human or chicken fragments from the corresponding germlines. The CDR sequences in this document were determined according to the IMGT® definitions for CDR1 and CDR2. For heavy and light chain CDR3, the definitions in this document include an additional amino acid residue upstream of IMGT-CDR3 (Cys) and an additional amino acid residue downstream (Trp for CDR3 of Vh, Phe for CDR3 of Vl). The chicken CDR and human framework regions were assembled using overlap extension PCR. The resulting humanized Vh and Vl PCR products were cloned into expression vectors (plasmids) harboring human heavy and light chain constant regions. To enhance the correct cleavage of the upstream signal peptide from the lambda chain, the second amino acid (Ser) of the IGLV3.19 lambda gene was replaced with another amino acid (Tyr) present in other human germlines, such as IGLV3.25. The heavy chain sequence contains the two "LALA" mutations (L234A / L235A) known to reduce the effector function of the Fc region of lgG1 antibodies (Armour et al., Eur J Immunol. 29 (8):2613-24 (1999); and Armour et al., Mol Immunol. 40 (9):585-93 (2003)).The expression vector also contained the necessary regulatory sequences, allowing simultaneous expression of light and heavy chains that assemble to provide complete antibodies after transfection of mammalian cells. Results The final humanized antibody sequences are shown below in Table 4, and the CDR sequences are shown separately in the sequences in Table 5. The CDR sequences are defined in the Tables according to the IMGT® numbering scheme. 4 All humanized antibodies comprised the IgG1 variant heavy chain constant region "LALA" and light chain constant region amino acid sequences shown below. Heavy chain constant region (SEQ ID NO: 67) : ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSSLG TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFPPKPKDTLMISRTPEVTCVVVDVSHEPCPAP EVKFNWYVDGVEVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNCALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVQQDKWKVFS CSVMHEALHNHYTQKSLSLSPGK Light chain constant region (SEQ ID NO: 68) : GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETKPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS Example 5: Screening of anti-PD-1 antibody candidates PD-1 is mainly expressed on the surface of activated T lymphocytes, where it negatively regulates T cell activity. To select the most functional anti-PD-1 antibody candidates, two different in vitro screening systems were established: a Staphylococcus Enterotoxin B (SEB) whole blood assay and a unidirectional lymphocyte mixed reaction assay. Materials and methods A repertoire of 69 unique humanized mAbs in the lgG1-LALA scaffold format, i.e., having the "LALA" mutations described in Example 4, was initially screened. These mAbs were cloned and humanized as described above to determine their functional activity in whole blood assays of SEB. SEB is a superantigen that binds to MHC class II molecules and specific Vp regions of T cell receptors (TCRs) and directs non-specific T cell stimulation. This results in the activation / proliferation of polyclonal T cells and the release of cytokines, including IL-2 and IFN-γ. To investigate the relevance of SEB assay for screening anti-PD-1 activity, PD-1 expression levels were assessed in different donors before and after SEB stimulation. PBMCs from six different donors were assayed for PD-1 expression by flow cytometry on day 0 and day 3 post-SEB stimulation. A relevant lymphocyte output was established for further analysis. Based on screening of SEB whole blood assays, using blood from at least three different donors, the top 10 anti-PD-1 antibody candidates were identified. The anti-PD-1 antibody candidates were subsequently titrated to obtain dose-response curves for each individual antibody compared to positive controls, reference analogues of the anti-PD-1 antibodies pembrolizumab (Merck) and nivolumab (Bristol-Myers Squibb); see Example 2. The functionality of the 10 selected top anti-PD-1 antibodies was validated in an alternative in vitro assay, the unidirectional lymphocyte mixed reaction (MLR) assay. In this assay, dendritic cells (DCs) from one donor were co-cultured with CD4+ T cells from another donor to achieve alloantigen-specific stimulation, induced in 10–15% of all T cells, leading to T cell activation / proliferation and cytokine secretion. Due to a protein stability issue for one of the candidates (12748.15381), alternative germline sequences were used for this specific antibody. One of the resulting antibodies, 12748.16124, is mentioned below. This variant has a different VL sequence but the same VH sequence as 12748.15381 (Table 1, above). Results The data in Figure 4 clearly show that the frequency of PD-1-expressing lymphocytes increases in all donors tested after SEB stimulation. These observations confirm the relevance of this assay for screening for anti-PD-1 antibodies. The titration of the most functional anti-PD-1 antibodies in the SEB assay, shown in Figures 5A-I, identified the best anti-PD-1 candidates with functionality similar to or better than the positive control antibody analogs pembrolizumab and nivolumab. In this assay, whole blood was stimulated with SEB for 48 h in the presence of the indicated antibodies, and IL-2 secretion was measured after 48 h by ELISA. Each data point represents an average of six replicates, with the bars indicating the time to first trial (TTT). Figures 5A-H show the results obtained with the humanized anti-PD-1 antibodies. Due to aggregation above 5% for one of the antibodies [12748.15381], an alternative framework for this antibody was tested. The data in Figure 5I show similar functionality between the original humanized antibody [12748.15381] and its germline variant (framework) [12748.16124]. The functionality of the anti-PD-1 antibodies was validated in a one-way MLR assay. In this assay, dendritic cells and CD4+ T cells (1:10 ratio) from two different donors were cultured together, and IFN-γ secretion was measured using MesoScale after 5 days. Each data point represents an average of six replicates, with bars indicating the time to metastasis (TTM). The data obtained from the one-way MLR assay and illustrated in Figures 6A–H show the same functionality and classification of the anti-PD-1 antibodies as the data obtained from the SEB assay. This consistency across different assays provides further confirmation that the selected antibodies are functional. The selected antibodies originate from two different families ("bins") of major epitopes, indicating that they bind to two different, non-overlapping epitopes. All the anti-PD-1 antibodies shown belong to Bin 1, except for antibodies 12760 and 13112, which belong to Bin 2. Anti-PD-1 antibodies from Bin 1 were found to exhibit the highest functionality in these in vitro assays. Example 6: Flow cytometry analysis of anti-PD-1 antibodies to determine PD-L1 ligand blocking activity This example illustrates how the anti-PD-1 antibody panel was tested to determine PD-L1 ligand blocking activity by performing a competitive analysis using flow cytometry with cell surface-expressed PD-1 and fluorochrome-labeled soluble PD-L1. Materials and methods The blocking activity of the PD-L1 ligand was investigated in multiplex cell assays, in which human and cynomolgus PD-1 were recombinantly expressed in CHO-S cells, and the binding of the R-PE (R-phycoerythrin)-labeled human PD-L1-Fc chimeric protein was analyzed by flow cytometry. The commercially available recombinant PD-L1-Fc chimeric protein (R&D Systems, USA) was conjugated to R-PE using the Lightning-Link® R-Phycoerythrin Conjugation Kit (Innova Biosciences, UK). CHO-S cells transiently transfected to express human PD-1 were mixed with CFSE-stained CHO-S cells transiently expressing cynomolgus PD-1. This cell mixture was then incubated with 50 pl of anti-PD-1 antibody at 20 pg / ml on ice, followed by the addition of 50 pl of R-PE-labeled PD-L1-Fc at approx.3.4 pg / ml (final concentration 16.4 nM) and subsequent incubation for an additional 20 min (final anti-PD-1 antibody concentration: 10 pg / ml). The bound antibody was detected using APC (allophycocyanin)-conjugated anti-human IgG light chain antibody. The binding of PD-L1 and anti-PD-1 antibodies was quantified by flow cytometry detecting R-PE and APC fluorescence, respectively. Results The results of the competition experiment are presented in Figures 7A–B and summarized in Table 6 below. All anti-PD-1 antibodies were tested at a final antibody concentration of 10 pg / ml (see above). Three of the assayed antibodies were able to inhibit PD-L1 binding by 83% or more, similar to the reference anti-PD-1 antibody lambrolizumab (Merck), which is the same as pembrolizumab and was included as a positive control. One antibody (12777.13362) only partially inhibited binding by 69%. One antibody (13112.13208) did not block PD-1 binding. PD-L1 binding to PD-1-expressing cells in the presence of the negative control anti-VEGFR2 antibody ramicirumab (Genentech) was set to 0%. Table 6 Inhibition of PD-L1 binding in the presence of anti-PD-1 antibodies The humanized variants shown in Table 6 have the same amino acid sequences as those in Table 1, sharing the first five digits in their names, except that the variants in Table 1 have "SY" amino acid residues at the N-terminus of the light chain. In some embodiments, the SY dipeptide enhances signal peptide processing during antibody light chain expression. The variants in Tables 1 and 6 are expected to have identical functional properties. Example 7: Measurement of the affinities of the PD-1 antibody against the EDC antigen of human PD-1 and cinomolau This example demonstrates that most anti-PD-1 antibodies show high picomolar (pM) affinity and good cross-reactivity against the extracellular domains (ECD) of PD-1 from both humans and cynomolges. Materials and methods Kinetic binding analysis of the purified anti-PD-1 antibody repertoire was performed on an XPR-36 surface plasmon resonance (SPR) biosensor (Bio-Rad, USA). Human or cynomolgus PD-1 anti-CD antigens labeled with His were acquired from Acro Biosystems, UK. Binding kinetics were measured under monovalent antigen conditions by immobilizing anti-PD-1 antibodies and maintaining monovalent PD-1 antigen in solution as previously described (Canziani et al., Anal Biochem 325(2):301–307 (2004)). The lowest possible density of anti-PD-1 antibodies was used to avoid nonspecific binding and bulk transport limitations. To measure antibody kinetics, anti-PD-1 antibodies were adjusted to a concentration of 1.0 µg / ml and captured on surfaces with human IgG anti-Fc generated by immobilizing approximately 1,000 UR of a human anti-Fc monoclonal antibody (Biacore, Denmark).Anti-PD-1 antibodies were assayed to determine the binding of human or cynomolgo PD-1 to endocrine-dissolving cells (EDCs) at a concentration range of 3 times higher, from 25 nM to 0.31 nM, followed by regeneration of the surfaces with 3 M MgCl2 regeneration buffer (Biacore, Denmark). A high flow rate of 20 J L / min, an association time of 3.33 min, and a dissociation time between 1.5 and 2.75 hours were used. The recorded binding responses were fitted to a simple 1:1 Langmuir binding model for the calculation of the association rate constant (kon or ka), dissociation rate constant (koff or kd), and affinity constant (Kd) using a double reference. Results The binding kinetics are tabulated in Table 7 below, which illustrates that the anti-PD-1 antibody panel binds to PD-1 with very high affinities in the pM range. All antibodies recognize human PD-1 with higher affinity than the nivolumab and pembrolizumab analogues. The highest affinity antibody [12819.15384] binds to human PD-1 with a Kd of 20 pM. Table 7 Kinetics of binding of anti-PD-1 antibodies to ECD of human or cynomolgo PD-1 measured by Surface Plasmon Resonance (SPR) Example 8: Establishment of families ("binnin") of anti-PD-1 antibody epitopes This example illustrates how PD-1 antibodies clustered into epitope families based on paired competition patterns. Antibodies belonging to different epitope locations recognize different epitopes on PD-1 ECDs. Methods The investigation of paired antibody competition was performed using Surface Plasmon Resonance (SPR) analysis with a Continuous Flow Microscope (CFM) (Wasatch Microfluidics, USA) combined with an IBIS MX96 SPR instrument (IBIS Technologies, Netherlands). Surface Plasmon Resonance imaging was performed on E2S SensEye® SPR sensors (Ssens BV, Netherlands). A total of ten anti-PD-1 antibodies (human, IgG1) were diluted to 10 pg / ml in 50 mM sodium acetate buffer, pH 4.5. The antibodies were applied to an E2S SensEye® and conjugated for 15 minutes using a continuous flow microspotter. After application, the SensEye® was placed on the IBIS MX96 biosensor and deactivated with 1 M ethanolamine, pH 8.5, for 10 minutes. Following sensor preparation, antibody competition analysis was performed using a classic sandwich assay.The monovalent PD-1 ECD antigen (Sino Biological, China) was diluted in HBS-EP migration buffer and injected at a concentration of 50 nM and captured by the anti-PD-1 antibody conjugate array. Subsequently, individual injections of each of the ten PD-1 antibodies, diluted to 100 nM in HBS-EP migration buffer, were performed to establish antibody competition patterns. After each competition cycle, the sensor surface was regenerated with 10 mM glycine HCl buffer, pH 2.0. Results The competition pattern of ten anti-PD-1 antibodies is shown in Figure 8. Antibodies 12866 and 12807 were not found to have functional activity in cell-based assays, but were included because they recognize distinct epitopes. The functional anti-PD-1 antibodies tested were found to bind to two non-overlapping epitope families. All functional antibodies belonging to epitope Bin 1 were cross-blocked by each other and included nivolumab analogue (“Nivo”), pembrolizumab analogue (“Pembro”), 12819, 12892, 12865, and 12777. These antibodies were found to significantly block PD-L1 and PD-L2 binding. It was found that 12760 and 13112 bind to a separate Bin 2 of epitopes because they cross-block each other, but do not block the binding of any of the antibodies in Bin 1 of epitopes.Therefore, 12760 and 13112 probably bind to a different site on PD-1 that does not overlap with the ligand-binding site of PD-L1 and PD-L2. The cross-blocking functional antibodies 12819, 12865, 12892, 12777, nivolumab, and pembrolizumab, which belong to epitope Bin 1, could be further subdivided into four subfamilies based on competition with 12866 and 12807 (Figure 8). 12819 (Bin 1C) was the only antibody that blocked the binding of both 12866 and 12807, while nivolumab (Bin 1D) blocked only 12866, and pembrolizumab (Bin 1F) blocked only 12807. The group of antibodies belonging to Bin 1E (12865, 12892, and 12777) was unique in that it did not block the binding of either 12866 or 12807. Finally, 12866 (Bin 1A) and 12807 (Bin 1B) joined families of unique epitopes. 12866 was blocked by 12819 and nivolumab, but not by other anti-PD-1 antibodies, and 12807 was blocked by 12819 and pembrolizumab, but not by other anti-PD-1 antibodies. Example 9: Measurement of the cross-reactivity of the PD-1 antibody against the anti-ECD of mouse and rat PD-1 This example demonstrates that the anti-PD-1 antibody 12819.15384 strongly cross-reacts with mouse PD-1, but does not bind to rat PD-1. Materials and methods Mouse and rat PD-1 ECDs tagged with His were acquired from Sino Biologicals. Kinetic binding analysis was performed as described in Example 7. Results Binding kinetics are tabulated in Table 8 below. Anti-PD-1 antibody 12819.15384 binds to mouse PD-1 with a Kd of 809 pM, but does not recognize rat PD-1. The affinity for the ECD of human PD-1 was similar to that measured in Example 7. Antibody 12865.17150 did not bind to mouse or rat PD-1. Neither of the reference analogues of nivolumab and pembrolizumab cross-reacted with mouse or rat PD-1 (data not shown). Table 8 Binding kinetics of the antibody PD-1 12819.15384 to ECD of human, mouse or rat PD-1 measured by Surface Plasmon Resonance (SPR) Example 10: Analysis of the PD-L1 and PD-L2 ligand blocking activity of PD-1 mAbs This example illustrates how the anti-PD-1 antibody panel was analyzed to determine PD-L1 or PD-L2 ligand blocking activity by performing a competition assay using Bio-Layer Interferometry analysis. Materials and methods The investigation of PD-L1 or PD-L2 ligand-blocking activity was performed using Bio-Layer Interferometry (BLI) analysis with an Octet QK384 instrument (Fortebio, USA). Commercially available human PD-1 Fc fusion protein (Sino Biological) at a concentration of 5 g / ml was captured onto human anti-Fc sensor chips (Fortebio, USA), and residual anti-Fc sites were blocked with Herceptin® negative control antibody. The antigen-coated surface was then saturated with anti-PD-1 antibody at a concentration of 10 pg / ml. After PD-1 saturation with anti-PD-1 antibody, the ligand-blocking activity of PD-L1 or PD-L2 was assessed by incubation with the assayed human PD-L1 or PD-L2 Fc fusion protein (Sino Biological) at 5 pg / ml. Results The results of the competition analysis are presented in Table 9 below. All antibodies completely blocked the binding of either PD-L1 or PD-L2 ligands, except for antibody 12760.13169, which showed no significant blockade of PD-L1 or PD-L2 (26% and 36%, respectively), and 13112.13208, which showed no blockade of PD-L1 and weak blockade of PD-L2 (27% and 53%, respectively). The results were in agreement with the epitope family establishment analysis (Example 8) and the epitope mapping analysis (Example 11), which showed that all antibodies except 12760 and 13112 bind to overlapping epitopes that map to the PD-L1 PD-L2 binding site on PD-1, while antibodies 12760 and 13112 bind to a separate PD-1 site and do not significantly cross-compete with PD-L1 and PD-L2. Table 9 Inhibition of PD-L1 and PD-L2 after saturation of anti-PD-1 antibodies Example 11: Mapping anti-PD-1 antibody epitopes using PD-1 mutagenesis Antibody epitopes can generally be characterized as linear epitopes (also called continuous epitopes) or conformational epitopes (also called discontinuous epitopes). While linear epitopes are defined based on a single continuous amino acid sequence, conformational epitopes can consist of many smaller discontinuous linear sequences or single contact fragments. A collection of contact fragments that cluster at the intermolecular protein interface between the antibody and antigen is also called a hotspot or core epitope (Moreira et al., Proteins 68(4):803-12 (2007)). It is now widely recognized that most B cell epitopes are discontinuous in nature (Sivalingam and Shepherd, Mol Immunol. 51(3-4):304-9 (2012), Kringelum et al., Mol Immunol.53 (1-2) :24-34 (2013) ) encompassing the middle epitope 15-22 amino acid residues of which 2-5 amino acids contribute most of the bond energy (Sivalingam and Shepherd, above). By classifying the binding affinity to 111 different PD-1 mutants, this example illustrates how the binding epitopes of antibodies 12819 and 12865 can be divided into linear epitopes and hot spots that are distinct from the epitopes recognized by nivolumab and pembrolizumab. Methods The human PD-1 receptor consists of a 268-amino-acid extracellular domain (remains 21-288). The extracellular domain spans amino acids 21-170, followed by a transmembrane domain (remains 171-191) and a cytoplasmic domain (remains 192-288). PD-1 belongs to the immunoglobulin superfamily and is composed of a two-layered p-sandwich formed by interactions of eight antiparallel p-chains arranged in two p-sheets, with GFCC' p-strands on one side and ABED p-strands on the opposite side. The two p-sheets are stabilized by a disulfide bond between C54-C123 residues. A crystal structure is available for human PD-1: human PD-L1 complex (PDB 4ZQK), but the C'D loop between the p C' and D chains was unstructured and missing, as well as part of the C-terminal sequence after remainder 146 (PDB 4ZQK, Zak et al., Structure 23 (12):2341-2348 (2015)).A crystal structure of the human PD-1:pembrolizumab complex (PDB 5JXE, Na et al., Cell Res. 2016 [Electronic Publication before Print], PMID:27325296) has recently been published. In this structure, the C'D loop is much more ordered, and the contact residues important for pembrolizumab binding were shown to cluster around a central epitope in this loop. The crystal structure of the human PD-1:human PD-L2 complex is not available. An NMR structure of human PD-1 in solution shows high structural similarity to the crystal structure of PDB 4ZQK (PDB 2M2D, Cheng et al., J Biol Chem 288 (17):11771-85 (2013)). Human PD-1 binds to human PD-L1 or PD-L2 ligands in a 1:1 stoichiometry and binding occurs mainly at overlapping binding sites mediated by the GFCC' p-sheet (Cheng et al., J Biol Chem 288 (17):11771-11785 (2013)) (Figure 9, panels A and B).Human PD-L1 binds to human PD-1 via contact remnants V64, N66, Y68 located on the p strand of C and G124, 1126, L128, A132, 1134 and E136 located on the p strands of F and G (Zak et al., Structure 23 (12):2341-8 (2015)). Human pD-L1 and PD-L2 bind to human PD-1 with Kd of 8 pM and 2 pM, respectively (Cheng et al., above). The human PD-1 protein sequence was downloaded from Uniprot (Accession No. Q15116, amino acid sequence is represented in SEQ ID NO: 1). The complete Macaca fascicularis protein sequence was downloaded from Uniprot (Accession No. B0LAJ3_MACFA (SEQ ID NO: 89)). The complete PD-1 protein sequences from Gallus gallus, Mus musculus, and Rattus norvegicus were downloaded from NCBI (Xp_422723 (SEQ ID NO: 90), NP_032824.1 (SEQ ID NO: 91), and XP_006245633.1 (SEQ ID NO: 92), respectively). The sequence identities of the different extracellular PD-1 amino acid sequences compared to human PD-1 are shown in Table 10 below. Table 10 Comparison of PD-1 ECD sequence between species A molecular model of human PD-1 was constructed by combining structural information from the crystal structure of the human PD-1:PD-L1 complex determined at a resolution of 2.45 Å (PDB 4ZQK) and an APO human PD-1 NMR structure (PDB 2M2D). The PDB 4ZQK structure was used as the basis for the model, with the missing CD loop and the C-terminal portion of PD-1 provided from the NMR structure. Surface-exposed amino acid residues were then highlighted, and 83 individual alanine substitutions on surface-exposed residues in the ECD of human PD-1 (alanine sweep) and 5 exposed residue positions that differed between human, mouse, and rat PD-1 were designed to trace PD-1 residues. To map linear antibody epitopes in the context of the native human PD-1 structure, 23 chimeric proteins were generated where 10 amino acids in the ECD sequence of human PD-1 were sequentially exchanged for chicken sequence in segments that overlapped by 5 amino acids. Sequence exchanges were performed in the extracellular domain of human PD-1 spanning amino acids 31–146, as the Gallus gallus protein sequence outside this segment did not align well with human PD-1 and was omitted. The PD-1 cDNA encoding the extracellular domain of human PD-1 was synthesized and cloned into a vector containing the CMV promoter and the human IgG1 Fc sequence (P101-K330 remnants), resulting in the fusion of the C-terminal IgG1 Fc to the cloned PD-1 ECD. Mutated human PD-1 Fc fusion constructs were generated using conventional genetic engineering and PCR techniques, and the protein was transiently expressed in 2 ml of culture using an ExpiCHO™ expression system. The human PD-1 Fc fusion constructs were harvested after 9 days, and the supernatants were assayed for anti-PD-1 Fab binding affinity by Surface Plasmon Resonance (sPr).Culture supernatants containing PD-1 fusion proteins were immobilized on a SensEye® (Ssens BV, The Netherlands) for Ga-hu-lgG Fc for 15 minutes using a Continuous Flow Microspotter (CFM, Wasatch Microfluidics, Salt Lake City, USA). After application, the SensEye® was placed on an IBIS MX96 biosensor, and the captured proteins were fixed to the surface using the FixIT kit (Ssens BV, The Netherlands). Kinetic analysis was performed using the so-called kinetic titration series (Karlsson R. 2006), where monomeric Fab fragments of the antibodies of the invention were injected at increasing concentrations from 1 nM to 50 nM without surface regeneration steps after each antigen injection. Fab association was performed for 15 minutes and antigen dissociation was performed for 30 minutes.The recorded binding responses were fitted to a simple Langmuir 1:1 binding model with the Scrubber 2 software support for calculating the association rate constants (kon or ka), dissociation rate constants (koff or kd), and affinity constants (Kd). Results The binding affinities of the anti-PD-1 Fabs 12819.17149 and 12865.17150 and the reference analogues nivolumab and pembrolizumab were evaluated. 12819.17149 and 12865.17150 have VH and VL amino acid sequences identical to 12819.15384 and 12865.15377, respectively, but are identified by different 10-digit numbers because the heavy and light chain sequences of each of the first two variants were co-expressed on the same plasmid rather than on separate plasmids in the host cells. Fab 13112.15380 and Herceptin®, which do not block PD-L1 and PD-L2 ligands, were included as controls. All 111 PD-1 mutants tested were well expressed. Only three chimeric constructs failed to bind to any of the tested antibodies, suggesting that the mutations introduced into these three constructs presumably resulted in major conformational perturbations that affected the binding of all tested PD-1 antibodies. The change in binding affinity of Fab antibodies binding to the mutant PD-1 constructs compared to the wild type was expressed as the mutant Kd / wild-type Kd ratio (normalized binding affinity). Table 11 below provides an overview of the linear epitope scan performed by inserting 10-amino-acid Gallus gallus sequences into the ECD of human PD-1. At least a 5-fold reduction in affinity was used as the cutoff criterion to detect the reduction in binding affinity to the mutant PD-1 constructs.In some cases, binding to specific antibodies could not be detected. These constructs were listed as SU (no binding). Single contact residues were also mapped by performing 83 alanine substitutions or 5 rat backmutations (Table 12 below). A description of the linear epitopes or contact remnants identified for assayed antibodies is presented in Table 13. Figure 9 shows an illustration of the mapped binding epitopes shown as density plots on the ECD structure of human PD-1. The analysis showed that the binding epitopes of the anti-PD-1 antibodies 12819 and 12865 were clearly distinct compared to the reference antibodies nivolumab and pembrolizumab (Tables 11–13, Figure 9). The core epitope of pembrolizumab (Figure 9, panel C) was located on the p-strand of C' and in the C'–D loop. Contact remnants / linear epitopes were also found on the p-strands of C and F, where contact remnants for PD-L1 are also present. The core epitope of nivolumab (Figure 9, panel D) was present at the end of the p-strand of F and on the entire p-strand of G, covering some of the reference PD-1 contact remnants used by human PD-L1. The core epitopes of 12819 and 12865 (Figure 9, panels E and F) were located on the p-strands of F and G, covering more area than nivolumab and overlapping with all contact rests reported for human PD-L1 in this region. 12865 was also highly sensitive to mutations at rests 69-75.12819 also shared a contact rest with pembrolizumab (V64) on the p-strand of C, which has also been reported as a contact rest for human PD-L1. Both 12819 and 12865 share linear epitopes that map to the p-strands of C and C' and part of the CD loop. Aside from the V64 rest, no other contact rests were shared among the assayed antibodies. The non-ligand-blocking antibody 13112 was shown by alanine scanning to map a region distant from the PD-L1 and PD-L2 ligand-blocking site (Figure 9, panel G). In summary, this example illustrates that, although 12819, 12865, nivolumab, and pembrolizumab bind to overlapping epitopes on human PD-1 that can block the binding of PD-L1 and PD-L2 ligands, each antibody has a distinct binding epitope, as evidenced by competitive binding analysis (epitope clustering, Example 8) and shown at the molecular level by mapping individual linear epitopes and contact remnants to a panel of 111 PD-1 mutants, as summarized in Table 13. 12819 is also the only antibody in the investigated anti-PD-1 panel that cross-reacts with mouse PD-1 ECD (Kd of 809 pM, Example 9), highlighting that this antibody's binding epitope is unique compared to the other PD-1 antibodies tested. Table 12 Binding affinity of Fab antibody to ECD remnants of human PD-1 swept with alanine* * The normalized union is listed as mutant Kd / wild-type Kü. Table 13 Anti-PD-1 antibody binding epitopes identified using mutated PD-1 Fc fusion constructs Example 12: In vivo efficacy of an antibody 12819 in four syngeneic models of murine tumors This example demonstrates the in vivo efficacy of an antibody 12819 in four syngeneic models of murine tumors. Methods Two x 10⁵ Sa1N cells (fibrosarcoma), one x 10⁶ CT26 cells (colon carcinoma), five x 10⁶ ASB-XIV cells (lung carcinoma), or eight x 10⁶ MC38 cells (colon carcinoma) were subcutaneously inoculated into the flank of 6–8 week-old female mice of A / J (Sa1N), BALB / cAnNRj (CT26 and ASB-XIV), or C57BL / 6 (MC38). Tumors were measured three times per week using a two-dimensional caliper, and tumor volume in mm³ was calculated according to the formula: (width)² x length x 0.5. With an average tumor size of 30–50 mm³, mice were randomly divided into two groups of ten animals, and treatment was initiated. Mice were treated three times a week with a total of six treatments by intraperitoneal injection of vehicle buffer or the monoclonal antibody 12819.17149, followed by an observation period. Antibody treatments were dosed at 10 mg / kg.Two-way ANOVA with Bonferroni multiple comparisons test was applied to compare tumor volumes at each time point between treatment groups. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.). Results The results show a profound tumor-inhibiting effect of antibody 12819.17149 in all syngeneic tumor models tested (P <0.001 vs. vehicle) (Figure 10). Antibody 12819.17149 induced regression of tumor growth in the Sa1N tumor model and resulted in delayed tumor growth in the CT26, MC38, and ASB-XIV tumor models. Example 13: In vivo efficacy of an antibody 12819 in a semi-humanized xenograft tumor model with a mixture of CD8+ / CD4+ T cells and A375 melanoma cells This example demonstrates the in vivo efficacy of an antibody 12819 in a semi-humanized xenograft tumor model, in which the human melanoma cell line A375 was mixed with purified human CD8+ and CD4+ T cells. Methods 4.5 x 10⁵ CD8+ and CD4+ T cells were isolated from a human PBMC donor and mixed with 2.05 x 10⁶ A375 cancer cells (human melanoma) prior to subcutaneous flank inoculation of 6–8 week-old female NODscd / mice. Treatment was initiated on the day of tumor inoculation, and mice were treated three times a week for a total of six treatments by intraperitoneal injection of vehicle buffer, Keytruda® (pembrolizumab) (10 mg / kg), or the monoclonal antibody 12819.17149 (10 mg / kg), followed by an observation period. Tumors were measured three times a week using a two-dimensional caliper and the tumor volume in mm3 was calculated according to the formula: (width) 2 x length x 0.5. Two-way ANOVA with Bonferroni multiple comparisons test was applied to compare tumor volumes at each time point between the treatment groups.Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.). Results In the semi-humanized tumor model, treatment with antibody 12819.17149 resulted in a significant delay of tumor growth (P <0.001 vs. vehicle), whereas Keytruda® showed a limited effect on tumor growth compared to the vehicle-treated group (Figure 11). Table 14 List of SEQ ID NO

Claims

1. An anti-PD-1 antibody or an antigen-binding portion thereof, binding to an epitope on human PD-1 comprising amino acid residues V64, L128, P130, K131, and A132 of SEQ ID NO:

1.

2. The anti-PD-1 antibody or antigen-binding portion of claim 1, wherein said antibody comprises H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NO: 18-20 and SEQ ID NO: 21-23, respectively.

3. The anti-PD-1 antibody or antigen-binding portion of claim 1, wherein said antibody comprises a Vh comprising the amino acid sequence SEQ ID NO: 2 and a Vl comprising the amino acid sequence SEQ ID NO:

3. 4.The anti-PD-1 antibody of any one of claims 1-3, wherein the antibody is an IgG1, optionally comprising a mutation in one or both of the amino acid positions 234 and 235 of the heavy chain, numbered according to the IMGT numbering scheme, optionally wherein one or both of the amino acid residues in positions 234 and 235 are mutated to Ala. 5.The anti-PD-1 antibody or antigen-binding portion of any one of claims 1-4, wherein the antibody or portion has at least one of the following properties: a) binds to human PD-1 with a Kd of 750 pM or less; b) binds to cynomolgo PD-1 with a Kd of 7 nM or less; c) binds to mouse PD-1 with a Kd of 1 nM or less; d) does not bind to rat PD-1; e) increases IL-2 secretion in a whole blood assay using SEB; f) increases IFN-γ secretion in a unidirectional lymphocyte mixed reaction assay; g) inhibits the interaction of PD-1 with PD-L1 by at least 60% at a concentration of 10 pg / ml in a competitive assay using flow cytometry; h) block the binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 pg / ml as determined by Bio-Layer interferometry analysis; ei) inhibit tumor growth in vivo. 6.An anti-PD-1 antibody comprising a heavy chain comprising the amino acid sequences of SEQ ID NO: 2 and 67 and a light chain comprising the amino acid sequences of SEQ ID NO: 3 and 68.

7. A pharmaceutical composition comprising an anti-PD-1 antibody or antigen-binding portion according to any one of claims 1-6 and a pharmaceutically acceptable excipient, optionally further comprising a chemotherapeutic agent, an antineoplastic agent, an antiangiogenic agent, a tyrosine kinase inhibitor, or a PD-1 pathway inhibitor.

8. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, or a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, or both, of the anti-PD-1 antibody of any one of claims 1-6. 9.A vector comprising the isolated nucleic acid molecule of claim 8, wherein said vector further comprises an expression control sequence.

10. A host cell comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, or a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, of the anti-PD-1 antibody of any one of claims 1-6.

11. A method for producing an anti-PD-1 antibody or an antigen-binding portion thereof, comprising providing a host cell according to claim 10, culturing said host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion. 12.A bispecific binding molecule having the binding specificity of an anti-PD-1 antibody according to any one of claims 1-6 and the binding specificity of a different antibody.

13. An anti-PD-1 antibody or antigen-binding portion according to any one of claims 1-6, a pharmaceutical composition according to claim 7, or a bispecific binding molecule according to claim 12 for use in enhancing immunity in a patient in need.

14. The anti-PD-1 antibody or antigen-binding portion according to any one of claims 1-6, a pharmaceutical composition according to claim 7, or a bispecific binding molecule according to claim 12, for use in the treatment of cancer in a patient. 15.The anti-PD-1 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule for the use of claim 14, wherein the cancer originates in a tissue selected from the group consisting of skin, lung, intestine, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus, and pancreas.

16. The anti-PD-1 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule for the use of claim 14, wherein the cancer is selected from the group consisting of advanced or metastatic melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, renal cell carcinoma, and Hodgkin lymphoma. 17.The anti-PD-1 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule for use according to any one of claims 13-16, wherein said antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule is administered with a chemotherapeutic agent, an antineoplastic agent, an antiangiogenic agent, a tyrosine kinase inhibitor, or a PD-1 pathway inhibitor.

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