Human monoclonal antibodies against programmed cell death 1 (PD1) and methods for the treatment of cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents

Abstract

An isolated monoclonal antibody, or antigen-binding portion thereof, comprising: a) a CDR1 of the variable region of the heavy chain comprising the amino acids having the sequence shown in SEC ID NO: 18; b) a CDR2 of the variable region of the heavy chain comprising the amino acids having the sequence shown in SEC ID NO: 25; c) a CDR3 of the heavy chain variable region comprising amino acids having the sequence shown in SEC ID NO: 32; d) a CDR1 of the variable region of the light chain comprising the amino acids having the sequence shown in SEC ID NO: 39; e) a CDR2 of the variable region of the light chain comprising the amino acids having the sequence shown in SEC ID NO: 46; and f) a CDR3 of the variable region of the light chain comprising the amino acids having the sequence shown in SEC ID NO: 53; where the antibody specifically binds to the human Programmed Death 1 (PD-1) protein.

Description

Human monoclonal antibodies against programmed death 1 (PD1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents technical field The present invention relates generally to immunotherapy in the treatment of human diseases and the reduction of adverse events related thereto. More specifically, the present invention relates to anti-PD-1 antibodies and the use of the antibodies and combined immunotherapy, including the combination of anti-CTLA-4 and anti-PD-1 antibodies, to treat cancer and / or to decrease the incidence or magnitude of adverse events related to treatment with said antibodies individually. Background of the Invention The Programmed Death 1 (PD-1) protein is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al, supra.; Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al (2003) J Immunol 170:711-8). The initial members of the family, CD28 and ICOS, were discovered by functional effects in increasing T cell proliferation after the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. al (1980) 10:247-260 Immunogenics). PD-1 was discovered through screening for differential expression in apoptotic cells (Ishida et al. (1992) EMBO J. 11:3887-95). The other members of the family, CTLA-4, and BTLA were discovered through screening for differential expression on cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS, and CTLA-4 have an unpaired cysteine ​​residue that allows homodimerization. In contrast, it is suggested that PD-1 exists as a monomer, lacking the characteristic unpaired cysteine ​​residue found in other members of the CD28 family. The PD-1 gene is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily (Agata et al. (1996) Int. Immunol 8:765-72). PD-1 contains an immunoreceptor proximal membrane tyrosine-based inhibitory motif (ITIM) and a distal membrane tyrosine-based activation "switch" motif (ITSM) (Thomas, M.L. (1995) J. Exp. Med 181:1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91). Although structurally similar to CTLA-4, PD-1 lacks the MYPPPY motif that is critical for the binding of B7-1 and B7-2. Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to down-regulate T cell activation upon PD-1 binding (Freeman et al. (2000) J. Exp Med 192:1027-34, Latchman et al (2001) Nat Immunol 2:261-8, Carter et al (2002) Eur J Immunol 32:63443). Both PD-L1 and PD-L2 are homologues of B7 that bind to PD-1, but do not bind to other members of the CD28 family. A PD-1 ligand, PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med 8:787-9). The interaction between PD-1 and PD-L1 results in a decrease in tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and immune evasion of cancer cells (Dong et al. (2003 ) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is also blocked (Iwai et al. (2002) Proc. Natl Acad Sci USA 99:12293-7 Brown et al (2003) J Immunol 170:1257-66). PD-1 is an inhibitory member of the CD28 family expressed on activated B cells, T cells, and myeloid cells (Agata et al, supra; Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al (2003) J Immunol 170:711-8). Animals lacking PD-1 develop various autoimmune phenotypes, including autoimmune cardiomyopathy and a lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51; Nshimura et al. (2001) Science 291: 319-22). Additionally, PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78, Prokunina and Alarcón-Riquelme (2004) Hum Mol Genet 13:R143, Nielsen et al (2004) Lupus 13:510). In a murine B-cell tumor line, the ITSM of PD-1 has been shown to be essential for blocking BCR-mediated Ca2+ influx and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001)). PNAS 98:13866-71). Accordingly, agents that recognize PD-1, and methods of using such agents, are desired. Description of the Invention The present invention provides isolated monoclonal antibodies, in particular human monoclonal antibodies, that bind to PD-1 and that exhibit numerous desirable properties. These properties include, for example, high affinity binding to human PD-1, but lack of substantial cross-reactivity with either human CD28, CTLA-4, or ICOS. Still further, the antibodies of the invention have been shown to modulate the immune response. The antibodies are used to inhibit the growth of tumor cells in vivo. Therefore, the present invention provides an isolated monoclonal antibody, comprising: a) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO: 4; and b) a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO: 11; wherein the antibody specifically binds to the human Programmed Death 1 (PD-1) protein. The antibody is a human antibody. Preferably, the antibody inhibits suppression of CD25-CD4+ T cells by regulatory T cells. The antibody may enhance the antigen-specific memory response to a tumor or pathogen or may confer persistent immunity to tumor relapse. The antibody may have an IgG4 isotype, optionally having neither ADCC activity nor CDC activity. The antibodies of the invention are, for example, whole antibodies, for example of an IgG1 or IgG4 isotype. The invention also provides an immunoconjugate product comprising an antibody of the invention, linked to a therapeutic agent, such as a cytotoxin or radioactive isotope. The invention also provides a bispecific molecule comprising an antibody, of the invention, linked to a second functional moiety having a different binding specificity than said antibody. Compositions comprising an antibody, or immunoconjugate or bispecific molecule of the invention, and a pharmaceutically acceptable carrier are also provided. An antibody of the invention may be provided for use in a method of modulating an immune response in a subject. Typically, the method comprises administering to the subject the antibody, of the invention, in such a manner as to modulate the immune response in the subject. Preferably, the antibody of the invention potentiates, stimulates or augments the immune response in the subject. An antibody of the invention may also be provided for use in a method of inhibiting the growth of tumor cells in a subject. Typically, the method comprises administering to a subject a therapeutically effective amount of the antibody. Other anti-PD-1 antibodies can be used in combination with an anti-PD-1 antibody of the invention. For example, a chimeric, humanized, or fully human anti-PD-1 antibody can be used in the method to inhibit tumor growth. An antibody of the invention for use in a method of treating an infectious disease in a subject may further be provided. Typically, the method comprises administering to a subject a therapeutically effective amount of the antibody. Other anti-PD-1 antibodies can be used in combination with an anti-PD-1 antibody of the invention. For example, a chimeric, humanized or fully human anti-PD-1 antibody can be used in the method of treating an infectious disease. Brief Description of the Drawings Figure 1A shows the nucleotide sequence (SEQ ID NO: 57) and amino acid sequence (SEQ ID NO: 1) of the heavy chain variable region of human monoclonal antibody 17D8. The CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO: 22) and CDR3 (SEQ ID NO: 29) regions are delineated and the germline derivations V, D and J are indicated. Figure 1B shows the nucleotide sequence (SEQ ID NO: 64) and amino acid sequence (SEQ ID NO: 8) of the light chain variable region of human monoclonal antibody 17D8. The CDR1 (SEQ ID NO: 36), CDR2 (SEQ ID NO: 43) and CDR3 (SEQ ID NO: 50) regions are delineated and the germline derivations V and J are indicated. Figure 2A shows the nucleotide sequence (SEQ ID NO: 58) and amino acid sequence (SEQ ID NO: 2) of the heavy chain variable region of human monoclonal antibody 2D3. The CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 30) regions are delineated and the germline derivations V and J are indicated. Figure 2B shows the nucleotide sequence (SEQ ID NO: 65) and amino acid sequence (SEQ ID NO: 9) of the light chain variable region of human monoclonal antibody 2D3. The CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 44) and CDR3 (SEQ ID NO: 51) regions are delineated and the germline derivations V and J are indicated. Figure 3A shows the nucleotide sequence (SEQ ID NO: 59) and amino acid sequence (SEQ ID NO: 3) of the heavy chain variable region of human monoclonal antibody 4H1. The CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 24) and CDR3 (SEQ ID NO: 31) regions are delineated and the germline derivations V and J are indicated. Figure 3B shows the nucleotide sequence (SEQ ID NO: 66) and amino acid sequence (SEQ ID NO: 10) of the light chain variable region of human monoclonal antibody 4H1. The CDR1 (SEQ ID NO: 38), CDR2 (SEQ ID NO: 45) CDR3 (SEQ ID NO: 52) regions are delineated and the germline derivations V and J are indicated. Figure 4A shows the nucleotide sequence (SEQ ID NO: 60) and amino acid sequence (SEQ ID NO: 4) of the heavy chain variable region of human monoclonal antibody 5C4. The CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 25) and CDR3 (SEQ ID NO: 32) regions are delineated and the germline derivations V and J are indicated. Figure 4B shows the sequence of nucleotides (SEQ ID NO: 67) and amino acid sequence (SEQ ID NO: 11) of the light chain variable region of human monoclonal antibody 5C4. The CDR1 (SEQ ID NO: 39), CDR2 (SEQ ID NO: 46) and CDR3 (SEQ ID NO: 53) regions are delineated and the germline derivations V and J are indicated. Figure 5A shows the sequence of nucleotides (SEQ ID NO: 61) and amino acid sequence (SEQ ID NO: 5) of the heavy chain variable region of human monoclonal antibody 4A11. The CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO: 26) CDR3 (SEQ ID NO: 33) regions are delineated and the germline derivations V and J are indicated. Figure 5B shows the nucleotide sequence (SEQ ID NO: 68) and amino acid sequence (SEQ ID NO: 12) of the light chain variable region of human monoclonal antibody 4A11. The CDR1 (SEQ ID NO: 40), CDR2 (SEQ ID NO: 47) and CDR3 (SEQ ID NO: 54) regions are delineated and the germline derivations V and J are indicated. Figure 6A shows the sequence of nucleotides (SEQ ID NO: 62) and amino acid sequence (SEQ ID NO: 6) of the heavy chain variable region of human monoclonal antibody 7D3. The CDR1 (SEQ ID NO: 20), CDR2 (SEQ ID NO: 27) and CDR3 (SEQ ID NO: 34) regions are delineated and the germline derivations V and J are indicated. Figure 6B shows the sequence of nucleotides (SEQ ID NO: 69) and amino acid sequence (SEQ ID NO: 13) of the light chain variable region of human monoclonal antibody 7D3. The CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 48) and CDR3 (SEQ ID NO: 55) regions are delineated and the germline derivations V and J are indicated. Figure 7A shows the sequence of nucleotides (SEQ ID NO: 63) and amino acid sequence (SEQ ID NO: 7) of the heavy chain variable region of human monoclonal antibody 5F4. The CDR1 (SEQ ID NO: 21), CDR2 (SEQ ID NO: 28) and CDR3 (SEQ ID NO: 35) regions are delineated and the germline derivations V and J are indicated. Figure 7B shows the sequence of nucleotides (SEQ ID NO: 70) and amino acid sequence (SEQ ID NO: 14) of the light chain variable region of human monoclonal antibody 5F4. The CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 49) and CDR3 (SEQ ID NO: 56) regions are outlined and the germline leads V and J are indicated. Figure 8 shows the alignment of the heavy chain variable region amino acid sequence of 17D8, 2D3, 4H1, 5C4 and 7D3 with the amino acid sequence 3-33 of human germline VH (SEQ ID NO: 71). Figure 9 shows the alignment of the light chain variable region amino acid sequence of 17D8, 2D3 and 7D3 with the L6 amino acid sequence of human germline Vk (SEQ ID NO: 73). Figure 10 shows the alignment of the amino acid sequence of the light chain variable region of 4H1 and 5C4 with the L6 amino acid sequence of human germline Vk (SEQ ID NO: 73). Figure 11 shows the alignment of the heavy chain variable region amino acid sequence of 4A11 and 5F4 with the amino acid sequence 4-39 of human germline VH (SEQ ID NO: 72). Figure 12 shows the alignment of the amino acid sequence of the light chain variable region of 4A11 and 5F4 with the amino acid sequence L15 of human germline Vk (SEQ ID NO: 74). Figures 13A-13B show the results of flow cytometry experiments demonstrating that human monoclonal antibodies 5C4 and 4H1, directed against human PD-1, bind to the cell surface of CHO cells transfected with whole human PD-1. Figure 13A shows the flow cytometry plot for 5C4.Figure 13B shows the flow cytometry plot for 4H1. Thin line represents binding to CHO cells and thick line represents binding to hPD-1 CHO cells. Figure 14 shows a graph demonstrating that human monoclonal antibodies 17D8, 2D3, 4H1, 5C4, and 4A11, directed against human PD-1, bind specifically to PD-1, and not to other members of the CD28 family. Figures 15A-15C show the results of flow cytometry experiments demonstrating that human monoclonal antibodies 4H1 and 5C4, directed against human PD-1, bind to PD-1 on the cell surface. Figure 15A shows binding to activated human T cells. Figure 15B shows binding to cynomolgus monkey T cells. Figure 15C shows binding to transfected CHO cells expressing PD-1. Figures 16A-16C show the results of experiments demonstrating that human monoclonal antibodies against human PD-1 promote T-cell proliferation, IFN-gamma secretion, and IL-2 secretion in a mixed lymphocyte reaction assay. . Figure 16A is a bar graph showing concentration-dependent proliferation of T cells; Figure 16B is a bar graph showing concentration dependent IFN-gamma secretion; Figure 16C is a bar graph showing concentration-dependent secretion of IL-2. Figures 17A-17B show the results of flow cytometry experiments demonstrating that human monoclonal antibodies against human PD-1 block the binding of PD-L1 and PD-L2 to transfected CHO cells expressing PD-1. Figure 17A is a graph showing inhibition of PD-L1 binding; Figure 17B is a graph showing the inhibition of PD-L2 binding. Figure 18 shows the results of flow cytometry experiments showing that human monoclonal antibodies against human PD-1 do not promote T cell apoptosis. Figure 19 shows the results of experiments showing that anti-PD-1 HuMAbs 1 have a concentration-dependent effect on IFN gamma secretion by PBMC from CMV-positive donors when PBMC were stimulated with CMV lysate and anti-PD-1. Figure 20 shows the results of tumor growth experiments in a mouse model system demonstrating that in vivo treatment of mouse tumors with anti-PD-1 antibodies inhibits tumor growth. Figures 21A to 21D show tumor volume over time in individual mice implanted with MC38 (PD-L1-) colon tumor cells and treated on the same day with one of the following therapies: (A) Mouse IgG (control), (B) anti-CTLA-4 antibody, (C) anti-PD-1 antibody, and (D) anti-CTLA-4 antibodies and anti-PD-1 antibodies. Mice received subsequent antibody treatments on days 3, 6, and 10, as described in Example 13, and tumor volume was monitored for 60 days. Figure 22 shows the mean tumor volume of the mice shown in Figure 21. Figure 23 shows the mean tumor volume of the mice shown in Figure 21. Figures 24A to 24D show the tumor volume over time in individual mice implanted with MC38 colon tumor cells (PD- L1-) and one week later they were treated with one of the following therapies: (A) mouse IgG (control), (B) anti-CTLA-4 antibody, (C) anti-PD-1 antibody, and (D) anti-CTLA4 antibody and anti-PD-1 antibody. The tumor volume on the first day of treatment was approximately 315 mm3. Mice received subsequent antibody treatments on days 3, 6, and 10, as described in Example 14. Figure 25 shows the mean tumor volume of the mice shown in Figure 24. Figure 26 shows the mean tumor volume of the mice shown in Figure 24. Figure 27 shows mean tumor volume over time in individual mice implanted with MC38 (PD-L1-) colon tumor cells (Day -7) and then treated on days 0, 3, 6. and 10 post-implantation (as described in Example 15) with one of the following therapies: (A) control mouse IgG (20 mg / kg, X20) (B) anti-PD-1 antibody (10 mg / kg) and mouse IgG (10 mg / kg) (P10X10), (C) anti-CTLA-4 antibody (10 mg / kg) and mouse IgG (10 mg / kg) (C10X10), (D) antibody anti-CTLA-4 and anti-PD-1 antibody (10 mg / kg each) (C10P10), (E) anti-CTLA-4 antibody and anti-PD-1 antibody (3 mg / kg each) (C3P3 ), and (F) anti-CTLA-4 antibody and anti-PD-1 antibody (1 mg / kg each) (C1P1). Two groups of mice were treated with each antibody successively as follows: (G) anti-CTLA-4 antibody (10 mg / kg, day 0), anti-CTLA-4 antibody (10 mg / kg, day 3), anti-PD-1 antibody (10 mg / kg, day 6), and anti-PD-1 antibody (10 mg / kg, day 10) (C10C10P10P10); and (H) anti-PD-1 antibody (10 mg / kg, day 0), anti-PD-1 antibody (10 mg / kg, day 3), anti-CTLA-4 antibody (10 mg / kg, day 6), and anti-CTLA-4 antibody (10 mg / kg, day 10) (10 mg / kg, day 10) (P10P10C10C10). Figure 28 shows the mean tumor volume of the mice shown in Figure 27. Figure 29 shows the mean tumor volume of the mice shown in Figure 27. Figures 30A to 30F show the tumor volume over time in individual mice implanted with SA1 / N fibrosarcoma cells (PD- L1-) and one day later they were treated with one of the following therapies: (A) PBS (control carrier), (B) mouse IgG (control antibody, 10 mg / kg), (C) anti-PD-1 antibody (10 mg / kg), (D) anti-CTLA antibody -4 (10 mg / kg), (E) anti-CTLA-4 antibody (0.2 mg / kg), and (F) anti-PD-1 antibody (10 mg / kg) and anti-CTLA-4 antibody (0.2mg / kg). Mice received subsequent antibody treatments on days 4, 7, and 11, as described in Example 16, and tumor volume was monitored for 41 days. Figure 31 shows the mean tumor volume of the mice shown in Figure 29. Figure 32 shows the mean tumor volume of the mice shown in Figure 29. Figures 33A to 33J show the tumor volume over time in individual mice implanted with SAl / N fibrosarcoma cells (PD- L1-) and then treated on days 7, 10, 13 and 17 post-implantation (as described in Example 17) with one of the following therapies: (A) PBS (carrier control), (B) IgG mouse (control antibody, 10 mg / kg), (C) anti-CTLA-4 antibody (0.25 mg / kg), (D) anti-CTLA-4 antibody (0.5 mg / kg), ( E) anti-CTLA-4 antibody (5 mg / kg), (F) anti-PD-1 antibody (3 mg / kg), (G) anti-PD-1 antibody (10 mg / kg), (H) anti-PD-1 antibody (10 mg / kg) and anti-CTLA-4 antibody (0.25 mg / kg), (I) anti-PD-1 antibody (10 mg / kg) and anti-CTLA-4 antibody (0 , 5 mg / kg), and (F) anti-PD-1 antibody (3 mg / kg) and anti-CTLA4 antibody (0.5 mg / kg). The tumor volume on the first day of treatment was approximately 110 mm3. Figure 34 shows the mean tumor volume of the mice shown in Figure 33. Figure 35 shows the mean tumor volume of the mice shown in Figure 33. Figures 36A and 36B show the tumor volume over time in individual mice implanted with Sal / N fibrosarcoma cells (PD- L1-) and then treated on days 10, 13, 16, and 19 post-implantation (as described in Example 17) with one of the following treatments: (A) Mouse IgG (control antibody, 10 mg / kg) or (B) anti-PD-1 antibody (10 mg / kg) and anti-CTLA-4 antibody (1 mg / kg). The tumor volume on the first day of treatment was approximately 250 mm3. Figure 37 shows the mean tumor volume of the mice shown in Figure 36. Figure 38 shows the mean tumor volume of the mice shown in Figure 36. Figure 39 shows the mean and median percent tumor inhibition calculated from the tumor volumes shown in Figures 33 and 36. Figures 40A to 40D show tumor volume in BALB / c mice implanted subcutaneously with RENCA renal adenocarcinoma cells (PD-L1+) (Murphy and Hrushesky (1973) J. Natl. Cancer Res. 50:1013 -1025) (day -12) and then treated intraperitoneally on days 0, 3, 6, and 9 post-implantation with one of the following therapies: (A) Mouse IgG (control antibody, 20 mg / kg ), (B) anti-PD-1 antibody (10 mg / kg), (C) anti-CTLA-4 antibody (10 mg / kg), and (D) anti-PD-1 antibody (10 mg / kg) combined with anti-CTLA-4 antibody (10 mg / kg). The tumor volume on the first day of treatment was approximately 115 mm3. Figure 41 shows that binding of mouse PD-L2-Fc fusion protein to mouse PD-1 (mPD-1) is blocked by anti-mPD-1 antibody 4H2 in a dose-dependent manner. Binding is detected by measuring the fluorescence of FITC labeled donkey anti-rat IgG by ELISA. The higher the MFI (mean fluorescence intensity) the greater the binding. Figure 42 shows binding curves of anti-mPD-1 antibodies to immobilized mPD-1-Fc fusion protein by ELISA. Figure 43 shows binding curves of rat anti-mPD-1 antibody 4H2.B3 to CHO cells expressing mPD-1. Binding was detected with FITC-conjugated donkey anti-rat IgG and measured by FACS (IMF). Figure 44 shows the binding curve of mPD-L1-hFc fusion protein to CHO cells expressing mPD-1 in the presence of increasing concentrations of anti-mPD-1 antibody 4H2.B3. Binding was detected with goat anti-human IgG, conjugated with FITC and measured by FACS (IMF). Figure 45 shows the binding curves of rat anti-mPD-1 4H2.B3 antibody to mPD-1 expressing CHO cells, compared to rat anti-mPD-1 4H2:chimeric mouse. Figure 46 shows the binding curves of mPD-L1-hFc fusion protein to CHO cells expressing mPD-1 in the presence of increasing concentrations of rat anti-mPD-1 4H2.B3 antibody or anti-mPD-1 4H2 antibody. 1 rat:chimeric mouse. Figure 47 shows the mean tumor volume of tumor-free mice previously treated with anti-PD1 antibody and resensitized with Sal / N (PD-L1-) fibrosarcoma cells. Also shown is the mean tumor volume of naive (control, non-sensitized, or pretreated) mice implanted with Sal / N fibrosarcoma cells. Figure 48 shows tumor volume over time in individual mice, which survived tumor-free after implantation of MC3 8 (PD-L1-) colon tumor cells and treatment with anti-PD1 antibody or a combination of anti-PD1 antibody with anti-CTLA-4 antibody), sensitized again with 10x more MC38 colon tumor cells than the initial treatment. Also shown is the mean tumor volume of naive (control, non-sensitized, or pretreated) mice implanted with MC38 colon tumor cells. Figure 49 shows the mean tumor volume of the mice shown in Figure 48. Figure 50 shows the mean tumor volume over time in individual mice implanted with CT26 colon tumor cells. Figures 51A-B show the results of experiments demonstrating that human monoclonal antibodies against human PD-1 promote T cell proliferation and IFN-gamma secretion in cultures containing regulatory T cells. Figure 50A is a bar graph showing concentration dependent T cell proliferation using HuMAb 5C4; Figure 50B is a bar graph showing the concentration dependent secretion of IFN-gamma from HuMAb 5C4. Figures 52A-B show the results of experiments demonstrating that human monoclonal antibodies against human PD-1 promote T cell proliferation and IFN-gamma secretion in cultures containing activated T cells.Figure 51A is a bar graph showing concentration dependent T cell proliferation using HuMAb 5C4; Figure 51B is a bar graph showing the concentration dependent secretion of IFN-gamma from HuMAb 5C4. Figure 53 shows the results of an antibody-dependent cellular cytotoxicity (ADCC) assay demonstrating that human monoclonal anti-PD-1 antibodies kill human activated T cells in an ADCC concentration-dependent manner relative to the region Anti-PD-1 antibody Fc. Figure 54 shows the results of a complement dependent cytotoxicity (CDC) assay demonstrating that human monoclonal anti-PD-1 antibodies do not kill human activated T cells in a concentration dependent CDC. Best Mode for Carrying Out the Invention The present invention relates to isolated monoclonal antibodies, which specifically bind to PD-1. The antibodies described herein exhibit one or more desirable functional properties, such as high affinity binding to PD-1, lack of cross-reactivity with other members of the CD28 family, ability to stimulate T cell proliferation, secretion IFN-γ and / or IL-2 in mixed lymphocyte reactions, ability to inhibit binding of one or more PD-1 ligands (eg, PD-L1 and / or PD-L2), responsiveness cross-linked with cynomolgus monkey PD-1, ability to stimulate antigen-specific memory responses, ability to stimulate antibody responses and / or ability to inhibit tumor cell growth in vivo. The antibodies of the invention may be derived from particular germline heavy and light chain sequences. Antibodies in particular comprise CDR regions comprising particular amino acid sequences. More specifically, antibodies comprise: a) a heavy chain variable region CDR1 comprising amino acids having the sequence shown in SEQ ID NO: 18; b) a heavy chain variable region CDR2 comprising amino acids having the sequence shown in SEQ ID NO: 25; c) a heavy chain variable region CDR3 comprising amino acids having the sequence shown in SEQ ID NO: 32; d) a light chain variable region CDR1 comprising amino acids having the sequence shown in SEQ ID NO: 39; e) a light chain variable region CDR2 comprising amino acids having the sequence shown in SEQ ID NO: 46; Y f) a light chain variable region comprising CDR3 of amino acids having the sequence shown in SEQ ID NO: 53. In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. The terms "Programmed Death 1", "Programmed Cell Death 1", "PD-1 protein", "PD-1," "PD1", "PDCD1", "hPD-1" and "hPD-I" are used interchangeably, and include variants, isoforms, species homologues of human PD-1, and analogs having at least one epitope in common with PD-1 The complete sequence of PD-1 can be found under GenBank accession number U64863. The terms "cytotoxic T-lymphocyte associated antigen-4", "CTLA-4", "CTLA4", "CTLA-4 antigen", and "CD152" (see, e.g., Murata, Am. J. Pathol. ( 1999) 155:453-460) are used interchangeably, and include variants, isoforms, species homologs of human CTLA-4, and analogs that have at least one epitope in common with CTLA-4 (see, p. eg, Balzano (1992) Int. J. Cancer Suppl. 7:28-32). The complete nucleic acid sequence of CTLA-4 can be found under GenBank Accession Number L15006. The term "immune response" refers to the action of, e.g. g., lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that result in selective damage to, destruction of, or elimination from the body from invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. A "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. As used herein, the phrase "cell surface receptor" includes, e.g. eg, molecules and complexes of molecules capable of receiving a signal and transmitting that signal across the plasma membrane of a cell. An example of a "cell surface receptor" of the present invention is the PD-1 receptor. The term "antibody" referred to herein includes whole antibodies. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of antibodies can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies that have different antigen specificities (eg, an isolated antibody that specifically binds PD- 1 is substantially free of antibodies that specifically bind to antigens other than PD-1). An isolated antibody that specifically binds to PD-1 can have, however, cross-reactivity with other antigens, such as PD-1 molecules from other species. On the other hand, an isolated antibody may be substantially free of other cellular material and / or chemical agents. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single specificity and binding affinity for a particular epitope. The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. The constant region is also derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies to which CDR sequences derived from the germ line of another mammalian species, such as mouse, have been grafted onto sequences. human structures. The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity having variable regions in which both the in-frame and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, e.g. eg, a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (eg, a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g. from a transfectoma, (c) antibodies isolated from a combinatorial library of recombinant human antibodies, and (d) antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using a transgenic animal for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH regions and VL of recombinant antibodies are sequences that, although derived from and related to the human germline VH and VL sequences, cannot occur naturally in the human germline antibody repertoire in vivo. As used herein, "isotype" refers to the class of antibody (eg, IgM or IgG) that is encoded by the heavy chain constant region genes. The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used herein interchangeably with the term "an antibody that specifically binds an antigen." The term "human antibody derivatives" refers to any modified form of the human antibody, e.g. eg, a conjugate of antibody and another agent or antibody. The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Other framework region modifications can be made within the human framework sequences. The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from from a mouse antibody and the constant region sequences are derived from a human antibody. As used herein, an antibody that "specifically binds human PD-1" is intended to refer to an antibody that binds human PD-1 with a KD of 1 x 10 -7 M or less, more preferably 5 x 10 -8 M or less, more preferably 1 x 10 -8 M or less, more preferably 5 x 10 -9 M or less. The term "Kasoc" or "Ka" as used herein is intended to refer to the rate of association of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" , as used herein, refers to the rate of dissociation of a particular antibody-antigen interaction. The term "KD" as used herein is intended to refer to the dissociation constant, which is derived from the ratio of Ka to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values ​​for antibodies can be determined using methods well established in the art. A preferred method of determining the KD of an antibody is by surface plasmon resonance, preferably using a biosensor system such as a Biacore® system. As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a KD of 10-8 M or less, more preferably 10-9 M or less, and even more preferably 10-10 M or less for a target antigen. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype: refers to an antibody having a KD of 10-7 M or less, more preferably 10-8 M or less, even more preferably 10-9 M or less. The term "treatment" or "therapy" refers to the administration of an active agent for the purpose of curing, healing, alleviating, soothing, altering, remedying, correcting, ameliorating, or affecting a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of symptoms, complications, biochemical clues of a disease, or otherwise arrest or further inhibit the development of the disease, condition, or disorder in a statistically significant way. An "adverse event" (AE), as used herein, is any unfavorable and generally undesirable, even undesirable, sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system or expansion of cells of the immune system (eg, T cells) in response to treatment. A medical treatment may have one or more associated AEs, and each AEs may have the same or a different level of severity. Reference to methods susceptible to "altering adverse events" means a treatment regimen that decreases the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen. As used herein, "hyperproliferative disease" refers to conditions in which cell growth is increased above normal levels. For example, hyperproliferative diseases or disorders include malignant diseases (eg, esophageal cancer, colon cancer, biliary cancer) and non-malignant diseases (eg, atherosclerosis, benign hyperplasia, benign prostatic hypertrophy). As used herein, "subtherapeutic dose" means a dose of a therapeutic compound (eg, an antibody) that is less than the normal or typical dose of the therapeutic compound when administered alone for the treatment of a disease. hyperproliferative (eg, cancer). For example, a subtherapeutic dose of CTLA 4 antibody is a single dose of the antibody at less than about 3 mg / kg, ie, the known dose of anti-CTLA-4 antibody. The use of the alternative (eg, "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite articles "a", "one", or "an" should be understood to refer to "one or more" of any of the recited or enumerated components. As used herein, "approximately" or "essentially comprising" means within an acceptable range of error for the particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined. , that is, the limitations of the measurement system. For example, "approximately" or "essentially comprising" may mean a standard deviation within 1 or more than 1 by practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 20%. On the other hand, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5 times a value. Where particular values ​​are given in the application and claims, unless otherwise indicated, the meaning of "approximately" or "essentially comprising" should be assumed to be within an acceptable margin of error for that particular value. As described herein, any concentration range, percentage range, ratio range, or whole number range is to be understood to include any whole number value within the recited range and, where appropriate, fractions thereof ( such as one-tenth and one-hundredth of a whole number), unless otherwise noted. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g. eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Except where noted, the terms "patient" or "subject" are used interchangeably. Various aspects of the invention are described in more detail in the following subsections. Anti-PD-1 antibodies The antibodies of the invention are characterized by particular features or functional properties of the antibodies. The antibodies specifically bind to PD-1 (eg, they bind to human PD-1 and may cross-react with PD-1 from other species, such as cynomolgus monkey). An antibody of the invention binds PD-1 with high affinity, with a KD of 1 x 10 -7 M or less. The anti-PD-1 antibodies described herein preferably exhibit one or more of the following characteristics: (a) binds to human PD-1 with a KD of 1 x 10 -7 M or less; (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-gamma production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses; (i) stimulates antibody responses; (j) inhibits tumor cell growth in vivo. Conventional assays for evaluating the ability of antibodies to bind to PD-1 are known in the art, including e.g. eg, ELISA, Western blots, and RIA. Binding kinetics (eg, binding affinity) of antibodies can also be assessed by standard assays known in the art, such as by Biacore assays. Assays suitable for evaluation of any of the characteristics described above are described in detail in the Examples. Monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 A preferred antibody of the invention is the human monoclonal antibody 5C4. That antibody and human monoclonal antibodies 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 which are not part of the present invention were isolated and structurally characterized as described in Examples 1 and 2. The VH amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7, respectively. The VL amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 are shown in SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14, respectively. Thus, antibodies comprising the heavy chain and light chain CDR1, CDR2, and CDR3 of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 are disclosed. The amino acid sequences of the VH CDR1s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21, respectively. The amino acid sequences of the VH CDR2s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28, respectively. The amino acid sequences of the VH CDR3s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35, respectively. The amino acid sequences of the Vk CDR1s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42, respectively. The amino acid sequences of the Vk CDR2s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49, respectively. The amino acid sequences of the Vk CDR3s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56, respectively. CDR regions are delineated using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) In addition to the antibodies according to the invention, therefore, also antibodies specifically binding PD-1, preferably human PD-1, and comprising: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 22; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 29; (d) a light chain variable region CDR1 comprising SEQ ID NO: 36; (e) a light chain variable region CDR2 comprising SEQ ID NO: 43; Y (f) a light chain variable region CDR3 comprising SEQ ID NO: 50; either (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 16; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 23; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 30; (d) a light chain variable region CDR1 comprising SEQ ID NO: 37; (e) a light chain variable region CDR2 comprising SEQ ID NO: 44; Y (f) a light chain variable region CDR3 comprising SEQ ID NO: 51; either (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 17; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 31; 5 (d) a light chain variable region CDR1 comprising SEQ ID NO: 38; (e) a light chain variable region CDR2 comprising SEQ ID NO: 45; Y (f) a light chain variable region CDR3 comprising SEQ ID NO: 52; either (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 19; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 26; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 33; (d) a light chain variable region CDR1 comprising SEQ ID NO: 40; (e) a light chain variable region CDR2 comprising SEQ ID NO: 47; Y (f) a light chain variable region CDR3 comprising SEQ ID NO: 54. Antibodies that have particular germline sequences The antibodies described herein comprise a heavy chain variable region of a particular germline heavy chain immunoglobulin gene and / or a light chain variable region of a germline light chain immunoglobulin gene. the particular germ line. For example, an isolated monoclonal antibody described herein, or an antigen-binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH 3-33 gene, wherein the antibody specifically binds to PD-1, preferably human PD-1. Also disclosed herein is an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from a human VK L6 gene, wherein the antibody is specifically binds PD-1, preferably human PD-1. Also disclosed is an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH 4-39 gene, wherein the antibody specifically binds to PD-1, preferably human PD-1. Also disclosed is an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from a human VK L15 gene, wherein the antibody specifically binds to PD-1, preferably human PD-1. Thus, an antibody that specifically binds to PD-1 may comprise: (a) a heavy chain variable region that is the product of or derived from a human VH 3-33 or 4-39 gene (gene encoding the amino acid sequence set forth in SEQ ID NOs: 71 or 73, respectively) ; Y (b) a light chain variable region that is the product of or derived from a human VK L6 or L15 gene (gene encoding the amino acid sequence set forth in SEQ ID NOs: 72 or 74, respectively). Examples of the antibodies having VH and VK of VH 3-33 and VK L6, respectively, are 17D8, 2D3, 4H1, 5C4 and 7D3. Examples of the antibodies having VH and VK of VH 4-39 and VK L15, respectively, are 4A11 and 5F4. As used herein, a human antibody comprises heavy or light chain variable regions that is "the product of" or "derived from" a particular germline sequence, if the variable regions of the antibody are derived from of a system using human germline immunoglobulin genes. Such systems include immunization of a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening of a library of human immunoglobulin genes displayed on phage 50 with the antigen of interest. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins. and selecting the human germline immunoglobulin sequence having the closest sequence (ie, the highest % identity) to the human antibody sequence. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, due to, e.g. eg, naturally occurring somatic mutations or the intentional introduction of site-directed mutation. However, a selected human antibody is typically at least 90% identical in amino acid sequence to an amino acid sequence encoded by a germline immunoglobulin gene. and contains amino acid residues that identify the human antibody as human when compared to germline immunoglobulin amino acid sequences from other species (eg, murine germline sequences). In certain instances, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the immunoglobulin gene of the line germinal. Typically, a human antibody derived from a sequence from the particular human germline will show no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may show no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene. Genetically Engineered and Modified Antibodies Genetically engineered antibodies include those in which modifications have been made to framework residues within VH and / or VK, e.g. eg, to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences with the germline sequences from which the antibody is derived. For example, Table 1 shows a series of amino acid changes in the framework regions of anti-PD-1 antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 that differ from the heavy chain parental germline sequence. . To return one or more of the amino acid residues in the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence, e.g. eg, by site-directed mutagenesis or PCR-mediated mutagenesis. Amino acid changes can occur in the framework regions of anti-PD-1 antibodies that differ from the parental germline sequence of the light chain. For example, for 17D8, amino acid residue No. 47 (within FR2) of VK is an isoleucine while this residue in the corresponding VK germline L6 sequence is a leucine. To return the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence, e.g. eg, by site-directed mutagenesis or PCR-mediated mutagenesis (eg, residue #47 (residue #13 of FR2) of 17D8 VK can be "back-mutated" from isoleucine to leucine). As another example, for 4A11, amino acid residue #20 (within FR1) of VK is a serine while this residue in the corresponding VK germline L15 sequence is a threonine. To return framework region sequences to their germline configuration, e.g. eg, 4A11 VK residue #20 can be "back-mutated" from serine to threonine. Such "back-mutated" antibodies are also intended to be encompassed by the invention. The alignment of the VH regions of 17D8, 2D3, 4H1, 5C4, and 7D3, against the parental germline VH 3-33 sequence is shown in Figure 8. The alignment of the VH regions for 4A11 and 5F4 against the sequence VH 4-39 of the parental germline is shown in Figure 11. Table 1. Antibody 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 modifications of heavy chain germline configuration. Ab Anti-PD-1 Amino acid position of Amino acid of antibody Original amino acid of germline configuration 17D8 10 D T 16 TR 27VF 28 AT 78MT 93MV 2D3 10 D T 27 L F 30T S 85 NS 98 TR 4H1 3 AND Q Ab Anti-PD-1 Amino acid position of Amino acid of antibody Original amino acid of germline configuration 84 T N 88 VAC 98sR 5C4 21DS 23K A 27 IF 80F Y 98 TR 4A11 29 L I 79QH 98 VAC 7D3 23 T A 24 T A 27 IF 70 L I 74 DN 97 VAC 98 TR 5F4 23 ST 29 L I 51 AT 77RK Another type of framework modification involves mutation of one or more residues within the framework region to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is described in more detail in US Patent Publication 20030153043 to Carr et al. In addition to or alternatively to modifications made within the framework regions, antibodies of the invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as half-life in serum, fixation complement, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, an antibody of the invention may be chemically modified (eg, one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of the residues in the Fc region is that of the Kabat EU index. In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g. i.e. increase or decrease. This approach is further described in US Patent No. 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered to, for example, facilitate the assembly of heavy and light chains or to increase or decrease antibody stability. In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment in such a way that the antibody has impaired protein A binding. Staphylococcal (SpA) with respect to binding of SpA to the native Fc-hinge domain. This approach is described in more detail in US Patent No. 6,165,745 to Ward et al. In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in US Patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, the antibody can be altered within the CH1 or CL region to contain a wild-type receptor-binding epitope taken from two loops of a CH2 domain of an IgG Fc region, as shown. described in US Pat. Nos. 5,869,046 and 6,121,022 to Presta et al. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be replaced by a different amino acid residue such that the antibody has altered affinity for a ligand. effector but retains the antigen-binding ability of the parent antibody. The effector ligand in which the affinity is altered may be, e.g. eg, an Fc receptor or the C1 component of complement. This approach is described in more detail in US Pat. Nos. 5,624,821 and 5,648,260, both to Winter et al. In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 may be replaced by a different amino acid residue such that the antibody has impaired C1q binding and / or reduced or abolished enzyme-dependent cytotoxicity. complement (CDC). This approach is described in more detail in US Patent No. 6,194,551 to Idusogie et al. In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the antibody's ability to fix complement. This approach is further described in PCT Publication WO 94 / 29351 by Bodmer et al. In another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids in the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is further described in PCT Publication WO 00 / 42072 from Presta. On the other hand, binding sites on human IgG1 for FcyR1, RII, RIII and FcRn have been mapped and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol.. Chem.. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 were shown to enhance FcRIII binding. In addition, the following combination mutants were shown to enhance FcRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A. In yet another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (ie, the antibody lacks glycosylation). Glycosylation can be altered to e.g. eg, increasing the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved by e.g. eg, alteration of one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more glycosylation sites from the variable framework region to thereby eliminate glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in more detail in US Pat. Nos. 5,714,350 and 6,350,861 to Co et al. Additionally or alternatively, an antibody can be made that has an altered glycosylation type, such as a hypofucosylated antibody that has reduced amounts of fucosyl residues or an antibody that has more bisecting GlcNAc structures. Glycosylation patterns have been shown to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved e.g. eg, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1, 6) fucosyltransferase), so antibodies expressed in cell lines Ms704, Ms705, and Ms709 lack fucose in their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see US Patent Publication No. 20040110704 to Yamane et al. and Yamane-Ohnuki et al (2004) Biotechnol Bioeng 87:614-22). As another example, in EP 1,176,195 to Hanai et al. A cell line with a functionally altered FUT8 gene, encoding a fucosyltransferase, is described, such that antibodies expressed from such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6-linkage-related enzyme. Hanai et al . also describe cell lines that have low enzyme activity for the addition of fucose to N-acetylglucosamine that binds to the Fc region of the antibody or does not have the activity of the enzyme, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 by Presta describes a variant of the CHO cell line, Lec13 cells, with reduced ability to anchor fucose to Asn-linked carbohydrates (297), also resulting in hypofucosylation of antibodies expressed on said cell. host (see also Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740 ). PCT Publication WO 99 / 54342 to Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (eg, beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased glycoprotein structures. bisecting GlcNAcs resulting in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody can be cleaved using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino, AL et al. (1975) Biochem. 14:5516-23). Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (eg, serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or fragment thereof. antibody. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as monoalkoxy- or aryloxy-(C1-C10)-polyethylene glycol or polyethylene glycol- maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See, p. eg, EP 0 154 316 to Nishimura et al. and EP 0 401 384 to Ishikawa et al. Nucleic Acid Molecules Encoding Antibodies of the Invention The nucleic acid molecules encoding the antibodies of the invention may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when it is purified apart from other cellular components or other contaminants, e.g. g., other nucleic acids or cellular proteins, by conventional techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See, F. Ausubel, et al., Ed. (1987), Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid can be, e.g. g., DNA or RNA and may or may not contain intronic sequences. The nucleic acid may be a cDNA molecule. Nucleic acids can be obtained using conventional molecular biology techniques. For antibodies expressed by hybridomas (eg, hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding antibody heavy and light chains made by the hybridoma are they can be obtained by conventional PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display techniques), nucleic acid encoding the antibody can be retrieved from the library. Preferred nucleic acid molecules are those that encode the VH and VL sequences of monoclonal antibody 5C4. A DNA sequence encoding the VH sequence of 5C4 is shown in SEQ ID NO: 60, a DNA sequence encoding the VL sequence of 5C4 is shown in SEQ ID NO: 67. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by conventional recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, in Fab fragment genes or in a scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame. Isolated DNA encoding the VH region can be converted into a complete heavy chain gene by operably linking the VH-encoding DNA with another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). Human heavy chain constant region gene sequences are known in the art (see e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services). United States Humans, NIH Publication No. 913242) and DNA fragments including these regions can be obtained by PCR amplification conventional. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably is an IgG1 or IgG4 constant region. For a heavy chain gene of a Fab fragment, the DNA encoding VH can be operably linked to another DNA molecule encoding only the constant region CH1 of the heavy chain. Isolated DNA encoding the VL region can be converted into a complete light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL . The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and US Human Services, NIH Publication No. 91-3242) and DNA fragments including these regions can be obtained by conventional PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is most preferably a kappa constant region. To create an scFv gene, the VH and VL encoding DNA fragments are operably linked to another fragment encoding a flexible linker, e.g. eg, encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see p Bird et al (1988) Science 242:423-426 Huston et al (1988) Proc Natl Acad Sci USA 85:5879-5883 McCafferty et al (1990) Nature 348 :552-554). Production of Monoclonal Antibodies of the Invention The antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies directed against PD-1 can be generated using transgenic or transchromosomal mice that carry parts of the human immune system in place of the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM™ mice, respectively, and are collectively referred to herein as "human Ig mice." The HuMAb® mouse (Medarex, Inc.) contains human immunoglobulin gene miniloci encoding unrearranged human immunoglobulin heavy (μ and γ) and light κ chain sequences, along with site-directed mutations that inactivate endogenous μ chain loci. and κ (see eg, Lonberg, et al. (1994) Nature 368(6474):856859). Thus, mice show reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human monoclonal IgGκ ( Lonberg, N. et al (1994), supra, reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101, Lonberg, N. and Huszar, D. (1995) Intern Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad Sci. 764:536-546). The preparation and use of HuMab mice, and the genomic modifications made by such mice, are described in more detail by Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad Sci. USA 90:3720-3724; Choy et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of which are specifically incorporated by reference in their entirety. See additionally, US Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, US Pat. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962, all to Lonberg and Kay, and PCT Publication No. WO 01 / 14424 to Korman et al. . In another embodiment, the human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences in the transgenes and transchromosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM™ mice", are described in detail in PCT Publication WO 02 / 43478 by Ishida et al. Additionally, other alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-PD-1 antibodies of the invention. For example, an alternative transgenic system known as XenoMouse (Abgenix, Inc.) can be used; such mice are described, e.g. eg, in US Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al. Alternatively, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-PD-1 antibodies of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice"; such mice are described by Tomizuka et to the. (2000) Proc. Natl. Acad. USA 97: 722-727. In addition, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to raise anti-PD-1 antibodies from cows. invention. Human monoclonal antibodies of the invention can also be prepared using phage display methods for screening of human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art. See, for example: United States Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; United States Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; United States Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and United States Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al. The human monoclonal antibodies of the invention can also be prepared using SCID mice in which the human immune cells have been reconstituted such that a human antibody response can be generated after immunization. Such mice are described, e.g. eg, in US Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al. Human Ig Immunization of Mice When human Ig mice are used to produce the human antibodies of the invention, such mice may be immunized with a purified or enriched preparation of recombinant PD-1 and / or PD-1 antigen, or a fusion protein. of PD-1, as described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; and PCT Publications WO 98 / 24884 and WO 01 / 14424. Preferably, the mice will be 6-16 weeks old after the first infusion. For example, a purified or recombinant preparation (5-50 g) of the PD-1 antigen can be used to immunize mice with human Ig intraperitoneally. Detailed procedures for generating fully human monoclonal antibodies against PD-1 are described in Example 1 below. Cumulative experience with various antigens has shown that transgenic mice respond when initially immunized intraperitoneally (IP) with antigen in Freund's complete adjuvant, followed by IP immunizations every other week (up to a total of 6) with antigen in incomplete adjuvant. of Freund. However, adjuvants other than Freund's are also found to be effective. Furthermore, whole cells in the absence of adjuvant are found to be highly immunogenic. The immune response can be monitored during the course of the immunization protocol with plasma samples obtained by retroorbital bleeds. Plasma can be screened by ELISA (as described below), and mice with sufficient anti-PD-1 human immunoglobulin titers can be used for fusions. Mice can be boosted intravenously with antigen 3 days prior to sacrifice and removal of the spleen. It is expected that 2-3 fusions may be necessary for each immunization. Typically between 6 and 24 mice are immunized for each antigen. Both strains HCo7 and HCo12 are generally used. Furthermore, the HCo7 and HCo12 transgenes can be generated together in a single mouse that has two different human heavy chain transgenes (HCo7 / HCo12). Alternatively or additionally, the KM™ strain mouse may be used, as described in Example 1. Generation of Human Monoclonal Antibody-Producing Hybridomas of the Invention To generate human monoclonal antibody-producing hybridomas of the invention, splenocytes and / or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a cell line. mouse myeloma cell. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to one-sixth the number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Alternatively, single cell suspensions of splenic lymphocytes from immunized mice can be fused by an electric field-based electrofusion method, using a Cyto Pulse large-chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, MD). Cells were plated at approximately 2 x 105 in a flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% ​​FetalClone serum, 18% "653" conditioned medium, origin (IGEN ) 5%, L-glutamine 4 mM, sodium pyruvate 1 mM, HEPES 5 mM, 2-mercaptoethanol 0.055 mM, penicillin 50 units / ml, streptomycin 50 mg / ml, gentamicin 50 mg / ml and 1X HAT (Sigma; HAT medium is added 24 hours after fusion). After about two weeks, the cells can be cultured in medium where the HAT medium is replaced by HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive growth of the hybridoma occurs, the medium can usually be observed after 10-14 days. Antibody-secreting hybridomas can be replated, screened again, and if still positive for human IgG, monoclonal antibodies can be subcloned at least twice by limiting dilution. Stable subclones can be grown at continued in vitro to generate small amounts of antibody in tissue culture medium for characterization. To purify human monoclonal antibodies, selected hybridomas can be grown in two-liter shake flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated prior to affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using the extinction coefficient of 1:43. Monoclonal antibodies can be aliquoted and stored at -80°C. Generation of Transfectomes Producing Monoclonal Antibodies of the Invention Antibodies of the invention can also be produced in a host cell transfectome using, e.g. eg, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (eg, Morrison, S. (1985) Science 229:1202). For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or complete heavy and light chains can be obtained by standard molecular biology techniques (eg, PCR amplification or cloning). cDNAs using a hybridoma expressing the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector such that the transcriptional and translational control sequences within the vector serve their intended function of regulating transcription and translation of the gene. of the antibody. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by conventional methods (eg, ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present). The heavy and light chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors that already encode the heavy chain constant and light chain constant regions. of the desired isotype such that the VH segment is operably linked to the CH segment(s) within the vector and the VK segment is operably linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain genes, recombinant expression vectors carry regulatory sequences that control expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, e.g. eg, by Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements that drive high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40) , adenovirus, (eg, the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or the β-globin promoter, can be used. Additionally, composite regulatory elements from sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the human T-cell leukemia virus type 1 long terminal repeat (Takebe, Y. et al (1988) Mol. Mobile Biol. 8:466-472). In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg, 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, eg, Axel et al., US Pat. Nos. All). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, in a host cell in which it is you have entered the vector. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection). For expression of the heavy and light chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g. eg, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably mammalian host cells, is most preferred because such eukaryotic cells are more likely to , and, in particular, mammalian cells, assemble and secrete a correctly folded and immunologically active antibody than prokaryotic cells. Prokaryotic expression of antibody genes has been reported to be inefficient for the production of high yields of active antibody (Boss, MA and Wood, C.R (1985) Immunology Today 6:12-13). Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary cells (CHO cells) (including dhfr-CHO cells, described by Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, eg, as described by R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells . In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system described in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody. antibody in the culture medium in which the host cells are grown. The antibodies can be recovered from the culture medium using conventional protein purification methods. Characterization of Antibody Binding to Antigen Antibodies of the invention can be assayed for binding to PD-1, e.g. eg, conventional ELISA. Briefly, microtiter plates are coated with purified PD-1 at 0.25 µg / ml in PBS, and then blocked with 5% bovine serum albumin in PBS. Antibody dilutions (eg, PD-1 plasma dilutions from immunized mice) are added to each well and incubated for 1-2 hours at 37°C. Plates are washed with PBS / Tween and then incubated with secondary reagent (eg, for human antibodies, a polyclonal goat anti-human IgG Fc-specific reagent) conjugated with alkaline phosphatase for 1 hour at 37° c. After washing, plates were developed with pNPP substrate (1 mg / ml), and tested at OD 405-650. Preferably, mice developing the highest titers will be used for fusions. An ELISA assay as described above can also be used for the detection of hybridomas showing positive reactivity with the PD-1 immunogen. Hybridomas that bind with high avidity to PD-1 were subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parental cells (by ELISA), can be chosen for making a 5-10 vial cell bank stored at -140°C, and for antibody purification. To purify anti-PD-1 antibodies, selected hybridomas can be grown in two-liter shake flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated prior to affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer can be exchanged into PBS, and the concentration determined by OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80°C. To determine if selected monoclonal anti-PD-1 antibodies bind unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using PD-1 coated ELISA plates as described above. Binding of biotinylated mAb can be detected with a streptavidin-alkaline phosphatase probe. To determine the isotype of purified antibodies, isotype ELISAs can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of microtiter plates can be coated with 1 µg / ml anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, plates are reacted with 1 µg / ml or less of purified test monoclonal antibodies or isotype controls, to room temperature for one to two hours. The wells can then be reacted with either human IgG1 or human IgM-specific alkaline phosphatase conjugated probes. Plaques are grown and analyzed as described above. Human anti-PD-1 IgG can be further tested for reactivity with PD-1 antigen by Western blotting. Briefly, PD-1 can be prepared and electrophoresed on sodium dodecyl sulfate polyacrylamide gel. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10% fetal calf serum, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO). Immunoconjugates In another aspect, the present invention relates to an anti-PD-1 antibody, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (eg, an immunosuppressant), or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are known as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental (eg, kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents also include, e.g. eg, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (eg, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine ( CCNU) , cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin) , anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (eg. , dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC) ), and anti-mitotic agents (eg, vincristine and vinblastine). Other preferred examples of therapeutic cytotoxins that can be conjugated to an antibody of the invention include duocarmycins, calicheamicins, maytansins, and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg™; Wyeth-Ayerst). Cytoxins can be conjugated to the antibodies of the invention using linker technology available in the art. Examples of the types of linkers that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, esters, thioethers, disulfides, and peptide-containing linkers. You can choose a connector that is e.g. e.g., susceptible to low pH cleavage within the lysosomal compartment or is susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue, such as cathepsins (eg, cathepsins B, C, D) . For a further discussion of cytotoxin types, linkers, and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al., (2003) Adv. Drug Deliver. Rev 55:199-215; Trail, PA et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T.M. (2002) Nat Rev Cancer 2:750-763; Pastan, I. and Kreitman, R.J. (2002) Curr. opinion Research Drugs 3:1089-1091; Senter, P.D. and Springer, C.J. (2001) Adv. Drug Deliver. Rev 53:247-264. The antibodies of the present invention can also be conjugated with a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, Iodine131, Indium111, Yttrium90, and Lutetium177. The method for the preparation of the radioimmunoconjugates is established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety should not be construed as being limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide that possesses a desired biological activity. Such proteins may include, e.g. eg, an enzymatically active toxin, or an active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or, biological response modifiers such as, e.g. eg, lymphokines, interleukin-1 ("IL1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GMCSF"), granulocyte colony-stimulating factor ("G-CSF"), or other growth factors. Techniques for conjugating such therapeutic moieties to antibodies are well known, see, p. eg, Arnon et al., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev. 62:119-58 (1982). Bispecific Molecules In another aspect, the present invention relates to bispecific molecules comprising an anti-PD1 antibody, of the invention. An antibody of the invention may be derivatised or linked to another functional molecule, e.g. eg, another peptide or protein (eg, another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention can in fact be derivatized or linked to more than one different functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention may be functionally linked (eg, by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule is produced. Thus, the present invention includes bispecific molecules comprising at least a first binding specificity for PD-1 and a second binding specificity for a second target epitope. In a particular embodiment of the invention, the second target epitope is an Fc receptor, e.g. eg, human FcγRI (CD64) or a human Fcα receptor (CD89). Thus, the invention includes bispecific molecules capable of binding both to effector cells expressing FcγR or FcαR (eg, monocytes, macrophages, or polymorphonuclear cells (PMN)), and to target cells expressing PD-1. These bispecific molecules target PD-1-expressing cells to the effector cell and trigger Fc receptor-mediated effector cell activities, such as phagocytosis of PD-1-expressing cells, antibody-dependent cell-mediated cytotoxicity ( ADCC), the release of cytokines, or the generation of superoxide anion. In an embodiment of the invention where the bispecific molecule is multispecific, the molecule may further include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-PD-1 binding specificity. In one embodiment, the third binding specificity is an anti-enhancing factor (EF) moiety, e.g. eg, a molecule that binds to a surface protein that is involved in cytotoxic activity and thereby enhances the immune response against the target cell. The "anti-enhancing factor portion" may be an antibody, functional antibody fragment, or ligand that binds to a given molecule, e.g. eg, an antigen or a receptor, and thus results in an enhancement of the effect of the binding determinants for the CF receptor or target cell antigen. The "anti-enhancing factor portion" can bind to a CF receptor or a target cell antigen. Alternatively, the anti-enhancing factor portion may bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancing factor portion can bind a cytotoxic T cell (eg, via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cell that results in a increased immune response against the target cell). In one embodiment, the bispecific molecules of the invention comprise as binding specificity at least one antibody, or antibody fragment thereof, including, e.g. g., a Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as an Fv or single chain construct as described in US Pat. No. Ladner et al. whose content is expressly incorporated by reference. In one embodiment, the binding specificity for an Fcy receptor is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of eight γ-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms that are grouped into three Fcγ receptor classes: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In a preferred embodiment, the Fcγ receptor is a high affinity human FcγRI. Human FcγRI is a 72 kDa molecule, showing high affinity for monomeric IgG (108-109 M-1). The production and characterization of certain preferred monoclonal anti-Fcγ antibodies are described by Fanger et al. in PCT Publication WO 88 / 00052 and in United States Patent No. 4,954,617, the teachings of which are fully incorporated by reference herein. These antibodies bind an epitope of FcγRI, FcγRII, or FcγRIII at a site that is distinct from the Fcγ binding site of the receptor, and therefore their binding is not substantially blocked by physiological levels of IgG. Specific anti-FcγRI antibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Hybridoma producing mAb 32 is available from the American Type Culture Collection, ATCC Accession No. HB9469. In other embodiments, the anti-Fcγ receptor antibody is a humanized form of monoclonal antibody 22 (H22). The production and characterization of the H22 antibody is described by Graziano, RF et al. (1995) J. Immunol 155(10):4996-5002 and PCT Publication WO 94 / 10332. The H22 antibody-producing cell line was deposited with the American Type Culture Collection under the designation HA022CL1 and has Accession No. CRL 11177. In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g. eg, an Fc-alpha receptor (FcαRI(CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of an α gene (FcαRI) located on chromosome 19. This gene is known to encode several alternatively spliced ​​transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) is constitutively expressed on monocytes / macrophages, eosinophils, and neutrophil granulocytes, but not on non-effector cell populations. FcαRI has a medium affinity (≈ 5 × 107 M-1) for both IgA1 and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology 16:423-440). Four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, that bind FcαRI outside the ligand-binding domain of IgA, have been described (Monteiro, RC. et al. (1992) J. Immunol. 148 : 1764). FcαRI and FcγRI are preferred activation receptors for use in the bispecific molecules of the invention, since they are (1) primarily expressed on immune effector cells, e.g. eg, monocytes, PMNs, macrophages, and dendritic cells, (2) expressed at high levels (eg, 5,000-100,000 per cell); (3) mediators of cytotoxic activities (eg, ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens, including self-antigens, directed to them. While human monoclonal antibodies are preferred, other antibodies that can be employed in the bispecific molecules of the invention are murine, chimeric, and humanized monoclonal antibodies. Bispecific molecules of the present invention can be prepared by conjugation of constitutive binding specificities, e.g. eg, anti-FcR and anti-PD-1 binding specificities, using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (OPDM), N -succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J Exp Med 160:1686;Liu, MA et al (1985) Proc Natl Acad USA 82:8648). Other methods include those described by Paulus (1985) Behring Ins. mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:8183), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL). When the binding specificities are antibodies, they may be conjugated through sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab') 2 or ligand x Fab fusion protein. A bispecific molecule of the invention may be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules can comprise at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in US Patent No. 5,260,203; US Patent No. 5,455,030; US Patent No. 4,881,175; US Patent No. 5,132,405; US Patent No. 5,091,513; US Patent No. 5,476,786; US Patent No. 5,013,653; US Patent No. 5,258,498; and US Patent No. 5,482,858. Binding of bispecific molecules to their specific targets can be confirmed, e.g. eg, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (eg, growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (eg, an antibody) specific for the complex of interest. For example, FcR-antibody complexes can be detected using e.g. eg, an enzyme-linked antibody or antibody fragment that specifically recognizes and binds to antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, eg, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means such as the use of a γ counter or scintillation counter or by autoradiography. Pharmaceutical Compositions In another aspect, the present invention provides a composition, e.g. eg, a pharmaceutical composition, containing a monoclonal antibody of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (eg, two or more) different antibodies, or immunoconjugates, or bispecific molecules of the invention. The pharmaceutical compositions of the invention can also be administered in combination therapy, that is, in combination with other agents. For example, the combination therapy can include an anti-PD-1 antibody of the present invention combined with at least one other anti-inflammatory or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on uses of the antibodies of the invention. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, ie, the antibody, immunoconjugate, or bispecific molecule, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The pharmaceutical compositions of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesirable toxicological effects (see eg, Berge, SM, et al. (1977) J. Pharm. Sci. 66:119). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as non-toxic organic amines, such as N,N-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine , ethylenediamine, procaine and the like. A pharmaceutical composition of the invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like, and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as such as olive oil, and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, e.g. by the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, e.g. eg, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable dosage form may be caused by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, e.g. eg, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, eg, sugars, polyols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, e.g. g., monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by microfiltration sterilization. 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 freeze drying (lyophilization) which produce a powder of the active ingredient plus any desired additional ingredients from a solution previously filtered in sterile conditions thereof. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, other than one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent active ingredient, preferably from about 0.1 percent to about 70 percent, more preferably from about 1 percent. to about 30 percent active ingredient in combination with a pharmaceutically acceptable carrier. Dosage regimens are adjusted to provide the optimal response desired (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technique of composition. of such active compound for the treatment of sensitivities in individuals. For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg, of host body weight. For example dosages may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or within from the range of 1-10 mg / kg. An illustrative treatment regimen involves administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. Preferred dosage regimens for an anti-PD-1 antibody of the invention include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, administering the antibody using one of the following: dosing regimens: (i) every four weeks for six doses, then every three months, (ii) every three weeks, (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each administered antibody is within the ranges indicated. The antibody is usually administered on multiple occasions. The intervals between the individual dosages can be, e.g. e.g., weekly, monthly, every three months or every year. The intervals may also be irregular as indicated by measurement of blood levels of antibody to the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 µg / ml and in some methods about 25-300 µg / ml. Alternatively, the antibody may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dose at relatively short intervals is sometimes required until disease progression slows or ends, and preferably until the patient shows partial or complete improvement of disease symptoms. Thereafter, the patient may be given a prophylactic regimen. The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt, or amide thereof, the route of administration, the time of administration, the rate of excretion of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific compositions used, the age, sex, weight, condition, general health and previous medical history of the patient being treated. treating, and similar factors well known in the medical arts. A "therapeutically effective dose" of an anti-PD-1 antibody of the invention preferably results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to disease affliction. For example, for the treatment of tumors, a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably at least about 80% relative to untreated subjects. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit such inhibition in vitro by assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise improve symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected. In another aspect, the present disclosure provides a pharmaceutical kit of parts comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody, as described herein. The kit may also further comprise instructions for use in the treatment of a hyperproliferative disease (such as cancer as described herein). In another embodiment, the anti-PD-1 and anti-CTLA-4 antibodies can be packaged simultaneously in a unit dosage form. In certain embodiments, two or more monoclonal antibodies with different binding specificities (eg, anti-PD-1 and anti-CTLA-4) are administered simultaneously, in which case the dosage of each administered antibody is within the ranges indicated. The antibodies can be administered as a single dose or, more commonly, can be administered multiple times. The intervals between the individual dosages can be, for example, weekly, monthly, every three months or every year. The intervals may also be irregular as indicated by measurement of blood levels of antibody to the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 µg / ml and in some methods about 25-300 µg / ml. A composition of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As the skilled artisan will appreciate, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac injection and infusion. , intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal. Alternatively, an antibody of the invention may be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g. g., intranasal, oral, vaginal, rectal, sublingual, or topical. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biocompatible, biodegradable polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are proprietary or generally known to those skilled in the art. See, p. Sustained and Controlled Release Drug Deliver and Systems, J.R Robinson, ed., Marcel Dekker, Inc., New York, 1978. The therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices described in US Pat. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: US Patent No. 4,487,603, which describes an implantable micro-infusion pump for dispensing medication at a controlled rate; US Patent No. 4,486,194, which describes a therapeutic device for administering drugs through the skin; US Patent No. 4,447,233, which describes a medication infusion pump for delivering medication at a precise infusion rate; US Patent No. 4,447,224, which describes a variable flow implantable infusion apparatus for continuous drug release; US Patent No. 4,439,196, which describes an osmotic drug delivery system having multiple chamber compartments; and US Patent No. 4,475,196, which describes an osmotic drug delivery system. These patents are incorporated herein by reference. Many other implants, delivery systems, and modules are known to those of skill in the art. In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure adequate distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, e.g. eg, in liposomes. For methods of making liposomes, see, p. eg, United States Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes may comprise one or more moieties that are selectively transported to specific cells or organs, thereby enhancing drug release (see, eg, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). . Exemplary targeting moieties include folate or biotin (see, eg, US Patent No. 5,416,016 to Low et al); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies; (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ killion; IJ Fidler (1994) Immunomethods 4:273. Uses and Methods of the Invention The antibodies, and antibody compositions of the present invention have numerous in vitro and in vivo utilities involving, e.g. eg, detection of PD-1 or enhancement of the immune response by blocking PD-1. The antibodies of the present invention are human antibodies. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g. g., in vivo, to enhance immunity in a variety of situations. Accordingly, the immune response in a subject can be modified by administering the antibody, of the invention, to the subject in such a manner as to modify the immune response in the subject. Preferably, the response is enhanced, stimulated or upregulated. As used herein, the term "subject" is intended to include human and non-human animals. Non-human animals include all vertebrates, e.g. mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses are preferred . Preferred subjects include human patients in need of enhancement of an immune response, particularly human patients who have a disorder that can be treated by augmentation of the T-cell mediated immune response. Cancer cells can be treated in vivo. To achieve antigen-specific enhancement of the immunity, anti-PD-1 antibodies can be administered in conjunction with an antigen of interest. When PD-1 antibodies are administered in conjunction with another agent, the two may be administered in any order or simultaneously. Additionally, the presence of human PD-1 antigen in a sample can be detected, or the amount of human PD-1 antigen measured, by contacting the sample, and a control sample, with a human monoclonal antibody, or a portion of antigen binding thereof, which specifically binds to human PD-1, under conditions that allow the formation of a complex between the antibody or portion thereof and human PD-1. Complex formation is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative of the presence of human PD-1 antigen in the sample. Taking into account the specific binding of the antibodies of the invention for PD-1, compared to CD28, ICOS and CTLA-4, the antibodies of the invention can be used to specifically detect the expression of PD-1 on the surface of cells and, on the other hand, can be used to purify PD-1 through immunoaffinity purification. Cancer Blocking PD-1 by antibodies can enhance the immune response against cancer cells in the patient. The PD-1 ligand, PD-L1, is not expressed in normal human cells, but is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 results in a decrease in tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and immunological evasion of cancer cells (Dong et al. (2003 ) J Mol Med 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 and the effect is additive when the interaction of PD-1 with PD-L2 is also blocked (Iwai et al. (2002) PNAS 99: 12293-7; Brown et al. (2003) J. Immunol. 170:1257-66). While previous studies have shown that T cell proliferation can be restored by inhibiting the interaction of PD-1 with PD-L1, there have been no reports of a direct effect on cancer tumor growth in vivo by blocking of the PD-1 / PDL1 interaction. However, a subject can now be treated in vivo using an anti-PD-1 antibody of the invention in such a way that the growth of cancerous tumors is inhibited. The anti-PD-1 antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-PD-1 antibody may be used in conjunction with other immunogenic agents, conventional cancer treatments, or other antibodies, as described below. Therefore, the growth of tumor cells in a subject can be inhibited by administering to the subject a therapeutically effective amount of an anti-PD-1 antibody of the invention. Preferred cancers whose growth can be inhibited using the antibodies of the invention include those that typically respond to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (eg, metastatic malignant melanoma), kidney cancer (eg, clear cell carcinoma), prostate cancer (eg, ., hormone-refractory adenocarcinoma of the prostate), breast cancer, colon cancer, and lung cancer (eg, non-small cell lung cancer). Furthermore, refractory or recurrent malignant tumors whose growth can be inhibited can be treated using the antibodies of the invention. Examples of other types of cancer that can be treated using the antibodies of the invention include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, endometrial carcinoma, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease , non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, acute or chronic leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma , bladder cancer, kidney or ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of such cancers. Metastatic cancers, especially PD-L1 expressing metastatic cancers, can also be treated (Iwai et al. (2005) Int. Immunol. 17:133-144). Optionally, the PD-1 antibodies can be combined with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding stimulatory cytokines. of the immune system (He et al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides from melanoma antigens, such as peptides from gp100, MAGE, Trp2, MART1 and / or tyrosinase antigens, or tumor cells transfected to express the cytokine GM-CSF (discussed further). below). In humans, some tumors have been shown to be immunogenic, for example melanomas. It is anticipated that by raising the threshold of T cell activation by blocking PD-1, tumor responses in the host can be expected to be activated. PD-1 blockade is likely to be more effective when combined with a vaccination protocol. Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428 Foon, K. 2000, ASCO Educational Book Spring: 730-738 See also Restifo, N. and Sznol, M., Cancer Vaccines, Ch 61, pp 30233043 in DeVita, V. et al (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be more effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad Sci. USA 90:3539-43). The study of gene expression and large-scale gene expression patterns in various tumors has led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed on the tumors and on the cell from which the tumor arose, for example melanocyte gp100 antigens, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targeted by tumor-specific T cells found in the host. PD-1 blockade can be used in conjunction with a library of recombinant proteins and / or peptides expressed in a tumor in order to generate an immune response to these proteins. These proteins are normally seen by the immune system as self-antigens and are therefore tolerant to them. Tumor antigen may also include the protein telomerase, which is required for the synthesis of chromosomal telomeres and is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim, N et al. (1994) Science 266: 2011-2013). (These somatic tissues can be protected from immune attack by various means.) Tumor antigens can also be "neo-antigens" expressed on cancer cells due to somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (i.e. bcr-abl on the Philadelphia chromosome), or idiotype of B cell tumors. Other tumor vaccines may include proteins from viruses implicated in human cancers such as human papilloma virus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpes virus (KHSV). Other forms of tumor-specific antigen that can be used in conjunction with PD-1 blockade are heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain protein fragments from tumor cells and these HSPs are highly effective in delivering to antigen presenting cells to elicit tumor immunity (Suot, R & Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997) Science 278:117-120). Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be produced ex vivo and loaded with various protein and peptide antigens, as well as extracts from tumor cells (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to also express these tumor antigens. DCs have also been fused directly with tumor cells for immunization purposes (Kugler, A. et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization can be effectively combined with PD-1 blockade to activate more potent antitumor responses. PD-1 blockade can also be combined with conventional cancer treatments. PD-1 blockade can be effectively combined with chemotherapy regimens. In these cases, it may be possible to reduce the dose of chemotherapeutic agent administered (Mokyr, M. et al. (1998) Cancer Research 58:5301-5304). An example of such a combination is an anti-PD-1 antibody combined with descarbazine for the treatment of melanoma. Another example of such a combination is an anti-PD-1 antibody combined with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale behind the combined use of PD-1 blockade and chemotherapy is that cell death, which is a consequence of the cytotoxic action of most chemotherapeutic agents, should result in increased levels of tumor antigen. in the antigen presentation pathway. Other combination therapies that may result in synergy with the block of PD-1 via cell death are radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with PD-1 blockade. Inhibition of angiogenesis leads to tumor cell death which can feed tumor antigen to host antigen presentation pathways. PD-1 blocking antibodies can also be used in combination with bispecific antibodies, which target effector cells expressing Fc alpha or Fc gamma receptors to tumor cells (see, e.g., US Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, bispecific anti-Fc receptor / anti-tumor antigen antibodies (eg, Her-2 / neu) have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses would be increased by the use of PD-1 blockade. Alternatively, the antigen can be delivered directly to DCs using bispecific antibodies that bind to tumor antigen and a dendritic cell-specific cell surface marker. Tumors evade host immune surveillance by a variety of mechanisms. Many of these mechanisms can be overcome by the inactivation of proteins that are expressed by tumors and that are immunosuppressive. These include but are not limited to TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. and O'Garra, A. (1992 ) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies against each of these entities can be used in combination with anti-PD-1 to counteract the effects of the immunosuppressive agent and promote immune responses against host tumors. Other antibodies that can be used to activate the host's immune responsiveness can be used in combination with anti-PD-1. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies are capable of effectively substituting helper T cell activity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in conjunction with antibodies against PD-1 (Ito, N et al (2000) Immunobiology 201(5) 527-40). Activation of antibodies against T cell costimulatory molecules such as CTLA-4 (eg, US Patent No. 5,811,097), OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) can also provide increased levels of T cell activation. Bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. Although graft-versus-host disease is a consequence of this treatment, therapeutic benefit can be derived from graft-versus-tumor responses. Blockade of PD-1 can be used to increase the efficacy of engrafted donor tumor-specific T cells. There are also several experimental treatment protocols involving ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients in order to elicit tumor antigen-specific T cells (Greenberg, R. and Riddell, S (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-PD-1 antibodies can be expected to increase the frequency and activity of adoptively transferred T cells. Infectious Diseases The antibodies of the invention can be used to treat patients who have been exposed to particular toxins or pathogens. Accordingly, an infectious disease in a subject can be treated by administering an anti-PD-1 antibody of the invention to the subject, such that the subject is treated for the infectious disease. As in its application to tumors discussed above, antibody-mediated blockade of PD-1 can be used alone, or as an adjuvant, combined with vaccines, to stimulate immune responses to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include pathogens for which no effective vaccine currently exists, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to HIV, Hepatitis (A, B, and C), Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. Blockade of PD-1 is particularly useful against established infections by agents such as HIV that present altered antigens throughout the course of infections. These novel epitopes are recognized as foreign at the time of anti-human PD-1 administration, thereby eliciting a strong T cell response that is not dampened by negative signals through PD-1. Some examples of pathogenic viruses that cause infections treatable by the antibodies of the invention include HIV, hepatitis (A, B, or C), herpes viruses (eg, VZV, HSV-1, 6-HAV, HSV -II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus , parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus. Some examples of pathogenic bacteria that cause infections treatable by the antibodies of the invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera , tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria. Some examples of pathogenic fungi that cause infections treatable by the antibodies of the invention include Candida (albicans, krusei, glabrata, tropicalis, etc), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc), Mucorales genera (mucor, absidia , rhizophus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum. Some examples of pathogenic parasites that cause infections treatable by the antibodies of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lamblia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei , Trypanosoma cruzi, Leishmania donovani, Toxoplasma Gondi, and Nippostrongylus brasiliensis. In all of the above methods, PD-1 blockade can be combined with other forms of immunotherapy such as cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2), or antibody therapy. bispecific, which is provided to improve presentation of tumor antigens (see, eg, Holliger (1993) Proc. Natl. Acad. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123). Autoimmune Reactions The anti-PD-1 antibodies of the invention can elicit and amplify autoimmune responses. Indeed, the induction of antitumor responses using tumor cell and peptide vaccines reveals that many of the antitumor responses include anti-autoreactivities (depigmentation observed in anti-CTLA-4 + GM-CSF-modified B16 melanoma in van Elsas et al. supra ; depigmentation in mice vaccinated with Trp-2 (Overwijk, W et al. (1999) Proc. Natl. Acad. US 96: 2982-2987) ; TRAMP tumor cell vaccine-induced autoimmune prostatitis (Hurwitz, A. (2000) supra), melanoma peptide antigen vaccination, and vitiligo observed in human clinical trials (Rosenberg, SA and White, DE (1996) J. Immunother Emphasis Tumor Immunol 19(1):81-4). Therefore, it is possible to consider the use of anti-PD-1 blockade together with various self proteins in order to design vaccination protocols to efficiently generate immune responses against these self proteins for the treatment of the disease. For example, Alzheimer's disease involves inappropriate accumulation of Aβ peptide in amyloid deposits in the brain; amyloid antibody responses are capable of clearing these amyloid deposits (Schenk et al, (1999) Nature 400: 173-177). Other self proteins can also be used as targets such as IgE for the treatment of allergy and asthma, and TNF for rheumatoid arthritis. Lastly, antibody responses to various hormones can be induced by the use of anti-PD-1 antibody. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody responses to hormones and other soluble factors that are required for the growth of particular tumors can also be considered as potential vaccination targets. Methods analogous to those described above for the use of anti-PD-1 antibody for the induction of therapeutic autoimmune responses can be used to treat patients who have inappropriate accumulation of other self-antigens, such as amyloid deposits, including Aβ in Alzheimer's disease. Alzheimer's, cytokines such as TNFa, and IgE. Vaccines The anti-PD-1 antibodies of the invention can be used to stimulate antigen-specific immune responses by co-administration of such anti-PD-1 antibody with an antigen of interest (eg, a vaccine). Accordingly, an immune response to an antigen in a subject can be enhanced by administering to the subject: (i) the antigen; and (ii) an anti-PD-1 antibody of the invention, or antigen-binding portion thereof, so as to enhance an immune response to the antigen in the subject. Preferably, the antibody is a human anti-human PD-1 antibody (such as any of the anti-human PD-1 antibodies described herein). Additionally or alternatively, the antibody may be a chimeric or humanized antibody. The antigen can be e.g. eg, a tumor antigen, a viral antigen, a bacterial antigen, or a pathogen antigen. Non-limiting examples of such antigens include those described in the sections above, such as the tumor antigens (or tumor vaccines) described above, or antigens from the viruses, bacteria, or other pathogens described above. Suitable routes of administration of the antibody compositions (eg, human monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates) of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. . For example, the antibody compositions can be administered by injection (eg, intravenous or subcutaneous). Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and / or formulation of the antibody composition. As described above, the anti-human PD-1 antibodies of the invention may be co-administered with one or more other therapeutic agents, e.g. eg, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. The antibody can be linked to the agent (in the form of an immune complex) or it can be administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after or in conjunction with the agent or it can be administered simultaneously with other known therapies, eg. g., a cancer therapy, e.g. g., radiation. Such therapeutic agents include, but are not limited to, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine and cyclophosphamide, hydroxyurea which, by themselves, are only effective at levels that are toxic or subtoxic. for a patient. Cisplatin is administered intravenously as a 100 mg / dose once every four weeks and adriamycin is administered intravenously as a dose of 60-75 mg / ml once every 21 days. Simultaneous administration of the human anti-PD-1, antibodies or antigen-binding fragments thereof, of the present invention with chemotherapeutic agents provides two anticancer agents that function through different mechanisms producing a cytotoxic effect for tumor cells. human. Such simultaneous administration can solve problems due to the development of drug resistance or changes in the antigenicity of tumor cells rendering them non-reactive with the antibody. Kits comprising the antibody compositions of the invention (eg, human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use are also described herein. The kit may additionally contain at least one additional reagent, or one or more additional human antibodies of the invention (eg, a human antibody having complementary activity that binds to an epitope on the PD-1 antigen other than the first human antibody). Kits typically include a label indicating the intended use of the kit contents. The term label includes any written, or printed material provided in or with the kit, or otherwise accompanying the kit. Combination Therapy The present invention is based, in part, on the following experimental data. Mouse tumor models (MC38 colon cancer and SA1 / N fibrosarcoma) were used to examine the in vivo effect of treating a tumor by the combination of anti-CTLA-4 and anti-PD-1 immunostimulatory therapeutics. The immunotherapeutic combination was given either simultaneously with the implantation of the tumor cells (Examples 14 and 17) or after the tumor cells had been implanted for a time sufficient to become an established tumor (Examples 15, 16 and 18). Regardless of the time of antibody treatment, it was found that treatment with anti-CTLA-4 antibody alone and treatment with anti-PD-1 antibody (chimeric antibody in which a rat mouse anti-PD-1 was modified with a mouse immunoglobulin Fc region, see Example 1) had only a modest effect on reducing tumor growth in the MC38 tumor model (see, eg, Figures 21, 24 and 27). Anti-CTLA 4 antibody alone was quite effective in the SA1 / N tumor model (see Figure 30D), requiring a lower anti-CTLA 4 antibody dose for combination studies in this model. However, the combined treatment of anti-CTLA-4 antibodies and anti-PD-1 antibody showed an unexpectedly significantly greater effect in reducing tumor growth compared to treatment with the antibody alone (see, e.g., Figures 21D, 24D, 30F and 33H-J). Furthermore, the results of Examples 14, 16 and 18 show that the combined treatment of anti-CTLA-4 antibody and anti-PD-1 antibody had a significant (synergistic) effect on tumor growth even at suboptimal therapeutic doses compared for treatment with either antibody alone (ie, combination therapy was surprisingly more effective at subtherapeutic doses than any monotherapy). Without wishing to be bound by theory, it is possible that raising the threshold of T cell activation by blockade of PD-1 and CTLA-4 may activate anti-tumor responses in a host. In one embodiment, therefore, a hyperproliferative disease can be treated by administering a PD-1 antibody of the invention and a CTLA-4 antibody to a subject. In other embodiments, the anti-PD-1 antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In another embodiment, an associated adverse event with treatment of a hyperproliferative disease with an immunostimulatory agent can be altered by administering an anti-PD-1 antibody of the invention and a subtherapeutic dose of an anti-CTLA-4 antibody to a subject. In certain embodiments, the subject is human. In certain embodiments, the anti-CTLA-4 antibody is the human sequence monoclonal antibody 10D1 and the anti-PD-1 antibody is a human sequence monoclonal antibody, such as 5C4. The anti-CTLA-4 antibody and anti-PD-1 monoclonal antibodies (mAb) and human sequence antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g. eg, the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256:495. Any technique can be used for the production of the monoclonal antibody, e.g. eg, viral or oncogenic transformation of B lymphocytes. An animal system for preparing hybridomas is the murine system. The production of hybridomas in the mouse is a very well established procedure. Immunization protocols and techniques for the isolation of immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion procedures are also known (see, p. Harlow and Lane (1988) Antibodies, A Laboratory and Manual, Cold Spring Harbor Laboratory and Press, Cold Spring Harbor, New York). Anti-CTLA-4 antibodies can bind an epitope on CTLA-4 in order to inhibit CTLA-4 from interacting with a human B7 counter-receptor. Because interaction of CTLA-4 with human B7 transduces a signal that leads to inactivation of T cells bearing the human CTLA-4 receptor, antagonism of the interaction effectively induces, enhances, or prolongs activation of T cells that bear the human CTLA-4 receptor, thereby prolonging or augmenting an immune response. Anti-CTLA-4 antibodies are described in US Pat. Nos. 5,811,097; 5,855,887; 6,051,227; in PCT Application Publication Nos. WO 01 / 14424 and WO 00 / 37504, and in US Patent Publication 2002 / 0039581. An illustrative clinical anti-CTLA-4 antibody is the human monoclonal antibody 10D1 described in WO 01 / 14424 and US Patent Application No. 09 / 644,668. The 10D1 antibody has been administered in single and multiple doses, alone or in combination with a vaccine, chemotherapy, or interleukin-2, to more than 500 patients diagnosed with metastatic melanoma, prostate cancer, lymphoma, renal cell cancer, breast cancer , ovarian cancer, and HIV. Other anti-CTLA-4 antibodies encompassed by the methods of the present invention include, e.g. eg, those described in: WO 98 / 42752; WO 00 / 37504; US Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17):1006710071; Camacho et al. (2004) J.Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206), and Mokyr et al. (1998) Cancer Res. 58:5301-5304. In certain embodiments, an anti-CTLA-4 antibody is used that is a human sequence antibody, preferably a monoclonal antibody. In another embodiment, monoclonal antibody 10D1 is used. In certain embodiments, the anti-CTLA-4 antibody binds human CTLA-4 with a KD of 5 x 10 -8 M or less, binds human CTLA-4 with a KD of 1 x 10 -8 M or less, binds human CTLA-4 with a KD of 5 x 10 -9 M or less, or binds human CTLA-4 with a KD between 1 x 10 -8 M and 1 x 10 -10 M or less. The antibody combination is useful for enhancement of an immune response against hyperproliferative disease by blocking PD-1 and CTLA-4. In a preferred embodiment, the antibodies of the present invention are human antibodies. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g. eg, in vivo, to enhance immunity in a variety of situations. Accordingly, an immune response in a subject can be modified by administering to the subject a combination of antibodies, or a combination of antigen-binding portions thereof, of the invention so as to modify the immune response in the subject. . Preferably, the response is enhanced, stimulated, or upregulated. In another embodiment, adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory therapeutic agent can be altered by administering an anti-PD-1 antibody of the invention and a subtherapeutic dose of anti-CTLA 4 antibody to a subject. . Blocking PD-1 and CTLA-4 by antibodies can enhance the immune response to cancer cells in the patient. Cancers whose growth can be inhibited by the antibodies of the present disclosure include cancers that normally respond to immunotherapy. Representative examples of cancer types for treatment with the combination therapy of the present disclosure include melanoma (eg, metastatic malignant melanoma), kidney cancer, prostate cancer, breast cancer, colon cancer, and lung cancer. Examples of other types of cancer that can be treated using the methods of the present disclosure include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease , non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, acute or chronic leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, carcinoma renal pelvis, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell cancer, squamous cell cancer, lymphoma T-cell cancers, environmentally induced cancers including those induced by asbestos, and combinations of such cancers. Metastatic cancers can also be treated. In certain embodiments, the combination of therapeutic antibodies discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or at the same time as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially. For example, an anti-CTLA-4 antibody and an anti-PD-1 antibody can be administered sequentially, for example by administering anti-CTLA-4 first and anti-PD-1 second, or by administering anti-PD-1 first. and anti-CTLA-4 second. In addition, if more than one dose of the combination therapy is administered sequentially, the order of the sequential administration may be reversed or maintained in the same order at each time of administration, the sequential administrations may be combined with the simultaneous administrations, or any combination of them. For example, the first administration of a combination of anti-CTLA-4 antibody and anti-PD-1 antibody may be concurrent, the second administration may be sequential with anti-CTLA-4 first and anti-PD-1 second, and the third administration can be sequential with anti-PD-1 first and anti-CTLA-4 second, etc. Another representative dosing schedule might involve a first administration that is sequential with anti-PD-1 first and anti-CTLA-4 second, and subsequent administrations may be concurrent. Optionally, the combination of anti-PD-1 and anti-CTLA-4 antibodies can be further combined with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells , and cells transfected with genes encoding immune-stimulating cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides from melanoma antigens, such as peptides from gp100, MAGE, Trp-2, MART1 and / or tyrosinase antigens, or tumor cells transfected to express the cytokine GM-CSF (discussed additionally below). A combined PD-1 and CTLA-4 blockade can be further combined with a vaccination protocol. Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S. (2000) Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. (2000) ASCO Educational Book Spring: 414-428; Foon, K. (2000) ASCO Educational Book Spring: 730-738; see also Restifo and Sznol, Cancer Vaccines, Ch. 61, pgs. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. Fifth Edition). In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be more effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad Sci. USA. 90: 3539-43). The study of gene expression and large-scale gene expression patterns in various tumors has led to the definition of so-called tumor-specific antigens (Rosenberg (1999) Immunity 10:281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed on the tumors and on the cell from which the tumor arose, for example melanocyte gp100 antigens, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targeted by tumor-specific T cells found in the host. In certain embodiments, a combined blockade of PD-1 and CTLA-4 using the antibody compositions described herein can be used in conjunction with a library of recombinant proteins and / or peptides expressed in a tumor in order to generate an immune response. to these proteins. These proteins are normally seen by the immune system as self-antigens and are therefore tolerant to them. Tumor antigen may also include the protein telomerase, which is required for the synthesis of chromosomal telomeres and is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994). ) Science 266: 2011-2013). (These somatic tissues can be protected from immune attack by various means.) Tumor antigens may also be "neo-antigens" expressed on cancer cells due to somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (i.e., bcr-abl on the Philadelphia chromosome), or the idiotype from B cell tumors. Other tumor vaccines may include proteins from viruses implicated in human cancers such as Human Papilloma Virus (HPV), Hepatitis Virus (HBV and HCV), and Kaposi's Sarcoma Herpes Virus (KHSV). Other forms of tumor-specific antigen that can be used in conjunction with PD-1 blockade are Heat Shock Proteins (HSPs) isolated from purified tumor tissue itself. These shock proteins HSPs contain protein fragments from tumor cells and these HSPs are highly efficient at delivering antigen presenting cells to elicit tumor immunity (Suot and Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120). Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be produced ex vivo and loaded with various protein and peptide antigens, as well as extracts from tumor cells (Nestle et al. (1998) Nature Medicine 4:328-332). DCs can also be genetically transduced to also express these tumor antigens. DCs have also been fused directly with tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization can be further effectively combined with a combined blockade of PD-1 and CTLA-4 to activate more potent antitumor responses. A combined blockade of PD-1 and CTLA-4 can also be additionally combined with conventional cancer treatments. For example, a combined blockade of PD-1 and CTLA-4 can be effectively combined with chemotherapeutic regimens. In these cases, as seen with the combination of anti-PD-1 and anti-CTLA-4 antibodies, it may be possible to reduce the dose of the other chemotherapeutic reagent administered with the combination of the present description (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is a combination of anti-PD-1 and anti-CTLA-4 antibodies further combined with dacarbazine for the treatment of melanoma. Another example is a combination of anti-PD-1 and anti-CTLA-4 antibodies further combined with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale behind the combined use of PD-1 and CTLA-4 blockade with chemotherapy is that cell death, which is a consequence of the cytotoxic action of most chemotherapeutic agents, should result in increased levels. of tumor antigen in the antigen presentation pathway. Other combination therapies that may result in synergy with a combined blockade of PD-1 and CTLA-4 through cell death include radiation, surgery, or hormonal deprivation. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with a combined PD-1 and CTLA-4 blockade. Inhibition of angiogenesis leads to tumor cell death, which can also be a source of tumor antigen to feed into host antigen presentation pathways. A combination of blocking antibodies against PD-1 and CTLA-4 can also be used in combination with bispecific antibodies that target effector cells expressing the Fcα or Fcy receptor to tumor cells (see, e.g., US Pat. US Nos. 5, 922, 845 and 5, 837, 243). Bispecific antibodies can be used to target two separate antigens. For example, bispecific anti-Fc receptor / anti-tumor antigen (eg, Her-2 / neu antibodies) have been used to target macrophages to tumor sites. This targeting can more efficiently activate specific tumor responses. The T cell arm of these responses would be augmented by the use of a combined PD-1 and CTLA-4 blockade. Alternatively, the antigen can be delivered directly to DCs using bispecific antibodies that bind to tumor antigen and a dendritic cell-specific cell surface marker. In another example, a combination of anti-PD-1 and anti-CTLA-4 antibodies can be used together with anti-neoplastic antibodies, such as Rituxan® (Rituximab), Herceptin® (Trastuzumab), Bexxar® (Tositumomab), Zevalin ® (Ibritumomab), Campath® (Alemtuzumab), Lymphocide® (Eprtuzumab), Avastin® (Bevacizumab), and Tarceva® (Erlotinib), and the like. By way of example and without wishing to be bound by theory, treatment with an anti-cancer antibody or a toxin-conjugated anti-cancer antibody may lead to the death of cancer cells (eg, tumor cells) which could enhance a immune response mediated by CTLA-4 or PD-1. In an illustrative embodiment, a treatment of a hyperproliferative disease (eg, a cancerous tumor) can include an anti-cancer antibody in combination with anti-PD-1 and anti-CTLA-4 antibodies, concurrently or sequentially or any combination. thereof, which can enhance an antitumor immune response by the host. Tumors evade host immune surveillance by a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins, which are expressed by tumors and are immunosuppressive. These include, but are not limited to, TGF-β (Kehrl, J. et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. and O'Garra, A. ( 1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). In another example, antibodies to each of these entities can be further combined with a combination of anti-PD-1 and anti-CTLA-4 to counteract the effects of immunosuppressive agents and promote immune responses against tumors by the host. . Other antibodies that can be used to activate the host's immune responsiveness can be further used in combination with a combination of anti-PD-1 and anti-CTLA-4. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies are capable of effectively substituting helper T cell activity (Ridge, J. et al. (1998) Nature 393:474-478) and can be used in conjunction with a combination of anti-PD-1 and anti-CTLA-4 (Ito, N. et al. (2000) Immunobiology 201(5) 527-40). Activation of antibodies against cell costimulatory molecules T, such as OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine 3: 682-685 (1997) , and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) may also provide increased levels of T cell activation. Bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. Although graft-versus-host disease is a consequence of this treatment, therapeutic benefit can be derived from graft-versus-tumor responses. A combined blockade of PD-1 and CTLA-4 can be used to increase the efficacy of engrafted donor tumor-specific T cells. There are also several experimental treatment protocols involving the ex vivo activation and expansion of antigen-specific T cells and the adoptive transfer of these cells to receptors in order to elicit T cell-specific antigens against the tumor (Greenberg, R. & Riddell , S. (1999) Science 285: 546-51). These methods can also be used to activate T cell responses against infectious agents such as CMV. Ex vivo activation in the presence of anti-PD-1 and anti-CTLA-4 antibodies can be expected to increase the frequency and activity of adoptively transferred T cells. As stated herein, organs may exhibit immune-related adverse events following immunostimulatory therapeutic antibody therapy, such as the GI tract (diarrhea and colitis) and the skin (rash and pruritus) following treatment with anti-CTLA-4 antibody. For example, immune-related non-colonic gastrointestinal adverse events have also been observed in the esophagus (esophagitis), duodenum (duodenitis), and ileum (ileitis) after anti-CTLA-4 antibody treatment. In certain embodiments, an adverse event associated with the treatment of a hyperproliferative disease with an immunostimulatory agent can be altered by administering an anti-PD-1 antibody of the invention and a subtherapeutic dose of anti-CTLA-4 antibody to a subject. . For example, the incidence of colitis or diarrhea induced by therapeutic immunostimulatory antibodies can be reduced by administering a non-absorbable steroid to the patient. Because any patient who is to receive an immunostimulatory therapeutic antibody is at risk of developing antibody-induced colitis or diarrhea, this entire patient population is suitable for therapy using antibodies of the present invention. Although steroids have been used to treat inflammatory bowel disease (IBD) and prevent IBD exacerbations, they have not been used to prevent (reduce the incidence of) IBD in patients who have not been diagnosed with IBD. Significant side effects associated with steroids, even nonabsorbable steroids, have discouraged prophylactic use. In further embodiments, a combination of PD-1 and CTLA-4 blockade (ie, therapeutic immunostimulatory anti-PD-1 and anti-CTLA-4 antibodies) may be further combined with the use of any non-absorbable steroid. As used herein, a "nonabsorbable steroid" is a glucocorticoid that exhibits extensive first-pass metabolism such that, after metabolism in the liver, the bioavailability of the steroid is low, ie, less than about twenty%. In one embodiment of the invention, the non-absorbable steroid is budesonide. Budesonide is a locally acting glucocorticosteroid that is extensively metabolized, primarily in the liver, after oral administration. ENTOCORT CE® (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and throughout the colon. ENTOCORT CE® is approved in the United States for the treatment of mild to moderate Crohn's disease affecting the ileum and / or ascending colon. The usual oral dose of ENTOCORT CE® for the treatment of Crohn's disease is 6 to 9 mg / day. ENTOCORT CE® is released in the intestines before being absorbed and retained in the mucosa of the intestine. Once it passes through the target tissue of the intestinal mucosa, ENTOCORT CE® is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Therefore, the bioavailability is low (approximately 10%). The low bioavailability of budesonide results in an improved therapeutic ratio compared to other glucocorticoids with less extensive first-pass metabolism. Budesonide results in fewer adverse effects, including less hypothalamic-pituitary suppression than systemically acting corticosteroids. However, chronic administration of ENTOCORT CE® may result in systemic glucocorticoid effects such as hypercortisolism and adrenal suppression. See PDR 58th ed. 2004; 608-610. In still other embodiments, a combined blockade of PD-1 and CTLA-4 (ie, therapeutic immunostimulatory anti-PD-1 and anti-CTLA-4 antibodies) combined with a non-absorbable steroid can be further combined with a salicylate. Salicylates include 5-ASA agents, such as, for example: sulfasalazine (AZULFIDINE®, Pharmacia & Upjohn); olsalazine (DIPENTUM®, Pharmacia & Upjohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.), and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay). Any overlapping or sequential administration of the salicylate and the nonabsorbable steroid can be used for the purpose of decreasing the incidence of immunostimulatory antibody-induced colitis. Thus, p. For example, the incidence of colitis induced by the immunostimulatory antibodies according to the present invention can be reduced by administering a salicylate and a nonabsorbable steroid at the same time or sequentially (eg, a salicylate is administered 6 hours after a non-absorbable steroid), or any combination thereof. In addition, a salicylate and a nonabsorbable steroid may be administered by the same route (eg, they are both administered orally) or by different routes (eg, a salicylate is administered orally and a steroid is not). absorbable is administered rectally), which may differ from the route(s) used to administer anti-PD-1 and anti-CTLA-4 antibodies. The present invention is further illustrated by the following Examples which are not to be construed as further limitation. Antibody 5C4 is an antibody according to the present invention. Examples Example 1: Generation of human monoclonal antibodies against PD-1 Antigen Immunization protocols used as antigen (i) a recombinant fusion protein comprising the extracellular portion of PD-1 and (ii) complete membrane-bound PD-1. Both antigens were generated by recombinant transfection methods in a CHO cell line. HuMab and KM™ transgenic mice Fully human monoclonal antibodies against PD-1 were prepared using HuMab transgenic mouse strain HCo7 and transchromosomal transgenic mouse strain KM, each expressing human antibody genes. In each of these mouse strains, the endogenous mouse kappa light chain gene has been homozygously altered as described by Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been homozygously altered as described in Example 1 of PCT Publication WO 01 / 09187. Each of these mouse strains carries a human kappa light chain transgene, KCo5, as described by Fishwild et al. (1996) Nature Biotechnology 14:845-851. The HCo7 strain carries the HCo7 human heavy chain transgene as described in US Pat. 5,545,806; 5,625,825; Y 5,545,807. Strain KM contains transchromosome SC20 as described in PCT Publication WO 02 / 43478. HuMab and KM immunizations: To generate fully human monoclonal antibodies against PD-1, HuMab and KM™ mice were immunized with purified recombinant PD-1 fusion protein and PD-1 transfected CHO cells as antigen. General immunization schedules for HuMab mice are described by Lonberg, N. et al (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851 and PCT Publication WO 98 / 24884. Mice were 6-16 weeks old after the first antigen infusion. A purified recombinant preparation (5-50 g) of PD-1 fusion protein antigen and 5-10x106 cells were used to immunize HuMab mice and KM™ mice intraperitoneally, subcutaneously (Sc), or by injection. foot pad injection medium. Transgenic mice were immunized twice with antigen in complete Freund's adjuvant or Ribi's adjuvant IP, followed by 3-21 days IP (for a total of 11 vaccinations) with the antigen in incomplete Freund's adjuvant or Ribi's adjuvant. The immune response was monitored by retroorbital bleeding. Plasma was screened by ELISA (as described below), and mice with sufficient anti-human PD-1 anti-immunogolobulin titers were used for fusions. Mice were intravenously boosted with antigen 3 days prior to sacrifice and spleen removal. Typically, 10-35 fusions were made for each antigen. Several tens of mice were immunized for each antigen. Selection of HuMab or KM™ Mice Producing Anti-PD-1 Antibodies: To select for HuMab or KM™ mice that produce antibodies that bind to PD-1, sera from immunized mice were assayed by ELISA as described by Fishwild, D. et al. (nineteen ninety six). Briefly, microtiter plates were coated with recombinant PD-1 fusion protein purified from transfected CHO cells at 1-2 µg / ml in PBS, 100 µl / well incubated at 4°C overnight then blocked with 200 µl / well of 5% fetal bovine serum in PBS / Tween (0.05%). Dilutions of sera from PD-1 immunized mice were added to each well and incubated for 1-2 hours at room temperature. Plates were washed with PBS / Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG polyclonal antibody for 1 hour at room temperature. After washing, the Plates were developed with ABTS substrate (Sigma, A-1888, 0.22 mg / ml) and analyzed by spectrophotometer at OD 415-495. The mice that developed the highest titers of anti-PD1 antibodies were used for the fusions. Fusions were made as described below and hybridoma supernatants assayed for anti-PD-1 activity by ELISA. Generation of hybridomas producing human monoclonal antibodies against PD-1: Mouse splenocytes, isolated from HuMab or KM mice, were fused with a mouse myeloma cell line using either PEG based on conventional protocols or electric field-based electrofusion using a Cyto Pulse large-chamber cell fusion electroporator. (Cyto Pulse Sciences, Inc., Glen Burnie, MD). The resulting hybridomas were then screened for the production of antibodies specific for the antigen. Single cell suspensions of splenocytes from immunized mice were fused to one fourth the number of SP2 / 0 non-secreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG (Sigma). Cells were plated at approximately 1x105 / well in a flat bottom microtiter plate, followed by approximately two weeks incubation in selective medium containing 10% fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63 ) conditioned medium, 3-5% origin (IGEN) in DMEM (Mediatech, CRL 10013, high glucose, L-glutamine, and sodium pyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg / ml of gentamicin and 1x HAT (Sigma, P CRL-7185). After 1-2 weeks, the cells were cultured in medium where HAT was replaced by HT. Individual wells were then screened by ELISA (described above) for anti-human PD-1 monoclonal IgG antibodies. Once extensive growth of the hybridoma had occurred, the medium was checked usually after 10-14 days. Antibody-secreting hybridomas were replated, screened again and, if still positive for human IgG, anti-PD-1 monoclonal antibodies were subcloned at least twice by limiting dilution. Stable subclones were then grown in vitro to generate small amounts of antibody in tissue culture medium for further characterization. Hybridoma clones 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 were selected for further analysis. Example 2: Structural Characterization of Human Monoclonal Antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 The cDNA sequences encoding the variable regions of the heavy and light chains of monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 were obtained from hybridomas 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4, respectively, using standard PCR techniques and sequenced using standard DNA sequencing techniques. The nucleotide and amino acid sequences of the 17D8 heavy chain variable region are shown in Figure 1A and SEQ ID NOs: 57 and 1, respectively. The nucleotide and amino acid sequences of the 17D8 heavy chain variable region are shown in Figure 1B and SEQ ID NOs: 64 and 8, respectively. Comparison of the 17D8 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 17D8 heavy chain utilizes a human germline VH segment 3-33 of VH, an indeterminate D segment, and a JH segment from human germline 4b of JH. The alignment of the VH 17D8 sequence with the germline VH 3-33 sequence is shown in Figure 8. Further analysis of the VH 17D8 sequence using Kabat's CDR region determination system led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 1A and 8, and in SEQ ID NOs: 15, 22 and 29, respectively. Comparison of the 17D8 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 17D8 light chain utilizes a VL segment from human germline VK L6 and a VK segment. JK of human germ line 4 of JK. The alignment of the 17D8 VL sequence with the germline VK L6 sequence is shown in Figure 9. Further detailed analysis of the 17D8 VL sequence using Kabat's CDR region determination system led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 1B and 9, and in SEQ ID NOs: 36, 43 and 50, respectively. The nucleotide and amino acid sequences of the 2D3 heavy chain variable region are shown in Figure 2A and SEQ ID NOs: 58 and 2, respectively. The nucleotide and amino acid sequences of the 2D3 light chain variable region are shown in Figure 2B and SEQ ID NOs: 65 and 9, respectively. Comparison of the heavy chain immunoglobulin sequence of 2D3 with known human germline immunoglobulin heavy chain sequences demonstrated that the 2D3 heavy chain utilizes a human germline VH segment 3-33 of VH, a D segment of human germline 7-27, and a JH segment of human germline 4b of JH. The alignment of the VH 2D3 sequence with the VH 3-33 germline sequence is shown in Figure 8. Further analysis of the VH 2D3 sequence using Kabat's CDR region determination system led to the delineation of the CDR1, CDR2 and CD3 regions of the heavy chain as shown in Figures 2A and 8, and in SEQ ID NOs: 16, 23 and 30, respectively. Comparison of the 2D3 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 2D3 light chain utilizes a VL segment from the human germline VK L6 and a VK segment. JK of human germ line 4 of JK. The alignment of the 2D3 VL sequence with the germline VK L6 sequence is shown in Figure 9. Further analysis of the 2D3 VL sequence using Kabat's CDR region determination system led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 2B and 9, and in SEQ ID NOs: 37, 44 and 51, respectively. The nucleotide and amino acid sequences of the 4H1 heavy chain variable region are shown in Figure 3A and SEQ ID NOs: 59 and 3, respectively. The nucleotide and amino acid sequences of the 4H1 light chain variable region are shown in Figure 3B and SEQ ID NOs: 66 and 10, respectively. Comparison of the 4H1 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 4H1 heavy chain utilizes a human germline VH segment 3-33 of VH, an indeterminate segment D, and a JH segment from human germline 4b of JH. The alignment of the 4H1 VH sequence with the 3-33 germline VH sequence is shown in Figure 8. Further analysis of the 4H1 VH sequence using Kabat's CDR region determination system led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 3A and 8, and in SEQ ID NOs: 17, 24 and 31, respectively. Comparison of the 4H1 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 4H1 light chain utilizes a VL segment from the human germline VK L6 and a VK segment. JK of human germ line 1 of JK. The alignment of the 4H1 VL sequence with the germline VK L6 sequence is shown in Figure 10. Further analysis of the 4H1 VL sequence using Kabat's CDR region determination system led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 3B and 10, and in SEQ ID NOs: 38, 45 and 52, respectively. The nucleotide and amino acid sequences of the 5C4 heavy chain variable region are shown in Figure 4A and SEQ ID NOs: 60 and 4, respectively. The nucleotide and amino acid sequences of the 5C4 light chain variable region are shown in Figure 4B and SEQ ID NOs: 67 and 11, respectively. Comparison of the 5C4 heavy chain immunoglobulin sequence with known human germline heavy chain immunoglobulin sequences demonstrated that the 5C4 heavy chain utilizes a human germline VH segment 3-33 of VH, an indeterminate segment D, and a JH segment from human germline 4b of JH. The alignment of the 5C4 VH sequence with the 3-33 germline VH sequence is shown in Figure 8. Further analysis of the 5C4 VH sequence using Kabat's CDR region determination system led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 4A and 8, and in SEQ ID NOs: 18, 25 and 32, respectively. Comparison of the 5C4 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 5C4 light chain utilizes a VL segment from the human germline VK L6 and a VK segment. JK of human germ line 1 of JK. The alignment of the 5C4 VL sequence with the germline VK L6 sequence is shown in Figure 10. Further analysis of the 5C4 VL sequence using Kabat's CDR region determination system led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 4B and 10, and in SEQ ID NOs: 39, 46 and 53, respectively. The nucleotide and amino acid sequences of the 4A11 heavy chain variable region are shown in Figure 5A and SEQ ID NOs: 61 and 5, respectively. The nucleotide and amino acid sequences of the 4A11 light chain variable region are shown in Figure 5B and SEQ ID NOs: 68 and 12, respectively. Comparison of the 4A11 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 4A11 heavy chain utilizes a human germline VH segment 4-39 of VH, a D segment of human germline 3-9, and a JH segment of human germline 4b of JH. The alignment of the 4A11 VH sequence with the 4-39 germline VH sequence is shown in Figure 11. Further analysis of the 4A11 VH sequence using Kabat's CDR region determination system led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 5A and 11, and in SEQ ID NOs: 19, 26 and 33, respectively. Comparison of the 4A11 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 4A11 light chain utilizes a VL segment from the human germline VK L15 and a VK segment. JK of human germ line 1 of JK. The alignment of the 4A11 VL sequence with the germline VK L6 sequence is shown in Figure 12. Further analysis of the 4A11 VL sequence using Kabat's CDR region determination system led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 5B and 12, and in SEQ ID NOs: 40, 47 and 54, respectively. The nucleotide and amino acid sequences of the 7D3 heavy chain variable region are shown in Figure 7A and SEQ ID NOs: 62 and 6, respectively. The nucleotide and amino acid sequences of the 7D3 light chain variable region are shown in Figure 7B and SEQ ID NOs: 69 and 13, respectively. Comparison of the 7D3 immunoglobulin heavy chain sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 7D3 heavy chain utilizes a VH segment from human germline VH 3-33, a segment 7-27 D of the human germ line, and a segment JH of the human germ line 4b of JH. The alignment of the 7D3 VH sequence with the 3-33 germline VH sequence is shown in Figure 8. Further analysis of the 7D3 VH sequence using Kabat's CDR region determination system led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 6A and 8, and in SEQ ID NOs: 20, 27 and 34, respectively. Comparison of the 7D3 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 7D3 light chain utilizes a human germline VL segment VK L6 and a human germline L6 segment. JK of human germ line 4 of JK. The alignment of the 7D3 VL sequence with the germline VK L6 sequence is shown in Figure 9. Further analysis of the 7D3 VL sequence using Kabat's CDR region determination system led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 6B and 9, and in SEQ ID NOs: 41, 48 and 55, respectively. The nucleotide and amino acid sequences of the 5F4 heavy chain variable region are shown in Figure 7A and SEQ ID NOs: 63 and 7, respectively. The nucleotide and amino acid sequences of the 5F4 light chain variable region are shown in Figure 7B and SEQ ID NOs: 70 and 14, respectively. Comparison of the 5F4 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 5F4 heavy chain utilizes a human germline VH segment 4-39 of VH, a D segment of human germline 3-9, and a JH segment of human germline 4b of JH. The alignment of the 5F4 VH sequence with the 4-39 germline VH sequence is shown in Figure 11. Further analysis of the 5F4 VH sequence using Kabat's CDR region determination system led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 7A and 11, and in SEQ ID NOs: 21, 28 and 35, respectively. Comparison of the 5F4 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 5F4 light chain utilizes a VL segment from the human germline VK L15 and a VK segment. JK of human germ line 1 of JK. The alignment of the 5F4 VL sequence with the germline VK L6 sequence is shown in Figure 12. Further analysis of the 5F4 VL sequence using Kabat's region determination system CDR led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 7B and 12, and SEQ ID NOs: 42, 49 and 56, respectively. Example 3: Characterization of the Binding Specificity and Binding Kinetics of Human Monoclonal Anti-PD-1 Antibodies In this example, the binding affinity and binding kinetics of anti-PD-1 antibodies were examined by means of a Biacore analysis. Binding specificity, and cross-competition were analyzed by flow cytometry. Affinity and Binding Kinetics Anti-PD-1 antibodies were characterized for their affinities and binding kinetics by Biacore assay (Biacore AB, Uppsala, Sweden). Purified recombinant human PD-1 fusion protein was covalently linked to a CM5 chip (Carboxymethyldextran-coated chip) via primary amines, using standard amine coupling chemistry and kit provided by Biacore. Binding was measured by passing the antibodies through HBS EP buffer (provided by Biacore AB) at a concentration of 267 nM, at a flow rate of 50 µl / min. The kinetics of antigen-antibody association was followed for 3 minutes and the kinetics of dissociation was followed for 7 minutes. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software (Biacore AB). To minimize the effects of avidity on the estimation of binding constants, only the initial segment of the data corresponding to the association and dissociation phases was used for adjustment. The KD, Kon and koff values ​​that were determined are shown in Table 2. Table 2. BIACORE binding data of human monoclonal antibodies to PD-1. Sample No. Sample ID Affinity KD x 109 (M) Activation speed kon x 105 (1 / ms) Deactivation speed koff x 10-4 1 / s 1 17D8 0, 16 2, 56 0, 45 2 2D3 1, 20 3, 77 4, 52 3 4H1 5, 46 3, 15 1, 72 4 5C4 0.73 4.32 3.15 5 4A11 0.13 0.76 0.099 6 7D3 2, 49 18, 2 4, 54 7 5F4 2, 91 8, 74 2, 54 Binding Specificity by Flow Cytometry Chinese hamster ovary (CHO) cell lines expressing recombinant human PD-1 on the cell surface were developed and used to determine the specificity of human monoclonal antibodies to PD-1 by flow cytometry. CHO cells were transfected with expression plasmids containing full length cDNA encoding the transmembrane forms of PD-1. The binding of the human anti-PD1 monoclonal antibodies 5C4 and 4H1 was assessed by incubating the transfected cells with the human anti-PD-1 monoclonal antibodies at a concentration of 20 µg / ml. Cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. Flow cytometric analyzes were performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results are depicted in Figures 13A (5C4) and 13B (4H1). Human anti-PD-1 monoclonal antibodies bound to PD-1 transfected CHO cells, but not to CHO cells that were not transfected with human PD-1. These data demonstrate the specificity of human anti-PD-1 monoclonal antibodies for PD-1. Binding specificity by ELISA against other members of the CD28 family A comparison of the binding of anti-PD-1 antibodies to members of the CD28 family was performed by standard ELISA using four different members of the CD28 family to examine the specificity of the binding for PD-1. Fusion proteins of the CD28 family members, ICOS, CTLA-4 and CD28 (R&D Biosystems) were tested for binding against human anti-PD-1 monoclonal antibodies 17D8, 2D3, 4H1, 5C4, and 4A11. Standard ELISA procedures were carried out. Human monoclonal anti-PD-1 antibodies were added at a concentration of 20 µg / ml. Goat anti-human IgG polyclonal antibody was used. (kappa chain specific) conjugated with horseradish peroxidase (HRP) as secondary antibody. The results are shown in Figure 14. Human anti-PD-1 monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 each bound with high specificity to PD-1, but not to the others. members of the CD28 family. Example 4: Characterization of Anti-PD-1 Antibody Binding PD-1 Expressed on the Surface of Human and Monkey Cells Anti-PD-1 antibodies were assayed for binding to PD-1 expressing cells on their cell surface by flow cytometry. Activated human T cells, monkey peripheral blood mononuclear cells (PBMC), and PD-1 transfected CHO cells were assayed for antibody binding. Human T cells and cynomolgus PBMCs were activated by anti-CD3 antibody to induce PD-1 expression on T cells prior to binding with a human anti-PD-1 monoclonal antibody. The binding of the human anti-PD-1 monoclonal antibodies 5C4 and 4H1 was assessed by incubating the transfected cells with either the IgG1 or IgG4 forms of the human anti-PD-1 monoclonal antibodies at different concentrations. Cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. Flow cytometric analyzes were performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figures 15A (activated human T cells), 15B (cynomolgus monkey PBMC) and 15C (PD-1 transfected CHO cells). Anti-PD-1 monoclonal antibodies 5C4 and 4H1 bound to activated human T cells, activated monkey PBMC, and human PD-1 transfected CHO cells, as measured by mean fluorescence intensity (MFI) of staining. These data demonstrate that anti-PD-1 HuMAbs bind to PD-1 on the surface of both human and cynomolgus monkey cells. Example 5: Effect of Human Anti-PD-1 Antibodies on Cell Proliferation and Cytokine Production in a Mixed Lymphocyte Reaction A mixed lymphocyte reaction was used to demonstrate the effect of blocking the PD-1 pathway to effector cells. of lymphocytes. Assay analysis T cells were assayed for proliferation, IFN-gamma secretion and IL-2 secretion in the presence or absence of an anti-PD-1 HuMAb antibody. Human T cells were purified from PBMC using a human CD4+ T cell enrichment column (R&D). Each culture contained 105 purified T cells and 104 allogeneic dendritic cells in a total volume of 200 µl. 1 monoclonal anti-PD-1 antibody 5C4, 4H1, 17D8, 2D3 or a portion of the Fab fragment 5C4 was added to each culture at different antibody concentrations. Either no antibody or an isotype control antibody was used as a negative control. Cells were cultured for 5 days at 37°C. After day 5, 100 µl of medium was taken from each culture for cytokine measurement. Levels of IFN-gamma and other cytokines were measured using OptEIA ELISA kits (BD Biosciences). Cells were labeled with 3H-thymidine, cultured for an additional 18 hours, and analyzed for cell proliferation. The results are shown in Figures 16A (T cell proliferation), 16B (IFN-γ secretion), and 16C (IL-2 secretion). Human anti-PD-1 monoclonal antibodies promoted T cell proliferation, IFN-gamma secretion, and IL-2 secretion in a concentration-dependent manner. The 5C4 Fab fragment also promoted T cell proliferation, IFN-gamma secretion, and IL-2 secretion in a concentration-dependent manner. In contrast, cultures containing the isotype control antibody did not show an increase in T cell proliferation, IFN-gamma secretion, or IL-2 secretion. Example 6: Blocking of Ligand Binding to PD-1 by Human Anti-PD-1 Antibodies Anti-PD-1 HuMAb were assayed for the ability to block the binding of PD-L1 and PD-L2 ligands to PD-1 expressed in transfected CHO cells using flow cytometric analysis. CHO cells expressing PD-1 were suspended in FACS buffer (PBS with 4% fetal calf serum). Various concentrations of the anti-PD-1 HuMAbs 5C4 and 4H1 were added to the cell suspension and incubated at 4°C for 30 minutes. Unbound antibody was washed away and either FITC-labeled PD-L1 fusion protein or FITC-labeled PD-L2 fusion protein was added to the tubes and incubated at 4°C for 30 minutes. Flow cytometry analyzes were performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results are depicted in Figures 17A (PD-L1 blockade) and 17B (PD-L2 blockade). Anti-PD-1 monoclonal antibodies 5C4 and 4H1 blocked the binding of PD-L1 and PDL2 to human PD-1 transfected CHO cells, as measured by mean fluorescence intensity (MFI) of staining. These data demonstrate that anti-PD-1 HuMAbs block ligand (both PD-L1 and PD-L2) binding to cell surface PD-1. Example 7: Effect of Human Anti-PD-1 Antibodies on Cytokine Release in Human Blood Anti-PD-1 HuMAbs were mixed with fresh human whole blood in order to determine whether anti-PD-1 HuMAbs alone stimulated the release of certain cytokines from human blood cells. 500 µl of fresh heparinized human whole blood was added to each well. 10 µg or 100 µg added of an anti-PD-1 HuMAb antibody (4H1 or SC4, the latter as an IgG1 or IgG4 isotype) to each well. Some wells were incubated with anti-CD3 antibody as a positive control, or human IgG1 or human IgG4 antibody as isotype-matched negative controls. Cells were incubated at 37°C for 6 or 24 hours. Cells were centrifuged and plasma collected for measurement of cytokines, IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-10, and IL-12 using bead array analysis. Cytokine cytometric assays (BD Biosciences). The concentration of each of the cytokines (pg / ml) is shown below in Tables 3a, with a 6 hour incubation, and 3b, with a 24 hour incubation. The results show that treatment with the human anti-PD-1 antibodies 5C4 and 4H1 alone did not stimulate human blood cells to release any of the cytokines IFN-gamma, TNF-alpha, IL-2, IL-4, IL- 6, IL-10 and IL-12. Table 3a. Cytokine production after 6 hours of incubation Ab IFN-gamma (pg / ml) TNF-alpha (pg / ml) IL-10 (pg / ml) IL-6 (pg / ml) IL-4 (pg / ml) IL-2 (pg / ml) NOAB 12, 3 2 3 5 3, 6 1, 9 10 mg / mL anti-CD3 5000 530 82, 6510, 4 37, 2467, 9 100 mg / mL anti-CD3 5000 571 91.3 530 43.9 551.5 hIgG1 10mg / mL 7 1, 8 2, 8 4, 4 2, 6 1, 5 hIgG1 100mg / mL 0 2, 2 2, 7 6 2, 6 1, 4 hIgG4 10mg / mL 5, 4 1, 4 2, 5 4, 5 2, 1 1, 3 hIgG4 100mg / ml 6, 4 2, 3 3 32, 6 2, 9 1, 4 10 mg / mL 4H1 6, 2 1, 8 2, 4 4, 1 2, 8 1, 6 100mg / mL 4H1 11.8 2 2.6 3.5 2.6 1.7 10mg / ml IgG1 5C4 4, 2 1, 6 2, 3 3, 9 2, 5 1, 3 100mg / mL IgG1 5C4 0 1, 4 2, 2 3, 6 2, 1 1, 2 5C4 IgG4 10mg / mL 8.3 2.5 1.9 4.8 1.6 1.5 100mg / mL 5C4 IgG4 3, 6 1, 7 2, 4 3, 9 2, 3 1, 5 Table 3b. Cytokine production after 24 hours of incubation 10 Ab IFN-gamma (pg / ml) TNF-alpha (pg / ml) IL-10 (pg / ml) I-6 (pg / ml) IL-4 (pg / ml) IL-2 (pg / ml) No Ab 11, 2 2 6, 1 5, 9 2, 6 1, 7 10 mg / mL anti-CD3 5000 565.9 432 5000 64.5 1265.3 100mg / mL anti-CD3 5000 535 461 5000 73.8 1334.9 10mg / mL MIG1 0 0 0 0 0 0 hIgG1 100mg / mL 11.5 1.7 7.9 60.8 2.9 1.5 hIgG4 10mg / mL 24, 6 3, 1 8, 3 63, 4 3, 1 2, 3 hIgG4 100mg / ml 11, 2 1, 8 8 27, 7 3, 1 2, 4 10mg / mL 4H1 27.3 2.9 8 13.9 5.3 2.6 100mg / mL 4H1 17.5 2.5 4.4 7 4 2.1 10mg / mL IgG1 5C4 9.1 2 7.6 68.5 3.5 1.8 100mg / ml IgG1 5C4 12, 9 1, 9 6, 1 25, 3 2, 9 1, 7 5C4 TgG4 10mg / mL 14 1.9 4.4 3.3 2.6 1.9 100mg / mL 5C4 IgG4 0 0 0 0 0 0 Example 8: Effect of anti-PD-1 antibodies on T cell apoptosis The effect of anti-PD-1 antibodies on the induction of T cell apoptosis was measured using an annexin V staining assay. T cells were cultured in a mixed lymphocyte reaction, as described above in Example 5. Anti-PD-1 antibody 5C4 was added to the tube at a concentration of 25 µg / ml. A non-specific antibody was used as a control. Annexin V and propidium iodide were added according to standard protocol (BD Biosciences). The mixture was incubated for 15 minutes in the dark at room temperature and then analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figure 18. Anti-PD-1 antibody 5C4 has no effect on T cell apoptosis. Example 9: Effect of Anti-PD-1 Antibodies on Cytokine Secretion by Virus-Stimulated PBMC Cells from a Virus-Positive Donor In this example, peripheral blood mononuclear cells (PBMCs) were isolated from a CMV-positive donor. and exposed to CMV lysate in the presence or absence of anti-PD-1 antibodies to examine the effect of the antibodies on antigen-stimulated cytokine secretion. 2 x 10 5 human PMBC from a CMV-positive donor were cultured in a total volume of 200 µl and added to each well along with a lysate from CMV-infected cells. Anti-PD15C4 HuMAb was added to each well at various concentrations for 4 days. After 4 days, 100 µl of medium was taken from each culture for cytokine measurement. IFN-gamma level was measured using OptEIA ELISA kits (BD Biosciences). Cells were labeled with 3 H-thymidine, cultured for an additional 18 hours, and analyzed for cell proliferation. Cell proliferation was analyzed using the Cell Titer-Glo reagent (Promega). The results are shown in Figure 19. Anti-PD-1 HuMab 5C4 increased IFN gamma secretion in a concentration-dependent manner. These results show that anti-PD-1 HuMAbs can stimulate IFN-gamma release in a memory T cell response from PBMC cells previously stimulated against an antigen. Example 10: Effect of anti-PD-1 antibodies on the secondary antibody response to an antigen Mice were immunized and re-sensitized with T1 antigen (DNP-Ficoll) and also treated with an anti-PD-1 antibody from rat mouse, or a control antibody to examine the effect of anti-PD-1 antibody on antibody titers. Female C57BL6 mice were divided into two groups, with 6 mice / group. One group was treated with a control rat IgG and the other with a rat anti-mouse PD-1 antibody. Mice were immunized with 5 µg DNP-Ficoll (a T1 antigen) in 50 µl CFA ip on day 0. Control rat IgG antibody or rat mPD-1 antibody (200 µg / mouse) was administered ip. on days -1, 0, and 2. Four weeks later, mice were sensitized again with 5 µg DNP-Ficoll in 50 µl IFA ip on day 0. Rat anti-mPD-1 antibody or Control antibodies (200 µg / mouse) were administered ip on days 0 and 1. Antibody titers were measured by standard ELISA assay on day 7 post-boost. The results are shown in Table 4 below. In mice treated with anti-mPD-1 antibody, both IgM and IgG3 isotypes showed the greatest increase in titer after T1 antigen challenge, compared to mice treated with a control antibody. These results demonstrate that anti-PD-1 treatment can increase antibody titers in response to T1 antigen. Table 4 Murine secondary response after treatment with anti-PD-1 antibody Antibody isotype Control group Rat mouse anti-PD-1 antibody P-value IgM 606 1200 0.026 IgG 9 15, 55 0, 18 IgG1 1, 2 1, 1 0, 83 IgG2b 5, 05 9, 26 0, 18 IgG3 21, 9 81, 2 0, 03 * Results shown are mean concentration of antibody isotype (µg / mL) Example 11: Treatment of in vivo tumor model using anti-PD-1 antibodies 43 Mice implanted with a cancerous tumor were treated in vivo with anti-PD-1 antibodies to examine the in vivo effect of the antibodies on tumor growth. As a positive control, an anti-CTLA-4 antibody was used, since such antibodies have been shown to inhibit tumor growth in vivo. In this experiment, the anti-PD-1 antibody used was a chimeric rat mouse anti-PD-1 antibody generated using well known laboratory techniques. To generate rat anti-mouse PD-1 antibody, rats were immunized with mouse cells transfected to express a recombinant mouse PD-1 fusion protein (R&D Systems Catalog No. 1021-PD) and antibodies were screened. monoclonals for binding to mouse PD-1 antigen by ELISA assay. The rat anti-PD-1 antibody V regions were then recombinantly linked to a murine IgG1 constant region using standard molecular biology techniques and rescreened for binding to mouse PD-1 by ELISA and FACS. The chimeric rat anti-mouse PD-1 antibody used herein is referred to as 4H2. For tumor studies, 6-8 week old female AJ mice (Harlan Laboratories) were randomized by weight into 6 groups. Mice were implanted subcutaneously in the right flank with 2 x 10 6 SA1 / N fibrosarcoma cells dissolved in 200 µl DMEM medium on day 0. Mice were treated with PBS vehicle, or antibodies at 10 mg / kg. Animals were administered by intraperitoneal injection with approximately 200 µl of PBS containing antibody or vehicle on days 1, 4, 8 and 11. Each group contained 10 animals and the groups consisted of: (i) a vehicle group, (ii) ) control mouse IgG, (iii) control hamster IgG, (iv) hamster anti-mouse CTL-4 antibody, and (v) the 4H2 chimeric anti-PD-1 antibody. Mice were monitored twice weekly for tumor growth for approximately 6 weeks. Using an electronic caliper, tumors were measured in three dimensions (height x width x length) and tumor volume was calculated. Mice were sacrificed when tumors reached the tumor endpoint (1,500 mm3) or when they showed greater than 15% weight loss. The results are shown in Figure 20. Anti-PD-1 antibody prolonged the median time to reach tumor endpoint volume (1500 mm 3 ) from -25 days in the control groups to ~40 days. Therefore, treatment with an anti-PD-1 antibody has a direct inhibitory effect in vivo on tumor growth. Example 12: Generation of chimeric (rat-mouse) anti-PD-1 antibody 4H2 Rat monoclonal antibodies against antibodies to mouse PD-1 (rat anti-mPD-1) were generated from rats immunized with mPD-1-hFc fusion protein using standard hybridoma production methods (see Kohler and Milstein ( 1975) Nature 256:495; and Harlow and Lane (1988) Antibodies, A Laboratory and Manual, Cold Spring Harbor Laboratory and Press, Cold Spring Harbor New York). Eight hybridomas were subcloned, and antibodies were isolated and screened for their ability to block mouse PD-L2 (MPD-L2) binding to mPD-1. Several anti-mPD-1 antibodies capable of blocking the binding of mPD-L2 to mPD-1 (see, e.g., 4H2 activity, Figure 41) were identified, and the binding affinity of several of these antibodies to mPD-1 was determined. mPD-1-Fc fusion protein by ELISA (FIG. 42). The 4H2.B3 antibody, which is interchangeably referred to herein as "4H2," was further characterized. CHO cells expressing mouse PD-1 were constructed and incubated with anti-mPD-1 antibody 4H2 at a concentration ranging from 200 µg / ml to 0.012 µg / ml to determine the binding affinity of 4H2 to PD- 1. Anti-mPD-1 antibody binding to PD-1 expressing CHO cells was detected by incubation with FITC-conjugated donkey anti-rat IgG and measured by FACS. The anti-mPD-1 antibody had an EC50 (50% effective concentration) of approximately 0.38 µg (FIG. 43) and a KD of 4.7 x 10 -9 M. To examine the inhibition of PD-L1 binding to PD-1, the same assay was performed with the exception that cells were also incubated with 0.16 µg of mPD-L1-hFc fusion protein, then , PD-L1 binding to PD-1 expressing CHO cells was detected by incubation with FITC-conjugated goat anti-human IgG (Fc-specific) and measuring the binding signal by FACS (IFM, intensity mean fluorescence). The anti-mPD-1 antibody had an EC50 of approximately 0.72 µg (FIG. 44). For use in mouse tumor models, the 4H2 rat anti-mPD-1 had to be modified so that the mouse immune system would not neutralize the immunotherapeutic antibody (i.e., so that the antibody would have better pharmacokinetics) and to prevent antibody-dependent cellular cytotoxicity (ADCC) by reducing Fc receptor interactions (ie, so that anti-PD-1 blockade could be assessed as being compromised by the effects of ADCC). The original rat anti-mPD antibody, 4H2, was determined to be a rat IgG2a isotype. Therefore, the Fc portion of the 4H2 antibody was replaced by an Fc portion of a mouse IgG1 isotype. Using the analysis described above, the binding affinity of rat-mouse chimeric 4H2 to mPD-1 was found to be comparable to that of the rat anti-mPD-1 antibody 4H2.B3 (FIG. 45). Similarly, the inhibition of PD-L1 binding to PD-1 was comparable for both antibodies (FIG. 46). Therefore, the rat-mouse anti-mPD-1 4H2 antibody was used to examine the therapeutic efficacy of anti-PD-1 combined with anti-CTLA-4. Example 13: In Vivo Efficacy of Combination Therapy (Anti-CTLA-4 and Anti-PD-1 Antibodies) on Tumor Establishment and Growth Colorectal cancer MC38 (PD-L1-) cells (available from Dr. N. Restifo, National Cancer Institute, Bethesda, MD; or Jeffrey Schlom, National Institutes of Health, Bethesda, MD) were implanted into C57BL / 6 mice (2 x 106 cells / mouse). On day 0 (ie, the day the MC38 cells were implanted into the mice), each of the four groups of 10 mice was injected intraperitoneally (IP) with one of the following: (1) IgG from mouse (control) , (2) anti-CTLA-4 monoclonal antibody 9D9 (mouse anti-CTLA-4, obtained from J. Allison, Memorial Sloan-Kettering Cancer Center, New York, NY), (3) anti-PD-monoclonal antibody 1 4H2 (chimeric antibody in which a rat anti-mouse PD-1 was modified with a mouse Fc region, as described in Example 6), or (4) anti-CTLA-4 9D9 antibody and anti -PD-1 4H2. Additional antibody injections were subsequently administered on days 3, 6, and 10. Single antibody treatments were dosed at 10 mg / kg, and the combination of anti-CTLA-4 antibody and anti-PD-1 antibody was dosed at 5 mg / kg. mg / kg of each antibody (ie, 10 mg / kg of antibody in total). Using an electronic caliper, tumors were measured in three dimensions (height x width x length) and tumor volume was calculated. Mice were sacrificed when tumors reached a designated tumor endpoint. The results are shown in Table 5 and Figure 21. Table 5. Percentage of tumor-free mice following treatment with anti-PD-1 and / or anti-CTLA-4. Treatment Total mice studied Tumor-free mice (%) mIgG1 10 0 anti-CTLA-4 10 1 (10) anti-PD-1 10 3 (30) anti-CTLA-4 + anti-PD-1 10 6 (60) Eight mice in the IgG group reached tumor endpoint at approximately day 30 and two mice (86066 and 87260) in the IgG group had ulcerated tumors (FIG. 21A). In the anti-CTLA-4 antibody alone group, seven mice reached tumor endpoint at approximately day 60, one mouse had an ulcerated tumor (84,952), one mouse had a tumor with a volume of less than 1500 mm3 (85,246), and one mouse was tumor free (86,057) (FIG. 21B). In the anti-PD-1 antibody-alone group, six mice reached tumor endpoint at approximately day 60, one mouse had an ulcerated tumor (86055), and three mice were tumor-free (84955, 85239, and 86750) ( Figure 21C). In the combined anti-CTLA-4 antibody and anti-PD-1 antibody group, four mice reached tumor endpoint at approximately day 40, and six mice were tumor-free (84596, 85240, 86056, 86071, 86082 and 86761) (FIG. 21D). Figure 22 shows that the mean tumor volume measured on day 21 was approximately 2955 mm 3 for the IgG control group; approximately 655 mm3 for the CTLA-4 antibody only group; approximately 510 mm3 for the PD-1 antibody alone group, and approximately 280 mm3 for the combined anti-CTLA-4 antibody and anti-PD-1 antibody group. Figure 23 demonstrates that the mean tumor volume measured on day 21 was approximately 2715 mm3 for the IgG group; approximately 625 mm3 for the CTLA-4 antibody alone group; approximately 525 mm3 for the PD-1 antibody alone group, and approximately 10 mm3 for the combined antibody to CTLA-4 and antibody to and PD-1 group (and up to 0 mm3 by day 32). This study indicates that, in a murine tumor model, CTLA-4 antibody treatment alone and PD-1 antibody treatment alone have a modest effect on tumor growth, and that combined CTLA-4 antibody treatment alone 4 and antibody to y PD-1 has a significantly greater effect on tumor growth. Interestingly, combined treatment with antibody to CTLA-4 and antibody to PD-1 had a more significant effect on tumor growth at a dose of 5 mg / kg of each antibody compared to the effect of either antibody alone when each is administered at a higher dose of 10 mg / kg. Example 14: Efficacy in vivo of the combined therapy (anti-CTLA-4 and anti-PD-1 antibodies) on the growth of established tumors Colorectal cancer cell MC38 (PD-L1-) were implanted in C57BL / 6 mice (2 x 10 6 cells / mouse) for a sufficient time (approximately 6 to 7 days) to allow tumor formation. On day 6 post-implantation (day -1), tumor measurements were taken and mice were randomized based on mean tumor volume (approximately 250 mm 3 ) into 11 groups for subsequent antibody therapy. On day 0 (ie, one week after the MC38 cells were implanted), mice were injected IP with (1) mouse IgG (control), (2) anti-CTLA-4 9D9 monoclonal antibody, (3) anti-PD-1 4H2 monoclonal antibody, or (4) anti-CTLA-4 9D9 monoclonal antibody and anti-PD-1 4H2 monoclonal antibody, at a concentration of 10 mg / kg per mouse. Antibody injections were also given on days 3, 6, and 10. Antibody compositions monoclonals used had low levels of endotoxin and did not aggregate significantly. Using an electronic caliper, tumors were measured in three dimensions (height x width x length) and tumor volume was calculated. Tumor measurements were taken at day 0 (tumors at the start of treatment had a volume of approximately 125 mm 3 ), and at days 3, 6, 10, 13, 17 and 20 after antibody injection. Mice were sacrificed when tumors reached a designated tumor endpoint (a particular tumor volume, such as 1500 mm 3 and / or when mice showed greater than about 15% weight loss). All eleven mice in the IgG group reached tumor endpoint at approximately day 17 (FIG. 24A). In the anti-CTLA-4 antibody alone group, seven of eleven mice reached tumor endpoint at approximately day 12 (FIG. 24B). In the anti-PD-1 antibody alone group, four mice reached tumor endpoint at approximately day 13 and two mice were tumor free (FIG. 24C). In the combined anti-CTLA-4 and anti-PD-1 antibody group, one mouse reached tumor endpoint at approximately day 17, one mouse reached tumor endpoint at approximately day 45, and nine mice were tumor-free on day 45 (FIG. 24D). Figure 25 demonstrates that the mean tumor volume measured on day 10 was approximately 1485 mm 3 for the IgG control group; approximately 1010 mm3 for the CTLA-4 antibody alone group; approximately 695 mm3 for the PD-1 antibody alone group; and approximately 80 mm3 for the group with combined anti-CTLA-4 and anti-PD-1 antibodies. Figure 26 demonstrates that the mean tumor volume measured on day 10 was approximately 1365 mm3 for the IgG group; approximately 1060 mm3 for the group with anti-CTLA-4 antibody alone; approximately 480 mm3 for the anti-PD-1 antibody group; alone and approximately 15 mm3 for the combined anti-CTLA-4 and anti-PD-1 antibody group (which had dropped to 0 mm3 by day 17). This study indicates that, in a murine tumor model, treatment with the combination of CTLA4 antibody and PD-1 antibody has a significantly greater effect on tumor growth than antibodies alone, even when a tumor is already well established. . Example 15: Dose Titration of Combination Therapy (Anti-CTLA-4 and Anti-PD-1 Antibodies) on Established Tumor Growth MC38 (PD-L1-) colorectal cancer cells were implanted into C57BL / 6 mice (2 x 10 6 cells / mouse) for a sufficient time (approximately 6 to 7 days) to allow tumor formation as described in Example 3. Groups of 10 mice were injected IP on day 0, 3, 6 and 10 as follows: Group (A) mouse IgG (control, 20 mg / kg), Group (B) anti-PD-1 4H2 monoclonal antibody (10 mg / kg), Group (C) anti-CTLA- 4 9D9 (10 mg / kg) and mouse IgG (10 mg / kg), Group (D) monoclonal anti-CTLA-4 antibody 9D9 (10 mg / kg) and anti-PD-1 monoclonal antibody 4H2 (10 mg / kg), Group (E) anti-CTLA-4 monoclonal antibody 9D9 (3 mg / kg) and anti-PD-1 monoclonal antibody 4H2 (3 mg / kg), or Group (F) anti-CTLA-4 monoclonal antibody 9D9 (1 mg / kg) and anti-PD-1 monoclonal antibody 4H2 (1 mg / kg). Using an electronic caliper, tumors were measured in three dimensions (height x width x length) and tumor volume was calculated. Tumor measurements were taken at the start of treatment (ie, on day 0 tumors had an average volume of approximately 90 mm3), and on days 3, 6, 10, 13, 17, and 20 after antibody treatment. Mice were sacrificed when tumors reached a designated tumor endpoint (a particular tumor volume, such as 1500 mm 3 and / or when mice showed greater than about 15% weight loss). Figure 27A demonstrates that all 10 control mice had reached a tumor endpoint. Figure 27B demonstrates that the group treated with 10 mg / kg anti-PD-1 antibody (Group B) had 6 mice that achieved tumor endpoint and 4 mice with tumors that were approximately 750 mm 3 or less in volume. Figure 27C demonstrates that the group treated with 10 mg / kg anti-CTLA-4 antibody (Group C) had 3 mice that achieved tumor endpoint and 7 mice with tumors that were approximately 1000 mm 3 or less in volume. Figure 27D demonstrates that the group treated with a combination of 10 mg / kg anti-PD-1 antibody with 10 mg / kg anti-CTLA-4 antibody (Group D) had 2 mice with tumors having a volume of approximately 1000 mm3 or less, and 8 mice that were tumor free. Figure 27E demonstrates that the group treated with a combination of 3 mg / kg anti-PD-1 antibody with 3 mg / kg anti-CTLA-4 antibody (Group E) had one mouse that had reached tumor endpoint. , 7 mice with tumors having a volume of approximately 500 mm 3 or less, and 2 mice that were tumor free. Figure 27F demonstrates that the group treated with a combination of 1 mg / kg anti-PD-1 antibody with 1 mg / kg anti-CTLA-4 antibody (Group F) had 4 mice that had achieved tumor endpoint. , 5 mice with tumors having a volume of approximately 1100 mm 3 or less, and one mouse that was tumor free. Figures 27G and 27H show tumor volumes in mice successively treated with anti-PD-1 antibody first and anti-CTLA-4 antibody second, and vice versa. Mice in Figure 27G first received 10 mg / kg anti-CTLA-4 on each of days 0 and 3, and then received 10 mg / kg anti-PD-1 antibody on each of days 6 and 10. Mice in Figure 27H first received 10 mg / kg of anti-PD-1 antibody on each of days 0 and 3, and then received 10 mg / kg of anti-CTLA antibody. -4 on each of days 6 and 10. For group G on day 27, 8 mice reached the tumor endpoint, one mouse had a very small tumor (which, after a significant delay, eventually grew) and one mouse was tumor free. For group H on day 27, 8 mice reached tumor endpoint and 2 were tumor free. Figure 28 demonstrates that the mean tumor volume measured on day 10 was approximately 1250 mm 3 for the IgG control group; approximately 470 mm3 for PD-1 antibody with control IgG; about 290 mm 3 for CTLA-4 antibody with control IgG (measured on day 6); about 40 mm3 for the combined anti-CTLA-4 antibody (10 mg / kg) and anti-PD-1 antibody (10 mg / kg) group; approximately 165 mm3 for anti-CTLA-4 antibody (3 mg / kg) and anti-PD-1 antibody (3 mg / kg) group combined, and approximately 400 mm3 for anti-CTLA-4 antibody (1 mg / kg) group / kg) and anti-PD-1 antibody (1 mg / kg) combined. Figure 29 demonstrates that the mean tumor volume measured on day 13 was approximately 1680 mm3 for the IgG control group; approximately 400 mm 3 for PD-1 antibody with control IgG; approximately 660 mm3 for CTLA-4 antibody with control IgG; 0 mm3 for the combined anti-CTLA-4 antibody (10 mg / kg) and anti-PD-1 antibody (10 mg / kg) group; approximately 90 mm3 for the group with anti-CTLA-4 antibody (3 mg / kg) and anti-PD-1 antibody (3 mg / kg) combined, and approximately 650 mm3 for the group with anti-CTLA-4 antibody (1 mg / kg) and anti-PD1 antibody (1 mg / kg) combined. For the combined treatment of anti-PD-1 antibody with anti-CTLA-4 antibody, the number of mice per group that were tumor-free on day 27 of the study was 8 / 10 (10 mg / kg), 2 / 10 (3 mg / kg) and 1 / 10 (1 mg / kg) (data not shown). This study indicates that, in a murine tumor model, treatment with the combination of antibody to CTLA4 and antibody to PD-1 works in a dose-dependent manner and has a significantly greater effect on tumor growth than both antibodies alone. themselves, even at a lower dose and even when a tumor is already well established. Alternatively, antibodies can be given successively (anti-CTLA-4 antibody first and anti-PD-1 antibody second, or vice versa) and the combination is still superior to antibody monotherapies. Example 16: In vivo efficacy of combined therapy (anti-CTLA-4 and anti-PD-1 antibodies) on the establishment and growth of fibrosarcoma SA1 / N (PD-L1-) fibrosarcoma cells were implanted (Leach et al. (1996) Science 271:1734-1736) subcutaneously in A / J mice (2 x 10 6 cells / mouse) on day 0. On days 1, 4, 7 and 11 after implantation, mice were injected IP as follows manner: Group (A) PBS alone (referred to as "vehicle"); Group (B) mouse IgG (control, 10 mg / kg per mouse), Group (C) anti-PD-1 4H2 monoclonal antibody (10 mg / kg per mouse), Group (D) anti-CTLA-4 monoclonal antibody 9D9 (10 mg / kg or 0.2 mg / kg per mouse), and Group (E) anti-PD-1 monoclonal antibody 4H2 (10 mg / kg per mouse) combined with anti-CTLA-4 monoclonal antibody 9D9 (0 , 2 mg / kg per mouse). The study lasted 41 days and tumor measurements were taken on different days throughout the course of the study (see Figure 29). Tumor volume was calculated by measuring tumors in three dimensions (height x width x length) using an electronic caliper. Mice were sacrificed when tumors reached a designated tumor endpoint-a volume of 1500 mm3 and / or an ulcerated tumor. Figures 30A and 30B demonstrate that 19 of 20 control mice (9 / 10 in group A and 10 / 10 in group B) had either reached tumor endpoint or developed ulcerated tumors. Figure 30C demonstrates that the group treated with 10 mg / kg anti-PD-1 antibody (Group C) had 6 mice that achieved a tumor endpoint (2 with a volume greater than 1500 mm3 and 4 with an ulcerated tumor). and 4 mice that were tumor free. Figure 30D demonstrates that the group treated with 10 mg / kg anti-CTLA-4 antibody (Group D) had 5 mice that achieved a tumor endpoint (2 with a volume greater than 1500 mm3 and 3 with an ulcerated tumor). , one mouse with a small tumor (approximately 70 mm 3 volume) and 4 mice that were tumor free. Figure 30E demonstrates that the group treated with 0.2 mg / kg anti-CTLA-4 antibody (Group E) had 10 mice that achieved a tumor endpoint (6 with a volume greater than 1500 mm3 and 4 with a tumor ulcerated). Figure 30F demonstrates that the group treated with a combination of 10 mg / kg anti-PD-1 antibody with 0.2 mg / kg anti-CTLA-4 antibody (Group F) had 2 mice that achieved an endpoint. tumor (one with a volume greater than 1500 mm3 and one with an ulcerated tumor) and 8 mice that were tumor free. Figures 31 and 32 show the mean and median tumor volume, respectively, that developed in treated and untreated mice during the course of this study. The inhibition of tumor growth in mice treated with these antibodies, compared to mice treated with the control mouse IgG antibody, is summarized in Table 6. Table 6. Inhibition of tumor growth and tumor-free mice after treatment with anti-PD-1 and / or anti-CTLA-4. These data further indicate that combination therapy comprising anti-PD-1 and anti-CTLA-4 antibodies is substantially more effective than treatment with the antibody alone. Indeed, the combination is even more effective than the individual antibody treatments, even when the combination therapy contains a subtherapeutic dose of anti-CTLA-4 antibody. These data also suggest that, surprisingly, the presence or absence of PD-L1 on the tumor may have no effect on the efficacy of treatment with this combination of antibodies, although the presence of PD-L1 may influence the effect of monotherapies. with antibodies as PD-L1 expression on the tumor can also lead to inhibition of anti-tumor T cell responses (see Figure 40). Example 17: Efficacy in vivo and dose titration of combined therapy (anti-CTLA-4 and anti-PD-1 antibodies) on the growth of PD-L1 fibrosarcoma SA1 / N (PD-L1-) fibrosarcoma cells were implanted subcutaneously in A / J mice (2 x 10 6 cells / mouse) on day 0 for a sufficient time (approximately 7 days) to allow tumor establishment. On days 7, 10, 13 and 16 post-implantation, ten groups of 8 mice having a mean tumor volume of 110 mm3 were injected IP as follows: Group (A) PBS alone (referred to as "vehicle ") ; Group (B) mouse IgG (control, 10 mg / kg per mouse); Group (C) anti-CTLA-4 monoclonal antibody 9D9 (0.25 mg / kg); Group (D) anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg / kg per mouse); Group (E) anti-CTLA-4 monoclonal antibody 9D9 (5 mg / kg); Group (F) anti-PD-1 monoclonal antibody 4H2 (3 mg / kg per mouse); Group (G) anti-PD-1 monoclonal antibody 4H2 (10 mg / kg per mouse); Group (H) anti-PD-1 monoclonal antibody 4H2 (10 mg / kg per mouse) combined with anti-CTLA-4 monoclonal antibody 9D9 (0.25 mg / kg per mouse); Group (I) anti-PD-1 monoclonal antibody 4H2 (10 mg / kg per mouse) combined with anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg / kg per mouse); and Group (J) anti-PD-1 monoclonal antibody 4H2 (3 mg / kg per mouse) combined with anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg / kg per mouse). On days 10, 13, 16 and 19 after implantation, two groups of 6 mice having a mean tumor volume of 255 mm3 were injected IP as follows: Group (K) mouse IgG (control, 10 mg / kg per mouse) ; and Group (L) anti-PD-1 monoclonal antibody 4H2 (10 mg / kg per mouse) combined with anti-CTLA-4 monoclonal antibody 9D9 (1 mg / kg per mouse). The study lasted 51 days and tumor measurements were taken on different days throughout the course of the study (see Figures 33-38). Tumor volume was calculated by measuring tumors in three dimensions (height x width x length) using an electronic caliper. Mice were sacrificed when tumors reached a designated tumor endpoint-a volume of 1500 mm3 and / or an ulcerated tumor. Figure 33 shows the response to immunostimulatory antibody treatment in mice with tumors that had an initial volume of approximately 110 mm3 (i.e., at the time of treatment with the first antibody). Figures 33A and 33B show that the 16 control mice (Groups A and B) achieved a tumor endpoint (15 with a tumor volume greater than 1500 mm3 and 1 with an ulcerated tumor.) Figures 33C33E demonstrate that tumor-bearing mice respond to anti-CTLA-antibody treatment. 4 in a dose-dependent manner (eg, Group C receiving 0.25 mg / kg had 7 / 8 reaching tumor endpoint and one mouse with tumor volume less than 200 mm3, whereas Group E receiving 5 mg / kg had 6 / 8 mice that achieved tumor endpoint and two mice that were tumor-free.) Figures 33F and 33G demonstrate that the mice responded in approximately the same manner, regardless of depending on the dose of anti-PD-1 antibody (Group F received 3 mg / kg and Group G received 10 mg / kg). In contrast, mice receiving a combined treatment of 10 or 3 mg / kg anti-PD-1 antibody with 0.25 or 0.5 mg / kg of anti-CTLA-4 antibody (Groups H, I and J) showed a significant reduction in tumor growth. For example, Figure 33J demonstrates that the group treated with a combination of 3 mg / kg anti-PD-1 antibody with 0.5 mg / kg anti-CTLA-4 antibody (Group J) had 2 mice that had tumors. ulcerated, 2 mice with a tumor volume less than 500 mm3 and 4 mice that were tumor free. The unexpected synergistic effect of an anti-PD-1 antibody combined with an anti-CTLA-4 antibody, together with the surprising efficacy of subtherapeutic levels of anti-CTLA-4 antibody in the combination, are shown in Figures 34 (mean tumor volume) and 35 (median tumor volume). Figure 36 shows the response to immunostimulatory antibody treatment in mice with larger tumors, those having an initial volume of approximately 250 mm 3 (ie, at the time of first antibody treatment). Figure 36A demonstrates that all 6 control mice (Group K) achieved a tumor endpoint (4 with a tumor volume greater than 1500 mm 3 and 2 with an ulcerated tumor). Figure 36B demonstrates that the group treated with a combination of 10 mg / kg anti-PD-1 antibody with 1 mg / kg anti-CTLA-4 antibody (Group L) had one mouse with an ulcerated tumor, 4 mice with a tumor volume greater than 1500 mm3 and a mouse that was tumor free. The mean and median tumor volumes are shown in Figures 37 and 38. The inhibition of tumor growth in mice treated with these antibodies, compared to mice treated with the control mouse IgG antibody, is summarized in Table 7 and Figure 39. Table 7. Inhibition of tumor growth after treatment with anti-PD-1 and / or anti-CTLA-4. Taken together, these data indicate that combination therapy comprising anti-PD-1 and anti-CTLA-4 antibodies is substantially more effective than treatment with one antibody alone. Furthermore, it is surprising that the dose of each antibody can be reduced without affecting the synergistic efficacy of this immunostimulatory therapeutic antibody combination. Combination therapy still appears to be effective even when the tumor mass is more mature (ie larger). Example 18: Tumor immunity in mice after treatment with anti-PD-1 antibody and re-sensitization with 30 PD-L1 fibrosarcoma cells Mice that survived tumor-free from priming with tumor cells and anti-PD-1 antibody treatment (i.e., treatment similar to the efficacy studies described in Examples 5 and 6) were then re-sensitized with tumor cells to investigate immunity to tumor formation after such treatment. Briefly, at initial sensitization, SA1 / N (PD-L1-) fibrosarcoma cells were implanted subcutaneously into A / J mice (1 x 10 6 cells / mouse) on day 0. On days 1, 4, 7 , 10, 14, 17, and 20 post-implantation, groups of mice were injected IP with mouse IgG (control, 10 mg / kg per mouse) or with one of several doses of anti-PD-1 4H2 monoclonal antibodies. (30, 10, 3.1 and 0.3 mg / kg per mouse). Tumor formation and volume were monitored with an electronic precision caliper twice a week until the study was complete. One group of 8 mice was tumor-free after anti-PD1 antibody treatment (4 treated with 30 mg / kg, 2 with 3 mg / kg, one with 1 mg / kg, and one with 0.3 mg / kg). kg). The eight treated, tumor-free A / J mice were re-sensitized subcutaneously by implanting 1 x 10 6 SA1 / N fibrosarcoma cells / mouse. As a control, 1 x 10 6 SA1 / N fibrosarcoma cells / mouse were implanted subcutaneously into nine naïve mice. Tumor formation and volume were monitored with an electronic precision caliper twice a week until day 62 after implantation. All nine pretreated (control) mice reached tumor endpoint around day 22 after implantation of the fibrosarcoma cells. In contrast, the eight tumor-free mice re-sensitized with fibrosarcoma cells did not develop tumors up to 62 days after implantation. Figure 47 shows the mean tumor volume for untreated and re-sensitized mice. These results demonstrate that treatment with an immunostimulatory antibody, such as anti-PD-1, provides the treated subject with immunity to new tumor formation, even in the presence of cells capable of forming a tumor. Example 19: Tumor Immunity in Mice After Single Antibody (Anti-PD-1) or Combined Antibody Therapy (Anti-CTLA-4 and Anti-PD-1) Re-sensitized with Colorectal Cancer Cells PD-L1 Mice that survived tumor-free to challenge with tumor cells and treatment with anti-PD-1 antibody alone or anti-PD-1 antibody combined with anti-CTLA-4 antibody (i.e., treatment similar to the efficacy studies described in Examples 2-4) were then re-sensitized with tumor cells to investigate immunity to tumor formation after such treatments. Briefly, at initial priming, MC38 (PD-L1-) colorectal cancer cells were implanted into C57BL / 6 mice (2 x 106 cells / mouse) on day 0. On days 0, 3, 6, and 10 after After implantation, groups of mice were injected IP with one of the following treatments: (1) mouse IgG (control, 10 mg / kg per mouse), anti-PD-1 4H2 monoclonal antibody, or (3) anti-PD-1 monoclonal antibody. -PD-1 4H2 combined with anti-CTLA-4 monoclonal antibody 9D9. Tumor growth was monitored with electronic precision calipers as described in Example 15. One group of 11 mice were tumor-free after treatment with anti-PD1 antibody (2 total) or combined treatment with anti-PD1 antibody- 1 / anti-CTLA-4 (9 total). The 11 treated, tumor-free C57BL / 6 mice were re-sensitized by implantation of 2 x 10 7 MC38 colorectal cancer cells / mouse (ie, a cell dose 10 x higher than the initial challenge). As a control, seven naïve mice were implanted with 2 x 10 7 MC38 colorectal cancer cells / mouse. Tumor formation and volume were monitored with a precision electronic caliper for the duration of the resensitization experiment (at least 20 days). Figure 48 demonstrates that all seven naïve (control) mice developed a tumor and reached tumor endpoint at approximately day 18 after colorectal cancer cell implantation. In contrast, the 11 tumor-free mice re-sensitized with colorectal cancer cells did not develop tumors up to 18 days after implantation. Figure 49 shows the mean tumor volume of naïve and re-sensitized mice. These data indicate that, similar to antibody monotherapy, combination antibody therapy that results in blockade of PD-1 and CTLA-4 produces persistent immunity to tumor relapse. Example 20: In Vivo Efficacy of Combination Therapy (Anti-CTLA-4 and Anti-FD-1 Antibodies) on Growth of Established Tumors CT26 colorectal cancer cells were implanted into BALB / C mice (2 x 10 6 cells / mouse) for a sufficient time (approximately 10 days) to allow tumor formation. On day 10 post-implantation, tumor measurements were taken and mice were randomly assigned to 5 groups based on mean tumor volume (approximately 250 mm 3 ) for subsequent antibody therapy. On day 0 (ie, 10 days after CT26 cells were implanted), mice were injected IP with (1) mouse IgG (control), (2) anti-CTLA-4 monoclonal antibody 9D9, (3) anti-PD-1 4H2 monoclonal antibody, or (4) anti-CTLA-4 and 9D9 monoclonal antibody and anti-PD-1 4H2 monoclonal antibody, at a concentration of 10 mg / kg per mouse. Antibody injections were also given on days 3, 6 and 10. The monoclonal antibody compositions used had low levels of endotoxin and did not aggregate significantly. Using an electronic caliper, tumors were measured in three dimensions (height x width x length) and tumor volume was calculated. Tumor measurements were taken on day 0 (tumors at the start of treatment had a volume of approximately 125 mm3), and on days 3, 6, 10, 13, 17 and 20 after injection with antibody. Mice were sacrificed when tumors reached a designated tumor endpoint (a particular tumor volume, such as 1500 mm 3 and / or when mice showed greater than about 15% weight loss). The results are shown in Figure 50. This study indicates that, in a mouse tumor model, treatment with the combination of antibody to CTLA-4 and antibody to PD-1 has a significantly greater effect on tumor growth than either treatment. antibody alone, even when a tumor is already well established. Example 21: Effect of Human Anti-PD-1 Antibody on Regulatory T Cell Function Regulatory T cells are lymphocytes that suppress the immune response. In this example, regulatory T cells were tested for their inhibitory function on the proliferation and IFN-gamma secretion of CD4+ CD25- T cells in the presence or absence of a human anti-PD-1 monoclonal antibody. Regulatory T cells were purified from PBMC using a CD4+ CD25 T cell isolation kit (Miltenyi Biotec). Regulatory T cells were added to a mixed lymphocyte reaction (see above) containing purified CD4+CD25-T cells and allogeneic dendritic cells in a 2:1 ratio of CD4+CD25-cells to regulatory T cells. Anti-PD-1 monoclonal antibody 5C4 was added at a concentration of 10 µg / ml. As a negative control either no antibody was used or an isotype control antibody was used. Culture supernatants were collected on day 5 for cytokine measurement using a Beadlyte cytokine detection system (Upstate). Cells were labeled with 3 H-thymidine, cultured for an additional 18 hours, and analyzed for cell proliferation. The results are shown in Figures 51A (T cell proliferation) and 51B (IFN-gamma secretion). The addition of anti-human PD-1 monoclonal antibody 5C4 partially released the inhibition imposed by Treg cells on the proliferation and IFN-gamma secretion of CD4+ CD25- T cells, indicating that anti-PD-1 antibodies have an effect on regulatory T cells. Example 22: Effect of human anti-PD-1 antibody on T cell activation In this example, the effect of blocking the PD-1 pathway by anti-PD-1 antibody 5C4 on T cell activation was examined. Purified human CD4+ T cells (Dynal CD4 T Cell Purification Kit) were activated with 1 µg / ml soluble anti-CD3 antibody (BD) in the presence of autologous monocytes or monocyte-derived dendritic cells (DC). Monocytes were purified using the Miltenyi CD14 monocyte purification kit, and DCs were generated in vitro after culturing monocytes with GM-CSF and IL-4 (PeproTech) for 7 days. After three days of activation in the presence or absence of anti-PD-1 antibody or irrelevant isotype control mAb, culture supernatants were collected for ELISA analysis of IFN secretion, while tritiated thymidine was added for 18 hours. end of analysis in order to measure T cell proliferation. The results shown in Figures 52A and 52B demonstrate that blocking PD-1 by anti-PD-1 antibody resulted in increased T cell proliferation and secretion. of IFN-γ. A synergistic effect of anti-PD-1 antibody and anti-CTLA-4 antibody on T cell activation (specifically on IFN-γ secretion) was also observed in the presence of monocytes. Example 23: Evaluation of the ADCC activity of the anti-PD-1 antibody In this example, an antibody-dependent cellular cytotoxicity (ADCC) assay was performed to assess whether anti-PD-1 antibody could induce ADCC for target cells. Two versions of 5C4, one with a human IgG1 Fc region (5C4-IgG1) and the other with a human IgG4 Fc region (5C4IgG4), were tested in the assay. The Perkin Elmer Delfia Cell Cytotoxicity kit was used for analysis. Briefly, purified human CD4 T cells (Dynal CD4 T cell purification kit) were activated by plate-bound anti-CD3 antibody (BD) to induce PD-1 expression. Target activated CD4 T cells were then labeled with BATDA reagent. Labeled CD4 T cells were added to a 96-well V-bottom plate, followed by the addition of human PBMC (an effector to target (E / T) cell ratio of 50:1) and the designed antibody. After incubation for 1 hour at 37°C, the plate was centrifuged. The supernatant was transferred to a 96 well flat bottom plate and the plate was read using a RubyStar plate reader. The results demonstrated that 5C4-IgG4 did not mediate ADCC on activated CD4 T cells, while 5C4-IgG1 mediated ADCC on activated CD4 T cells (FIG. 53), indicating that ADCC activity is related to its Fc region. anti-PD-1 antibody. Example 24: Evaluation of the complement-dependent cytotoxicity of the anti-PD-1 antibody In this example, the complement dependent cytotoxicity (CDC) of the anti-PD-1 antibody was examined. Two versions of 5C4 were tested, one with the Fc region of human IgG1 (5C4-IgG1) and the other with the Fc region of human IgG4 (5C4-IgG4). Briefly, purified human CD4 T cells (Dynal CD4 T Cell Purification Kit) were activated by plate-bound anti-CD3 antibody (BD) to induce expression of PD-1. Serial dilutions of anti-PD-1 antibody (5C4) and control antibodies from 50 µg / ml to 640 pg / ml were tested for CDC in the presence of human complement (Quidel-A113). Alamar Blue (Biosource International) was used to measure cytotoxicity. The plate was read on a fluorescence plate reader (EX530 EM590). Viable cell counts are proportional to fluorescence units. The results demonstrated that neither 5C4-IgG1 nor 5C4-IgG4 mediated CDC on activated CD4 T cells, while the positive control antibody (anti-HLA-ABC antibody) did (FIG. 54). Example 25: Assessment of PD-1 Expression in Human T Cells In this example, human PBMC from different donors were screened for PD-1 expression in various cell subsets by FACS. Biotinylated anti-PD-1 antibody was used in this assay, which has shown much higher sensitivity than commercially available anti-PD-1 antibody in detecting PD-1 molecules on the cell surface. Bound antibody was detected using a PE-conjugated streptavidin. Flow cytometric analyzes were performed using a FACScan flow cytometer (Becton Dickinson) and FlowJo software (Tree Star). PD-1 expression was detected on some peripheral human T cells, but not on B cells or monocytes. Further examination of T-cell subsets indicates that PD-1 is expressed on memory and effector CD4 and CD8 T cells, but is absent on naïve CD4 or CD8 T cells. The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The invention, therefore, should be limited only by the terms of the appended claims together with the full scope of equivalents to which the claims are entitled. LIST OF SEQUENCES <110> Medarex, Inc. <110> Ono Pharmaceutical Co., LTD. <120> HUMAN MONOCLONAL ANTIBODIES AGAINST PROGRAMMED DEATH 1 (PD-1) AND METHODS OF TREATMENT OF CANCER USING ANTI-PD-1 ANTIBODIES ALONE OR COMBINED WITH OTHER IMMUNOTHERAPEUTIC AGENTS <130> GEOP-50 <150> US 60 / 679,466 <151> 2005-05-09 <150> US 60 / 738,434 <151> 2005-11-21 <150> US 60 / 748, 919 <151> 2005-12-08 <160> 74 <170> PatentIn version 3.1 <210> 1 <211> 113 <212> PRT <213> Homo sapiens <400> 1 <210> 2 <211> 113 <212> PRT <213> Homo sapiens <400> 2 <210> 3 <211> 113 <212> PRT <213> Homo sapiens <400> 3 <210> 4 <211> 113 <212> PRT <213> Homo sapiens <400> 4 <210> 5 <211> 121 <212> PRT <213> Homo sapiens <400> 5 <210> 6 <211> 113 <212> PRT <213> Homo sapiens <400> 6 <210> 7 <211> 121 <212> PRT 5 <213> Homo sapiens <400> 7 <210> 8 <211> 107 <212> PRT <213> Homo sapiens <400> 8 <210> 9 <211> 107 <212> PRT <213> Homo sapiens 20 <400> 9 <210> 10 <211> 107 <212> PRT <213> Homo sapiens <400> 10 <210> 11 <211> 107 <212> PRT <213> Homo sapiens <400> 11 <210> 12 <211> 107 <212> PRT <213> Homo sapiens <400> 12 <210> 13 <211> 107 <212> PRT <213> Homo sapiens <400> 13 <210> 14 <211> 107 <212> PRT <213> Homo sapiens <400> 14 <210> 15 15 <211> 5 <212> PRT <213> Homo sapiens <400> 15 <210> 16 <211> 5 <212> PRT <213> Homo sapiens <400> 16 <210> 17 <211> 5 <212> PRT <213> Homo sapiens 30 <400> 17 <210> 18 <211> 5 <212> PRT <213> Homo sapiens <400> 18 <210> 19 <211> 7 <212> PRT <213> Homo sapiens <400> 19 <210> 20 15 <211> 5 <212> PRT <213> Homo sapiens <400> 20 <210> 21 <211> 7 <212> PRT <213> Homo sapiens <400> 21 <210> 22 <211> 17 <212> PRT <213> Homo sapiens 30 <400> 22 <210> 23 <211> 17 <212> PRT <213> Homo sapiens <400> 23 <210> 24 <211> 17 <212> PRT <213> Homo sapiens <400> 24 <210> 25 45 <211> 17 <212> PRT <213> Homo sapiens <400> 25 <210> 26 <211> 16 <212> PRT 58 <213> Homo sapiens <400> 26 <210> 27 <211> 17 <212> PRT <213> Homo sapiens <400> 27 <210> 28 <211> 16 <212> PRT <213> Homo sapiens <400> 28 <210> 29 <211> 4 <212> PRT <213> Homo sapiens 20 <400> 29 <210> 30 <211> 4 <212> PRT <213> Homo sapiens <400> 30 <210> 31 <211> 4 <212> PRT <213> Homo sapiens <400> 31 <210> 32 <211> 4 <212> PRT <213> Homo sapiens 40 <400> 32 <210> 33 <211> 11 <212> PRT <213> Homo sapiens <400> 33 <210> 34 <211> 4 <212> PRT <213> Homo sapiens <400> 34 <210> 35 <211> 11 59 <212> PRT <213> Homo sapiens <400> 35 <210> 36 <211> 11 <212> PRT <213> Homo sapiens <400> 36 <210> 37 <211> 11 <212> PRT <213> Homo sapiens 15 <400> 37 <210> 38 <211> 11 <212> PRT <213> Homo sapiens <400> 38 <210> 39 <211> 11 <212> PRT <213> Homo sapiens <400> 39 <210> 40 30 <211> 11 <212> PRT <213> Homo sapiens <400> 40 <210> 41 <211> 11 <212> PRT <213> Homo sapiens <400> 41 <210> 42 <211> 11 <212> PRT <213> Homo sapiens 45 <400> 42 <210> 43 <211> 7 <212> PRT <213> Homo sapiens <400> 43 <211> 7 <212> PRT <213> Homo sapiens <400> 44 <210> 45 <211> 7 <212> PRT <213> Homo sapiens <400> 45 <210> 46 10 <211> 7 <212> PRT <213> Homo sapiens <400> 46 <210> 47 <211> 7 <212> PRT <213> Homo sapiens <400> 47 <210> 48 <211> 7 <212> PRT <213> Homo sapiens 25 <400> 48 <210> 49 <211> 7 <212> PRT <213> Homo sapiens <400> 49 <210> 50 <211> 9 <212> PRT <213> Homo sapiens <400> 50 <210> 51 40 <211> 9 <212> PRT <213> Homo sapiens <400> 51 <210> 52 <211> 9 <212> PRT <213> Homo sapiens <400> 52 <210> 53 <211> 9 <212> PRT <213> Homo sapiens 55 <400> 53 <210> 54 <211> 9 <212> PRT <213> Homo sapiens <400> 54 <210> 55 <211> 9 <212> PRT <213> Homo sapiens <400> 55 <210> 56 15 <211> 9 <212> PRT <213> Homo sapiens <400> 56 <210> 57 <211> 339 <212> DNA <213> Homo sapiens <220> 25 <221> CDS <222> (1).. (339) <400> 57 <210> 58 <211> 339 <212> DNA <213> Homo sapiens <220> 62 <221> CDS <222> (1).. (339) <400> 58 <210> 59 <211> 339 <212> DNA <213> Homo sapiens <220> 10 <221> CDS <222> (1).. (339) <400> 59 <210> 60 <211> 339 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (339) <400> 60 <210> 61 <211> 363 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (363) <400> 61 <210> 62 <211> 339 15 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (339) 20 <400> 62 <210> 63 <211> 363 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (363) <400> 63 <210> 64 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (321) <400> 64 <210> 65 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (321) <400> 65 <210> 66 <211> 324 <212> DNA <213> Homo sapiens <220> 15 <221> CDS <222> (1).. (321) <400> 66 <210> 67 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (321) <400> 67 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (321) <400> 68 <210> 69 <211> 321 <212> DNA <213> Homo sapiens 10 <220> <221> CDS <222> (1).. (321) <400> 69 <210> 70 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1).. (321) <400> 70 <210> 71 <211> 98 <212> PRT <213> Homo sapiens 15 <400> 71 <210> 72 <211> 95 <212> PRT <213> Homo sapiens <400> 72 <210> 73 <211> 99 <212> PRT <213> Homo sapiens <400> 73 <210> 74 <211> 95 <212> PRT <213> Homo sapiens <400> 74

Claims

1. An isolated monoclonal antibody, or antigen-binding portion thereof, comprising: a) a heavy-chain variable region comprising the amino acids having the sequence shown in SEC ID NO: 4; and b) a light-chain variable region comprising the amino acids having the sequence shown in SEC ID NO: 11; wherein the antibody specifically binds to human Programmed Death 1 (PD-1) protein.

2. The antibody of claim 1, wherein the antibody is an IgG4 isotype.

3. A composition comprising the antibody of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. The antibody of claim 1 or 2, for use in a method for modulating an immune response.

5. The antibody of claim 1 or 2, for use in a method for inhibiting the growth of tumor cells.

6. The antibody of claim 5,wherein the tumor cells are from a cancer selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, and lung cancer.

7. The antibody of claim 5, wherein the tumor cells are from a cancer selected from the group consisting of bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer,Chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of such cancers.

8. The antibody of claim 1 or 2 for use in a method of treating an infectious disease.

9. The antibody of claim 8, wherein the infectious disease is caused by a virus selected from the group consisting of HIV, Influenza, Herpes, Giardia, Malaria, Leishmania, Hepatitis A, B, or C, a herpes virus,VZV, HSV-1, 6-HAV, HSV-II, CMV, Epstein-Barr virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arbovirus encephalitis virus, chlamydia, Rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague leptospirosis, Lyme disease bacteria, Candida albicans, krusei, glabrata or tropicalis, Cryptococcus neoformans, Aspergillus fumigatus or niger, Mucorales mucor, absidia or rhizophus, Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis,Histoplasma capsulatum, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lamblia, Crohn's disease and Ptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Toxoplasma gondii and Panosoma brucei, Toxoplasma cruzi and Panosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

10. An immunoconjugate product comprising the antibody of claim 1 or 2 connected to a therapeutic agent.

11. A composition comprising the immunoconjugate product of claim 10 and a pharmaceutically acceptable carrier.

12. The immunoconjugate product of claim 10, wherein the therapeutic agent is a cytotoxin.

13. A composition comprising the immunoconjugate of claim 12 and a pharmaceutically acceptable carrier.

14. The immunoconjugate product of claim 10,wherein the therapeutic agent is a radioactive isotope.

15. A composition comprising the immunoconjugate of claim 14 and a pharmaceutically acceptable vehicle.

16. A bispecific molecule comprising the antibody of claim 1 or 2, connected to a second functional radical having a different binding specificity than said antibody, or antigen-binding portion thereof.

17. A composition comprising the bispecific molecule of claim 14 and a pharmaceutically acceptable carrier. 15 Binding of AbHuMab anti-hPD-1 to activated human T cells Concentration [nM] Figure 15a Binding of AbHuMab anti-hPD-1 to activated Cynomolgus monkey PBMC cells Concentration [nM] Figure 15b,