Anti-PD-1 antibodies, antigen-binding fragments thereof and their medical uses

Abstract

Anti-PD-1 antibodies, antigen-binding fragments thereof, and pharmaceutical uses thereof are provided. Specifically, humanized anti-PD-1 antibodies and antigen-binding fragments thereof comprising specific CDR regions, pharmaceutical compositions comprising the anti-PD-1 antibodies and antigen-binding fragments thereof, and their use as medicaments are provided. In particular, use of the humanized anti-PD-1 antibodies in the preparation of medicaments for treating PD-1-associated diseases or disorders is provided.

Description

[Technical field]

[0001] The present disclosure is in the field of biotechnology. More specifically, the present disclosure relates to an anti-PD-1 antibody and its use. [Background technology]

[0002] The statements made herein are merely intended to provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Tumor immunotherapy is a treatment method that fully utilizes and mobilizes the killer T cells of tumor patients to kill tumors. At the same time, tumor cell evasion is a major obstacle for tumor immunotherapy. Tumor cells use their own suppressive effect on the immune system to promote the rapid growth of tumors. There is a very complex relationship between tumor immune evasion mechanisms and the body's immune response to tumors. In the early stage of tumor immunotherapy, tumor-specific killer T cells have biological activity, but as the tumor grows, they lose their killing function in the later stage.

[0004] The activation of T cells in the human body employs a two-signaling pathway system. In addition to the first signal provided to T cells by the presentation of MHC-antigen peptides by antigen-presenting cells, a second signal provided by a set of costimulatory molecules is also necessary to enable T cells to generate a normal immune response. This dual signaling pathway system plays a key role in the balance of the immune system in the body. It tightly regulates the body's different immune responses to self- and non-self-antigens. In the absence of the second signal provided by costimulatory molecules, it causes non-responsiveness or persistent specific immune responses of T cells, thus resulting in immune tolerance. Thus, the second signaling pathway plays a very important regulatory role in the entire process of the body's immune response.

[0005] Programmed death-l (PD-l) is a protein receptor expressed on the surface of T cells, discovered in 1992, and is involved in the process of cell apoptosis. PD-l belongs to the CD28 family. PD-l shares 23% amino acid homology with cytotoxic T-lymphocyte antigen 4 (CTLA-4), but its expression differs from that of CTLA, and it is mainly expressed on activated T cells, B cells, and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed on T cells, B cells, macrophages, and dendritic cells (DCs), and its expression on cells can be upregulated after activation. PD-L2 expression is relatively restricted and is mainly expressed on antigen-presenting cells such as activated macrophages and dendritic cells.

[0006] PD-L1 inhibits the immune system by binding to PD-1 and B7-1. Many tumor cells and immune cells in the tumor tissue microenvironment express PD-L1. New research has revealed that high expression of PD-L1 protein has been detected in breast cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, intestinal cancer, kidney cancer, melanoma, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer and other human tumor tissues, and the expression level of PD-L1 is closely related to the clinical symptoms and prognosis of patients.

[0007] Anti-PD-1 monoclonal antibodies can maximize the patient's own immune system's response to tumors by blocking the binding of PD-L1 / PD-1, achieving the goal of killing tumor cells. Currently, the anti-PD-1 antibodies pembrolizumab (also known as Merck Keytruda, Keytruda, Merck-Pemb, Merck-Keytruda, Merck-PD-1, pembrolizumab) and novilumab (also known as BMS Opdivo, Opdivo, BMS-Nivolumab, novilumab) are approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with melanoma, Hodgkin's lymphoma, non-small cell lung cancer and other tumors. In addition, patent documents such as WO200139722, WO2006121168, WO2010036959, WO2010089411, WO2011110604, WO2013173223, WO2013181634, US2014335093, US6803192B1, US8617546B2, and WO2015085847 also disclose various anti-PD-1 monoclonal antibodies. Summary of the Invention

[0008] The present disclosure provides novel anti-PD-1 antibodies, antigen-binding fragments thereof, and their medical uses.

[0009] In some alternative embodiments, the disclosure provides an anti-PD-1 antibody, or antigen-binding fragment thereof, selected from any one of the following i)-iii): i) an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises an HCDR1 as set forth in SEQ ID NO:8 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:8, an HCDR2 as set forth in SEQ ID NO:9 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:9, and an HCDR3 as set forth in SEQ ID NO:10 or having up to 8, 3, 2 or 1 amino acid mutations relative to SEQ ID NO:10; and the light chain variable region comprises an LCDR1 as set forth in SEQ ID NO:11 or having up to 4, 3, 2 or 1 amino acid mutations relative to SEQ ID NO:11, an LCDR2 as set forth in SEQ ID NO:12 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:12, and an LCDR3 as set forth in SEQ ID NO:13 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:13; ii) an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises an HCDR1 as set forth in SEQ ID NO:14 or having up to 3, 2, or 1 amino acid mutations relative to SEQ ID NO:14, an HCDR2 as set forth in SEQ ID NO:15 or having up to 3, 2, or 1 amino acid mutations relative to SEQ ID NO:15, and an HCDR3 as set forth in SEQ ID NO:16 or having up to 8, 3, 2, or 1 amino acid mutations relative to SEQ ID NO:16; and the light chain variable region comprises an LCDR1 as set forth in SEQ ID NO:17 or having up to 4, 3, 2, or 1 amino acid mutations relative to SEQ ID NO:17, an LCDR2 as set forth in SEQ ID NO:12 or having up to 3, 2, or 1 amino acid mutations relative to SEQ ID NO:12, and an LCDR3 as set forth in SEQ ID NO:18 or having up to 3, 2, or 1 amino acid mutations relative to SEQ ID NO:18; and iii) an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises an HCDR1 as set forth in SEQ ID NO:21 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:21, an HCDR2 as set forth in SEQ ID NO:22 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:22, and an HCDR3 as set forth in SEQ ID NO:23 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:23; and the light chain variable region comprises an LCDR1 as set forth in SEQ ID NO:24 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:24, an LCDR2 as set forth in SEQ ID NO:25 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:25, and an LCDR3 as set forth in SEQ ID NO:26 or having up to 3, 2 or 1 amino acid mutations relative to SEQ ID NO:26.

[0010] In some embodiments, the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof of the present disclosure are -7 It binds to human PD-1 with a dissociation equilibrium constant of less than or equal to 10 M. In some embodiments, it -8 M, 10 -9 M, 10 -10 M or 10 -11 It binds to human PD-1 with a dissociation equilibrium constant of less than M

[0011] In some alternative embodiments, the disclosure provides anti-PD-1 antibodies or antigen-binding fragments thereof, wherein the heavy chain variable region comprises an HCDR1 set forth in SEQ ID NO:65, an HCDR2 set forth in SEQ ID NO:66, and an HCDR3 set forth in SEQ ID NO:67, and the light chain variable region comprises an LCDR1 set forth in SEQ ID NO:68, an LCDR2 set forth in SEQ ID NO:12, and an LCDR3 set forth in SEQ ID NO:69, the sequences of which are set forth in Table 1 below:

[0012] [Table 1]

[0013] In some alternative embodiments, in the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, the heavy chain variable region comprises an HCDR1 set forth in SEQ ID NO:8, an HCDR2 set forth in SEQ ID NO:9, and an HCDR3 set forth in SEQ ID NO:10, and the light chain variable region comprises an LCDR2 set forth in SEQ ID NO:12, an LCDR3 set forth in SEQ ID NO:13, and an HCDR4 set forth in the general formula RSSQSX 13 VHSX 14 X 15 X 16 TYLE (SEQ ID NO: 68), wherein: X 13 is L and X 14 is selected from the group consisting of N, Q, L, T and D; X 15 is selected from the group consisting of G, A and V; X 16 is N.

[0014] In some alternative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from any one of the following: (a) an anti-PD-1 antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively, and LCDR2 and LCDR3 set forth in SEQ ID NO:12 and SEQ ID NO:13, respectively, and LCDR1 set forth in SEQ ID NO:11, 47, 48, 49, 50, 51 or 52; (b) an anti-PD-1 antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively, and LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:17, SEQ ID NO:12, and SEQ ID NO:18, respectively; (c) an anti-PD-1 antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; (d) an anti-PD-1 antibody or antigen-binding fragment thereof, comprising an HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively, and an LCDR2 and LCDR3 set forth in SEQ ID NO:12 and SEQ ID NO:13, respectively, and an LCDR1 set forth in SEQ ID NO:11, 47, 48, 49, 50, 51, or 52; and (e) an anti-PD-1 antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, and LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:17, SEQ ID NO:12, and SEQ ID NO:18, respectively.

[0015] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:49, SEQ ID NO:12, and SEQ ID NO:13, respectively.

[0016] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively.

[0017] In some alternative embodiments, the disclosure provides an anti-PD-1 antibody, or antigen-binding fragment thereof, selected from any one of the following: iv) an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the same sequences as the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in the sequence of SEQ ID NO:4, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the same sequences as the LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in the sequence of SEQ ID NO:5; v) an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the same sequences as the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in the sequence of SEQ ID NO:6, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the same sequences as the LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in the sequence of SEQ ID NO:7; and vi) An anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the same sequences as the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in the sequence of SEQ ID NO:19, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the same sequences as the LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in the sequence of SEQ ID NO:20.

[0018] In some embodiments of the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

[0019] In some embodiments of the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, the anti-PD-1 antibodies or antigen-binding fragments thereof are humanized antibodies or antigen-binding fragments thereof.

[0020] In some embodiments, the humanized antibody comprises framework regions derived from a human antibody or framework region variants thereof.

[0021] In some embodiments, the framework region variants have up to 11 amino acid backmutations in each of the light chain framework regions and / or the heavy chain framework regions of a human antibody.

[0022] In some embodiments, the framework region variant comprises a mutation selected from any one of the following: (f) a 2G amino acid back mutation in the light chain variable region, and / or one or more amino acid back mutations selected from the group consisting of 27Y, 48I, 67T, 69L, 82F, and 93T in the heavy chain variable region; (g) 2V amino acid backmutations in the light chain variable region, and / or one or more amino acid backmutations selected from the group consisting of 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F, and 93T in the heavy chain variable region; and (h) one or more amino acid backmutations selected from the group consisting of 42G, 44V and 71Y in the light chain variable region, and / or 1K and / or 94S amino acid backmutations in the heavy chain variable region.

[0023] In some embodiments of the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, the anti-PD-1 antibodies or antigen-binding fragments thereof comprise an antibody variable region selected from the group consisting of: (a2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively, and one or more amino acid back mutations of 27Y, 48I, 67T, 69L, 82F and 93T contained in the heavy chain framework region; a light chain variable region comprising an LCDR2 and LCDR3 as set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and an LCDR1 as set forth in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52, and 2G amino acid back mutations contained within said light chain framework regions; (b2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and one or more amino acid back mutations selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T contained in the heavy chain variable region; a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:17, SEQ ID NO:12 and SEQ ID NO:18, respectively, and 2 V amino acid backmutations contained within said light chain framework regions; (c2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively, and a 1K and / or 94S amino acid back mutation contained in the heavy chain framework region; A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively, and one or more amino acid back mutations selected from 42G, 44V and 71Y contained in the light chain framework region.

[0024] In some embodiments of the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, the anti-PD-1 antibodies or antigen-binding fragments thereof comprise an antibody variable region selected from any one of the following: (i) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO:4 or has at least 90% sequence identity to SEQ ID NO:4; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO:5 or has at least 90% sequence identity to SEQ ID NO:5; (j) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO:6 or has at least 90% sequence identity to SEQ ID NO:6; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO:7 or has at least 90% sequence identity to SEQ ID NO:7; (k) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO: 19 or has at least 90% sequence identity to SEQ ID NO: 19; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO:20 or has at least 90% sequence identity to SEQ ID NO:20; (l) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO: 27, 30, 31 or 32, or has at least 90% sequence identity to SEQ ID NO: 27, 30, 31 or 32, respectively; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO: 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, or has at least 90% sequence identity to SEQ ID NO: 28, 29, 34, 35, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, respectively; (m) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO: 33, 36, 37, 38, 39 or 40, or has at least 90% sequence identity to SEQ ID NO: 33, 36, 37, 38, 39 or 40, respectively; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, or has at least 90% sequence identity to SEQ ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, respectively; (n) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO: 41, 45 or 46, or has at least 90% sequence identity to SEQ ID NO: 41, 45 or 46, respectively; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO: 42, 43 or 44, or has at least 90% sequence identity to SEQ ID NO: 42, 43 or 44, respectively; (o) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO: 70 or has at least 90% sequence identity to SEQ ID NO: 70; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO:71 or has at least 90% sequence identity to SEQ ID NO:71; and (p) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO: 27, 30, 31 or 32, or has at least 90% sequence identity to SEQ ID NO: 27, 30, 31 or 32, respectively; and / or a light chain variable region, the sequence of which is as set forth in SEQ ID NO: 34 or 35, or has at least 90% sequence identity to SEQ ID NO: 34 or 35, respectively; Here, the sequences of SEQ ID NO: 70 and SEQ ID NO: 71 are represented by the general formula sequences shown in Table 2.

[0025] [Table 2]

[0026] In some embodiments, the heavy chain variable region of the anti-PD-1 antibody is as set forth in SEQ ID NO:27, or has at least 90% sequence identity to SEQ ID NO:27, and the sequence of the light chain variable region of the anti-PD-1 antibody is as set forth in SEQ ID NO:55, or has at least 90% sequence identity to SEQ ID NO:55.

[0027] In some embodiments, the heavy chain variable region of the anti-PD-1 antibody is as set forth in SEQ ID NO:46, or has at least 90% sequence identity to SEQ ID NO:46, and the sequence of the light chain variable region of the anti-PD-1 antibody is as set forth in SEQ ID NO:43, or has at least 90% sequence identity to SEQ ID NO:43.

[0028] The foregoing "at least 90% identity" includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0029] In some other embodiments of the aforesaid anti-PD-1 antibodies or antigen-binding fragments thereof, the antibodies further comprise an antibody constant region; in some other embodiments, the heavy chain constant region of the antibody constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region of the antibody constant region is selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof; in some other embodiments, the antibody constant region comprises an IgG4 heavy chain constant region into which one or more of S228P, F234A, and L235A mutations have been introduced, for example, an IgG4 heavy chain constant region having three amino acid mutations, S228P, F234A, and L235A; in some other embodiments, the antibody comprises a heavy chain constant region set forth in SEQ ID NO:72 or 79, and a light chain constant region set forth in SEQ ID NO:73.

[0030] In some embodiments of the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, the anti-PD-1 antibodies comprise a light chain set forth in SEQ ID NO:78 and a heavy chain set forth in SEQ ID NO:77 or 82, or the anti-PD-1 antibodies comprise a light chain set forth in SEQ ID NO:75 and a heavy chain set forth in SEQ ID NO:74, 76, 80, or 81.

[0031] In some embodiments, the anti-PD-1 antibody is a heavy chain as set forth in SEQ ID NO: 74 and a light chain as set forth in SEQ ID NO: 75; or Heavy chain shown in SEQ ID NO: 77 and light chain shown in SEQ ID NO: 78 Includes.

[0032] In some embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof is provided, wherein the antibody binds to human PD-1 competitively with or binds to the same human PD-1 epitope as any one of the foregoing anti-PD-1 antibodies or antigen-binding fragments thereof.

[0033] In some embodiments of the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, the antibodies are bispecific or multispecific antibodies.

[0034] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment thereof, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a F(ab')2, a single chain antibody (scFv), a dimerized V region (diabody), and a disulfide bond stabilized V region (dsFv).

[0035] In some embodiments, an isolated monoclonal antibody or antigen-binding fragment thereof is disclosed, wherein the antibody binds to human PD-1 competitively with any one of the anti-PD-1 antibodies or antigen-binding fragments thereof described in any one of the preceding.

[0036] In some embodiments, the present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the foregoing anti-PD-1 antibodies or antigen-binding fragments thereof, or a therapeutically effective amount of the foregoing isolated monoclonal antibodies or antigen-binding fragments thereof, and one or more pharma- ceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the foregoing therapeutically effective amount refers to a unit dose of a composition comprising 0.1-3000 mg of the anti-PD-1 antibody or antigen-binding fragment thereof.

[0037] In some embodiments, the disclosure also provides a nucleic acid molecule encoding an anti-PD-1 antibody or antigen-binding fragment thereof described in any one of the foregoing, or encoding the foregoing isolated monoclonal antibody or antigen-binding fragment thereof.

[0038] In some embodiments, the present disclosure also provides host cells comprising the aforementioned nucleic acid molecules.

[0039] In some embodiments, the disclosure also provides a method for immunodetection or determination of PD-1 comprising using any one of the anti-PD-1 antibodies or antigen-binding fragments thereof described above, or using the isolated monoclonal antibody or antigen-binding fragment thereof described above.

[0040] In some embodiments, the disclosure also provides a kit comprising the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof, or the aforementioned isolated monoclonal antibodies or antigen-binding fragments thereof.

[0041] In some embodiments, there is provided a use of the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof in the preparation of a diagnostic agent for a PD-1-related disease.

[0042] In some embodiments, the disclosure also provides a method for treating a disease comprising administering to a subject a therapeutically effective amount of any one of the anti-PD-1 antibodies or antigen-binding fragments thereof described above, or any of the isolated monoclonal antibodies or antigen-binding fragments thereof, or any of the pharmaceutical compositions, or any of the nucleic acid molecules described above.

[0043] In some embodiments, the disease is a tumor.

[0044] In some other embodiments, the disease is head and neck squamous cell carcinoma, head and neck cancer, brain tumor, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, osteosarcoma, chondrosarcoma, myeloma, multiple myeloma, ... In some of these embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma. and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia; in some other embodiments, the disease is selected from the group consisting of PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, intestinal cancer and colon cancer.

[0045] In some embodiments, the disclosure also provides for the use of an anti-PD-1 antibody or antigen-binding fragment thereof described in any one of the preceding, or an isolated monoclonal antibody or antigen-binding fragment thereof, or an aforementioned pharmaceutical composition, or an aforementioned nucleic acid molecule, in the preparation of a medicament for the treatment or prevention of a disease.

[0046] In some embodiments, the disease is a tumor.

[0047] In some other embodiments, the disease is head and neck squamous cell carcinoma, head and neck cancer, brain tumor, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, osteosarcoma, chondrosarcoma, myeloma, multiple myeloma, ... In some of these embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma. and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia; in some other embodiments, the disease is selected from the group consisting of PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, intestinal cancer and colon cancer.

[0048] In some embodiments, the disclosure also provides an anti-PD-1 antibody or antigen-binding fragment thereof described in any one of the preceding, or an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated nucleic acid molecule, or a pharmaceutical composition, for use as a medicament.

[0049] In some embodiments, the medicament is used for the treatment or prevention of a PD-1 associated disease.

[0050] In some embodiments, the disease is a tumor.

[0051] In some other embodiments, the disease is head and neck squamous cell carcinoma, head and neck cancer, brain tumor, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, osteosarcoma, chondrosarcoma, myeloma, multiple myeloma, ... In some of these embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma. and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia; in some other embodiments, the disease is selected from the group consisting of PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, intestinal cancer and colon cancer. [Brief description of the drawings]

[0052] [Figure 1] Results from testing anti-PD-1 antibodies that block the binding of PD-1 to its ligands. [Diagram 2] Effect of anti-PD-1 antibody on IFNγ secretion from PBMC cells. [Diagram 3] Efficacy of anti-PD-1 antibodies against colon cancer MC38 xenograft tumors in mice. [Figure 4] Effect of anti-PD-1 antibody on tumor volume of colon cancer MC38 in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] <Summary> In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0054] The terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PDCD1" and "hPD-1" are used interchangeably and include human PD-1 variants, isotypes, species homologs, and analogs that share at least one epitope in common with PD-1. The complete PD-1 sequence can be found at GenBank accession number U64863.

[0055] The term "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), hPD-L1 variants, isotypes and interspecies homologs, and five analogs that have at least one epitope in common with hPD-1. The complete hPD-L1 sequence can be found at GenBank Accession No. Q9NZQ7.

[0056] The term "cytokine" is a general term for proteins released by a population of cells and acting on other cells as intercellular mediators. Examples of such cytokines include lymphokines, monokines, chemokines and traditional polypeptide hormones. Exemplary cytokines include human IL-2, IFN-γ, IL-6, TNFα, IL-17 and IL-5.

[0057] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, 3558 (1968).

[0058] "Antibody" as described in this disclosure generally refers to immunoglobulins. Natural intact antibodies are tetrapeptide chain structures composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Their antigenicity differs due to differences in the amino acid composition and sequence of the immunoglobulin heavy chain constant region. This allows immunoglobulins to be classified into five classes or named immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, whose corresponding heavy chains are μ, δ, γ, α and ε chains, respectively. Igs of the same class can be classified into different subclasses depending on the differences in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3 and IgG4. Light chains are classified into κ or λ chains due to differences in the constant region. Each of the five classes of Ig can have κ or λ chains. The antibody referred to in this disclosure includes an antibody or antigen-binding fragment thereof, including an antibody or antigen-binding fragment thereof modified based on immunoglobulin while retaining the ability to bind to an antigen; including a monospecific antibody, a bispecific antibody, or a multispecific antibody; also including a monovalent antibody, a bivalent antibody, or a multivalent antibody. An antigen-binding fragment of an antibody can, for example, comprise at least one VH-CH1 and at least one VL-CL structure, where the VH and VL structures can approach each other based on interchain interactions and retain antigen-binding ability. In some embodiments, an antigen-binding fragment of an antibody can be a monovalent Fab fragment (Fab1 fragment), a bivalent Fab fragment (F(ab)2), a trivalent Fab fragment (F(ab)3), a multivalent (two or more valent) Fab fragment, or other monospecific, bispecific, or multispecific antigen-binding fragment comprising at least one Fab fragment.

[0059] "Bispecific antibodies" refers to antibodies (including antibodies or antigen-binding fragments thereof, e.g., single-chain antibodies) that can specifically bind to two different antigens or two different epitopes of the same antigen. The prior art discloses bispecific antibodies with various structures. Depending on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Depending on the number and configuration of the antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or higher polyvalent bispecific antibodies. Depending on whether the structure is symmetric, they can be classified into symmetric structure bispecific antibodies and asymmetric structure bispecific antibodies. Among them, antibody fragment-based bispecific antibodies, e.g., Fab fragments lacking Fc fragments, form bispecific antibodies by binding to two or more Fab fragments in one molecule. These antibodies have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type are bispecific antibodies such as F(ab)2, scFv-Fab, (scFv)2-Fab; in the case of IgG-like bispecific antibodies (e.g., containing Fc fragments), this type of antibody has a larger molecular weight. The Fc fragment aids in the purification of the antibody at a later stage and improves its solubility and stability. The Fc portion can also bind to the receptor FcRn, increasing the serum half-life of the antibody.Exemplary bispecific antibody structural models include, for example, KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, and other bispecific antibodies (Aran F. Labrijn et al., Nature Reviews Drug Discovery Volume 18, pp. 585-608 (2019); Chen S1 et al., J Immunol Res. February 11, 2019; 2019: p. 4516041).

[0060] The terms "monovalent", "bivalent", "trivalent" or "multivalent" refer to the presence of a particular number of antigen-binding sites on an antibody or polypeptide complex. For example, a "monovalent antibody" means that the antibody has one antigen-binding site, a "monovalent polypeptide complex" means that the polypeptide complex has one antigen-binding site; a "bivalent antibody" means that the antibody has two antigen-binding sites, a "bivalent polypeptide complex" means that the polypeptide complex has two antigen-binding sites; a "trivalent antibody" means that the antibody has three antigen-binding sites, a "trivalent polypeptide complex" means that the polypeptide complex has three antigen-binding sites; a "multivalent antibody" means that the antibody has multiple (three or more) antigen-binding sites, and a "multivalent polypeptide complex" means that the polypeptide complex has multiple (two or more) antigen-binding sites.

[0061] The term "antibody fusion protein" refers to a biologically active fusion protein formed by linking a protein (polypeptide) of interest to an immunoglobulin. The fusion protein has the biological activity of both the linked protein and the immunoglobulin.

[0062] Approximately 110 amino acid sequences near the N-terminus of the heavy and light chains of an antibody are highly variable and are the variable regions (Fv regions); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions. The variable regions contain three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conservative sequences. The three hypervariable regions that determine the specificity of the antibody are also known as complementarity determining regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions. The order from the amino terminus to the carboxyl terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCL2 and HDR3.

[0063] The antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies.

[0064] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human PD-1 prepared according to the knowledge and techniques of the art. During preparation, a test subject is injected with PD-1 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present disclosure, the mouse anti-PD-1 antibody or antigen-binding fragment thereof can further comprise a light chain constant region of mouse kappa, lambda chain or variants thereof, or can further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3 or variants thereof.

[0065] A "chimeric antibody" is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by mouse antibodies. Establishing a chimeric antibody requires first establishing a hybridoma secreting a specific mouse monoclonal antibody, then cloning the variable region genes from the mouse hybridoma cells, then cloning the constant region genes of a human antibody if necessary, linking the mouse variable region genes to the human constant region genes to form a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the PD-1 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, and preferably comprises a heavy chain constant region of human IgG1, IgG2, or IgG4, or an IgG1, IgG2, or IgG4 variant having amino acid mutations (e.g., the L234A and / or L235A mutation, and / or the S228P mutation).

[0066] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences into the framework of a human antibody variable region, i.e., an antibody produced with a different type of human germline antibody framework sequence. Humanized antibodies can overcome the xenogeneic reaction induced by chimeric antibodies because they retain a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase) and Kabat, EA, et al. 1991 Sequences of Proteins of Immunological Interest, 5th Edition. To avoid a decrease in activity caused by a concomitant decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal back mutations or reverse mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that have been further subjected to CDR affinity maturation by yeast display.

[0067] Due to the residues in contact with the antigen, CDR grafting may result in a decrease in the affinity of the produced antibody or its antigen-binding fragment to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of somatic hypermutation. Therefore, it may still be necessary to graft such donor framework amino acids into the framework of the humanized antibody. The amino acid residues involved in antigen binding and derived from the non-human antibody or its antigen-binding fragment can be identified by examining the sequence and structure of the animal monoclonal antibody variable region. Residues of the CDR donor framework that are different from the germline can be considered to be related. If the closest germline cannot be determined, the sequence can be compared to the consensus sequence of animal antibody sequences with a subclass or high similarity percentage. Rare framework residues are considered to be the result of somatic hypermutation and therefore play an important role in binding.

[0068] In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a human or mouse kappa, lambda chain or a variant thereof, or may further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably a heavy chain constant region of human IgG1, IgG2 or IgG4, or an IgG1, IgG2 or IgG4 variant having amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0069] The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in this disclosure refer to variants of the heavy or light chain constant region that have been disclosed in the prior art and do not change the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-specific modifications and amino acid substitutions in the heavy chain constant region. Specific substitutions include YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations to obtain knob-into-hole structures (such that the antibody heavy chain has a knob-Fc and hole-Fc combination) known in the art. These mutations have been identified to provide new properties to the antibody but do not change the function of the antibody variable region.

[0070] "HuMAb", "human antibody", "fully human antibody" and "complete human antibody" can be used interchangeably and can refer to antibodies derived from humans or from genetically modified organisms that have been "engineered" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, where the endogenous heavy and light chain loci have been targeted disrupted by targeting the endogenous heavy and light chain loci contained in these cells. The transgenic organisms can synthesize human antibodies specific to human antigens, and the organisms can be used to produce hybridomas that secrete human antibodies. Human antibodies can also be antibodies in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by genetic or chromosomal transfection methods and phage display techniques, or from in vitro activated B cells, all of which are known in the art.

[0071] The terms "full-length antibody", "intact antibody", "complete antibody" and "whole antibody" are used interchangeably herein and refer to an antibody in its substantially intact form, as distinguished from an antigen-binding fragment, as defined below. These terms specifically refer to antibodies in which the heavy chain comprises an Fc region.

[0072] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, which comprises a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment, which consists of the VH and CH1 domains; (iv) an Fv fragment, which is composed of the VH and VL domains of one arm of an antibody; (v) a single domain or dAb fragment, which consists of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked using recombinant techniques by a synthetic linker to produce a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained by using conventional techniques known in the art, and the functionality of the fragments is screened in the same way as that of intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.The antibodies can be of different isotypes, for example IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0073] Antigen-binding fragments of the present disclosure include Fab, F(ab')2, Fab', single chain antibodies (scFv), dimerized V regions (diabodies), disulfide bond stabilized V regions (dsFv), CDR-containing peptides, and the like.

[0074] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity; it is obtained by treating an IgG antibody molecule with the protease papain (which cleaves the amino acid residue at position 224 in the H chain); approximately the N-terminal half of the H chain and the entire L chain are linked by disulfide bonds.

[0075] The Fab of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure with papain. Additionally, the Fab can be produced by inserting DNA encoding the Fab of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab.

[0076] F(ab')2 is an antibody fragment having a molecular weight of about 100,000, having antigen-binding activity, and containing two Fab regions linked at the hinge position, and can be obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0077] The F(ab')2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure with pepsin.Furthermore, the F(ab')2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.

[0078] Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, and can be obtained by cleaving the disulfide bond in the hinge region of F(ab')2. The Fab' of the present disclosure can be produced by treating the F(ab')2 of the present disclosure, which specifically recognizes PD-1 and binds to the amino acid sequence of the extracellular domain or its conformation, with a reducing agent such as dithiothreitol.

[0079] Furthermore, Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryotic or eukaryotic organism to express Fab'.

[0080] The term "single chain antibody", "single chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker. Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of a repeating GGGGS amino acid sequence or a variant thereof, such as a variant having 1 to 4 repeats (Holliger et al. (1993), Proc Natl Acad Sci USA. 90:6444-6448). Other linkers that can be used in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur J Immuno. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J Mol Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0081] The scFv of the present disclosure can be produced by the following steps: obtaining cDNA encoding the VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure, constructing DNA encoding the scFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the scFv.

[0082] A diabody is an antibody fragment formed by dimerization of scFv and has bivalent antigen-binding activity, in which the two antigens may be the same or different.

[0083] The diabodies of the present disclosure can be produced by the following steps: obtaining cDNA encoding the VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure, constructing DNA encoding the scFv such that the peptide linker is 8 amino acid residues or less in length, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the diabody.

[0084] dsFv can be obtained by linking polypeptides in which one amino acid residue in each of VH and VL is replaced with a cysteine ​​residue via a disulfide bond between the two cysteine ​​residues. The amino acid residue to be replaced with a cysteine ​​residue can be selected according to a known method (Protein Engineering, 7, 697 (1994)) based on prediction of the three-dimensional structure of an antibody.

[0085] The dsFv of the present disclosure can be produced by the following steps: obtaining cDNA encoding the VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure, constructing DNA encoding the dsFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the dsFv.

[0086] A CDR-containing peptide is constructed by one or more regions containing the CDRs of VH or VL. Peptides containing multiple CDRs can be linked directly or via suitable peptide linkers.

[0087] The CDR-containing peptides of the present disclosure can be produced by the following steps: constructing DNA encoding the CDRs of VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the peptide. The CDR-containing peptides can also be produced by chemical synthesis, such as the Fmoc method or the tBoc method.

[0088] The term "amino acid difference" or "amino acid mutation" refers to the presence of an amino acid change or mutation in a protein or polypeptide variant compared to an original protein or polypeptide, including having one, two, three or more amino acid insertions, deletions or substitutions based on the original protein or polypeptide.

[0089] The term "antibody framework" or "FR region" refers to the part of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without the CDRs.

[0090] The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contributes to antigen binding. Generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2 and HCDR3) and each light chain variable region has three CDRs (LCDR1, LCDR2 and LCDR3). The amino acid sequence boundaries of the CDRs can be determined using any one of the well-known schemes, including the "Kabat" numbering standard (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, Md.), the "Chothia" numbering standard (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImmunoGenTics (IMGT) numbering standard (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP, et al., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, in the classical format, according to the Kabat standard, the numbering of the CDR amino acid residues of the heavy chain variable domain (VH) is 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HDR3), and the numbering of the CDR amino acid residues of the light chain variable domain (VL) is 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to the Chothia standard, the numbering of the CDR amino acid residues of the VH is 26-32 (HDR1), 52-56 (HDR2) and 95-102 (HDR3), and the numbering of the amino acid residues of the VL is 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). According to a combination of both the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCL1), 50-65 (HCR2), and 95-102 (HDR3) of the human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of the human VL.According to the IMGT standard, the numbering of the CDR amino acid residues of the VH is approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the numbering of the CDR amino acid residues of the VL is approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). According to the IMGT standard, the CDR regions of an antibody can be determined using the IMGT / DomainGap Align program.

[0091] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a particular site on a PD-L1 molecule). An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by GE Morris (1996).

[0092] The terms "specifically binds," "selectively binds," "binds selectively," and "binds specifically" refer to the binding of an antibody to an epitope on a given antigen. Generally, an antibody binds to an epitope on a given antigen at a concentration of about 10 -8 Less than m, e.g., about 10 -9 M, 10 -10 M or 10 -11 It binds with an affinity (KD) of less than or equal to M.

[0093] "KD" or "K D The term "antibody-antigen dissociation equilibrium constant" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Generally, the antibodies of the present disclosure have a dissociation equilibrium constant of about 10 as measured on a BIACORE instrument using surface plasmon resonance (SPR) technology. -7 Less than M, e.g., about 10 -8 M or 10 -9It binds to PD-1 with a dissociation equilibrium constant (KD) less than M.

[0094] When the term "competition" is used in the context of competing antigen binding proteins (e.g., neutralizing antigen binding proteins or antibodies) for the same epitope, it refers to competition between the antigen binding proteins and can be determined by an assay in which the antigen binding protein being tested (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., PD-1 or a fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen binding protein competes with another antigen binding protein. These assays include, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J. Immunol. 137: 3614-3619), solid-phase direct label assays, solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see, e.g., Morel et al, 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176: 546-552); and direct labeling RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). In general, the assays involve using purified antigen bound to a solid surface or cells bearing either the unlabeled antigen-binding protein to be tested or a labeled reference antigen-binding protein.Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen-binding protein being tested. Typically, the antigen-binding protein being tested is present in excess. Antigen-binding proteins identified by competitive assays (competitor antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an adjacent epitope close enough to the binding epitope of the reference antigen-binding protein (where the two epitopes sterically prevent binding to each other). Further details regarding the methods used to determine competitive binding are provided in the Examples herein. Typically, when a competitor antigen-binding protein is present in excess, it inhibits (e.g., reduces) the specific binding of the reference antigen-binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or greater than 97%.

[0095] The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and are preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the coding sequence.

[0096] The term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be linked. In another embodiment, the vector is a viral vector to which additional DNA segments can be linked to the viral genome. The vectors disclosed herein can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors), or can be integrated into the genome of the host cell after being introduced into the host cell and replicate together with the host genome (e.g., non-episomal mammalian vectors).

[0097] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art. See, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor, Chapters 5-8 and 15. For example, mice can be immunized with human PD-1 or a fragment thereof, and the resulting antibodies can be regenerated, purified, and amino acid sequenced by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. By using genetic engineering methods, one or more human FR regions are added to the non-human CDR regions of the antibody or antigen-binding fragment of the present invention. Human FR germline sequences can be obtained from the ImmunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from The Immunoglobulin Facts Book, 2001 ISBN012441351.

[0098] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plant or animal cells. Bacteria that can be easily transformed include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella strains; Bacillaceae, such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.

[0099] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and incorporated into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can result in glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region. Stable clones are obtained by expressing an antibody that specifically binds to human PD-1. The positive clones are grown in serum-free medium in a bioreactor to produce the antibody. The culture medium into which the antibody is secreted can be purified by conventional techniques. For example, using an A or G Sepharose FF column with a buffer adjusted for purification. Non-specifically bound components are washed away. The bound antibody is then eluted by a pH gradient method, and the antibody fragments are detected and recovered by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0100] "Administer," "give," and "treat," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of a reagent with the cell, and contact of a reagent with a fluid, where the fluid is in contact with the cell. "Administer," "give," and "treat" also refer to treating a cell, for example, with a reagent, diagnostic, binding composition, or with another cell in vitro and ex vivo. "Treat," when applied to a human, veterinary, or experimental subject, refers to therapeutic treatment, preventing or preventive measures, research, and diagnostic applications.

[0101] "Treatment" refers to administering an internal or external therapeutic agent, such as a composition comprising any one of the binding compounds of the present disclosure, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. In general, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, to induce regression of such symptoms or to suppress the onset of such symptoms to any clinically measured extent. The amount of the therapeutic agent effective to alleviate any particular disease symptom (also called a "therapeutically effective amount") may vary depending on various factors, such as the patient's medical condition, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical testing method commonly used by physicians or other medical professionals to evaluate the severity or progression of the condition. Although embodiments of the present disclosure (e.g., treatment methods or products) may not be effective in alleviating each target disease symptom, they should alleviate the target disease symptom in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0102] "Conservative modification" or "conservative substitution" refers to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that the change can be made frequently without changing the biological activity of the protein. Those skilled in the art recognize that, generally speaking, single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Publishers, p. 224 (4th ed.)). Moreover, the substitution of amino acids with similar structure or function is unlikely to destroy biological activity. Exemplary conservative substitutions are shown in the following table "Exemplary Amino Acid Conservative Substitutions".

[0103] Table 3. Exemplary Amino Acid Conservative Substitutions [Table 3]

[0104] "Effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition required to obtain any one or more beneficial or desired therapeutic results. In the case of prophylactic use, beneficial or desired results include eliminating or reducing the risk, reducing the severity or delaying the onset of a disease, including the biochemical, histological and behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes occurring during the developmental process of the disease. In the case of therapeutic use, beneficial or desired results include clinical results, such as reducing the incidence of or improving one or more symptoms of various target antigen-related disorders of the present disclosure, reducing the dosage of other agents required to treat the disorder, enhancing the effect of another agent, and / or slowing the progression of the target antigen-related disorder of the present disclosure in a patient.

[0105] "Exogenous" refers to a substance that is produced outside an organism, cell, or body, as the case may be. "Endogenous" refers to a substance that is produced inside a cell, organism, or body, as the case may be.

[0106] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If the positions in the two sequences being compared are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology percentage between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, and then multiplied by 100 (x100). For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences are matching or homologous, then the two sequences are 95% homologous. In general, when two sequences are aligned, the comparison is performed to give the maximum homology percentage. For example, comparison can be performed by BLAST algorithm, in which the parameters of the BLAST algorithm are selected to maximize the match between each sequence over the entire length of each reference sequence.The following references relate to the BLAST algorithm that is commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol.215:403-410; Gish, W. et al., (1993) Nature Genet.3:266-272; Madden, TL et al., (1996) Meth. Enzymol.266:131-141; Altschul, SF et al., (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. et al., (1997) Genome Res.7:649-656. Other conventional BLAST algorithms, such as that provided by NCBI BLAST, are also familiar to those of skill in the art.

[0107] The terms "cell", "cell line" and "cell culture" as used herein may be used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of passages. It should also be understood that not all progeny are completely identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the original transformed cell are included. When alternative designations are referenced, it is clearly understood from the context.

[0108] As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of nucleic acid, DNA and / or specific portions of DNA are amplified, as described, for example, in U.S. Pat. No. 4,683,195. Generally speaking, sequence information must be obtained from the ends or outside of the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotides of the two primers can be identical to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor, Symp. Ouant. Biol. 51:263; Erlich, ed. (1989) PCR TECHNOLOGY (Stockton Press, New York). As used herein, PCR is considered an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which involves using known nucleic acids as primers and a nucleic acid polymerase to amplify or generate a specific portion of a nucleic acid.

[0109] "Isolated" refers to a purified state, in this case meaning that the specified molecule is substantially free of other biological molecules (e.g., nucleic acids, proteins, lipids, carbohydrates) or other materials (e.g., cell debris and growth medium). In general, the term "isolated" does not imply the complete absence of these materials, or the absence of water, buffers, or salts, unless present in amounts that would significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0110] "Any" or "optionally" means that the subsequently described event or circumstance may occur, but does not necessarily occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but do not have to be present.

[0111] "Pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein, or physiologically / pharmaceutical acceptable salts or prodrugs thereof, and other chemical components (e.g., physiologically / pharmaceutical acceptable carriers and excipients). The purpose of a pharmaceutical composition is to facilitate administration to an organism, which facilitates absorption of the active ingredient, thereby exerting its biological activity.

[0112] The term "pharmaceutical acceptable carrier" refers to any inert substance suitable for use in a formulation for delivery of an antibody or antigen-binding fragment. Carriers can be anti-adhesives, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial or antifungal agents), sweeteners, absorption retardants, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutical acceptable carriers include water, ethanol, polyols (such as glycerol, propanediol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents (such as sugars, polyols, sorbitol, and sodium chloride).

[0113] Furthermore, the present disclosure includes a pharmaceutical agent for treating a disease associated with target antigen (e.g., PD-1) positive cells, the pharmaceutical agent comprising an anti-PD-1 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.

[0114] The disease associated with PD-1 in the present disclosure is not limited as long as it is a disease associated with PD-1.For example, the therapeutic response induced by the molecule of the present disclosure can be achieved by binding to human PD-1, then inhibiting the binding of PD-1 and its ligands PD-L1 and PD-L2, or killing the cells that overexpress PD-1.Therefore, in preparations and formulations suitable for therapeutic use, the molecule of the present disclosure is very useful for people with tumor or cancer, preferably melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.

[0115] Furthermore, the present disclosure relates to a method for immunodetection or determination of a target antigen (e.g., PD-1), a reagent for immunodetection or determination of a target antigen (e.g., PD-1), a method for immunodetection or determination of cells expressing a target antigen (e.g., PD-1), and a diagnostic agent for diagnosing a disease associated with target antigen (e.g., PD-1)-positive cells, comprising an antibody or antibody fragment of the present disclosure that specifically recognizes the target antigen (e.g., human PD-1) and binds to an amino acid sequence of the extracellular domain or its three-dimensional structure, and is used as an active ingredient.

[0116] In the present disclosure, the method used to detect or measure the amount of a target antigen (e.g., PD-1) can be any known method, such as an immunodetection or measurement method.

[0117] Immunodetection or measurement methods are methods that use labeled antigens or antibodies to detect or measure the amount of antibodies or antigens. Examples of immunodetection or measurement methods include radioimmunoassays (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescence immunoassays, Western blotting, physicochemical methods, etc.

[0118] The aforementioned diseases associated with PD-1 positive cells can be diagnosed by detecting or measuring PD-1 expressing cells using the antibodies or antibody fragments of the present disclosure.

[0119] To detect cells expressing a polypeptide, known immunodetection methods can be used, preferably immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. Furthermore, fluorescent antibody staining using the FMAT8100HTS system (Applied Biosystem) can also be used.

[0120] In the present disclosure, the in vivo sample used to detect or measure a target antigen (e.g., PD-1) is not particularly limited, so long as it may contain cells expressing the target antigen (e.g., PD-1), and may be, for example, histiocytes, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

[0121] Depending on the required diagnostic method, the diagnostic agent containing the monoclonal antibody or its antibody fragment of the present disclosure may also contain a reagent for carrying out an antigen-antibody reaction or a reagent for detecting the reaction. The reagents used for carrying out the antigen-antibody reaction include buffers, salts, etc. The reagents used for detection include reagents commonly used in immunodetection or measurement methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the label.

[0122] <Preparation of antigen> 1. Construction of antigen Human PD-1-IgG1Fc fusion protein is designed and synthesized to contain 150 amino acids of the extracellular domain of human PD-1 at the N-terminus and the Fc fragment of human IgG1 (hIgG1Fc) at the C-terminus. High-purity recombinant PD-1-Fc protein can be obtained after purification with a protein A affinity column and used to detect the binding of anti-PD-1 antibody to antigen.

[0123] Human PD-1-IgG1Fc (SEQ ID NO:1): [ka]

[0124] NOTE: The underlined part is the signal peptide, the normal part is the extracellular region of human PD-1, and the italicized part is hIgG1Fc (signal peptide + extracellular region + hIgG1Fc).

[0125] Human PD-1-his (SEQ ID NO:2): [ka]

[0126] PD-1 antigen encoded by nucleic acid transfected into cells (SEQ ID NO:3): [ka]

[0127] <Preparation of antibodies> Anti-human PD-1 antibodies can be produced by immunizing mice and can also be obtained from an anti-human PD-1 phage mouse immunization library.

[0128] The method for preparing anti-human PD-1 antibodies by immunizing mice is as follows:

[0129] 1. Immunization: Laboratory SJL white mice, female, 6-8 weeks old, and Balb / c white mice, female, 6-8 weeks old. Rearing environment: SPF level. After purchase, the mice were reared in a laboratory environment for one week under the control of temperature 20-25°C, humidity 40-60%, and 12 / 12 hours light / dark cycle. The environmentally adapted mice were immunized according to different protocols, using 6-10 mice in each group. The immunization antigen could be purified recombinant protein PD-1-IgG1Fc (see SEQ ID NO:1), PD-1-his (see SEQ ID NO:2), or Jurkat / CHO-PD-1 cells transfected with PD-1 as antigen (see SEQ ID NO:3). Single antigens combined with different immune adjuvants or different types of immunogens can be used for cross-immunization. The immunization site can be the abdominal cavity or under the skin on the back, or immunization can be performed alternately at two sites. Immune adjuvant TiterMax (登録商標) Gold Adjuvant (hereafter referred to as Titermax, purchased from Sigma, catalog number T2684) and Imject Alum Adjuvant (hereafter referred to as adjuvant, purchased from Pierce, catalog number 77161) were used for cross-immunization. The antigen-to-adjuvant (Titermax) ratio was 1:1, and the antigen-to-adjuvant (Alum) ratio was 3:1, with 25–50 μg / animal (primary immunization), 50 μg / animal (booster immunization), or 1 × 10 7 Jurkat / CHO-PD-1 cells / animal. Emulsified antigen was injected intraperitoneally at 25–50 μg / animal on day 0, and after the first immunization, Titermax and Alum were used alternately once a week or once every two weeks for a total of 5–8 doses.

[0130] 2. Cell fusion: Mice with high antibody titers in serum were selected for spleen cell fusion. 72 hours after sprint immunization, mice were bled from the eyes. The mice were sacrificed by pulling the neck and placed in 75% ethanol for disinfection. Spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (Chinese Academy of Sciences), and an optimized PEG-mediated fusion procedure was applied to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1x HAT and 1x OPI) and plated in 96-well cell culture plates (1x10 5 The cells were then aliquoted at 100 μl / well (150 μl / well) and incubated at 37°C, 5% CO2, and seeded onto approximately 10–30 plates in total. On day 5 after fusion, HAT complete medium was added at 50 μl / well and incubated at 37°C, 5% CO2. From day 7 to day 8 after fusion, depending on cell growth density, the medium was completely replaced with 200 μl / well and incubated at 37°C, 5% CO2.

[0131] 3. Hybridoma cell screening: On the 7th to 9th day after fusion, depending on the cell growth density, ELISA method was performed to detect the binding of antibodies to PD-1, and the cells in the positive wells were further detected by ELISA to detect the blocking of PD-1 / PDL1 binding. The medium in the positive wells was replaced, and the cells were grown in time to 24-well plates depending on the cell density. The cell lines transferred to the 24-well plates were retested, then stored and subcloned for the first time. Those that were positive in the first subcloning screening were stored and subcloned for the second or third time until a single cell clone was obtained. Hybridoma cells with the effect of blocking the binding of PD-1 and PDL1 were obtained by multiple fusions.

[0132] The method for obtaining anti-human PD-1 antibodies via anti-human PD-1 phage mouse immune library is as follows:

[0133] 1. Construction of anti-human PD-1 phage mouse immune library: The spleens of mice with high serum antibody titers were selected, and total RNA from the tissue was extracted using Trizol (Invitrogen, Cat. No. 15596-018). PrimeScript (商標) cDNA was obtained using II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A) for reverse transcription. Primers for constructing the library were designed and synthesized according to the IMGT database. Single-chain antibody fragments were obtained by three rounds of PCR reactions. The single-chain antibody fragments and the modified library construction vector pCantab5E (Amersham Biosciences / GE, Cat. No. 27-9400-01) were digested with Sfi1 (NEB, Cat. No. R0123L), purified and recovered with EZNA® Gel Extraction Kit (Omega, Cat. No. D2500-02) after electrophoresis. Then, they were ligated using T4 DNA ligase (NEB, Cat. No. M0202L) at 16°C for 16-18 hours, purified and recovered using the above kit, and finally eluted with deionized water. 1 μg of the ligation product was taken and mixed with one vial of electroporation competent TG1 (Lucigen, Cat. No. 60502-2) and electroporated using an electroporator (Bio Rad Micropulser) with parameters set to 2.5 kV, 200 Ω and 25 uF. The transformation was repeated 10 times. The product was spread on a plate and grown upside down at 37°C for 16-18 hours. All colonies were then isolated and mixed, glycerol was added to a final concentration of 15%, and stored at -80°C for use.

[0134] 2. Screening of the anti-human PD-1 phage mouse immune library: Screening the packaged anti-human PD-1 phage immune library (1 × 10 12 ~1×10 13) and 100 μl of streptomycin microbeads (Milenvi Biotec, Auburn, CA) were added to 1 ml of phosphate buffered saline containing 2% skim milk (MPBS), incubated at room temperature for 1 h, placed on a magnetic stand, and the supernatant was collected. 10 μg / ml of biotinylated human PD-1-ECD-his protein (purchased from Sino Biological) was added to the supernatant and incubated at room temperature for 1 h. Next, 100 μl of streptavidin-coated magnetic beads (pre-incubated with 1 ml of MPBS) were added and incubated at room temperature for 1 h. This was then loaded onto the magnetic stand system for sorting, and the supernatant was aspirated. 1 ml of PBST (phosphate buffer containing 0.1% Tween-20) was added and inverted several times. After complete aspiration, fresh wash solution was added, and this was repeated 11 times to remove unbound antibody fragments, and 0.5 ml of elution solution (450 μl PBS plus 50 μl of 10 mg / ml trypsin stock solution) was added. This was shaken for 15 minutes at room temperature, placed on a magnetic stand, and the supernatant was aspirated into a new EP tube. TG1 was inoculated into 2YT medium and grown to a culture density of OD600=0.4. 1.75 ml of TG1 (OD600=0.4) was added to each tube, 250 μl of eluted phage (phage) was added, incubated in a 37°C water bath for 30 minutes, and gradient diluted and spread on plates to test titers. The remaining TG1 solution was centrifuged, spread on plates, and incubated at 37°C overnight.

[0135] The phage-mouse immune library was screened for 2-3 rounds using biotinylated human PD-1-ECD-his antigen with MACS screening (streptomycin magnetic beads, Invitrogen), and monoclonal antibodies that could bind to PD-1 and block the binding of PD-1 to PD-L1 were finally obtained and verified by sequencing. The variable region sequences of the antibodies were obtained.

[0136] <Recombinant antigen protein / antibody purification> 1. Hybridoma Supernatant / Protein G Affinity Chromatography Separation and Purification: Protein G for affinity chromatography was the first choice for purifying mouse hybridoma supernatants. The cultured hybridomas were centrifuged to collect the supernatant, and the pH of the supernatant was adjusted by adding 10-15% of 1M Tris-HCl (pH 8.0-8.5) depending on the volume of the supernatant. For protein G columns, 6 M guanidine hydrochloride was used to wash 3–5 times the column volume, followed by washing 3–5 times the column volume using pure water; the column was equilibrated with 3–5 times the column volume using a buffer system such as 1× PBS (pH 7.4) as the equilibration buffer; the cell supernatant was loaded and bound using a controlled low flow rate such that the retention time was approximately 1 min or more; the chromatography column was washed with 3–5 times the column volume using 1× PBS (pH 7.4) until the UV absorption dropped to the baseline; 0.1 M acetic acid / sodium acetate (pH 3.0) buffer was used for sample elution, the elution peak was collected according to UV detection, and the pH of the eluted product was quickly adjusted to 5–6 using 1 M Tris-HCl (pH 8.0) for temporary storage. For the elution product, solution exchange can be performed by methods well known to those skilled in the art, such as using ultrafiltration tubing for ultrafiltration and concentration and exchanging the solution into the required buffer system, or using molecular exclusion such as G-25 to desalt and exchange into the required buffer system, or using a high resolution molecular exclusion column such as Superdex200 to remove aggregated components in the elution product and improve the purity of the sample.

[0137] 2. Protein A affinity chromatography purification of proteins or antibodies: First, the supernatant of the cell culture expressing the antigen protein or antibody was centrifuged at high speed to collect the supernatant. For the protein A affinity column, 6 M guanidine hydrochloride was used to wash 3-5 times the column volume, followed by washing 3-5 times the column volume using pure water. The chromatography column was equilibrated with 3-5 times the column volume using a buffer system such as 1x PBS (pH 7.4) as the equilibration buffer. The cell supernatant was loaded and bound using a controlled low flow rate such that the retention time was approximately 1 min or more. After the binding was completed, 1x PBS (pH 7.4) was used to wash the chromatography column with 3-5 times the column volume until the UV absorption dropped to the baseline. 0.1 M acetic acid / sodium acetate (pH 3.0-3.5) buffer was used for sample elution, the elution peak was collected according to UV detection, and the pH of the eluted product was quickly adjusted to 5-6 using 1 M Tris-HCl (pH 8.0) for temporary storage. For the elution product, solution exchange can be performed by methods well known to those skilled in the art, such as using ultrafiltration tubing for ultrafiltration and concentration and exchanging the solution into the required buffer system, or using molecular exclusion such as G-25 to desalt and exchange into the required buffer system, or using a high resolution molecular exclusion column such as Superdex200 to remove aggregated components in the elution product and improve the purity of the sample. EXAMPLES

[0138] The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. The experimental methods for which conditions are not specifically shown in the examples of the present disclosure generally follow conventional conditions such as Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, or follow the conditions recommended by the manufacturers of raw materials or products. Reagents whose origins are not specifically shown are commercially available conventional reagents.

[0139] Example 1: Obtaining anti-human PD-1 mouse antibody The anti-human PD-1 mouse antibodies obtained by the above method were subjected to antigen binding experiments and screened to obtain several strains of antibodies with good activity, including M23, M32 and M33. Single cell clones were grown and cultured, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using degenerate primers of mouse Ig to obtain the variable region sequences of the antibodies. The variable region sequences of the mouse antibodies were linked to the constant region sequences of human antibodies. Chimeric antibodies of mouse monoclonal antibodies were cloned and recombinantly expressed, and in vitro activity experiments were performed to confirm that the variable region sequences of the obtained monoclonal antibodies were correct.

[0140] The variable region sequences of murine antibodies M23, M32 and M33 are determined as follows:

[0141] Heavy chain variable region of murine antibody M23 (SEQ ID NO:4): [ka]

[0142] Light chain variable region of murine antibody M23 (SEQ ID NO:5): [ka]

[0143] Heavy chain variable region of murine antibody M32 (SEQ ID NO:6): [ka]

[0144] Light chain variable region of murine antibody M32 (SEQ ID NO:7): [ka]

[0145] Heavy chain variable region of murine antibody M33 (SEQ ID NO:19): [ka]

[0146] Light chain variable region of murine antibody M33 (SEQ ID NO:20): [ka]

[0147] NOTE: In the heavy and light chain variable region sequences of the above antibodies, underlined are the CDR sequences as determined by the Kabat numbering system, in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0148] Table 4. Heavy and light chain CDR region sequences of murine antibodies M23, M32 and M33 [Table 4]

[0149] NOTE: Antibody CDR sequences in the table are determined according to the Kabat numbering system.

[0150] Example 2: Humanization of anti-human PD-1 monoclonal antibody By aligning against the IMGT human antibody heavy and light chain variable region germline gene database and analyzing with MOE software, the human germline heavy and light chain variable region germline genes with high identity to the M23, M32 and M33 light and heavy chain sequences were selected as templates. The CDRs of these three mouse antibodies were grafted into the corresponding human antibody templates to construct the corresponding humanized antibodies.

[0151] 1. Humanization of mouse antibody M23 1.1 Selection of humanized frameworks for the murine antibody M23 The humanized light chain templates for the murine antibody M23 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The sequences of the variable regions after humanization are as follows (underlined are CDR sequences):

[0152] Hu23VH-CDR-grafted: (SEQ ID NO: 27) [ka] Hu23VL-CDR-grafted: (SEQ ID NO: 28) [ka]

[0153] 1.2 Humanization template selection and back mutation design of mouse antibody M23

[0154] Table 5. Reversion of humanized mouse antibody M23 [Table 5]

[0155] Note: Grafted means that the mouse antibody CDRs are grafted onto the human germline FR region sequences. The amino acid residues are determined and annotated according to the Kabat numbering system. For example, I2G means that the I at position 2 of the Kabat numbering is mutated back to G according to the Kabat numbering system.

[0156] The light / heavy chain variable region sequences of the M23 humanized antibody are as follows: [ka]

[0157] 1.3 Combination of humanized sequences of mouse antibody M23 The antibodies obtained by humanizing the mouse antibody M23 and the combinations of their variable regions are shown in the table below.

[0158] Table 6. Combinations of humanized Hu23 antibody variable regions [Table 6]

[0159] NOTE: For example, "Hu23-1" refers to an antibody whose light chain variable region is Hu23VL1 and whose heavy chain variable region is Hu23VH1, and so on.

[0160] The antibody light / heavy chain variable region combinations referenced in the tables above (e.g., Hu23-1) can each be linked to an antibody light / heavy chain constant region to form a full length antibody; unless otherwise specified in this disclosure, when forming a full length antibody, the light chain variable region is linked to a kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to an IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72 or an IgG4-P heavy chain constant region set forth in SEQ ID NO: 79 to form the antibody heavy chain, and the names in the tables that reference an antibody light / heavy chain variable region combination (e.g., Hu23-1) + the suffix ".IgG4AA" refers to the full length antibody formed by ligation with an IgG4-AA heavy chain constant region, and + the suffix ".IgG4P" refers to the full length antibody formed by ligation with an IgG4-P heavy chain constant region. For example, "Hu23-1.IgG4AA" refers to a full-length antibody formed by linking a heavy chain formed by linking the Hu23VH1 heavy chain variable region with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and a light chain formed by linking the Hu23VL1 light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73. "Hu23-1.IgG4P" refers to a full-length antibody formed by linking a heavy chain formed by linking the Hu23VH1 heavy chain variable region with the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and a light chain formed by linking the Hu23VL1 light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73.

[0161] 2. Humanization of mouse antibody M32 2.1 Selection of humanized frameworks for mouse antibody M32 The humanized light chain templates for murine antibody M32 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The sequences of the humanized variable regions are as follows (underlined are CDR sequences): [ka]

[0162] 2.2 Humanization template selection and back mutation design of mouse antibody M32

[0163] Table 7. Reversion of humanized mouse antibody M32 [Table 7]

[0164] Note: Grafted means that the mouse antibody CDRs are grafted onto the human germline FR region sequences. The amino acid residues are determined and annotated according to the Kabat numbering system. For example, I2V means that the I at position 2 of the Kabat numbering is mutated back to V according to the Kabat numbering system.

[0165] The light / heavy chain variable region sequences of the humanized antibody of murine antibody M32 are as follows: [ka]

[0166] 2.3 Combination of humanized sequences of mouse antibody M32 An antibody obtained by humanizing the mouse antibody M32 and a combination of its variable regions.

[0167] Table 8. Combinations of light and heavy chain variable regions of humanized antibody Hu32 [Table 8]

[0168] Note: In the table, for example, "Hu32-1" refers to an antibody having a combination of an antibody light chain variable region of Hu32VL1 and a heavy chain variable region of Hu32VH1, and so on.

[0169] The antibody light / heavy chain variable region combinations referenced in the tables above (e.g., Hu32-1) can be linked with antibody light / heavy chain constant regions, respectively, to form full length antibodies; unless otherwise specified in this disclosure, when forming full length antibodies, the light chain variable region is linked to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72 or the IgG4-P heavy chain constant region set forth in SEQ ID NO: 79 to form the antibody heavy chain, and the names in the tables that refer to the antibody light / heavy chain variable region combinations (e.g., Hu32-1) + the suffix ".IgG4AA" refer to the full length antibody formed by ligation with the IgG4-AA heavy chain constant region, + the suffix ".IgG4P" refer to the full length antibody formed by ligation with the IgG4-P heavy chain constant region. For example, "Hu32-1.IgG4AA" refers to a full-length antibody formed by linking a heavy chain formed by linking the Hu32VH1 heavy chain variable region with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and a light chain formed by linking the Hu32VL1 light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73. "Hu32-1.IgG4P" refers to a full-length antibody formed by linking a heavy chain formed by linking the Hu32VH1 heavy chain variable region with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and a light chain formed by linking the Hu32VL1 light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73. Hu32VH1 a heavy chain formed by linking a heavy chain variable region and an IgG4-P heavy chain constant region shown in SEQ ID NO: 79; Hu32VL1 It refers to a full-length antibody formed by linking a light chain formed by linking a light chain variable region with the kappa chain constant region shown in SEQ ID NO:73.

[0170] 3. Humanization of Mouse Antibody M33 3.1 Selection of humanized frameworks for mouse antibody M33 The humanized light chain templates for the murine antibody M33 are IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV3-7 and IGHJ6*01. The sequences of the humanized variable regions are as follows (underlined are CDR sequences): [ka]

[0171] 3.2 Humanization template selection and back mutation design of mouse antibody M33

[0172] Table 9. Reversion of humanized mouse antibody M33 [Table 9]

[0173] Note: Grafted means that the mouse antibody CDRs are grafted to human germline FR region sequences. The amino acid residues are determined and annotated according to the Kabat numbering system. For example, F71Y means that the F at position 71 of the Kabat numbering is mutated back to Y according to the Kabat numbering system.

[0174] The sequences of the light and heavy chain variable regions of the humanized antibody of mouse antibody M33 are as follows: [ka]

[0175] 3.3 Combination of humanized sequences of mouse antibody M33

[0176] Table 10. Combinations of light and heavy chain variable regions of humanized antibodies [Table 10]

[0177] Note: In the table, for example, "Hu33-6" refers to an antibody having a combination of an antibody light chain variable region of Hu33VL2 and a heavy chain variable region of Hu33VH3, and so on.

[0178] The antibody light / heavy chain variable region combinations referenced in the above tables (e.g., Hu33-6) can each be linked to an antibody light / heavy chain constant region to form a full-length antibody; unless otherwise specified in this disclosure, when forming a full-length antibody, the light chain variable region is linked to a kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to an IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72 or an IgG4-P heavy chain constant region set forth in SEQ ID NO: 79 to form the antibody heavy chain, and the names in the tables that reference an antibody light / heavy chain variable region combination (e.g., Hu33-6) + the suffix ".IgG4AA" refers to a full-length antibody formed by ligation with an IgG4-AA heavy chain constant region, and + the suffix ".IgG4P" refers to a full-length antibody formed by ligation with an IgG4-P heavy chain constant region. For example, "Hu32-6.IgG4AA" refers to Hu33VH3 a heavy chain formed by linking a heavy chain variable region and an IgG4-AA heavy chain constant region shown in SEQ ID NO: 72; Hu33VL2 "Hu33-6.IgG4P" refers to a full-length antibody formed by linking a light chain formed by linking a light chain variable region with a kappa chain constant region shown in SEQ ID NO: 73. Hu33VH3 a heavy chain formed by linking a heavy chain variable region and an IgG4-P heavy chain constant region shown in SEQ ID NO: 79; Hu33VL2 It refers to a full-length antibody formed by linking a light chain formed by linking a light chain variable region with the kappa chain constant region shown in SEQ ID NO:73.

[0179] 4. Humanized antibody variants 4.1 Mutations of Hu23 humanized antibody By computer simulation, amino acids at specific sites in the light chain LCDR1 (SEQ ID NO: 11) of the Hu23 humanized antibody were subjected to site-directed mutation, and the specific mutations are shown in Table 11.

[0180] Table 11. Mutation sequences of Hu23 light chain LCDR1: [Table 11]

[0181] NOTE: Hu23LCDR1(N28Q) means the LCDR1 mutant sequence of Hu23 humanized antibody light chain variable region Hu23VL1 or Hu23VL2 in which N at position 28 according to the Kabat numbering standard is mutated to Q, and Hu23LCDR1(G29A) means the LCDR1 mutant sequence of Hu23 humanized antibody light chain variable region Hu23VL1 or Hu23VL2 in which G at position 29 according to the Kabat numbering standard is mutated to A (CDRs are determined by the Kabat numbering system).

[0182] The sequence of the Hu23 humanized antibody light chain variable region after LCDR1 mutation is as follows: [ka] [ka]

[0183] Table 12. Combinations of light and heavy chain variable regions of Hu23 humanized antibody [Table 12-1] [Table 12-2]

[0184] Note: In the table, for example, "Hu23-11" refers to an antibody having a combination of the antibody light chain variable region of Hu23VL1(N28T) and the heavy chain variable region of Hu23VH1, and so on.

[0185] The antibody light / heavy chain variable region combinations referenced in the tables above (e.g., Hu23-11) can be linked to antibody light / heavy chain constant regions, respectively, to form full length antibodies; unless otherwise specified in this disclosure, when forming full length antibodies, the light chain variable region is linked to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72 or the IgG4-P heavy chain constant region set forth in SEQ ID NO: 79 to form the antibody heavy chain, and the names in the tables that refer to the antibody light / heavy chain variable region combinations (e.g., Hu23-11) + the suffix ".IgG4AA" refer to the full length antibody formed by ligation with the IgG4-AA heavy chain constant region, and + the suffix ".IgG4P" refer to the full length antibody formed by ligation with the IgG4-P heavy chain constant region. For example, "Hu23-11.IgG4AA" refers to a full-length antibody formed by linking a heavy chain formed by linking the Hu23VH1 heavy chain variable region with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and a light chain formed by linking the Hu23VL1(N28T) light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73. "Hu23-11.IgG4P" refers to a full-length antibody formed by linking a heavy chain formed by linking the Hu23VH1 heavy chain variable region with the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and a light chain formed by linking the Hu23VL1(N28T) light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73.

[0186] The experimental results showed that after site mutation, humanized antibodies Hu23LCDR1(N28Q), Hu23LCDR1(N28L), Hu23LCDR1(N28T), Hu23LCDR1(N28D), Hu23LCDR1(G29A) and Hu23LCDR1(G29V) all maintained the ability to bind to PD-1 (Table 16).

[0187] 4.2 Mutations of Hu32 humanized antibody A series of humanized antibodies Hu23 derived from M23 and Hu32 derived from M32 had high sequence identity by sequence analysis. The Hu23 light chain variable region and the Hu32 heavy chain variable region were combined into a new combination of light chain and heavy chain variable region. The experimental results showed that the humanized antibodies containing the new combination of light chain and heavy chain variable region maintained the ability to bind to the PD-1 antigen (Table 16).

[0188] Table 13. General formulas for Hu32 and Hu23 antibody variable region consensus sequences [Table 13]

[0189] Table 14. Combinations of Hu32 heavy chain variable region and Hu23 light chain variable region [Table 14]

[0190] Note: In the table, for example, "Hu32a-85" refers to an antibody having an antibody light chain variable region of Hu23VL1(N28T) and a heavy chain variable region of Hu32VH6, and so on.

[0191] The antibody light / heavy chain variable region combinations referenced in the tables above (e.g., Hu32a-85) can be linked to antibody light / heavy chain constant regions, respectively, to form full length antibodies; unless otherwise specified in this disclosure, when forming full length antibodies, the light chain variable region is linked to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to the IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72 or the IgG4-P heavy chain constant region set forth in SEQ ID NO: 79 to form the antibody heavy chain, and the names in the tables that reference the antibody light / heavy chain variable region combinations (e.g., Hu32a-85) + the suffix ".IgG4AA" refer to the full length antibody formed by ligation with the IgG4-AA heavy chain constant region, + the suffix ".IgG4P" refer to the full length antibody formed by ligation with the IgG4-P heavy chain constant region. For example, "Hu32a-85.IgG4AA" refers to a full-length antibody formed by linking a heavy chain formed by linking a Hu32VH6 heavy chain variable region with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and a light chain formed by linking a Hu23VL1(N28T) light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73. "Hu32a-85.IgG4P" refers to a full-length antibody formed by linking a heavy chain formed by linking a Hu32VH6 heavy chain variable region with the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and a light chain formed by linking a Hu23VL1(N28T) light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73.

[0192] Table 15. Combinations of Hu23 heavy chain variable region and Hu32 light chain variable region [Table 15]

[0193] Note: In the table, for example, "Hu23a-57" refers to an antibody having a combination of the antibody light chain variable region of Hu32VL1 and the heavy chain variable region of Hu23VH1, and so on.

[0194] The antibody light / heavy chain variable region combinations referenced in the tables above (e.g., Hu23a-57) can each be linked to an antibody light / heavy chain constant region to form a full length antibody; unless otherwise specified in this disclosure, when forming a full length antibody, the light chain variable region is linked to a kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, the heavy chain variable region is linked to an IgG4-AA heavy chain constant region set forth in SEQ ID NO: 72 or an IgG4-P heavy chain constant region set forth in SEQ ID NO: 79 to form the antibody heavy chain, and the names in the tables that reference an antibody light / heavy chain variable region combination (e.g., Hu32a-85) + the suffix ".IgG4AA" refers to a full length antibody formed by ligation with an IgG4-AA heavy chain constant region, + the suffix ".IgG4P" refers to a full length antibody formed by ligation with an IgG4-P heavy chain constant region. For example, "Hu23a-57.IgG4AA" refers to a full-length antibody formed by linking a heavy chain formed by linking a Hu23VH1 heavy chain variable region with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, and a light chain formed by linking a Hu32VL1 light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73. "Hu23a-57.IgG4P" refers to a full-length antibody formed by linking a heavy chain formed by linking a Hu23VH1 heavy chain variable region with the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and a light chain formed by linking a Hu32VL1 light chain variable region with the kappa chain constant region shown in SEQ ID NO: 73.

[0195] 5. Screening of humanized antibodies Affinity detection of various humanized antibodies was performed by Biacore (see Example 3 for method), and the results are shown in Table 16. The results showed that various humanized antibodies maintained the ability to bind to PD-1, and some humanized antibodies even had affinities substantially close to those of their murine antibodies.

[0196] Table 16. Affinity of Hu23 humanized antibody to human PD-1 [Table 16]

[0197] Example 3: Construction and expression of a humanized PD-1 antibody Each humanized antibody VH / VK gene fragment was constructed using designed primers via PCR and then used to construct the full-length antibody expression vector VH-CH1-Fc-pHr / VK-CL-pHr by homologous recombination with the expression vector pHr (containing the signal peptide and constant region genes (CH1-Fc / CL) fragment). IgG4-P represents the S228P (corresponding to position 108 of SEQ ID NO:72 or SEQ ID NO:79) mutation, and IgG4-AA represents the F234A (corresponding to position 114 of the sequence of SEQ ID NO:72 or SEQ ID NO:79), L235A (corresponding to position 115 of SEQ ID NO:72 or SEQ ID NO:79) and S228P (corresponding to position 108 of SEQ ID NO:72 or SEQ ID NO:79) mutations. IgG4-AA and IgG4-P antibody formats can be obtained by simple point mutations in the IgG4 antibody format.

[0198] The sequence of the IgG4-AA heavy chain constant region is as follows (SEQ ID NO:72): [ka]

[0199] The sequence of the antibody light chain (kappa chain) constant region is as follows (SEQ ID NO:73): [ka]

[0200] The sequence of the constructed full-length antibody in IgG4AA format is exemplified as follows: [ka] [ka]

[0201] The sequence of the IgG4-P heavy chain constant region is as follows (SEQ ID NO: 79): [ka]

[0202] The sequence of the constructed full-length antibody of IgG4-P form is exemplified as follows: [ka] [ka] Test Example

[0203] (Test Example 1) ELISA experiment of binding and binding block of anti-PD-1 antibody to PD-1 ligand in vitro PD-L1 on the surface of tumor cells binds to PD-1 on the surface of T cells, thereby suppressing the proliferation of T cells. PD-1 antibodies can block the PD-L1 / PD-1 signaling pathway by binding to PD-1, thereby stimulating the proliferation of T cells. PD-1 / PD-L1 binding blocking experiments were used to detect the blocking activity of anti-PD-1 antibodies on the signaling pathway.

[0204] In this experiment, PD-1-His protein (Cat. No. 10377H08H, Sino Biological) was coated on a 96-well plate, and then anti-PD-1 antibodies to be tested (including antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO2015085847)) were added separately and the reaction was incubated; then, HRP-labeled goat anti-human IgG(H+L) antibody (Cat. No. 109-035-003, Jackson ImmunoResearch) was added and incubated. After washing the plate, the binding amount of HRP-labeled goat anti-human IgG(H+L) was detected to determine the EC of binding of anti-PD-1 antibodies to the ligand PD-1. 50 The value was calculated.

[0205] In this experiment, PD-1 protein fused to Fc at the extracellular domain (PD-1-Fc, see SEQ ID NO:1 for sequence) was coated onto a 96-well plate, and then anti-PD-1 antibodies to be tested (including antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO2015085847)) were added separately and incubated; then biotin-labeled PD-L1 / PD-L2 were added and incubated. After washing the plate, the binding amount of biotin-labeled PD-L1 / PD-L2 was detected, and the IC of anti-PD-1 antibodies that block the binding of ligands to PD-L1 / PD-L2 was calculated. 50 The value was calculated.

[0206] PD-1-Fc was diluted to 1 μg / ml using CB buffer at pH 9.6 (1.59 g Na2CO3 and 2.93 g NaHCO3 dissolved in 1 L distilled water), which was added to a 96-well plate at a volume of 100 μl / well and left at 4°C for 16-20 hours. PBS buffer was aspirated from the 96-well plate and the plate was washed once with PBST (PBS at pH 7.4 containing 0.05% Tween 20) buffer. 120 μl / well of PBST / 1% milk was added and incubated at room temperature for 1 hour for blocking. The blocking solution was removed and washed once with PBST buffer. 90 μl of anti-PD-1 antibody to be tested diluted to the appropriate concentration in sample diluent (PBS at pH 7.4 containing 5% BSA, 0.05% Tween 20) was added and pre-incubated at 4°C for 1 hour. 10x concentration of biotin-labeled PD-L1 / PD-L2 (Beijing Sino Biological Co., Ltd.) (10μg / ml) was added at a volume of 10μl / well, thoroughly shaken and mixed on a shaker, and incubated at 37℃ for 1h. The reaction system was removed and the plate was washed six times with PBST. 100μl / well of streptavidin-peroxidase polymer 1:400 diluted in PBST buffer was added and incubated at room temperature for 50min with shaking. The plate was washed six times with PBST. 100μl / well of TMB was added and incubated at room temperature for 5-10min. The reaction was stopped by adding 100μl / well of 1M H2SO4. The absorbance value at 450nm was read using a microplate reader to determine the IC of anti-PD-1 antibody blocking the binding of ligand to PD-L1 / PD-L2. 50 Values ​​were calculated and the data is detailed in Table 17 below.

[0207] Table 17. ELISA of anti-PD-1 antibodies of the disclosure that bind to PD-1 and block ligand binding to PD-L1 / PD-L2 [Table 17]

[0208] The exemplary anti-PD-1 antibodies of the present disclosure, Hu23-11.IgG4AA, Hu32a-85.IgG4AA, and Hu33-6.IgG4AA, can all effectively block the binding of PD-1 to PD-L1 / PD-L2, and their blocking activities are similar to that of the positive control antibody.

[0209] (Test Example 2) Ligand blocking test of exemplary antibodies The blocking effect of antibodies on the binding of PD-1 and PD-L1 was investigated. The experimental process is briefly described as follows:

[0210] CHOK1 / PD-L1 cells (Promega) were digested and added to a 96-well plate at 100μl / well and incubated in a 37℃, 5% CO2 incubator for 24 hours. Controls and samples were diluted to the desired concentrations using PBS. Jurkat / PD-1 cells (Jurkat cells stably transfected with PD-1) were counted and seeded at a fixed ratio (90μl / well) into the cell culture plate containing CHOK1 / PD-L1 cells, and 10μl / well of diluted antibodies (antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibodies: H005-1, negative control: IgG4 protein, antibody gradient dilution concentrations: 0.3mg / ml, 3mg / ml, 30mg / ml) were added and incubated in a 37℃, 5% CO2 incubator for 5 hours. The cell culture plate was removed and placed at room temperature for 5 minutes. Then, 50 μl of Bio-Glo (商標) Reagent was added to each well and incubated at room temperature for 5 minutes before the plate was read. The experimental results are shown in Figure 1.

[0211] The results showed that the exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA in this disclosure could effectively block the binding of PD-1 to PD-L1.

[0212] (Test Example 3) BIAcore antibody affinity experiments of exemplary antibodies IgG was affinity captured using a Protein A biosensor chip (catalog number 29127556, GE). Human PD-1 antigen (catalog number 10377H08H, Sino Biological) and Cyno PD-1 antigen (purchased from Sino Biological) were flowed across the surface of the chip, and binding and dissociation curves were obtained by real-time detection of PD-1 antibody and antigen PD-1 reaction signals by a Biacore T200 instrument. After the dissociation of each experimental cycle was completed, the biosensor chip was washed and regenerated with 10 mM glycine-HCl (pH 1.5) buffer. The experimental buffer system was 1×HBS-EP buffer solution (catalog number BR-1001-88, GE). After the experiment, the data was fitted to a (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software to obtain affinity values. The results are shown in Table 18.

[0213] Table 18. Affinity of anti-PD-1 antibodies to human PD1 and monkey PD-1 [Table 18]

[0214] The results showed that the exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA of the present disclosure were all capable of binding to human PD-1 and monkey PD-1.

[0215] (Test Example 4) Effect of antibodies on IFNγ secretion by cells in PBMC-T lymphocyte activation experiment To investigate the effect of anti-PD-1 antibody on the function of human primary T lymphocytes, human peripheral blood mononuclear cells (PBMCs) were collected and purified, stimulated with tuberculin (TB) for 5 days, and the secretion level of cytokine IFNγ was detected. The experimental process is briefly described as follows:

[0216] PBMCs were obtained from fresh blood using Ficoll-Hypaque (17-5442-02, GE) for density gradient centrifugation (Stem Cell Technologies) and cultured in RPMI1640 (SH30809.01, GE) medium supplemented with 10% (v / v) FBS (10099-141, Gibco) at 37°C and 5% CO2.

[0217] Freshly isolated and purified PBMCs were cultured at 2 × 10 6 The density was adjusted to 1.1 × 10 cells / ml. 40 μl of tuberculin (97-8800, Synbiotics) was added to 20 mL of cell suspension and cultured for 5 days in a 37 °C, 5% CO2 incubator. On the 5th day, the cultured cells were collected by centrifugation, resuspended in fresh RPMI 1640 medium, and diluted to 1.1 × 10 6 The cells were adjusted to a density of 1000 cells / ml and seeded in a 96-well cell culture plate at 90 μl / well. At the same time, antibody samples (including antibodies of the present disclosure: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody H005-1, and negative control IgG4 protein, at antibody gradient dilution concentrations of 0.3 mg / ml, 3 mg / ml and 30 mg / ml) gradient diluted with PBS (B320, Shanghai BasalMedia Technologies Co., Ltd.) were added at 10 μl / well. The cell culture plate was placed in an incubator at 37° C. and 5% CO2 and incubated for 3 days. The cell culture plate was removed and the cell culture supernatant was collected by centrifugation (4000 rpm, 10 min). The IFN-γ level was detected using an ELISA method (Human IFN-γ Detection Kit (EHC102g.96, Neobioscience)). For specific procedures, refer to the instructions for the reagents.

[0218] The test results are shown in Figure 2. The results showed that the anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA of the present disclosure were all able to effectively activate IFN-γ secretion.

[0219] (Test Example 5) Effect of anti-PD-1 antibody on colon cancer model MC38 in transgenic PD-1 mice MC38 cells 5 x 10 5 90 hPD-1 TG mice (Biocytogen) were inoculated subcutaneously at the right rib with 100 μl of cells / mouse. After 10 days, animals with tumors that were too large or too small were excluded. 3 Based on the average tumor volume of the mice, the mice were randomly divided into a total of seven groups, each group containing eight animals: blank control vehicle (PBS), positive control H005-1 3mpk, Hu32a-85.IgG4AA 1mpk, Hu32a-85.IgG4AA 3mpk, Hu23-11.IgG4AA 1mpk, Hu23-11.IgG4AA 3mpk and Hu33-6.IgG4AA 3mpk. The antibodies in each group were intraperitoneally injected three times a week from day 0. From the first week of administration onwards, the tumors were found to be significantly inhibited. In the second and third weeks, the administration frequency was adjusted to once a week for a total of five administrations. The tumor volume and the body weight of the animals were monitored twice a week and the data were recorded. When the tumor volume reached 2000 mm 3 Tumor-bearing animals were euthanized as the experimental endpoint when the tumor volume exceeded 100 mg / kg, or when most of the tumors appeared ulcerated, or when there was a 20% loss in body weight. Tumor volume (TV) = 1 / 2 × L long ×L short 2 Tumor growth rate (T / C%)=(T-T0) / (C-C0)×100% Tumor growth inhibition rate (TGI%)=1-T / C%

[0220] In the formula, T and T0 represent the tumor volumes at the end and start of the test in the antibody-administered group, respectively, and C and C0 represent the tumor volumes at the end and start of the test in the blank control group, respectively.

[0221] The test results are shown in Table 19 and Figure 3. The test results showed that, compared with the blank control, all of the antibodies of the present disclosure could significantly inhibit the growth of mouse colon cancer MC38 xenograft tumors. Among them, the Hu32a-85.IgG4AA-3mpk group showed the highest tumor inhibition rate, with a tumor inhibition rate of 77.64% at the final measurement. When the administration frequency was three times a week for three doses, at the measurement on the 7th day, the results showed that the tumor inhibition rates of all of the antibodies of the present disclosure were significantly superior to the tumor inhibition rate of the positive control antibody H005-1; then, the administration frequency was reduced to once a week, and after two doses (21 days), the difference in efficacy of the antibodies of the present disclosure gradually increased and showed a dose-dependent manner, in which Hu32a-85.IgG4AA was significantly superior to H005-1 at the same dose (p<0.05). Moreover, tumor-bearing mice tolerated all anti-PD-1 antibodies well, their body weight steadily increased throughout the administration process, and no obvious drug-induced weight loss or other symptoms occurred.

[0222] Table 19. Mouse colon cancer MC38s (mm 3 Effect of anti-PD-1 antibody on tumor growth inhibition rate of [Table 19]

[0223] (Test Example 6) Effect of anti-PD-1 antibody on colon cancer model MC38 in transgenic PD-1 mice Transgenic PD-1 mice were purchased from ISIS INNOVATION LIMITED, University Offices, Wellington Square, Oxford OX1 2JD, England, and bred at Cephrim Biosciences Co., Ltd. to obtain fifth-generation mice. MC38 cells were cultured at 5 × 10 5 Cells / 100 μl / animal were inoculated subcutaneously into the right hind rib of hPD-1 transgenic mice (half female and half male). The mice had a mean tumor volume of 80–100 mm. 3When the tumor volume reached 100 mg / kg, animals with body weight or tumors that were too large or too small were excluded. According to the tumor volume, the tumor-bearing mice were randomly divided into five groups (8 animals in each group): negative control hIgG control 30mpk, H005-1 10mpk, H005-1 30mpk, Hu33-6.IgG4AA 10mpk and Hu33-6.IgG4AA 30mpk. The day of grouping and administration was set as day 0. After grouping, each drug was administered intraperitoneally once every 2 days for 22 days, a total of 11 times. Tumor volumes were measured twice a week, body weights were measured, and data were recorded. All body weights and tumor volumes of animals in each group were expressed as mean ± standard deviation (Mean ± SEM), graphing was performed using Graphpad Prism 5 and Excel software, and statistical analysis was performed using Student's t-test. Tumor volume (TV) = 0.5236 × L long ×L short 2 Tumor growth rate T / C%=(T-T0) / (C-C0)×100% Tumor growth inhibition rate %TGI=1-T / C%

[0224] In the formula, T and T0 represent the tumor volumes at the end and start of the test in the antibody-administered group, respectively, and C and C0 represent the tumor volumes at the end and start of the test in the blank control group, respectively.

[0225] The test results are shown in Table 20 and Figure 4. The test results showed that the antibody of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 xenograft tumors compared with the control group. Among them, the Hu33-6.IgG4AA-30mpk group showed the highest tumor inhibition rate, with a tumor inhibition rate of 80.4% on day 20. In the low-dose group (10mpk), the efficacy of Hu33-6.IgG4AA-10mpk is superior to that of the positive control H005-1-10mpk.

[0226] Table 20. Effect of anti-PD-1 antibody on tumor volume of mouse colon cancer MC38 [Table 20]

[0227] Note: The unit of the mean tumor volume for each group in the table is mm 3 It is.

[0228] (Test Example 7) Pharmacokinetic study of anti-PD-1 antibody in cynomolgus monkeys The cynomolgus monkeys used in the experiment were six males, aged 2–5 years and weighing 2–5 kg, purchased from Guangdong Frontier Biotechnology Co., Ltd., license number: SCXK (Guangdong) 2015-0037, animal certificate number: 44613900000219.

[0229] Housing environment: Room temperature was controlled at 18°C ​​to 26°C, relative humidity was 40% to 70%, and lighting was alternating 12 h of light and dark. Mice were allowed free access to food and water, except when fasting was required.

[0230] Before administration, the animals were weighed and the body weight was 2.81-3.52 kg. Administration was performed using an injection pump for subcutaneous intravenous infusion into the forelimbs or hindlimbs. The dose in each group was 1 mg / kg (1 mpk), and was administered by single intravenous injection at a rate of 0.1 mL / kg / min over a dosing period of approximately 30 minutes. Before administration and 5 minutes, 0.25 hours, 0.5 hours (immediately after the end of administration), 1 hour, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 13 days, 14 days, 21 days, and 28 days after the start of intravenous infusion, the whole blood of the animals was collected from the hindlimb vein, and the serum was separated. Of these, approximately 2 mL of whole blood was collected before administration and on the 14th, 21st, and 28th days after the start of intravenous infusion, and approximately 1 mL of whole blood was collected at the remaining blood collection points. Blood drug concentrations in the serum were detected by ELISA, and PK analysis was performed. The results are shown in Table 21.

[0231] Table 21. Pharmacokinetics of humanized anti-PD1 antibodies in cynomolgus monkeys [Table 21]

[0232] The results showed that Hu23-11.IgG4AA and Hu33-6.IgG4AA have good pharmacokinetic activity.

[0233] For clarity of understanding, the above invention has been described in detail with the aid of figures and examples, which should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are fully and expressly incorporated by reference.

Claims

1. An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the combination of the heavy chain variable region and the light chain variable region is as shown below: (a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; A light chain variable region comprising an LCDR2 and LCDR3 set forth in SEQ ID NO:12 and SEQ ID NO:13, respectively, and an LCDR1 set forth in SEQ ID NO:11, 47, 48, 49, 50, 51 or 52.

2. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, comprising a combination of the heavy chain variable region and the light chain variable region shown below: (a1) the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; The light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:49, SEQ ID NO:12 and SEQ ID NO:13, respectively.

3. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1 or 2, which is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

4. 4. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 3, wherein the anti-PD-1 antibody is a humanized antibody, the humanized antibody comprising a framework region or a framework region variant thereof derived from a human antibody; The framework region variants include the following mutations: (f) 2G amino acid back mutations in the light chain variable region, and / or An amino acid back-mutation in the heavy chain variable region selected from any one of the following (f1) to (f3): (f1) the 27Y and 69L amino acid back mutations contained in the heavy chain variable region; or (f2) 27Y, 48I, 67T, 69L, and 82F amino acid back mutations in the heavy chain variable region; or (f3) 27Y, 48I, 67T, 69L, 82F, and 93T amino acid back mutations in the heavy chain variable region; wherein the amino acid positions are numbered and determined according to the Kabat criteria.

5. 4. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 3, wherein the combination of the heavy chain variable region and the light chain variable region of the antibody comprises the following: (i) and (l): (i) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO:4; and a light chain variable region, the sequence of which is as set forth in SEQ ID NO:5; (l) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO: 27, 30, 31, or 32; and a light chain variable region, the sequence of which is as set forth in SEQ ID NO: 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64; An anti-PD-1 antibody, or an antigen-binding fragment thereof, selected from:

6. 6. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain variable region and the light chain variable region are as shown below: (p) a heavy chain variable region, the sequence of which is as set forth in SEQ ID NO:27; and A light chain variable region, the sequence of which is as set forth in SEQ ID NO:

55.

7. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody comprises an antibody constant region.

8. 8. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region of the antibody constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region of the antibody constant region is selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof.

9. 9. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 8, wherein the antibody comprises the heavy chain constant region set forth in SEQ ID NO: 72 or 79, and the light chain constant region set forth in SEQ ID NO:

73.

10. 10. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the anti-PD-1 antibody comprises a light chain set forth in SEQ ID NO: 75 and a heavy chain set forth in SEQ ID NO: 74 or 80.

11. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 10, wherein the anti-PD-1 antibody comprises a heavy chain set forth in SEQ ID NO:74 and a light chain set forth in SEQ ID NO:

75.

12. The antigen-binding fragment may be Fab, Fab', F(ab') ２ 12. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody (scFv), a dimerized V region (diabody), and a disulfide bond stabilized V region (dsFv).

13. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the antibody is a bispecific antibody, a multispecific antibody, or an antibody fusion protein.

14. A pharmaceutical composition comprising a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 13, and one or more pharma- ceutically acceptable carriers, diluents, buffers, or excipients.

15. A nucleic acid molecule encoding the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 13.

16. A host cell comprising the nucleic acid molecule of claim 15.

17. A kit comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 13.

18. The pharmaceutical composition according to claim 14, for treating a disease associated with PD-1.

19. The pharmaceutical composition of claim 18, wherein the disease is a tumor.

20. The disease is head and neck squamous cell carcinoma.

19. The pharmaceutical composition of claim 18, wherein the cancer is selected from the group consisting of: human, human ovarian, human esophageal, human thyroid, human ovarian, human thyroid, human thyroid carcinoma, human esophageal, human thyroid cancer ...

21. 21. The pharmaceutical composition of claim 20, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

22. The pharmaceutical composition according to claim 18, wherein the disease is selected from the group consisting of PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, intestinal cancer and colon cancer.

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