Antigen-binding protein that binds to PD-1

Abstract

To provide antigen binding proteins that bind PD-1.SOLUTION: There are disclosed, compositions and methods relating to or derived from anti-PD-1 antibodies. More specifically, there is disclosed fully human antibodies that bind PD-1, PD-1-binding fragments and derivatives of such antibodies, and PD-1 binding polypeptides comprising such fragments. Further, there is disclosed nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having PD-1 related disorders or conditions, including various inflammatory disorders and various cancers.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides compositions and methods related to or derived from anti-PD-1 antibodies. More specifically, the present invention provides human antibodies that bind to PD-1, PD-1-binding fragments and derivatives of the antibodies, and PD-1-binding polypeptides that include the fragments. Additionally, the present invention provides nucleic acids encoding the antibodies, antibody fragments and derivatives, and polypeptides, cells containing the polynucleotides, methods of making the antibodies, antibody fragments and derivatives, and polypeptides, and methods of using such antibodies, antibody fragments and derivatives, and polypeptides, such as for treating or diagnosing subjects exhibiting disorders or conditions associated with PD-1, including various inflammatory disorders and various cancers. [Background technology]

[0002] Programmed cell death protein-1 (PD-1) is a 268 amino acid type I membrane protein and a member of the extended CD28 / CTLA-4 family of T cell regulators (TheEMBOJournal (1992), vol. 11, issue 11, p. 3887-3895). Human PD-1 cDNA is available from EMBL / GenBank Acc.No. NM_005018 The mouse PD-1 cDNA consists of the base sequence shown in Acc. No. NM_008798. Its expression is seen when thymocytes differentiate from CD4-CD8- cells to CD4+CD8+ cells (International Immunology (1996), vol. 18, issue 5, p. 773-780., J. Experimental Med. (2000),vol. 191,issue 5, p. 891-898.). PD-1 expression in the periphery It has been reported that it is found in bone marrow cells, such as T cells or B lymphocytes activated by stimulation from antigen receptors, or activated macrophages (International Immunology (1996), vol. 18, issue 5, p. 765-772.).

[0003] PD-1 is a member of the CD28 family of receptors, which includes , CTLA-4, ICOS, PD-1 and BTLA. One member, CD28, was discovered by showing a functional effect on promoting T cell proliferation after the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260). The glycoprotein ligands, PD-1 and PD-L2, have been identified and shown to downregulate T cell activation and cytokine secretion associated with 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. 32:634-43; Ohigashi et al.(2005) Clin. Cancer Res. 11:2947-53). PD-1 (B7-H1) and PD-L2 (B7-D C) is a B7 homolog that binds to PD-1. It has also been shown that cell surface PD-1 expression is upregulated by IFN-γ stimulation. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] The EMBO Journal (1992), vol. 11, issue 11, p. 3887-3895. [Non-Patent Document 2] International Immunology (1996), vol. 18, issue 5, p. 773-780. [Non-Patent Document 3] J. Experimental Med. (2000), vol. 191, issue 5, p. 891-898. [Non-Patent Document 4] International Immunology (1996), vol. 18, issue 5, p. 765-772. [Non-Patent Document 5] Hutloff et al. (1999) Nature 397:263-266 [Non-Patent Document 6] Hansen et al. (1980) Immunogenics 10:247-260 [Non-Patent Document 7] Freeman et al. (2000) J. Exp. Med. 192:1027-34 [Non-Patent Document 8] Latchman et al. (2001) Nat. Immunol. 2:261-8 [Non-Patent Document 9] Carter et al. (2002) Eur. J. Immunol. 32:634-43 [Non-Patent Document 10] Ohigashi et al. (2005) Clin. Cancer Res. 11:2947-53 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention relates to at least 10 -6A fully human antibody of the IgG class that binds to a PD-1 epitope with a binding affinity of M, comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; Column number 8, sequence number 10, sequence number 12, sequence number 14, sequence number 16, sequence number 18, sequence number 20 The present invention provides a fully human antibody having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof. Preferably, the fully human antibody has both a heavy chain and a light chain, and the antibody is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2 (referred to herein as GA1), SEQ ID NO:3 / SEQ ID NO:4 (referred to herein as GA2), SEQ ID NO:5 / SEQ ID NO:6 (referred to herein as GA3), SEQ ID NO:7 (referred to herein as GA4), SEQ ID NO:8 (referred to herein as GA5), SEQ ID NO:9 (referred to herein as GA6), SEQ ID NO:10 (referred to herein as GA7), SEQ ID NO:11 (referred to herein as GA8), SEQ ID NO:12 (referred to herein as GA9), SEQ ID NO:13 (referred to herein as GA1), SEQ ID NO:14 (referred to herein as GA1), SEQ ID NO:15 (referred to herein as GA1), SEQ ID NO:16 (referred to herein as GA1), SEQ ID NO:17 (referred to herein as GA1), SEQ ID NO:18 (referred to herein as GA1), SEQ ID NO:19 (referred to herein as GA2), SEQ ID NO:20 (referred to herein as GA3), SEQ ID NO:21 (referred to herein as GA1), SEQ ID NO:22 (referred to herein as GA1), SEQ ID NO:24 (referred to herein as GA1), SEQ ID NO:2 Row No. 6 (referred to herein as GB1), SEQ ID NO:7 / SEQ ID NO:8 (referred to herein as GB6) , SEQ ID NO:9 / SEQ ID NO:10 (referred to herein as GH1), SEQ ID NO:11 / SEQ ID NO:12 (referred to herein as GH3), SEQ ID NO:13 / SEQ ID NO:14 (referred to herein as C7), SEQ ID NO:15 / SEQ ID NO: Sequence No. 16 (referred to herein as H7), SEQ ID NO: 17 / SEQ ID NO: 18 (referred to herein as SH-A4) (referred to herein as SH-A9), SEQ ID NO:19 / SEQ ID NO:20 (referred to herein as SH-A9), SEQ ID NO:21 / SEQ ID NO:22 (referred to herein as RG1B3), SEQ ID NO:23 / SEQ ID NO:24 (referred to herein as RG1H10), SEQ ID NO:25 / SEQ ID NO:26 (referred to herein as RG1H11), SEQ ID NO:27 / SEQ ID NO:28 (referred to herein as RG2H7), SEQ ID NO:29 / SEQ ID NO:30 (referred to herein as RG2H10), SEQ ID NO:31 / SEQ ID NO:32 (referred to herein as RG3E12), SEQ ID NO:33 / SEQ ID NO:34 (referred to herein as RG4A6), SEQ ID NO:35 / SEQ ID NO:36 (referred to herein as RG5D9), SEQ ID NO:37 / SEQ ID NO:24 (referred to herein as RG1H10-H2A-22-15), SEQ ID NO:38 / SEQ ID NO:24 (referred to herein as RG2H7), SEQ ID NO:39 / SEQ ID NO:30 (referred to herein as RG2H10), SEQ ID NO:31 / SEQ ID NO:32 (referred to herein as RG3E12), SEQ ID NO:33 / SEQ ID NO:34 (referred to herein as RG4A6), SEQ ID NO:35 / SEQ ID NO:36 (referred to herein as RG5D9), SEQ ID NO:37 / SEQ ID NO:24 (referred to herein as RG1H10-H2A-22-15), SEQ ID NO:38 / SEQ ID NO:24 (referred to herein as RG SEQ ID NO:39 / SEQ ID NO:24 (referred to herein as RG1H10-3C), SEQ ID NO:40 / SEQ ID NO:24 (referred to herein as RG1H10-16C), SEQ ID NO:41 / SEQ ID NO:24 (referred to herein as RG1H10-17C), SEQ ID NO:42 / SEQ ID NO:24 (referred to herein as RG1H10-19C), SEQ ID NO:43 / SEQ ID NO:24 (referred to herein as RG1H10-21C), SEQ ID NO:44 / It has a heavy / light chain variable region sequence selected from the group consisting of SEQ ID NO: 24 (referred to herein as RG1H10-23C2), and combinations thereof.

[0006] The present invention relates to a Fab fully human antibody fragment having a variable region derived from a heavy chain and a variable region derived from a light chain, and the fragments are represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO The present invention provides a Fab fully human antibody fragment having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and combinations thereof, and a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 and combinations thereof. Preferably, the fully human antibody Fab fragment has both a heavy chain variable region and a light chain variable region, and the antibody has a sequence similar to that of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO: No. 37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO: A heavy / light chain variable region selected from the group consisting of SEQ ID NO: 44 / SEQ ID NO: 24 and combinations thereof It has an array.

[0007] The present invention relates to a single chain human antibody having a variable region derived from a heavy chain and a variable region derived from a light chain, and a peptide linker connecting the variable regions of the heavy and light chains, the single chain human antibody being represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO: The present invention provides a single chain human antibody having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and combinations thereof, and a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 and combinations thereof. Preferably, the fully human single chain antibody has both heavy and light chain variable regions and is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO: Row number 14, SEQ ID NO: 15 / SEQ ID NO: 16, SEQ ID NO: 17 / SEQ ID NO: 18, SEQ ID NO: 19 / SEQ ID NO: 20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34 , SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and combinations thereof The heavy / light chain variable region sequences are selected from the following:

[0008] The present invention further relates to a method for treating a wide range of mammalian cancers or a wide range of inflammatory and autoimmune diseases comprising administering an effective amount of an anti-PD-1 polypeptide, wherein the anti-PD-1 polypeptide has an activity of at least 10- 6 a fully human antibody of the IgG class that binds to a PD-1 epitope with a binding affinity of M; a Fab fully human antibody fragment having a variable region derived from a heavy chain and a variable region derived from a light chain; a single chain human antibody having a variable region derived from a heavy chain and a variable region derived from a light chain and a peptide linker connecting the variable regions of the heavy and light chains, and combinations thereof; The fully human antibodies are represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof; The Fab fully human antibody fragments are represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof; The single chain human antibody is represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof, and has a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof.

[0009] Preferably, the fully human antibody has both heavy and light chains and is represented by SEQ ID NO: 1 / SEQ ID NO: 2 (the present invention). SEQ ID NO:3 / SEQ ID NO:4 (herein referred to as GA2), SEQ ID NO:5 / SEQ ID NO:6 (herein referred to as GB1), SEQ ID NO:7 / SEQ ID NO:8 (herein referred to as GB6) SEQ ID NO:9 / SEQ ID NO:10 (referred to herein as GH1), SEQ ID NO:11 / SEQ ID NO:12 (referred to herein as A2), SEQ ID NO:13 / SEQ ID NO:14 (referred to herein as C7), SEQ ID NO:15 / SEQ ID NO:16 (referred to herein as H7), SEQ ID NO:17 / SEQ ID NO:18 (referred to herein as SH-A4) SEQ ID NO:19 / SEQ ID NO:20 (referred to herein as SH-A9), SEQ ID NO:21 / SEQ ID NO:22 (referred to herein as RG1B3), SEQ ID NO:23 / SEQ ID NO:24 (referred to herein as RG1H10), SEQ ID NO:25 / SEQ ID NO:26 (referred to herein as RG1H11), SEQ ID NO:27 / SEQ ID NO:28 (referred to herein as RG2H7), SEQ ID NO:29 / SEQ ID NO:30 (referred to herein as RG2H10), SEQ ID NO:31 / SEQ ID NO:32 (referred to herein as RG3E12), SEQ ID NO:33 / SEQ ID NO:34 (referred to herein as RG4A6), SEQ ID NO:35 / SEQ ID NO:36 (referred to herein as RG5D9), SEQ ID NO:37 / SEQ ID NO:24 (referred to herein as RG1H10-H2A-22-15), SEQ ID NO:38 / SEQ ID NO:24 ...39 / SEQ ID NO:40 (referred to herein as RG2H7), SEQ ID NO:41 / SEQ ID NO:42 (referred to herein as and combinations thereof. Preferably, the fully human antibody Fab fragment has a heavy chain variable region sequence and a light chain variable region sequence selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2 (referred to herein as GA1), SEQ ID NO:3 / SEQ ID NO:4 (referred to herein as GA2), SEQ ID NO:5 / SEQ ID NO:6 (referred to herein as GA3), SEQ ID NO:7 (referred to herein as GA4), SEQ ID NO:8 (referred to herein as GA5), SEQ ID NO:9 (referred to herein as GA6), SEQ ID NO:10 (referred to herein as GA7), SEQ ID NO:11 (referred to herein as GA8), SEQ ID NO:12 (referred to herein as GA9), SEQ ID NO:13 (referred to herein as GA1), SEQ ID NO:14 (referred to herein as GA1), SEQ ID NO:15 (referred to herein Row No. 6 (referred to herein as GB1), SEQ ID NO:7 / SEQ ID NO:8 (referred to herein as GB6) , SEQ ID NO:9 / SEQ ID NO:10 (referred to herein as GH1), SEQ ID NO:11 / SEQ ID NO:12 (referred to herein as GH3), SEQ ID NO:13 / SEQ ID NO:14 (referred to herein as C7), SEQ ID NO:15 / SEQ ID NO: Sequence No. 16 (referred to herein as H7), SEQ ID NO: 17 / SEQ ID NO: 18 (referred to herein as SH-A4) (referred to herein as SH-A9), SEQ ID NO:19 / SEQ ID NO:20 (referred to herein as RG1B3), SEQ ID NO:23 / SEQ ID NO:24 (referred to herein as RG1H10), SEQ ID NO:25 / SEQ ID NO:26 (referred to herein as RG1H11), SEQ ID NO:27 / SEQ ID NO:28 (referred to herein as RG2H7), SEQ ID NO:29 / SEQ ID NO:30 (referred to herein as RG2H10), SEQ ID NO:31 / SEQ ID NO:32 (referred to herein as RG3E12), SEQ ID NO:33 / SEQ ID NO:34 (referred to herein as RG4A6), SEQ ID NO:35 / SEQ ID NO:36 (referred to herein as RG5D9), SEQ ID NO:37 / SEQ ID NO:24 (referred to herein as RG1H10-H2A-22-15), SEQ ID NO:38 / SEQ ID NO:24 (referred to herein as RG1H10-H2A-27-25), SEQ ID NO:39 / SEQ ID NO:24 (referred to herein as RG1H10-3C), SEQ ID NO:40 / SEQ ID NO:24 (referred to herein as RG1H10-16C), SEQ ID NO:41 / SEQ ID NO:24 (referred to herein as RG1H10-17C), SEQ ID NO:42 / SEQ ID NO:24 (referred to herein as RG1H10-19C), SEQ ID NO:43 / SEQ ID NO:24 (referred to herein as RG1H10-21C), SEQ ID NO:44 / The fully human single chain antibody has a heavy / light chain variable region sequence selected from the group consisting of SEQ ID NO:24 (referred to herein as RG1H10-23C2) and combinations thereof. Preferably, the fully human single chain antibody has both a heavy chain variable region and a light chain variable region, and is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO: No. 12, SEQ ID NO: 13 / SEQ ID NO: 14, SEQ ID NO: 15 / SEQ ID NO: 16, SEQ ID NO: 17 / SEQ ID NO: 18, SEQ ID NO: No. 19 / SEQ ID NO:20, No. 21 / SEQ ID NO:22, No. 23 / SEQ ID NO:24, No. 25 / SEQ ID NO:26, No. 27 / SEQ ID NO:28, No. 29 / SEQ ID NO:30, No. 31 / SEQ ID NO:32, Column number 33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and combinations thereof The heavy chain / light chain variable region sequences are selected from the group consisting of:

[0010] Preferably, the broad range of mammalian cancers to be treated include ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastoma-derived CNS tumors, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, and the like. , B cell derived lymphoma, T cell derived lymphoma, mast cell derived tumor and combinations thereof. Preferably, the autoimmune disease or inflammatory disease is selected from the group consisting of intestinal mucosal inflammation, colitis-associated wasting disease, multiple sclerosis, systemic lupus erythematosus, viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, Crohn's disease and inflammatory bowel disease. [Brief description of the drawings]

[0011] [Figure 1] 1 shows a mixed lymphocyte reaction (MLR) used to assess the effect of antibody RG1H10 (SEQ ID NO:23 / SEQ ID NO:24) on lymphocyte activation by anti-PD-1 on lymphocyte effector cells. T cell activity was measured in the presence or absence of anti-PD-1 monoclonal antibody. [Diagram 2]Mixed lymphocyte reaction (MLR) to evaluate the effect of RG1H10 antibody on lymphocyte activity in lymphocyte effector cells. IL-2 secretion was measured in the presence or absence of anti-PD-1 human monoclonal antibody. Vertical axis is IFNγ (pg / ml). [Diagram 3] 1 shows a mixed lymphocyte reaction (MLR) to demonstrate the effect of blockade of the PD-L1 / PD-1 pathway by the anti-PD-1 antibody RG1H10 described herein on lymphocyte effector cells. IFN-γ secretion was measured in the presence or absence of anti-PD-1 human monoclonal antibody. The vertical axis is IL-2 (pg / ml). [Figure 4] 1 shows dose-dependent cellular binding of anti-PD-1 antibodies to CHO-PD-1 cells. [Diagram 5] 1 shows the extent of inhibition of recombinant PD-1 binding to recombinant PD-L1 by anti-PD-1 antibodies. [Figure 6] 1 shows the binding specificity of anti-PD-1 antibody RG1H10 to recombinant PD-1 protein. [Figure 7] 1 shows a mixed lymphocyte reaction (MLR) used to compare the effect of antibody RG1H10 (SEQ ID NO:23 / SEQ ID NO:24) as IgG1 or IgG4 (S228P) on lymphocyte activation by anti-PD-1 on lymphocyte effector cells. T cell activation was measured as increased expression of the receptor for interleukin-2 (IL-2) (CD25) in the presence or absence of anti-PD-1 human monoclonal antibody. [Figure 8] Mixed lymphocyte reaction (MLR) to assess the effect of RG1H10 antibody as IgG1 or IgG4 (S228P) on lymphocyte activation in lymphocyte effector cells. IL-2 secretion was measured in the presence or absence of anti-PD-1 human monoclonal antibody. [Figure 9]1 shows a mixed lymphocyte reaction (MLR) to demonstrate the effect of blockade of the PD-L1 / PD-1 pathway with the anti-PD-1 antibody RG1H10 described herein, as either IgG1 or IgG4 (S228P), on lymphocyte effector cells. IFN-γ secretion was measured in the presence or absence of an anti-PD-1 human monoclonal antibody. [Figure 10] 1 shows the presence or absence of cross-reactivity between the human and mouse anti-PD-1 antibodies described herein. [Figure 11] Not mentioned in the original text [Figure 12] Not mentioned in the original text DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention provides a method for determining whether a PD-1 epitope is involved in -6 A fully human antibody of the IgG class, which binds with a binding affinity of M or less, and which is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, The present invention provides fully human antibodies having a heavy chain variable region that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof, and a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof. Preferably, the fully human antibody has both heavy and light chains and is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2 (referred to herein as GA1), SEQ ID NO:3 / SEQ ID NO:4 (referred to herein as GA2), SEQ ID NO:5 / SEQ ID NO:6 (referred to herein as GB1), SEQ ID NO:7 / SEQ ID NO:8 (referred to herein as GB6), SEQ ID NO:9 / SEQ ID NO:10 (referred to herein as GH1), SEQ ID NO:11 / SEQ ID NO:12 (referred to herein as A2), SEQ ID NO:13 / SEQ ID NO:14 (referred to herein as C7), SEQ ID NO:15 / SEQ ID NO:16 (referred to herein as H7), SEQ ID NO:17 / SEQ ID NO:18 (referred to herein as SH-A4), SEQ ID NO:19 / SEQ ID NO:20 (referred to herein as SH-A9), SEQ ID NO:21 / SEQ ID NO:22 (referred to herein as RG1B3). , SEQ ID NO:23 / SEQ ID NO:24 (referred to herein as RG1H10), SEQ ID NO:25 / SEQ ID NO:26 (referred to herein as RG1H11), SEQ ID NO:27 / SEQ ID NO:28 (referred to herein as RG2H7), SEQ ID NO:29 / SEQ ID NO:30 (referred to herein as RG2H10), SEQ ID NO:31 / SEQ ID NO:32 (referred to herein as RG3E12), SEQ ID NO:33 / SEQ ID NO:34 (referred to herein as RG4A6), SEQ ID NO:35 / SEQ ID NO:36 (referred to herein as RG5D9), SEQ ID NO:37 / SEQ ID NO:24 (referred to herein as RG1H10-H2A-22-15), SEQ ID NO:38 / SEQ ID NO:24 (referred to herein as RG1H10-H2A-27-25), ), SEQ ID NO:39 / SEQ ID NO:24 (referred to herein as RG1H10-3C), SEQ ID NO:40 / SEQ ID NO:24 (referred to herein as RG1H10-16C), SEQ ID NO:41 / SEQ ID NO:24 (referred to herein as RG1H10-17C), SEQ ID NO:42 / SEQ ID NO:24 (referred to herein as RG1H10-19C), SEQ ID NO:43 / SEQ ID NO: and having a heavy / light chain variable region sequence selected from the group consisting of SEQ ID NO:24 (referred to herein as RG1H10-21C), SEQ ID NO:44 / SEQ ID NO:24 (referred to herein as RG1H10-23C2), and combinations thereof.

[0013] The present invention relates to a Fab fully human antibody fragment having a variable region derived from a heavy chain and a variable region derived from a light chain, and the fragments are represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: The present invention provides a Fab fully human antibody fragment having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and combinations thereof, and a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 and combinations thereof. Preferably, the fully human antibody Fab fragment has both a heavy chain variable region and a light chain variable region, and the antibody has a sequence similar to that of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO: No. 37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO: A heavy / light chain variable region selected from the group consisting of SEQ ID NO: 44 / SEQ ID NO: 24 and combinations thereof It has an array.

[0014] The present invention provides a single-chain human antibody having a variable region derived from a heavy chain, a variable region derived from a light chain, and a peptide linker connecting the variable regions of the heavy chain and the light chain, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, The present invention provides single chain human antibodies having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof, and a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof. Preferably, the fully human single chain antibody has both a heavy chain variable region and a light chain variable region and is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO: No. 12, SEQ ID NO: 13 / SEQ ID NO: 14, SEQ ID NO: 15 / SEQ ID NO: 16, SEQ ID NO: 17 / SEQ ID NO: 18, SEQ ID NO: No. 19 / SEQ ID NO:20, No. 21 / SEQ ID NO:22, No. 23 / SEQ ID NO:24, No. 25 / SEQ ID NO:26, No. 27 / SEQ ID NO:28, No. 29 / SEQ ID NO:30, No. 31 / SEQ ID NO:32, Column number 33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and combinations thereof The heavy chain / light chain variable region sequences are selected from the group consisting of:

[0015] The present invention further provides methods for treating a wide range of mammalian cancers or inflammatory or autoimmune diseases comprising administering an effective amount of an anti-PD-1 polypeptide, wherein the anti-PD-1 polypeptide has a potency of at least 10- 6 a fully human antibody of the IgG class that binds to a PD-1 epitope with a binding affinity of M; a Fab fully human antibody fragment having a variable region derived from a heavy chain and a variable region derived from a light chain; a single-chain human antibody having a variable region derived from a heavy chain and a variable region derived from a light chain and a peptide linker connecting the variable regions of the heavy chain and the light chain; and combinations thereof; The fully human antibodies are represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: having a heavy chain variable region that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; and having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof; The Fab fully human antibody fragments are represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; Column number 8, sequence number 10, sequence number 12, sequence number 14, sequence number 16, sequence number 18, sequence number 20 , having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof; The single chain human antibody is represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and a combination thereof.

[0016] Preferably, the fully human antibody has both heavy and light chains and is represented by SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:11, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:19 Sequence number 3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, sequence no. No. 31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, Preferably, the fully human antibody Fab fragment has both a heavy chain variable region and a light chain variable region, and the antibody has a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 41 / SEQ ID NO: 24, SEQ ID NO: 42 / SEQ ID NO: 24, SEQ ID NO: 43 / SEQ ID NO: 24, SEQ ID NO: 44 / SEQ ID NO: 24, and combinations thereof. Sequence number 7 / SEQ ID NO:8, sequence number 9 / SEQ ID NO:10, sequence number 11 / SEQ ID NO:12, sequence number 13 / SEQ ID NO: No. 14, SEQ ID NO: 15 / SEQ ID NO: 16, SEQ ID NO: 17 / SEQ ID NO: 18, SEQ ID NO: 19 / SEQ ID NO: 20, Sequence number 21 / SEQ ID NO:22, sequence number 23 / SEQ ID NO:24, sequence number 25 / SEQ ID NO:26, sequence number 27 / SEQ ID NO:28, sequence number 29 / SEQ ID NO:30, sequence number 31 / SEQ ID NO:32, sequence number 33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24 , SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and combinations thereof. Preferably, the fully human single chain antibody has both a heavy chain variable region and a light chain variable region and is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO: No. 12, SEQ ID NO: 13 / SEQ ID NO: 14, SEQ ID NO: 15 / SEQ ID NO: 16, SEQ ID NO: 17 / SEQ ID NO: 18, Sequence number 19 / SEQ ID NO:20, sequence number 21 / SEQ ID NO:22, sequence number 23 / SEQ ID NO:24, sequence number 25 / SEQ ID NO:26, sequence number 27 / SEQ ID NO:28, sequence number 29 / SEQ ID NO:30, sequence number 31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and The heavy / light chain variable region sequences are selected from the group consisting of:

[0017] Preferably, the broad range of mammalian cancers to be treated include ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastoma-derived CNS tumors, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, and the like. , B cell derived lymphoma, T cell derived lymphoma, mast cell derived tumor and combinations thereof. Preferably, the autoimmune disease or inflammatory disease is selected from the group consisting of intestinal mucosal inflammation, colitis-associated wasting disease, multiple sclerosis, systemic lupus erythematosus, viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, Crohn's disease and inflammatory bowel disease.

[0018] An "antigen binding protein" is a protein that contains an antigen binding site and, optionally, a scaffolding or framework region that enables the antigen binding site to adopt a structure that facilitates binding of the antigen binding protein to an antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., antigen binding sites of antibodies), antibody derivatives, and antibody analogs. Antigen binding proteins The scaffolds may include, for example, alternative protein scaffolds or artificial scaffolds that include grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds, including, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, as well as entirely synthetic scaffolds, including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. Additionally, peptide antibody mimetics ("PAMs") as well as synthetic scaffolds using fibronectin components have been proposed. Scaffolds based on antibody mimetics may also be used as scaffolds.

[0019] The antigen-binding protein may have, for example, a naturally occurring immunoglobulin structure. An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains, each having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The C-terminal portion of each chain defines a constant region that is primarily responsible for effector functions. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as μ, δ, γ, α, or ε, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J region" of about 12 or more amino acids, and the heavy chains also contain a "D region" of about 10 or more amino acids. For a general discussion, see Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Intact immunoglobulins have two binding sites. Thus, the variable regions of each light / heavy chain pair form the antibody binding site.

[0020] The variable regions of immunoglobulin chains of natural origin exhibit relatively conserved framework regions (FR) of the same general structure linked by three hypervariable regions, also called complementarity determining regions or CDRs. Both light and heavy chains contain, from the N-terminus to the C-terminus, the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain follows the definition in Kabat et al. (Sequences of Proteins of Immunological Interest, 5.sup.th Ed., US Dept.of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991). Other numbering systems for the amino acids of immunoglobulin chains include the IMGT.RTM. (International ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0021] Antibodies may be obtained from sources such as serum or plasma that contain immunoglobulins with various antigen specificities. If the antibodies are subjected to affinity purification, they can be enriched for a particular antigen specificity. Such antibody enriched products are usually made from less than about 10% of the antibodies having specific binding activity for a particular antigen. These products can be subjected to multiple rounds of affinity purification to increase the percentage of antibodies with specific binding activity for that antigen. Antibodies made in this way are often referred to as "monospecific". Monospecific antibody products can be enriched for approximately 10%, 20%, 30%, 40%, 50%, 60%, 70% , 75%, 80%, 85%, 90%, 95%, 97%, 99% or 99.9% of a specific antigen It may comprise an antibody having specific binding activity against the

[0022] "Antibody" refers to an intact immunoglobulin or an antigen-binding portion thereof that competes with the intact antibody for specific binding, unless otherwise specified. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibody (dAb) and complementarity determining region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, bispecific antibodies, Included are trispecific antibodies, tetraspecific antibodies, and polypeptides that contain at least a sufficient portion of an immunoglobulin to bind a particular antigen to the polypeptide.

[0023] The Fab fragment is V L , V H , C L and C H1 F(ab')2 fragment, a monovalent fragment containing the F(ab')2 domain is a bivalent fragment having two Fab fragments linked by a disulfide bond at the hinge region; the Fd fragment is a V H and C H1 an Fv fragment has the V domain of a single arm of an antibody; L Area and V H region; the dAb fragment has a V H area, V L Area or V H Or V L Area antigen Binding fragments (U.S. Pat. Nos. 6,846,634 and 6,696,245; U.S. Patent Application Publication Nos. 20 / 0202512; 2004 / 0202995; 2004 / 0038291; 2004 / 0009507; 2003 / 0039958 specification and Ward et al., Nature 341:544-546, 1989).

[0024] Single chain antibodies (scFv) are LArea and V H The region may be linked by a linker (e.g., a synthetic sequence of amino acid residues). Bispecific antibodies are antibodies in which the two polypeptide chains are linked by a linker (a linker sequence) to form a continuous protein chain, the linker being long enough to allow the protein to fold over and form a monovalent antigen-binding site (see, e.g., Birdet al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Bispecific antibodies comprise two polypeptide chains, each of which is linked by a linker sequence consisting of two polypeptide chains on the same chain. The V domains are connected by linkers that are too short for pairing between the two domains. H and V L Bispecific antibodies are bivalent antibodies that contain domains such that each domain can pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48 and Poljak et al., 1994, Structure 2:1121-23). ​​When the two polypeptide chains of a bispecific antibody are identical, the bispecific antibody that results from their pairing has two identical antigen-binding sites. Polypeptide chains with different sequences can be used to generate bispecific antibodies with two different antigen-binding sites. Similarly, trispecific and tetraspecific antibodies are antibodies that contain three and four polypeptide chains, respectively, and form three and four antigen-binding sites, which may be identical or different.

[0025] The complementarity determining regions (CDRs) and framework regions (FRs) of a particular antibody are described in detail in Kabat et al., supra. al.; Lefranc et al., and / or Honegger and Pluckthun One or more CDRs may be covalently or non-covalently incorporated into a molecule to identify The antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may have the CDR(s) covalently linked to another polypeptide chain, or may be an antigen binding protein. The CDRs may be incorporated non-covalently into the antigen-binding protein to provide the desired specificity. This allows the antibody to specifically bind to a specific antigen.

[0026] An antigen-binding protein may have one or more binding sites. When there is more than one binding site, the binding sites may be identical to one another or different. For example, naturally occurring human immunoglobulins typically have two identical binding sites, and "bispecific" or "bifunctional" antibodies have two different binding sites.

[0027] The term "human antibody" includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, the variable and constant regions are all derived from human immunoglobulin sequences (fully human antibodies). These antibodies may be produced in a variety of ways, including by immunizing with the desired antigen mice that have been genetically modified to express antibodies derived from genes encoding human heavy and / or light chains, as described below.

[0028] A humanized antibody has a sequence that differs from an antibody sequence from a non-human species by substitution, deletion, and / or addition of one or more amino acids such that the humanized antibody is less likely to elicit an immune response and / or elicits a less severe immune response when administered to a human subject compared to the non-human species antibody. In one embodiment, specific amino acids in the framework and constant regions of the heavy and / or light chains of a non-human species antibody are mutated to generate a humanized antibody. In another embodiment, a constant region of a human antibody is fused to a variable region of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are mutated to reduce the immunogenicity of the non-human antibody when administered to a human subject, where the mutated amino acid residues are not important for the immunospecific binding of the antibody to the antigen or the amino acid sequence mutations are conservative mutations such that the binding of the humanized antibody to the antigen is not significantly less than that of the non-human antibody. Methods for making humanized antibodies are described, for example, in U.S. Patent Nos. 6,054,297; 5,886,152; and No. 5,877,293.

[0029] The term "chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more CDRs are from a human anti-PD-1 antibody. In another embodiment, all of the CDRs are from a human anti-PD-L1 antibody. In another embodiment, the CDRs from two or more human anti-PD-1 antibodies are mixed and matched in a chimeric antibody. For example, a chimeric antibody may contain CDR1 from the light chain of a first human anti-PD-1 antibody, CDR2 and CDR3 from the light chain of a second human anti-PD-1 antibody, etc. and may include the CDRs from the heavy chain of a third anti-PD-1 antibody. Other combinations are possible.

[0030] Furthermore, the framework regions may be from one of the same anti-PD-1 antibodies or from one or more different antibodies, e.g., from a human antibody or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to, homologous to, or derived from an antibody from another species or belonging to another antibody class or subclass. The desired biological activity (i.e., specific binding to PD-1) may be achieved by combining the heavy and / or light chains. Also included are fragments of said antibodies exhibiting different binding abilities.

[0031] A "neutralizing antibody" or "inhibitory antibody" is an antibody that inhibits proteolytic activation of PD-1 when an excess of anti-PD-1 antibody reduces the amount of activation by at least about 20% in the assays described in the Examples herein. In various embodiments, the antigen binding protein reduces the amount of proteolytic activation of PD-1 by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

[0032] Antibody fragments or analogs can be readily produced by one of skill in the art following the description herein and using techniques known in the art. The N- and C-termini of the fragment or analog are preferably near the boundaries of the functional domains. Structural and functional domains can be identified by comparing the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Computerized comparison methods may also be used to identify sequence motifs or predicted protein conformations found in other proteins of known structure and / or function. Methods are known to identify protein sequences that fold into known three-dimensional structures. See Bowie et al., 1991, Science 253:164.

[0033] A "CDR grafted antibody" is an antibody derived from a specific species or isotype. The antibody comprises one or more CDRs derived from a human antibody of the same or different species or isotype.

[0034] A "multispecific antibody" is an antibody that recognizes two or more epitopes on one or more antigens. A subclass of this type of antibody is the "bispecific antibody," which recognizes two different epitopes on the same or different antigens.

[0035] An antigen binding protein is considered to "specifically bind" an antigen (e.g., human PD-L1) if it binds to an antigen with a dissociation constant of 1 nM or less.

[0036] An "antigen-binding domain," "antigen-binding region," or "antigen-binding site" is a part of an antigen-binding protein that contains the amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen-binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to an antigen, this includes at least a portion of at least one CDR domain.

[0037] An "epitope" is a site of a molecule to which an antigen-binding protein (e.g., an antibody) binds. Epitopes are non-contiguous sites of the molecule (e.g., in a polypeptide, sites that are closely spaced in their primary sequence). The amino acid sequence may include amino acid residues that are not in contact with each other but are close enough to each other in the tertiary and quaternary structures to allow binding of the antigen-binding protein.

[0038] The "percent identity" of two polynucleotide or two polypeptide sequences can be determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) with its default parameters. The decision shall be made based on the following:

[0039] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout this specification and refer to a DNA molecule (e.g., cDNA or genomic DNA). , RNA molecules (e.g., mRNA), nucleic acid analogs (e.g., peptide nucleic acids and nucleic acids of non-natural origin) The nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding an antibody, a fragment, derivative, mutein or variant thereof.

[0040] If the sequences of two single-stranded polynucleotides can be arranged in an antiparallel orientation without introducing gaps, such that every nucleotide in one polynucleotide is opposite to the complementary nucleotide in the other polynucleotide, and no unpaired nucleotides occur at the 5' or 3' ends of either sequence, then these polynucleotides are "complementary" to each other. If the two polynucleotides can hybridize to each other under reasonably stringent conditions, then the polynucleotides are "complementary" to another polynucleotide. Thus, a polynucleotide may be complementary to another polynucleotide, without being its complement.

[0041] A "vector" is a nucleic acid that can be used to introduce another nucleic acid linked thereto into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Other types of vectors include viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that can introduce additional DNA segments into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors that contain a bacterial origin of replication and episomal mammalian vectors). An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide. Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0042] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or site of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or site of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can affect the nucleic acid it regulates, for example, directly or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include, for example, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences can be found, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology185, Academic Press, SanDiego, Calif, and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

[0043] A "host cell" is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the invention. A host cell can be a prokaryote, e.g., Escherichia coli (E. Coli), or a eukaryote, e.g., a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Examples of host cells include monkey kidney cells, COS-7 cell line (ATCCCRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCCCRL 163), Chinese hamster egg cells, and the like. CHO cells or their derivatives, such as Veggie CHO and cells grown in serum-free medium related cell lines (see Rasmussen et al., 1998, Cytotechnology 28:31) or DHFR-deficient In vitro cultures of CHO line DX-B11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line derived from African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, and primary tissue cultures. Host cells include cell lines derived from the host cell line, primary artificial cultures, HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The term "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid, but does not express the nucleic acid at a desired level unless a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. It is understood that the term host cell refers not only to a particular subject cell, but also to the progeny or potential progeny of the cell. Such progeny may not actually be identical to the parent cell, since certain changes may occur during successive generations, for example due to mutations or environmental influences, but are within the scope of the term as used herein.

[0044] Preferably, the mammalian cancer to be treated is an ovarian cancer, a colon cancer, a breast cancer or a liver cancer cell line, a myeloma, a neuroblastoma-derived CNS tumor, a monocytic leukemia, a B-cell derived leukemia, a T-cell derived leukemia. The tumor is selected from the group consisting of lymphomas derived from B cell, lymphomas derived from T cell, mast cell derived tumors and combinations thereof.

[0045] The polypeptides of the present invention may be produced using any standard method known in the art. As an example, the polypeptides are produced by recombinant DNA techniques by inserting a nucleic acid sequence (e.g., cDNA) encoding the polypeptide into a recombinant expression vector and expressing the DNA sequence under conditions promoting expression.

[0046] Nucleic acids encoding any of the various polypeptides described herein may be chemically synthesized. Codon usage may be selected to increase expression in cells. The codon usage depends on the type of cell selected. Specific codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See, for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 200112(5):446-9; Makrides et al. Microbiol. Rev. 199660(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

[0047] General techniques for nucleic acid manipulation are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and their latest editions, which are incorporated herein by reference in their entirety. The DNA encoding the polypeptide is operably linked to suitable transcription or translation regulatory elements derived from mammalian, viral or insect genes. Such regulatory elements include a transcription promoter, an optional operator sequence that controls transcription, a sequence that codes for a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation. The ability to replicate in a host, usually provided by a replication origin, and a selection gene to facilitate the recognition of transformants may also be incorporated.

[0048] The recombinant DNA may also contain any type of protein tag sequence that may be useful for protein purification. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described in Cloning Vectors: A Laboratory Manual, (Elsevier, NY, 1985).

[0049] The expression construct is introduced into the host cell by using a method suitable for the host cell.The method of introducing nucleic acid into the host cell is known in the art, and includes, but is not limited to, electroporation; gene transfer using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; particle bombardment; lipofection; and infection (if the vector is infectious).Suitable host cells include prokaryotic, yeast, mammalian cell or bacterial cell.

[0050] Suitable bacteria include gram-negative or gram-positive organisms such as E. coli or Bacillus spp. Yeast, preferably Saccharomyces spp., such as S. cerevisiae, may be used for the production of polypeptides. A variety of mammalian or insect cell culture systems may be used for the expression of recombinant proteins. Baculovirus systems for the production of heterologous proteins in insect cells have been reviewed by Luckow and Summers (Bio / Technology, 6:47, 1988). Examples of suitable mammalian host cell lines include endogenous baculovirus (EBV) and baculovirus (Bio / Technology, 6:47, 1988). epithelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary cells These include CHO, HEK (human embryonic kidney) cells, HeLa, 293, 293T and BHK cell lines. Purified polypeptides are produced by culturing an appropriate host / vector system to express the recombinant protein. For many applications, expression in E. coli is the preferred method of expression due to the small size of many of the polypeptides described herein. The protein is then purified from the culture medium or cell extracts.

[0051] The proteins described herein may also be produced using cellular translation systems. For such purposes, the nucleic acid encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and cell-free translation of said mRNA in the particular cell-free system used (eukaryotic, e.g., mammalian or yeast, cell-free translation systems or prokaryotic, e.g., bacterial, cell-free translation systems).

[0052] PD-1 binding polypeptides may also be made using chemical synthesis (e.g., the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications of the protein may also be made by chemical synthesis.

[0053] The polypeptides of the present invention may be purified by protein isolation / purification methods commonly known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., salting out with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination thereof. After purification, the polypeptides may be exchanged for buffer media and / or concentrated by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis.

[0054] The purified polypeptide is preferably at least 85%, more preferably at least 95%, and most preferably at least 98% pure. Regardless of the exact purity level, the polypeptide is sufficiently pure for use as a pharmaceutical product.

[0055] Post-translational Modification of Polypeptides In certain embodiments, the binding polypeptides of the invention may further comprise post-translational modifications. Examples of post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation, or addition of polypeptide side chains or hydrophobic groups. As a result, the modified soluble polypeptide may contain non-amino acid elements, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which attaches one or more sialic acid moieties to the polypeptide. Sialic acid moieties improve the solubility and serum half-life of the protein while reducing immunogenicity. See Rajuetal. Biochemistry. 2001 31; 40(30):8868-76. The effect of the non-amino acid elements on the functionality of the polypeptide may be tested for its antagonistic effect on PD-L1 or PD-1 function, such as an inhibitory effect on angiogenesis or tumor growth.

[0056] In one particular embodiment, the modified form of the subject soluble polypeptide comprises linking the subject soluble polypeptide to a nonproteinaceous polymer, as described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; The modified polypeptide may be polyethylene glycol ("PEG"), polypropylene glycol or polyoxyalkylene, as described in US Pat. No. 4,179,337 or US Pat. No. 4,179,337. An example of a modified polypeptide is PEGylated VK-B8.

[0057] PEG is a water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol by methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, p 138-161). The term "PEG" refers to is used broadly to refer to any polyethylene glycol molecule, regardless of size or PEG end modification; Formula: X--O(CH2CH2O) n -1CH2CH2OH(1) wherein n is 20 to 2300, and X is H or a terminal modification, such as C 1-4 In one embodiment, the PEG of the present invention is hydroxy or methoxy at one end, i.e., X is H or CH3 ("methoxy PEG"). PEG may further contain chemical groups necessary for conjugation reactions; it can be prepared by chemical synthesis of the molecule. or a spacer for optimizing the distance between the molecules. EG may consist of one or more PEG side chains linked together. PEGs with more than one PEG chain are called multiarmed or branched PEGs. Branched PEGs can be made, for example, by adding polyethylene oxide to various polyols, such as glycerol, pentaerythriol, and sorbitol. For example, a four-branched PEG can be made from pentaerythriol and ethylene oxide. Branched PEGs are described, for example, in EP 0 473 084 and U.S. Pat. No. 5,932,462. One form of PEG is formed by binding to the primary amino groups of lysine. and 2PEG side chains (PEG2) linked via (Monfardini et al., Bioconjugate Chem. 6 (1995)62-69).

[0058] PEG is well known, but 10F To our knowledge, this is the first time that it has been shown that n3 polypeptides can be PEGylated and retain ligand binding activity. In a preferred embodiment, the PEGylated 10FThe n3 polypeptide is prepared by site-specific PEGylation, in particular by attachment of PEG to a cysteine ​​at the N-terminus or C-terminus. Thus, the present invention provides target-binding polypeptides with improved pharmacological properties. 10F n3 polypeptide, 10F At least one loop of the n3 domain functions in target binding, and the loop is about 80 to about 150 amino acids long. 10F Contains an n3 domain and a covalently attached PEG moiety 10F n3poly A peptide is provided, wherein the 10F The n3 polypeptide has a K of less than 100 nM in mammals. D and exhibits a clearance rate of less than 30 mL / hr / kg. 10F The n3 polypeptide may be linked by site-specific PEGylation, e.g., by linkage to a Cys residue, wherein the Cys residue is 10F N-terminus or N-terminal of n3 polypeptide between the N-terminal and the N-terminal β-strand or β-like strand, or 10F The Cys residue may be located at the C-terminus of the n3 polypeptide or between the C-terminus and the C-most β-strand or β-like strand. , specifically on any loop that is not involved in target binding. The PEG moiety may also be attached by other chemical reactions, for example, by coupling to an amine.

[0059] Conjugation of PEG to a peptide or protein generally involves activation of PEG and direct conjugation of the activated PEG intermediate to the target protein / peptide or to a linker, which The PEG is then activated and conjugated to the target protein / peptide (see Abuchowski et al., J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem., 252, 3582 (1977); Zalipsky, et al., and Harris et al., in: Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; (JM Harris ed.) Plenum Press: New York, 1992; Chap. 21 and 22). Conjugated polypeptides containing PEG molecules are known as conjugated proteins, while conjugated polypeptides are known as non-conjugated proteins. It should be noted that a protein lacking a conjugated PEG molecule may also be referred to as unconjugated.

[0060] PEGs of various molecular weights may be selected for attachment to PD-1 binding polypeptides, e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20-2300). The number of repeating units in the PEG, "n," approximates the molecular weight in Da. It is preferred that the combined molecular weight of the PEG on the activated linker is appropriate for pharmaceutical use. Thus, in one embodiment, the molecular weight of the PEG molecule does not exceed 100,000 Da. For example, if three PEG molecules are attached to the linker, each PEG molecule has a molecular weight of 12,000. 0 Da (each n is about 270), where the total molecular weight of the PEG on the linker is about 36,000 Da (total of n is about 820). The molecular weights of the PEGs attached to the linker may be different, for example, of the three molecules on the linker, two PEG molecules are each 5,000 Da (each n is about 110) and one PEG molecule is 12,000 Da (n may be about 270).

[0061] In certain embodiments of the invention, the PD-1 binding polypeptide comprises Formula:--CO--(CH2) x--(OCH2CH2) m --OR one of the poly(ethylene glycol) groups of the --CO ( (i.e., carbonyl) is arranged to form an amide bond with one of the amino groups of the binding polypeptide. R is lower alkyl; x is 2 or 3; m is about 450 to about 950; and n and m are selected such that the molecular weight of the conjugate minus the binding polypeptide is about 10 to 40 kDa. The 6-amino group of is an available (free) amino group.

[0062] The above conjugate more specifically has the formula (II): P--NHCO--(CH2) x --(OCH2CH2) m --OR(II) where P is a group of binding polypeptides as described herein (i.e. i.e., it does not contain an amino group that forms an amide bond with the carbonyl shown in formula (II); R is a lower alkyl; x is 2 or 3; m is about 450 to about 950, and is selected so that the molecular weight of the conjugate minus the binding polypeptide is about 10 to about 40 kDa. In the formula (I), the specific range of "m" has significance for the orientation. The range of "m" in each case is strictly determined by the molecular weight of the PEG group.

[0063] One of skill in the art can select an appropriate molecular weight of PEG based on, for example, how the PEGylated binding polypeptide will be used therapeutically, the desired dose, circulation time, resistance to proteolysis, immunogenicity, and other considerations. For a discussion of PEG and its use to enhance protein properties, see Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993).

[0064] In one embodiment, the PEG molecule is activated to attach to amino groups on the binding polypeptide, e.g. It may react with lysine (Bencham et al., Anal.Biochem., 131, 25 (1983); Veronese et al.,Appl. Biochem., 11, 141 (1985).;Zalipsky et al., Polymeric Drugs and Drug Delivery Systems, adrs 9-110 ACSSymposium Series 469 (1999); Zalipsky et al.,Europ. Polym. J., 19, 1177-1183(1983); Delgado et al., Biotechnology andApplied Biochemistry, 12, 119-128(1990)).

[0065] In one particular embodiment, a carbonate ester of PEG is used to form the PEG-linked polypeptide conjugate. N,N'-disuccinimidyl carbonate (DSC) is used to react with PEG to form an activated mixed PEG-succinimidyl carbonate which may then react with a nucleophilic group on a linker or an amino group on a binding polypeptide (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similar type of reaction, 1,1'-(dibenzotriazolyl) carbonate and di-(2-pyridyl) carbonate may be reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed carbonates, respectively (U.S. Pat. No. 5,382,657).

[0066] 10FPEGylation of the n3 polypeptide may be carried out by state-of-the-art methods, for example by reaction of the binding polypeptide with electrophilically activated PEG (supplier: ShearwaterCorp., USA, www.shearwatercorp.com). Preferred PEG reagents of the present invention are, for example, N-hydroxysuccinimidyl propionate (PEG-SPA), butanoate (PEG-SBA), PEG- ... PEGylation at the f-amino groups of lysines or at the amino group at the N-terminus of the binding polypeptide.

[0067] In other embodiments, PEG molecules may be attached to sulfhydryl groups on the binding polypeptide (Sartore et al., Appl. Biochem.Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368 (1996); Goodson et al., Bio / Technology (1990)8, 343; (U.S. Patent No. 5,766,897). U.S. Patent Nos. 6,610,281 and 5,766,897 describe examples of reactive PEG species that may be attached to sulfhydryl groups.

[0068] In embodiments where the PEG molecule is attached to a cysteine ​​residue of the binding polypeptide, the cysteine ​​residue may be native to the binding polypeptide, while in other embodiments, one or more cysteine ​​residues may be engineered into the binding polypeptide. Mutations may be introduced into the binding polypeptide coding sequence to generate cysteine ​​residues. This may be accomplished, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutating to cysteine ​​residues include serine, threonine, alanine and other hydrophilic residues. The residues mutated to cysteine ​​are preferably surface-exposed residues. Algorithms for predicting surface accessibility of residues based on primary sequence or protein are well known in the art. Alternatively, where the crystal structure of the framework on which the binding polypeptide is designed and developed has been solved, surface residues can be predicted by comparing the amino acid sequence of the binding polypeptide (see Himanene et al., Nature. (2001) 20-27; 414(6866):933-8), thereby identifying surface-exposed residues. In one aspect, Cysteine ​​residues are introduced into the binding polypeptide at or near the N-terminus and / or C-terminus, or within loop regions.

[0069] In some embodiments, the PEGylated binding polypeptide comprises a PEG molecule covalently attached to the alpha amino group of the N-terminal amino acid. Site-specific N-terminal reductive amination is described by Pepinsky et al. et al., (2001) JPET, 297, 1059 and U.S. Patent No. 5,824,784. The use of PEG-aldehydes for the reductive amination of proteins with other available nucleophilic amino groups is described in U.S. Patent No. 4,002,531, Wieder et al., (1979) J. Biol. Chem. 254, 12579 and Chamow et al., (1994) Bioconjugate Chem. 5, 133 .

[0070] In other embodiments, the PEGylated binding polypeptide comprises one or more PEG molecules covalently attached to a linker, which is itself attached to the alpha amino group of the N-terminal amino acid residue of the binding polypeptide. Such methods are described in U.S. Patent Application Publication No. 2002 / 0044921 and WO 094 / 01451.

[0071] In one embodiment, the binding polypeptide is PEGylated at the C-terminus. In one particular embodiment, the protein is PEGylated at the C-terminus by introduction of a C-terminal azidomethionine followed by attachment of a methyl-PEG-triarylphosphine compound via the Staudinger reaction. This C-terminal attachment method is described in Cazalis et al., Bioconjug. Chem. 2004; 15(5):1005-1009.

[0072] MonoPEGylation of binding polypeptides may also be made by the general method described in WO 94 / 01451. WO 94 / 01451 describes a method for making recombinant polypeptides with modified terminal amino acid alpha carbon reactive groups. The steps of the method include forming the recombinant polypeptide and protecting the polypeptide with one or more protecting groups biologically added to the N-terminal alpha amine and C-terminal alpha carboxyl. The polypeptide may then be reacted with a chemical protecting agent to selectively protect reactive side groups, thereby preventing the side chains from being modified. The polypeptide is then cleaved with a cleavage reagent specific for the biological protecting group to form unprotected terminal amino acid alpha carbon reactive groups. Such unprotected terminal amino acid alpha carbon reactive groups are modified with a chemical modifying agent. The side chain protected terminal modified single copy polypeptide is then deprotected at the side chain groups to form a terminal modified recombinant single copy polypeptide. The number and order of the method steps may be varied to achieve selective modification at the N-terminal and / or C-terminal amino acids of the polypeptide.

[0073] The ratio of conjugated polypeptide to activated PEG during the conjugation reaction is about 1:0.5 to 1:50. , about 1:1 to 1:30, or about 1:5 to 1:15. A variety of aqueous buffers may be used in the methods of the invention for catalyzing the covalent attachment of PEG to a binding polypeptide. In one embodiment, the pH of the buffer used is about 7.0 to 9.0. In other embodiments, the pH is in the slightly basic range, for example, about 7.5 to 8.5. A pKa close to the neutral pH range may be used. A buffer having the formula:

[0074] Conventional separation and purification techniques known in the art may be used to purify the PEGylated binding polypeptide, such as size exclusion chromatography (e.g., gel filtration) and ion exchange chromatography. Products may be separated using SDS-PAGE. Products that may be separated include mono-, di-, tri-, poly-PEGylated and non-PEGylated binding polypeptides, as well as free PEG. The percentage of mono-PEG conjugates may be adjusted to a higher percentage of mono-PEG in the composition by pooling broader fractions around the elution peak. A good balance of yield and activity is achieved when the mono-PEG conjugates are about 90%. For example, compositions in which at least 92% or at least 96% of the conjugates are mono-PEG species may be desirable. In one embodiment of the invention, the percentage of mono-PEG conjugates is 90%-96%.

[0075] In one embodiment, the PEGylated binding polypeptides of the invention comprise one, two or more PEG moieties. In one embodiment, the PEG moieties are attached to amino acid residues on the surface of the protein and / or away from the surface that contacts the targeting ligand. In one embodiment, the combined or total molecular weight of the PEGs in the PEG-binding polypeptide is from about 3,000 Da to 60,000 Da, and in some cases from about 10,000 Da to 36,000 Da. In one embodiment, the PEG in the PEGylated binding polypeptide is a substantially linear, straight-chain PEG.

[0076] In one embodiment of the invention, the PEG in the PEGylated binding polypeptide is stable, e.g., not hydrolyzed from the PEGylated amino acid residue using a hydroxylamine assay with 450 mM hydroxylamine, pH 6.5, for 8-16 hours at room temperature. In one embodiment, more than 80%, more preferably at least 90%, of the composition is PEGylated. 0%, and most preferably at least 95% are stable mono-PEG-linked polypeptides.

[0077] In other embodiments, the pegylated binding polypeptides of the invention preferably retain at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of the biological activity of the unmodified protein. on or k off In one particular embodiment, the PEGylated binding polypeptide protein exhibits increased binding to PD-1 compared to non-PEGylated binding polypeptide.

[0078] The serum clearance rate of the PEG-modified polypeptide may be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the unmodified binding polypeptide. Half-life (t 1 / 2) may be increased as compared to the half-life of the unmodified protein. The half-life of the PEG-linked polypeptide may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, or 500% or 1000% as compared to the unmodified linked polypeptide. In some embodiments, the half-life of the protein is measured in vitro. In another embodiment, the half-life of the protein is determined in vivo, e.g., in buffered saline or serum. In another embodiment, the half-life of the protein is an in vivo half-life, e.g., the half-life in serum or other bodily fluid of an animal.

[0079] Therapeutic Formulations and Methods of Administration The present invention features a method for treating a condition or preventing a pre-condition responsive to inhibition of the biological activity of PD-1. Preferred examples are conditions characterized by inflammation or cellular hyperproliferation. Administration techniques and dosages will vary depending on the specific polypeptide and the specific condition being treated, and can be readily determined by one of skill in the art. Regulatory agencies generally require that protein formulations used as therapeutic agents be formulated to have acceptably low levels of pyrogens. Thus, therapeutic agents generally differ from other formulations in that they are essentially pyrogen-free or contain only pyrogens at or below acceptable levels as determined by the appropriate regulatory agency (e.g., FDA).

[0080] The therapeutic composition of the present invention may be administered in unit dosage form together with a pharma- ceutically acceptable diluent, carrier or excipient. Administration may be, but is not limited to, parenteral (e.g., intravenous, subcutaneous), oral or topical administration. In addition, any gene therapy technique using a nucleic acid encoding the polypeptide of the present invention may be used, such as delivery of DNA itself, recombinant genes and vectors, cell-based delivery, such as ex vivo manipulation of the patient's cells, and the like.

[0081] The compositions may be in the form of a pill, tablet, capsule, liquid, or sustained release tablet for oral administration; a liquid for intravenous, subcutaneous, or parenteral administration; a gel, lotion, ointment, cream, or polymer or other sustained release vehicle for topical administration.

[0082] Methods for preparing formulations, which are well known in the art, are described, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. AR Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). Formulations for parenteral administration can be prepared, for example, by For example, excipients may include sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactic acid polymers, lactic acid / glycolic acid copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used for sustained release of the compounds. Nanoparticulate formulations (e.g., Biodegradable nanoparticles, solid lipid nanoparticles, liposomes) to control the biodistribution of the compound Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems and liposomes. The concentration of the compound in the formulation varies depending on a number of factors, such as the dose of the drug to be administered and the route of administration.

[0083] The polypeptide may be administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids such as tannic acid or carboxymethylcellulose; inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid.Metal complexes include zinc and iron.In one example, the polypeptide is formulated in the presence of sodium acetate to enhance thermal stability.

[0084] Formulations for oral use include tablets containing the active ingredient mixed with non-toxic pharma- ceutically acceptable excipients, which may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, glidants and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils or talc).

[0085] Formulations for oral use may also be provided as chewable tablets or hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium.

[0086] The term therapeutically effective dose refers to the dose for which it is administered that produces the therapeutic effect. The exact dose will vary depending on the disorder being treated, but can be ascertained by one of skill in the art using known techniques. In general, the polypeptide is administered at about 0.01 μg / kg to about 50 mg / kg per day, preferably 0.01 mg / kg to about 30 mg / kg per day, and most preferably 0.1 mg / kg to about 20 mg / kg per day. The polypeptide is administered daily (e.g., once or twice a day, The dose may be administered once a day, once every three weeks, once every four weeks, once every five weeks, once every six months, once every seven days, once every eight months ... Adjustments may be necessary depending on age and weight, general health, sex, diet, timing of administration, drug interactions and severity of the disease, which can be determined by routine testing methods by one of ordinary skill in the art.

[0087] Pharmaceutical Formulations of the Antibodies and Tumor Vaccines Described Herein Combination therapeutic products or formulations of the anti-PD-1 antibodies and therapeutic vaccines described herein provide synergistic tumor treatment benefits. For example, the present invention provides a combination of the anti-PD-1 antibodies described herein with "Neuvax," a 9-mer synthetic peptide derived from E75 isolated from HER2 / neu, combined with GM-CSF as an adjuvant, as described in U.S. Patent No. 8,222,214, the disclosure of which is incorporated herein by reference. Additionally, the invention provides a combination of an anti-PD-1 antibody as described herein and the ALVAC-CEA vaccine, which is a canarypox virus in combination with a carcinoembryonic antigen.

[0088] Usage example The PD-1 binding proteins and related variants thereof described herein are useful in a number of therapeutic and diagnostic applications. These include inhibiting the biological activity of PD-1 by competing with or blocking binding to PD-1, and delivering cytotoxic or imaging agents to cells, particularly PD-1 expressing cells. The small size and structural stability of these molecules may be particularly useful for the manufacture of drugs, their rapid clearance from the body in certain applications where rapid clearance is desirable, or for formulation into suitable or improved novel delivery systems using molecules with such characteristics.

[0089] Based on its effectiveness as an inhibitor of PD-1 biological activity, the polypeptide of the present invention is effective against many cancer symptoms and complications caused by cancer, such as pleural effusion and ascites. Preferably, the PD-1 binding polypeptide of the present invention may be useful for treating or preventing hyperproliferative diseases or cancer and the spread of cancer through metastasis. Suitable indications for the anti-PD-1 antibodies described herein include colorectal cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer (NSCLC) and pancreatic cancer. Non-limiting examples of cancer include bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cartilage cancer, colon cancer, kidney cancer, liver cancer, lung cancer, lymph node cancer, neural tissue cancer, ovarian cancer, pancreatic cancer, prostate cancer, skeletal muscle cancer, skin cancer, spinal cancer, spleen cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, tracheal cancer, genitourinary cancer, ureter cancer, urethral cancer, uterine cancer or vaginal cancer.

[0090] Additionally, the anti-PD-1 binding polypeptides described herein may treat a variety of inflammatory disorders, including, for example, intestinal mucosal inflammation, debilitating colitis-associated diseases, multiple sclerosis, systemic lupus erythematosus, viral infections, rheumatoid arthritis, osteoarthritis, psoriasis, and Crohn's disease.

[0091] The PD-1 binding polypeptides may be administered alone or in combination with one or more additional treatments, such as chemotherapy, radiation therapy, immunotherapy, surgical intervention, or any combination thereof. As noted above, long-term treatment is also possible as an adjunct to other treatment strategies.

[0092] The anti-PD-L1 antibody agents described herein may be used alone or in combination with other conventional anti-cancer therapies for the treatment or prevention of proliferative disorders (e.g., tumors).For example, the methods may be used to prevent cancer, prevent cancer recurrence and metastasis after surgery, and may be used as an adjunct to other conventional cancer therapies.It can be seen from the description herein that the effect of conventional cancer therapies (e.g., chemotherapy, radiotherapy, phototherapy, immunotherapy, and surgery) can be enhanced by the subject polypeptide therapeutic agents.

[0093] A wide range of known compounds have been shown to exhibit antitumor activity. These compounds are used as medicines in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant cells in leukemic or myeloid malignancies. Although chemotherapy is effective in treating various types of malignancies, many antitumor compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, such treatments act synergistically, allowing the dose of each treatment to be reduced, thereby reducing the harmful side effects that each compound causes at high doses. In other examples, malignancies that are refractory to treatment may respond to the combination of two or more different therapies.

[0094] When the polypeptide therapeutic agent is administered in combination with other conventional anti-tumor agents, either simultaneously or sequentially, it may be shown that the therapeutic agent can enhance the therapeutic effect of the anti-tumor agent or overcome cellular resistance to the anti-tumor agent, making it possible to reduce the dose of the anti-tumor agent, thereby reducing undesirable side effects, or to restore the effect of the anti-tumor agent in resistant cells.

[0095] Pharmaceutical compounds that can be used in combination antitumor therapy include, for example: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, sirolimus, buserelin, busulfan, camptothecin, capecitabine, calcitabine Boplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leukotriene, These include volin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine and vinorelbine.

[0096] Specific chemotherapeutic antitumor compounds can be divided according to their mechanism of action, for example into the following groups: Antimetabolites / Anticancer drugs, for example pyrimidine analogues (5-fluorouracil, floxuridine, etc.); cyclosporine, capecitabine, gemcitabine and cytarabine) and purine analogues, folate antagonists and and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, e.g., natural products, e.g., vinca Alkaloids (vinblastine, vincristine and vinorelbine), microtubule disruptors such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilone and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamidis, iphosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin , plicamycin (mithramycin) and mitomycin; enzymes (total L-asparagine L-asparaginase, which actively metabolizes and removes asparagine from cells that do not have the ability to synthesize it themselves; antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil, Sil), ethylenimine and methylmelamine (hexamethylmelamine and thiotepa), Alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, e.g. folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, amino Glutethimide; hormones and hormone analogues (estrogen, tamoxifen, goserelin , bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); thrombolytic agents (e.g. tissue plasminogen activator, streptokinase and urokinase), amyloidosis aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine) porin, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil; antiangiogenic compounds (TNP-470, genistein) and growth factor inhibitors (e.g. VEGF inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, ibuprofen Darubicin and mitoxantrone, topotecan, irinotecan); corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone and prednisolone); growth factor signaling kinase inhibitors; mitoxantrone mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0097] Depending on the nature of the combination therapy, administration of the polypeptide therapeutic may continue during and / or after administration of the other therapeutic agent. Administration of the polypeptide therapeutic may be in a single dose or in multiple doses. Administration of the polypeptide therapeutic may begin at least several days prior to the conventional therapy, or may begin immediately prior to or at the time of administration of the conventional therapy.

[0098] The PD-1 binding polypeptide is further conjugated to a detectable label (e.g., For example, the active moiety may be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. can be, for example, a radioactive agent, such as a radioactive heavy metal, for example an iron chelate, a radioactive chelate of gadolinium or manganese, a positron emitter of oxygen, nitrogen, iron, carbon or gallium, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 132 I or 99 The binding agent attached to such a moiety may be Tc. Binding agents attached to such moieties may be used as imaging agents, and the localization and accumulation of the imaging agent detected after administration of an effective amount for diagnostic use in a mammal, e.g., a human. The localization and accumulation of the imaging agent may be detected by radioscintigraphy, nuclear magnetic resonance imaging, computed tomography, or positron emission tomography. Immunoscintigraphy using PD-1 binding polypeptides directed against PD-1 may be used to detect and / or diagnose cancer and the vasculature. For example, 99 technetium, 111 Indium or 125Any binding polypeptide to the PD-1 marker labeled with iodine may be usefully used for such imaging. As will be apparent to one of skill in the art, the amount of radioisotope administered will depend on the radioisotope. One of skill in the art can readily formulate the amount of imaging agent administered based on the specific activity and energy of the particular radionuclide used as the active moiety. Typically, 0.1 to 100 millicuries per dose of imaging agent, Preferably, 1 to 10 millicuries are administered, most frequently 2 to 5 millicuries. Thus, compositions of the invention useful as imaging agents comprising a targeting moiety linked to a radioactive moiety are administered in the range of 0.1 to 100 millicuries, and in some embodiments, preferably 1 to 5 millicuries. 10 millicuries, in some embodiments preferably 2 to 5 millicuries, and in some embodiments more preferably 1 to 5 millicuries.

[0099] The PD-1-binding polypeptides may also be used to deliver additional therapeutic agents, including but not limited to drug compounds, chemotherapeutic compounds, and radiotherapeutic compounds, to cells or tissues that express PD-1. In one example, the PD-1-binding polypeptides are fused to chemotherapeutic agents for targeted delivery to tumor cells or tissues that express PD-1.

[0100] The present PD-1-binding polypeptides are useful in a variety of research, diagnostic and therapeutic applications, for example, for isolating and / or purifying the receptor or portions thereof, and for studying receptor structure (e.g., conformation) and function.

[0101] The present invention also provides a method for detecting susceptibility of a mammal to a particular cancer. For example, the method can be used to detect susceptibility of a mammal to developing disease based on the amount of PD-1 present on cells and / or the number of PD-1 positive cells in the mammal.

[0102] Polypeptide sequences are represented using standard one-letter or three-letter abbreviations.Unless otherwise stated, each polypeptide sequence is N-terminal on the left and C-terminal on the right; each single-stranded nucleotide sequence and the upper strand of each double-stranded nucleic acid sequence is 5'-terminal on the left and 3'-terminal on the right.A particular polypeptide sequence can also be represented by describing how it differs from a reference sequence.

[0103] The following terms, unless otherwise specified, shall be understood to have the following meanings:

[0104] The terms "PD-1 inhibitor" and "PD-1 antagonist" are used interchangeably. Each is a molecule that detectably inhibits at least one function of PD-1. Conversely, a "PD-1 agonist" is a molecule that detectably increases at least one function of PD-1. The inhibition caused by a PD-1 inhibitor need not be complete, as long as it is detectable using an assay. Any assay for PD-1 function may be used, examples of which are described herein. Examples of PD-1 functions that may be inhibited by a PD-1 inhibitor or increased by a PD-1 agonist include cancer cell proliferation or apoptosis (programmed cell death). Examples of types of PD-1 inhibitors and PD-1 agonists include, but are not limited to, PD-1 binding polypeptides, such as antigen binding proteins (e.g., PD-1 inhibitory antigen binding proteins), antibodies, antibody fragments, and antibody derivatives.

[0105] The terms "peptide," "polypeptide," and "protein" each refer to a molecule comprising two or more amino acid residues joined together by peptide bonds. These terms include, for example, naturally occurring and artificial proteins, protein fragments, and polypeptide analogs of protein sequences (e.g., muteins, variants, and fusion proteins), as well as proteins that are post-translationally or otherwise covalently or non-covalently modified. A peptide, polypeptide, or protein may be monomeric or polymeric.

[0106] "Variants" of a polypeptide (e.g., an antibody) include amino acid sequences in which one or more amino acids have been inserted, deleted, and / or substituted in the amino acid sequence compared to another polypeptide sequence. Variants as described herein include, for example, fusion proteins.

[0107] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation to other chemical moieties (e.g., polyethylene glycol or albumin, e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise specified, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments and muteins thereof, examples of which are described below.

[0108] An "antigen binding protein" is a protein that contains an antigen binding site and, optionally, a scaffolding or framework region that enables the antigen binding site to adopt a structure that facilitates binding of the antigen binding protein to an antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., antigen binding sites of antibodies), antibody derivatives, and antibody analogs. Antigen binding proteins The scaffolds may include, for example, alternative protein scaffolds or artificial scaffolds that include grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds, including, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, as well as entirely synthetic scaffolds, including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. Additionally, peptide antibody mimetics ("PAMs") as well as antibody mimetics using fibronectin components have been proposed. Mimetics based scaffolds may be used as scaffolds.

[0109] The antigen-binding protein may have, for example, a naturally occurring immunoglobulin structure. An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains, each having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The C-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or gamma light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Preferably, the anti-PD-1 antibodies described herein have a heavy chain V H and V for light chains L They are characterized by the variable region sequence of amino acid sequence. A preferred antibody is A6, a kappa IgG antibody. In the light and heavy chains, the variable and constant regions are connected by a "J region" of about 12 or more amino acids, and the heavy chain also contains a "D region" of about 10 or more amino acids. For general information, see Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Intact immunoglobulins have two binding sites. Together, the variable regions of each light / heavy chain pair form the antibody binding site.

[0110] An antigen binding protein is considered to "specifically bind" an antigen (e.g., human PD-1) if it binds to an antigen with a dissociation constant of 1 nM or less.

[0111] An "antigen-binding domain," "antigen-binding region," or "antigen-binding site" is a part of an antigen-binding protein that contains the amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen-binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to an antigen, this includes, at least in part, at least one CDR domain.

[0112] An "epitope" is a site of a molecule to which an antigen-binding protein (e.g., an antibody) binds. Epitopes are non-contiguous sites of the molecule (e.g., in a polypeptide, sites that are closely spaced in their primary sequence). The amino acid sequence may include amino acid residues that are not in contact with each other but are close enough to each other in the tertiary and quaternary structures to allow binding of the antigen-binding protein.

[0113] The "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) with its default parameters.

[0114] A "host cell" is a cell that can be used to express a nucleic acid. A host cell can be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell) or a hybridoma. Examples of host cells include monkey kidney cells, COS-7 cell line (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells or derivatives thereof, For example, Veggie CHO and related cell lines grown in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31) or the DHFR-deficient CHO line DX-B11 (Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCCCRL 10) cell lines, the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCCCC CL 70) (McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells. Host cells include cell lines derived from in vitro culture of primary tissues, primary artificial tissues, HL-60, U937, HaK or Jurkat cells. Typically, host cells are cultured cells that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The term "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid, but does not express the nucleic acid at a desired level unless a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell, but also to the progeny or potential progeny of the cell. Such progeny may not actually be identical to the parent cell, since certain changes may occur during successive generations, for example due to mutations or environmental influences, but are within the scope of the term as used herein.

[0115] Antigen binding proteins (e.g., antibodies, antibody fragments, antibody derivatives, antibody muteins and antibody variants) Antigen binding (PD-1) is a polypeptide that binds to PD-1 (preferably human PD-1). The proteins include antigen-binding proteins that inhibit the biological activity of PD-1.

[0116] The term "Fc polypeptide" includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of said polypeptides, including the hinge region that promotes dimerization. Fusion proteins containing Fc portions (and multimers formed therefrom) offer the advantage that they can be readily purified by affinity chromatography on Protein A or Protein G columns.

[0117] Antigen-binding fragments of the antigen-binding proteins of the present invention may be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments.

[0118] The present invention provides monoclonal antibodies that bind to PD-1. The monoclonal antibodies may be produced by any technique known in the art, for example, by immortalizing spleen cells harvested from the transgenic animals after the completion of the immunization schedule. The spleen cells may be immortalized by any technique known in the art, for example, by fusing them with myeloma cells to produce hybridomas. The myeloma cells used in the hybridoma production fusion technique preferably do not produce antibodies, have high fusion efficiency, and have enzyme deficiencies that make them unable to grow in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for use in mouse fusions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XX0 Bul; examples of cell lines for use in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F, and 48210. Other cell lines useful for cell fusions include U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0119] Antigen binding proteins for PD-1 may be used in assays to detect the presence of PD-1 polypeptide, for example in vitro or in vivo. The antigen binding proteins may also be used to purify PD-1 protein by immunoaffinity chromatography. Blocking antigen binding proteins may be used in the methods described herein. The antigen binding proteins that function as PD-1 antagonists may be used to treat any PD-1-induced condition, including, but not limited to, various cancers.

[0120] The antigen binding proteins may be used in in vitro procedures or administered in vivo to inhibit PD-1-induced biological activity. Thus, disorders caused or exacerbated (directly or indirectly) by proteolytic activation of PD-1 may be treated, such as those exemplified herein. In one embodiment, the invention provides a method of treatment comprising administering an effective amount of a PD-1-blocking antigen binding protein in vivo to a mammal in need thereof to reduce PD-1-induced biological activity.

[0121] The antigen binding proteins include fully human monoclonal antibodies that inhibit the biological activity of PD-1.

[0122] Antigen binding proteins may be produced by any of a number of conventional techniques, for example, isolated from cells in which they are naturally expressed (e.g., antibodies may be purified from hybridomas that produce them) or produced in recombinant expression systems using any technique known in the art. See, e.g., Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0123] Any expression system known in the art may be used to produce the recombinant polypeptides of the present invention. Generally, a host cell is transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Host cells that may be used include prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include insect cells and yeast cells of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells, and HeLa cells. , BHK (ATCC CRL 10) cell line and the method described in McMahan et al., 1991, EMBO J. 10: 2821. The CV1 / EBNA cell line, derived from the African green monkey kidney cell line CV1 (ATCC CCL 70), has been Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian host cells are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0124] The transformed cells may be cultured under conditions which promote expression of the polypeptide, which is then recovered by conventional protein purification techniques. One such purification technique involves the use of affinity chromatography, such as affinity chromatography on a matrix having all or a portion of PD-1 (e.g., the extracellular domain) bound thereto. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian anti-PD-1 antibody polypeptides that are substantially free from contaminating endogenous material.

[0125] Any of a number of known techniques may be used to produce antigen binding proteins and screen for desired properties. Particular techniques involve isolating a nucleic acid encoding the polypeptide chain (or a portion thereof) of a desired antigen binding protein (e.g., an anti-PD-1 antibody) and manipulating the nucleic acid by recombinant DNA techniques. The nucleic acid may also be fused to another nucleic acid as desired, or mutated (e.g., by mutagenesis or other conventional techniques), e.g., by adding, deleting or substituting one or more amino acid residues.

[0126] Single chain antibodies can be formed by linking heavy and light chain variable region (Fv region) fragments via an amino acid bridge (short peptide linker) to obtain a single polypeptide chain. The single chain Fv (scFv) is composed of two variable region polypeptides (V L and V H These antibodies are created by fusing DNA encoding a peptide linker between DNA encoding the two variable regions. The resulting polypeptides can fold to form antigen-binding monomers or multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable regions (Korttetal., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). L and V H By combining polypeptides containing the following, multimeric scFvs that bind different epitopes can be formed (Kriangkumetal., 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single chain antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf et al., 2002, Methods Mol.Biol.178:379-87.

[0127] Techniques for obtaining antibodies of a different subclass or isotype from a desired antibody, i.e. subclass switching, are known. Thus, for example, it is possible to obtain IgG antibodies from IgM antibodies and vice versa. Such techniques make it possible to generate novel antibodies that have the antigen-binding properties of a particular antibody (parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be used. Cloned DNA encoding a particular antibody polypeptide, for example DNA encoding the constant region of an antibody of a desired isotype, may be used in such techniques (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Furthermore, IgG4 antibodies may be generated. Inter-heavy chain disulfide bonds that can lead to heterogeneity in IgG4 antibodies It may also be desirable to introduce a point mutation in the hinge region (CPSCP→CPPCP) to reduce the propensity for the formation of .alpha.-aminobutyric acid (Bloom et al., 1997, Protein Science 6:407).

[0128] In certain embodiments, the antigen binding protein of the present invention has an affinity for PD-1 of at least 10 6 Binding affinity (K a In other embodiments, the antigen binding protein has at least 10 7 , at least 10 8 , at least 10 9 Or at least 10 10 K of M a Other aspects The antigen-binding protein has substantially the same K as the antibodies described in the Examples herein. a Shows.

[0129] In another embodiment, the present invention provides an antigen binding protein that has a slow dissociation rate from PD-1. -4 ~1X10 -1 M or lower IK off In another embodiment, K off is 5X10 -5 ~5X10 -1 M or it In other embodiments, K off In another embodiment, the antigen binding protein has a K that is substantially the same as the antibodies described herein. off It binds to PD-1.

[0130] In another aspect, the present invention provides an antigen binding protein that inhibits the activity of PD-1. In one embodiment, the antigen binding protein has an IC 50 In another embodiment, the IC 50 is 100 nM or less; in other embodiments, the IC 50 In other embodiments, the IC 50 is the IC of the antibody described in the Examples herein. 50 In other embodiments, the antigen binding protein has an IC substantially the same as the antibodies described herein. 50 inhibits the activity of PD-1.

[0131] In another aspect, the invention provides antigen binding proteins that bind to human PD-1 expressed on the surface of a cell and, upon such binding, inhibit PD-1 signaling activity within the cell without significantly decreasing the amount of PD-1 on the cell surface. Any method for determining or assessing the amount of PD-1 on the surface and / or inside a cell may be used. In another embodiment, binding of the antigen binding protein to a PD-1 expressing cell reduces internalization of cell surface PD-1 by less than about 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1% or 0.1%. Reduce.

[0132] In another aspect, the present invention provides antigen binding proteins having a half-life in vitro or in vivo (e.g., when administered to a human subject) of at least one day. In one embodiment, the antigen binding protein has a half-life of at least three days. In another embodiment, the antigen binding protein has a half-life of four days or more. In yet another embodiment, the antigen binding protein has a half-life of eight days or more. In yet another embodiment, the antigen binding protein is derivatized or modified to enhance its half-life relative to the underivatized or unmodified antigen binding protein. In yet another embodiment, the antigen binding protein is modified with one or more properties to enhance serum half-life, e.g., as described in WO 00 / 09560. including mutations therein, which is incorporated herein by reference.

[0133] The present invention further provides multispecific antigen-binding proteins, e.g., bispecific antigen-binding proteins, e.g., antigen-binding proteins that bind to two different epitopes of PD-1, or to an epitope of PD-1 and an epitope of another molecule, via two different antigen-binding sites or regions. Additionally, the bispecific antigen-binding proteins described herein may contain a PD-1 binding site from one of the antibodies described herein and a second PD-1 binding region from another antibody described herein, including the antibodies incorporated herein by reference. Alternatively, the bispecific antigen-binding proteins may contain an antigen-binding site from one of the antibodies described herein and a second antigen-binding site from another PD-1 antibody known in the art, or an antibody made by methods known in the art or methods described herein.

[0134] Many methods for producing bispecific antibodies are known in the art. The use of hybrid-hybridomas and antibodies described by E. et al., 1983, Nature 305:537. Bispecific antibodies can also be constructed by recombinant methods, such as by chemical coupling of antibody fragments (Brenna et al., 1985, Science 229:81; Glennie et al., 1987, J. Immunol. 139:2367; U.S. Patent No. 6,010,902). Additionally, bispecific antibodies can be constructed by recombinant methods, such as by coupling leucine zipper moieties (i.e., preferentially heterodimers). The leucine zipper moieties from the Fos and Jun proteins form the .alpha.-aminobutyric acid esterase (AAS) complex; Kostelny et al., 1992, J. Immunol. 148:1547) or other important methods such as those described in U.S. Pat. No. 5,582,996. Such interacting domain structures may also be used to generate complexes. Additional useful techniques include those described in U.S. Patent Nos. 5,959,083 and 5,807,706.

[0135] In other aspects, the antigen binding protein includes a derivative of an antibody. Such a derivative antibody may contain any molecule or substance that provides the antibody with a desired property, such as extended half-life in a particular application. Such a derivative antibody may include, for example, a detectable (or labeled) moiety (e.g., a radioactive, chromogenic, antigenic or enzymatic molecule), a detectable bead (e.g., Magnetic or electrodense beads (e.g. gold beads) or other molecules The antibody may comprise a molecule that binds to a nucleotide sequence (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic or pharma- ceutically active moiety), or a molecule that enhances the suitability of the antibody for a particular use (e.g., administration to a subject, e.g., a human subject, or other in vivo or in vitro use). Examples of molecules that can be used to derivatize an antibody include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylated derivatives of antibodies can be made using techniques well known in the art. In one embodiment, the antibody may be conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant may be chemically modified, for example, with a chemical selected from the group consisting of dextran, poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, and polyvinyl alcohol.

[0136] Indications The present invention provides methods of treating a subject to treat, prevent, cure, alleviate or ameliorate ("treatment") a disease, disorder, condition or illness ("condition"). The conditions to be treated include those characterized by inappropriate expression or activity of PD-1. Such conditions include those in which the level of expression or activity is too high, and treatment includes administering a PD-1 antagonist. The disorder or condition is associated with cancer. In particular, such cancers include, but are not limited to, lung, ovarian and colon cancers and various myelomas.

[0137] Particular medical conditions and diseases that can be treated or prevented with the antigen binding proteins described herein include various cancers.

[0138] Methods of Treatment and Administration of Antigen Binding Proteins Certain methods described herein involve administering to a subject an antigen binding protein that binds PD-1, thereby reducing a PD-1-induced biological response that functions in a particular condition. In certain embodiments, the methods of the invention involve contacting endogenous PD-1 with an antigen binding protein that binds PD-1, e.g., by administration to a subject or by ex vivo techniques.

[0139] The term "treatment" includes alleviation or prevention of at least one symptom or other aspect of a disorder, or reducing the severity of a disease, etc. An antigen binding protein need not effect a complete cure or eradicate all symptoms or manifestations of a disease to constitute a viable therapeutic. As recognized in the relevant art, a drug used as a therapeutic need only reduce the severity of a particular disease state, not eradicate all manifestations of the disease, to be considered a useful therapeutic. Similarly, a treatment administered prophylactically need not be completely effective in preventing the onset of symptoms to constitute a viable prophylactic. It is sufficient to simply reduce the impact of the disease (e.g., by reducing the number or severity of symptoms, or by increasing the effectiveness of other treatments, or by producing other beneficial effects) or reduce the likelihood that the disease will develop or worsen in a subject. One embodiment of the present invention relates to a method comprising administering to a patient a PD-1 antagonist in an amount and for a duration sufficient to cause a sustained improvement over baseline in an indicator reflecting the severity of such a particular disorder.

[0140] As understood in the relevant art, the pharmaceutical compositions of the present invention, including antibodies and fragments thereof, are administered to a subject in a manner appropriate for the indication. The pharmaceutical compositions may be administered by any suitable technique, including, but not limited to, parenteral, topical, or inhalation administration. When injected, the pharmaceutical compositions may be administered, for example, via intraarticular, intravenous, intramuscular, intralesional, intraperitoneal, or subcutaneous routes, by bolus injection or continuous infusion. Local administration, for example, to the site of suspected disease or injury, is transdermal administration and sustained release from implants. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antagonist in aerosol form, and the like. Other alternatives include eye drops; oral formulations including pills, syrups, lozenges, or chewing gum; and topical formulations such as lotions, gels, sprays, and ointments.

[0141] The use of antigen binding proteins in ex vivo procedures is also contemplated. For example, a patient's blood or other bodily fluid may be contacted with an antigen binding protein that binds to PD-1 ex vivo. The antigen binding protein may be bound to a suitable insoluble matrix or solid support material.

[0142] The antigen binding protein is advantageously administered in the form of a composition that includes one or more additional components, such as a physiologically acceptable carrier, excipient, or diluent. The composition may include one or more physiologically effective agents, such as a second inflammatory or immune inhibitor, an anti-angiogenic agent, an analgesic agent, etc., non-limiting examples of which are described herein. In various specific embodiments, the composition includes one, two, three, four, five, or six physiologically effective agents in addition to the PD-1 binding antigen binding protein.

[0143] Combination therapy The present invention provides methods of treating a subject with a PD-1 inhibitory antigen binding protein and one or more other treatments. Such combination therapy can achieve synergistic or additive effects, for example, by attacking multiple sites or molecular targets in a tumor. Types of combination therapy that can be used include inhibiting or activating a single disease-related pathway, multiple pathways in a target cell, and multiple nodes in multiple cell types within a target tissue (as appropriate). Examples include those who are not good at English.

[0144] A method of combination therapy includes administering to a subject two, three, four, five, six or more PD-1 agonists or antagonists described herein. In other embodiments, the method includes administering to a subject two or more treatments that together inhibit or activate (directly or indirectly) PD-1 mediated signaling. Exemplary methods include administering to a subject two or more PD-1 inhibitory antigen binding proteins, one PD-1 inhibitory antigen binding protein and one or more other anti-cancer therapeutic agents (e.g., cytotoxic agents and / or immunomodulatory agents), or one or more other anti-cancer therapeutic agents. and one or more other treatments (e.g., surgery or radiation). In addition, one or more anti-PD-1 antibodies or antibody derivatives may be used in combination. , may be used in combination with one or more molecules or other treatments, where such other molecules and / or treatments do not directly bind or affect PD-1, but in combination are effective in treating or preventing the condition being treated. In one embodiment, the one or more molecules and / or treatments treat or prevent symptoms caused by the other one or more molecules or treatments during the course of treatment, such as nausea, fatigue, hair loss, cachexia, insomnia, etc. In all cases where a combination of molecules and / or other treatments is used, the individual molecules and / or treatments may be given in any order and for any period that is effective, such as simultaneously, sequentially, or alternating. In one embodiment, the method of treatment includes initiating a second course of treatment after a first course of treatment with one molecule or other treatment is completed. The period between the end of the first course of treatment and the start of the second course of treatment may be any period during which the courses of treatment may be generally effective, such as seconds, minutes, hours, days, weeks, months, or years.

[0145] Example 1 The ability of anti-PD-1 antibodies to modulate immune responsiveness was evaluated using mixed lymphocyte reactions (MLR). This assay measured the effect of anti-PD-1 antibody (RG1H10) on cell activation and the production of both IL-2 and interferon gamma. Purified human CD4+ cells x 10 5 10 monocyte-derived dendritic cells prepared from another donor 4 To prepare dendritic cells, purified monocytes were cultured with GM-CSF (1,000 U / ml) and IL-4 (500 U / ml) for 7 days. The -1 antibody or a control antibody was added to the allogeneic MLR culture at 10 μg / ml. ELISA kits (Biolegend) were used to measure IL-2 and IFNγ, respectively. Plates were set up in parallel to allow for collection of supernatants on days 2 or 3, and day 5. Remaining cells on day 5 were assessed for CD25 expression as an indicator of cell activation.

[0146] The results of cell activation are shown in Figure 1. For all anti-PD-1 antibodies, cell activation In Figure 1, data were obtained without any added antibody. The values ​​are expressed as percentages of the values ​​in the table. The vertical axis values ​​are %CD25+, which represents the activation of cells as a % increase over the medium control. By this method, the % increase in cell activation was expressed. Supernatants were harvested from the cultures and either IL-2 was measured on day 3 (Figure 2) or interferon gamma was measured on day 5 (Figure 3). In Figure 2, the vertical axis values ​​are IFNgamma (pg / ml). In Figure 3, The vertical axis indicates IL-2 (pg / ml). The production of both cytokines was increased by the addition of anti-PD-1 antibody.

[0147] Example 2 This example shows in vitro data for cell binding EC50 measurements and cell binding specificity. More specifically, this example shows the binding characteristics of anti-PD-1 antibodies (shown in FIG. 4) for specific cell binding and the concentration at which 50% binding saturation (EC50) is reached. In this example, anti-PD-1 antibodies The D-1 antibody was compared with BMS5C4, a commercially available therapeutic anti-PD-1 antibody. The procedure was as follows: CHO-PD-1 and CHO-PD-L1 cells were cultured with CHO-K1 cells. PD-1 cDNA ORF clone (Origene RG210364) and PD-L1 cDNA The ORF clones (Origene RG213071) were obtained by stably transducing the ORF clones, respectively. After selection with neomycin for 2 weeks, the clones were isolated by fluorescence activated cell sorting (FACS) and further expanded. The ES-2 cell line was established from ovarian cancer (ATCC CRL-1978), which showed For cell binding assays, 50,000 CHO-PD-1 cells, CHO-PD-L1 cells and ES-2 cells were added to a 96-well, v-bottom plate in 100μl FACS buffer (PBS+2% FBS). 12-point dilutions of each anti-PD-1 antibody were The curve (x3) was run at 50 μg / ml (3.33x10 -7 M) was used as a starting point. Spin down, wash twice with FACS buffer, and resuspend in 25 μl of antibody solution. After 0.5 h of incubation, cells were washed once with FACS buffer. After washing, the cells were incubated with a PE-conjugated goat anti-human IgG (γ-chain specific) secondary antibody (Southern Biotech Catalog The cells were then resuspended in 50 μl of PBS (No. 2040-09). The cells were then incubated for an additional 0.5 h. Afterwards, the cells were washed once with FACS buffer. The cells were resuspended in 25 μl of FACS buffer. The median fluorescence intensity (MFI) in the FL2-H channel was then determined using an Intellicyt HTFC flow cytometer. Data were analyzed using nonlinear regression fits. Analysis and plotting was performed in GraphPad Prism.

[0148] The antibodies used were GA2, RG1B3, RG1H10 and RG2A7, RG2H10, SH-A4, RG4A6 and RG6B5, as disclosed herein, and were compared with 5C4 (Bristol-Myers-Squibb / Medarex), an antibody described in the published literature that was produced in-house based on antibody sequences described in the published literature (U.S. Patent Application Publication No. 2009 / 0217401, the contents of which are incorporated herein by reference).

[0149] Results: As shown in Figure 4 and Table 1, the anti-PD-1 antibody was inhibited in CHO-PD-1 cells. The binding EC50 was determined to range from 36 pM to 2802 pM. Data points on the graph represent antibody concentrations. The mean fluorescence intensity (MFI) of positively labeled cells is shown ± standard error. For cells that do not express PD-L1 (CHO-PD-L1 cells and ES-2 cells), no nonspecific binding was observed. I couldn't. [Table 1]

[0150] Example 3 This example demonstrates the analysis of the inhibition of recombinant PD-1 binding to recombinant PD-L1 by anti-PD-1 antibodies (Figure 5). Briefly, ELISA plates were coated with PD-L1-His and blocked with casein in PBS, followed by pre-incubation with a mixture of anti-PD-1 antibodies (scFv) and recombinant human PD-1 protein. The remaining binding of PD-1 protein to PD-L1 protein was measured.

[0151] The antibodies used were GA1, GA2, GB1, GB6, GH1, A2, C7, H7, SH-A4, SH-A9, RG1B3, RG1H10 and RG1H11, as described herein, and were compared to the published antibody 5C4 (Bristol-Myers-Squibb / Medarex), which was produced in-house from a published antibody sequence (U.S. Patent Application Publication No. 2009 / 0217401, the contents of which are incorporated herein by reference).

[0152] Results: The anti-PD-1 antibody has the ability to block the interaction between recombinant human PD-1 and PD-L1 proteins.

[0153] Example 4 This example provides a comparison of affinity measurements of a particular anti-PD-1 antibody, RG1H10, as immunoglobulin gamma 1 (IgG1) and immunoglobulin gamma 4 (IgG4) with a stabilizing mutation that changes the serine residue at position 228 in the antibody hinge region to a proline (S228P) using BIAcore methodology. Briefly, a CM5 chip was coated with anti-human Fc antibody, the anti-PD1 mAb was captured, and serially diluted. Recombinant human PD1 (His-tagged) protein was used for the analysis.

[0154] Results: As shown in Table 2, the human anti-PD-1 antibody RG1H10 has excellent kinetic properties for recognizing and binding to human PD-1 as IgG1 or IgG4(228P). [Table 2]

[0155] Example 5 This example shows in vitro data for cell binding EC50 measurements. More specifically, this example shows the binding characteristics of anti-PD-1 antibodies as IgG1 or IgG4 (S228P) for cell binding and the concentration that reaches 50% binding saturation (EC50). The procedure is as follows: Peripheral blood mononuclear cells (PBMCs) were added to a 24-well plate and stimulated with anti-CD3 (3ng / ml). After culturing the cells for 3 days, they were harvested and added to wells of a 96-well v-bottom plate in 100μl FACS buffer (PBS + 2% FBS). A 12-point dilution curve (x3) of each anti-PD-1 antibody was run in FACS buffer at 10μg / ml (0.6x10 -7 M). Cells were spun down, washed twice with FACS buffer, and resuspended in 25 μl of antibody solution in triplicate. After 0.5 h incubation, cells were washed once with FACS buffer and incubated with PE-conjugated goat antibody. The cells were resuspended in 50 μl of anti-human IgG (γ-chain specific) secondary antibody (Southern Biotech Catalog No. 2040-09). The cells were incubated for a further 0.5 h and then washed once with FACS buffer. The cells were resuspended in 25 μl of FACS buffer and analyzed using an Intellicyt HTFC flow cytometer. The MFI of the FL2-H channel was determined using a chromatograph. The data was analyzed using a nonlinear regression fit. , Analysis and plotting were performed in Graph Pad Prism.

[0156] Results: As shown in Table 3, the cell binding of anti-PD-1 antibodies to activated peripheral blood mononuclear cells (PBMCs) was The combined EC50 was determined to be 47 (pM) for the IgG4 (S228P) isotype and 670 pM for the IgG1 version. [Table 3]

[0157] Example 6 This example demonstrates the ability of RG1H10 to specifically bind to recombinant PD-1 but not to other related proteins (Figure 6). Briefly, ELISA plates coated with the appropriate recombinant proteins, i.e., human PD-1, cynomolgus PD-1, human CTLA-4, human CD28, and human ICOS, were blocked with casein in PBS and then incubated with an anti-PD-1 antibody (RG1H10). Binding of RG1H10 was measured.

[0158] Results: The anti-PD-1 antibody RG1H10 shows excellent specificity for (human or cynomolgus monkey) PD-1 but does not recognize PD-1-associated proteins.

[0159] Example 7 Using a mixed lymphocyte reaction (MLR), we measured the ability of two isotypes of RGH10, IgG1 and IgG4(S228P), to modulate immune responses. The effects on cell activation and IL-2 and interferon gamma production were measured. The MLR consisted of 10 purified human CD4+ cells from a single donor. 5 10 monocyte-derived dendritic cells prepared from another donor 4 To prepare dendritic cells, The purified monocytes were incubated with GM-CSF (1,000 U / ml) and IL-4 (500 U / ml) for 7 days. Unless otherwise stated, anti-PD-1 antibodies or control antibodies were administered to allogeneic MLR cultures. IL-2 and IL-3 were detected using a commercially available ELISA kit (Biolegend). Plates were set up in parallel so that supernatants could be harvested on days 2 or 3, and 5, to measure IFNγ, respectively. The remaining cells on day 5 were assessed for CD25 expression, as an indicator of cell activation.

[0160] The results for cell activation are shown in Figures 7, 8 and 9. Both anti-PD-1 antibody isotypes For the treatment with anti-PD-1 antibody, an increase in cell activation was observed (Figure 7). The data are expressed as a percentage of the test values ​​obtained without adding any antibody. In this way, the % increase in cell activation was expressed. Supernatants were collected from the cultures and on day 3, IL-2 (Figure 8) or interferon gamma was measured (Figure 9). The production of both cytokines was similarly increased by the addition of anti-PD-1 antibody.

[0161] Example 8 The heavy chain sequences were further optimized by an affinity maturation process: specifically, affinity maturation of antibody RG1H10 (heavy chain SEQ ID NO: 23) yielded 12 additional heavy chain sequences (SEQ ID NO: 37-49) as listed in the sequence listing at the end, together with the light chain SEQ ID NO: 24.

[0162] Example 9 This example demonstrates a PD-1 blocking ELISA assay using the antibodies shown in Figure 5. ELISA plates were coated with PD-L1-His and blocked with casein in PBS, followed by pre-incubation mixtures of scFv and PD1 / Fc (12.5 μl 10 μg / ml PD1 / Fc+ 12.5 μl A phage soup (1:200 dilution of anti-human Fc-HRP in casein) was added. Incubation was for 1 hour and washed 3 times with PBS. Anti-human Fc-HRP diluted 1:200 in casein was added. Incubation was for 30 minutes and washed 3 times with PBS. TMB was used as substrate and the reaction was stopped with 2MH2SO4. OD 450nm was measured. All antibodies tested, except GA2 and H7, blocked the PD-1 receptor. .

[0163] Example 10 This example shows a PD-1 blocking assay using the antibodies shown in Figure 5. Plates were coated with PD-L1-His and blocked with casein in PBS, followed by the addition of scFv and A preincubation mixture of 0.7 μg / ml PD1 / Fc and 12.5 μl phage soup was added. The cells were incubated for 1 hour and washed three times with PBS. Anti-human Fc-HRP diluted 1:500 was added to the medium. The mixture was incubated for 30 minutes and washed three times with PBS. TMB was used as a substrate and the reaction was stopped with 2M H2SO4. The OD450nm values ​​were Measured.

[0164] Example 11 This example demonstrates the presence or absence of cross-reactivity between human and mouse anti-PD-1 antibodies shown in Figure 10. 50 μl of 1 μg / ml human PD-L1 / His or mouse PD1 / His (control: PBS) was loaded onto Ni-NTA plate, incubated for 1 hour, washed 3 times with PBS, and diluted with casein. IgG diluted 1:300 in casein was added. Incubated for 30 minutes and washed 3 times with PBS. Goat anti-human Fc-HRP diluted 1:300 in casein was added. Incubated for 30 minutes and washed 3 times with PBS. The reaction was stopped with 2MH2SO4 using TMB as substrate and OD 450nm was measured.

[0165] Example 12 This example shows a comparison of the epitopes of antibodies 5C4 and RG1H10. The PD1 / His was captured on the sensor. The sensor was then immersed in a well containing PBS and RG1H10.

[0166] Example 12 This example shows a comparison of the affinities of the anti-PD-1 antibodies described herein. Anti-human Fc antibodies were immobilized on a CM5 sensor chip at approximately 1000 RU. Antibodies (approximately 10 μg / ml) were captured for 60 seconds at a flow rate of 10 μl / min. PD1 / His was serially diluted in running buffer (HBS-EP). Measurements were performed. All runs were performed at a flow rate of 30 μL / min. Surfaces were regenerated with 3M MgCl2 for 60 seconds. 1:1 (Langmuir) A binding model was fitted to the data. [Table 4]

[0167] Example 13 This example demonstrates the target (PD-1) specificity of the RG1H10 antibody relative to three competing anti-PD-1 antibodies. Using an in vitro ELISA assay with either an anti-human IgG4 heavy chain or an anti-human λ light chain detection reagent, RG1H10 was specifically shown to be specific to recombinant human and cynomolgus monkey PD-1. It can be confirmed that RG1H10 binds to human and cynomolgus monkey PD-1 but not to mouse PD-1. 50The values ​​were 0.391 nM and 0.840 nM, respectively, and were significantly lower than the three reference antibodies tested, Competitive Antibody 1 (human EC 50 0.419 nM, Crab Kuizal EC 50 1.02 nM), competitor antibody 2 (human EC 50 0.495nM, cynomolgus EC 50 0.773 nM) and competitor antibody 3 (human EC 50 0.390 nM, cynomolgus EC 50 1.295nM) was essentially equivalent. In addition, RG1H10 binds other structurally related family members, such as human CTLA4. , but not to human CD28 or human ICOS. These results indicate that RG1H10 has unique targeting specificity for PD-1.

[0168] Example 14 This example demonstrates the affinity characterization of three antibodies using Biacore affinity characterization and cell-based binding assays. These studies demonstrate the target (PD-1) affinity of the RG1H10 antibody against competitor PD-1 antibodies. For Biacore affinity measurements, anti-PD-1 antibodies were captured on a Protein-A immobilized CM5 sensor chip, and then monomeric recombinant human PD-1 was flowed over the chip. Under these conditions, RG1H10 showed an affinity (KD) of 3.2 nM against competitor 1 (7.0 nM), competitor 2 (3 nM), and competitor 3 (42 nM). These studies demonstrate that RG1H10 has comparable affinity to competitors 1 and 2. It exhibits target affinity, with an affinity approximately 10-fold higher than that of competitor molecule 3.

[0169] In addition, we measured the binding of RG1H10 and three reference antibodies to human PD-1 expressed on the surface of T cells using a human Jurkat T cell line that stably expresses high levels of human PD-1. Binding of the primary antibody to Jurkat PD-1 cells was detected by flow cytometry using either anti-heavy or anti-light chain antibodies directly conjugated to fluorochromes (identical binding curves were obtained with either detection reagent). In this assay, RG1H10 bound to membrane-expressed PD-1 with an EC50 of 25.76 nM compared to competitor 1 (5.18 nM), competitor 2 (4.57 nM), and competitor 3 (8.83 nM). 50 It was bound by value.

[0170] During these studies, we observed a shift in the maximum binding of PD-1 receptor molecules to various anti-PD-1 antibodies. RG1H10 showed a C of 1103 mean fluorescence intensity (MFI) units. max which is higher than the value observed for competing antibody 2 (C max : 1110 MFI) and higher than that observed for competitor antibody 3 (C max : 789 MFI), approximately half the value observed for competitor antibody 1 (C max In summary, RG1H10 was as potent as all the reference antibodies tested. , binds to membrane-expressed human PD-1.

[0171] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0172] Furthermore, the present invention includes the following aspects. Item 1. A fully human antibody of the IgG class that binds to a PD-1 epitope with a binding affinity of at least 10-6M, comprising any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; A fully human antibody having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof.

[0173] Item 2. The fully human antibody according to claim 1, comprising SEQ ID NO:1 / SEQ ID NO:2 (referred to as GA1), SEQ ID NO:3 / SEQ ID NO:4 (referred to as GA2), SEQ ID NO:5 / SEQ ID NO:6 (referred to as GB1), SEQ ID NO:7 / SEQ ID NO:8 (referred to as GB6), SEQ ID NO:9 / SEQ ID NO:10 (referred to as GH1), SEQ ID NO:11 / SEQ ID NO:12 (referred to as A2), SEQ ID NO:13 / SEQ ID NO:14 (referred to as C7), SEQ ID NO:15 / SEQ ID NO:16 (referred to as H7), SEQ ID NO:17 / SEQ ID NO:18 (referred to as SH-A4), SEQ ID NO:19 / SEQ ID NO:20 (referred to as SH-A9) (referred to as RG1B3), SEQ ID NO:23 / SEQ ID NO:24 (referred to as RG1H10), SEQ ID NO:25 / SEQ ID NO:26 (referred to as RG1H11), SEQ ID NO:27 / SEQ ID NO:28 (referred to as RG2H7), SEQ ID NO:29 / SEQ ID NO:30 (referred to as RG2H10), SEQ ID NO:31 / SEQ ID NO:32 (referred to as RG3E12) , SEQ ID NO:33 / SEQ ID NO:34 (referred to as RG4A6), SEQ ID NO:35 / SEQ ID NO:36 (referred to as RG5D9), SEQ ID NO:37 / SEQ ID NO:24 (referred to as RG1H10-H2A-22-15), SEQ ID NO:38 / SEQ ID NO:24 (referred to as RG1H10-H2A-27-25), SEQ ID NO:39 / SEQ ID NO:24 (referred to as RG1H10-3C), SEQ ID NO:40 / SEQ ID NO: No. 24 (referred to as RG1H10-16C), SEQ ID NO: 41 / SEQ ID NO: 24 (referred to as RG1H10-17C), SEQ ID NO: 42 / SEQ ID NO: 24 (referred to as RG1H10-19C), SEQ ID NO: 43 / SEQ ID NO: 24 (referred to as RG1H10-21C), SEQ ID NO: 44 / SEQ ID NO: 24 (referred to as RG1H10-23C2) and combinations thereof. A fully human antibody having heavy chain / light chain variable region sequences as set forth above.

[0174] Item 3. A Fab fully human antibody fragment having a variable region sequence derived from a heavy chain and a variable region sequence derived from a light chain, comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; A Fab fully human antibody fragment having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof.

[0175] Item 4. The fully human antibody Fab fragment according to claim 3, wherein the antibody is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, Sequence number 17 / SEQ ID NO:18, sequence number 19 / SEQ ID NO:20, sequence number 21 / SEQ ID NO:22, sequence number 23 / SEQ ID NO:24, sequence number 25 / SEQ ID NO:26, sequence number 27 / SEQ ID NO:28, sequence number 29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24 A fully human antibody Fab fragment having heavy chain / light chain variable region sequences selected from the group consisting of SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and combinations thereof.

[0176] Item 5. A single-chain human antibody having a variable region derived from a heavy chain and a variable region derived from a light chain, and a peptide linker connecting the variable region sequences of the heavy chain and the light chain, comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; A single chain human antibody having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof.

[0177] Item 6. The fully human single chain antibody according to claim 5, comprising SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO: No. 11 / SEQ ID NO: 12, No. 13 / SEQ ID NO: 14, No. 15 / SEQ ID NO: 16, No. 17 / SEQ ID NO: 18, No. 19 / SEQ ID NO: 20, No. 21 / SEQ ID NO: 22, No. 23 / SEQ ID NO: 24, Sequence number 25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 A fully single-chain human antibody having a heavy chain / light chain variable region sequence selected from the group consisting of:

[0178] Item 7. A fully human single-chain antibody according to claim 5, which has both a heavy chain variable region and a light chain variable region, comprising SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28. , SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24, and combinations thereof.

[0179] Item 8. A method for treating a wide range of mammalian cancers or inflammatory or autoimmune diseases, comprising administering an effective amount of an anti-PD-1 polypeptide, the anti-PD-1 polypeptide is selected from the group consisting of a fully human antibody of the IgG class, a Fab fully human antibody fragment having a variable region from a heavy chain and a variable region from a light chain, a single chain human antibody having a variable region from a heavy chain and a variable region from a light chain and a peptide linker connecting the variable regions of the heavy and light chains, and combinations thereof, which bind to a PD-1 epitope with a binding affinity of at least 10-6M; The fully human antibody is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof; The Fab fully human antibody fragment is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; having a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and combinations thereof; The single chain human antibody is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, having a heavy chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof; The method of treatment has a light chain variable region sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 and combinations thereof.

[0180] Item 9. The method for treating a wide range of mammalian cancers according to claim 8, The fully human antibody is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20 , SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and combinations thereof.

[0181] Item 10. The method for treating a wide range of mammalian cancers according to claim 8, The fully human antibody Fab fragment has both a heavy chain variable region and a light chain variable region, and the antibody is selected from the group consisting of SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, S No. 8, SEQ ID NO: 9 / SEQ ID NO: 10, SEQ ID NO: 11 / SEQ ID NO: 12, SEQ ID NO: 13 / SEQ ID NO: 14, SEQ ID NO: No. 15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO: SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, A heavy chain selected from the group consisting of SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO:44 / SEQ ID NO:24 and combinations thereof. / A method of treatment comprising administering to said patient a light chain variable region sequence.

[0182] Item 11. The method for treating a wide range of mammalian cancers according to claim 8, The fully human single chain antibody has both a heavy chain variable region and a light chain variable region, and is SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO: No. 37 / SEQ ID NO:24, SEQ ID NO:38 / SEQ ID NO:24, SEQ ID NO:39 / SEQ ID NO:24, SEQ ID NO:40 / SEQ ID NO:24, SEQ ID NO:41 / SEQ ID NO:24, SEQ ID NO:42 / SEQ ID NO:24, SEQ ID NO:43 / SEQ ID NO:24, SEQ ID NO: A heavy / light chain variable region selected from the group consisting of SEQ ID NO: 44 / SEQ ID NO: 24 and combinations thereof Methods of treatment having sequences.

[0183] Item 12. The method for treating a wide range of mammalian cancers or inflammatory or autoimmune diseases according to claim 8, wherein the wide range of mammalian cancers to be treated are selected from the group consisting of ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastoma-derived CNS tumors, monocytic leukemia, B cell-derived leukemia, T cell-derived leukemia, B cell-derived lymphoma, T cell-derived lymphoma, mast cell-derived tumors, and combinations thereof.

[0184] Item 13. The method for treating a wide range of mammalian cancers or inflammatory or autoimmune diseases according to claim 8, wherein the wide range of autoimmune or inflammatory diseases is selected from the group consisting of intestinal mucosal inflammation, wasting diseases associated with colitis, multiple sclerosis, systemic lupus erythematosus, viral infections, rheumatoid arthritis, osteoarthritis, psoriasis, Crohn's disease and inflammatory bowel disease.

Claims

1. (i) an antibody or antigen-binding fragment thereof that binds to a PD-1 epitope, the antibody or antigen-binding fragment thereof comprising: The heavy chain variable region sequence of SEQ ID NO:33; and Light chain variable region sequence of SEQ ID NO:34 wherein the PD-1 epitope is a human PD-1 epitope. An antibody or antigen-binding fragment thereof; (ii) an isolated anti-PD-1 antibody or antigen-binding fragment thereof, A heavy chain variable region comprising the complementarity determining regions set forth in the heavy chain variable region amino acid sequence of SEQ ID NO: 33, A light chain variable region comprising the complementarity determining region shown in the light chain variable region amino acid sequence of SEQ ID NO:

34. Including, the complementarity determining regions in the heavy chain variable region and the complementarity determining regions in the light chain variable region are identified using the Kabat numbering system or the IMGT numbering system, and the PD-1 is human PD-1; An anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) an anti-PD-1 single chain human antibody comprising a heavy chain variable region and a light chain variable region connected by a peptide linker, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34; the PD-1 epitope is a human PD-1 epitope; anti-PD-1 single chain human antibody; and (iv) an isolated anti-PD-1 antibody or antigen-binding fragment thereof, a heavy chain variable region comprising a complementarity determining region as set forth in the heavy chain variable region amino acid sequence of SEQ ID NO: 33, wherein the heavy chain variable region is at least 95% identical to the amino acid sequence of SEQ ID NO: 33; a light chain variable region comprising a complementarity determining region as set forth in the light chain variable region amino acid sequence of SEQ ID NO: 34, wherein said light chain variable region is at least 95% identical to the amino acid sequence of SEQ ID NO: 34; the complementarity determining regions in the heavy chain variable region and the complementarity determining regions in the light chain variable region are identified using the Kabat numbering system or the IMGT numbering system, and the PD-1 is human PD-1; Anti-PD-1 antibody or antigen-binding fragment thereof An anti-PD-1 binding agent selected from the group consisting of:

2. 2. The anti-PD-1 binding agent of claim 1, wherein the antibody that binds to a PD-1 epitope or the isolated anti-PD-1 antibody is a fully human antibody of the IgG class.

3. 2. The anti-PD-1 binding agent of claim 1, wherein the antigen-binding fragment of an antibody that binds to a PD-1 epitope, or the isolated antigen-binding fragment of an anti-PD-1 antibody, is a Fab fragment of a fully human antibody.

4. 13. A pharmaceutical composition comprising the anti-PD-1 binding agent of claim 1 and a pharma- ceutically acceptable excipient.

5. The pharmaceutical composition according to claim 4 for treating a subject with cancer.

6. 6. The pharmaceutical composition of claim 5, wherein the cancer is selected from the group consisting of ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastoma-derived CNS tumors, monocytic leukemia, B cell-derived leukemia, T cell-derived leukemia, B cell-derived lymphoma, T cell-derived lymphoma, skin cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, colorectal cancer, thyroid cancer, prostate cancer, kidney cancer, gastric cancer, and testicular cancer.

7. 13. One or more nucleic acids encoding the anti-PD-1 binding agent of claim 1.

8. The antibody or antigen-binding fragment thereof that binds to the PD-1 epitope, or the anti-PD-1 single chain antibody, a heavy chain variable region comprising the complementarity determining regions (CDRs) set forth in the heavy chain variable region amino acid sequence of SEQ ID NO:33; and A light chain variable region comprising the CDRs shown in the light chain variable region amino acid sequence of SEQ ID NO:

34.

8. The one or more nucleic acids of claim 7, comprising:

9. 9. One or more vectors comprising one or more nucleic acids according to claim 8.

10. A host cell comprising one or more nucleic acids according to claim 8.

11. A host cell comprising one or more vectors according to claim 9.

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