CN110790839B

CN110790839B - anti-PD-1 antibody, antigen binding fragment thereof and medical application thereof

Info

Publication number: CN110790839B
Application number: CN201910710377.9A
Authority: CN
Inventors: 顾晓玲; 叶鑫; 葛虎; 胡齐悦; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2018-08-03
Filing date: 2019-08-02
Publication date: 2023-05-12
Anticipated expiration: 2039-08-02
Also published as: CN110790839A

Abstract

The present disclosure relates to anti-PD-1 antibodies, antigen-binding fragments thereof, and pharmaceutical uses thereof. In particular, the disclosure relates to humanized anti-PD-1 antibodies and antigen-binding fragments thereof that comprise specific CDR regions, as well as pharmaceutical compositions comprising anti-PD-1 antibodies and antigen-binding fragments thereof, and their use as medicaments. In particular, the disclosure relates to the use of a humanized anti-PD-1 antibody in the manufacture of a medicament for the treatment of a tumor.

Description

anti-PD-1 antibody, antigen binding fragment thereof and medical application thereof

Technical Field

The present disclosure is in the field of biotechnology, more specifically, the disclosure relates to anti-PD-1 antibodies and uses thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Tumor immunotherapy is a long-term hot spot in the field of tumor therapy, in which T-cell tumor immunotherapy is in its core position. Tumor immunotherapy is a method for fully utilizing and mobilizing killer T cells in a tumor patient to kill tumors, and is probably the most effective and safer way for treating tumors. At the same time, tumor cell escape is a great obstacle facing tumor immunotherapy, and tumor cells promote rapid growth of tumors by utilizing their own inhibitory effect on immune system.

There is a very complex relationship between the immune escape mechanism of a tumor and the immune response of the body to the tumor. Tumor-specific killer T cells are biologically active in early stages of tumor immunotherapy, but lose killing function with late stage tumor growth. Therefore, the tumor immunotherapy aims to maximally improve the immune response of a patient to the tumor, and not only needs to activate the original immune system response in vivo, but also needs to maintain the duration and the response intensity of the immune system response, which is the key of the immunotherapy.

In vivo activation of T cells takes place in two signal pathway systems, requiring a series of co-stimulatory molecules to provide a second signal in addition to the first signal provided to T cells by antigen presenting cell presenting MHC-antigen peptides, to allow the T cells to mount a normal immune response. This dual signal pathway system plays a vital role in the balance of the immune system in vivo, and it tightly regulates the body's different immune responses to self and non-self antigens. Absent the second signal provided by the co-stimulatory molecule, a non-responsive or sustained specific immune response of the T cell will result, thereby producing tolerance. Thus, the second signaling pathway plays a very critical regulatory role throughout the immune response of the body.

Programmed death molecule 1 (PD-l) is a protein receptor expressed on the surface of T cells found in 1992 and is involved in the apoptosis process of cells. PD-l belongs to the CD28 family, has 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (cytotoxic T Iymphocyte antigen, CTLA-4), but its expression is different from CTLA, mainly on activated T cells, B cells and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2, respectively. PD-L1 is expressed primarily on T cells, B cells, macrophages and Dendritic Cells (DCs), and expression on activated cells can be up-regulated. Whereas PD-L2 expression is relatively limited, primarily on antigen presenting cells, such as activated macrophages and dendritic cells.

PD-L1 inhibits the immune system by binding to PD-1 and B7-1, and many tumor cells and immune cells of the tumor tissue microenvironment express PD-L1. New researches find that the expression of high PD-L1 protein is detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, liver cancer and the like, and the expression level of PD-L1 is closely related to the clinic and prognosis of patients. Since PD-L1 plays a role in inhibiting T cell proliferation through a second signal path, blocking the combination of PD-L1/PD-1 becomes a very potential emerging target in the field of tumor immunotherapy.

At present, a plurality of nationwide pharmaceutical companies are developing monoclonal antibodies aiming at PD-L1/PD-1, and the monoclonal antibodies can furthest improve the immune system response of patients to tumors by blocking the combination of PD-L1/PD-1, so as to achieve the aim of killing tumor cells. The FDA has approved 5 PD-1/PD-L1 antibody drugs on the market, including antibodies against PD1 (Pembrolizumab, novilumab) and antibodies against PD-L1 (Atezolizumab, avelumab, durvalumab), have been approved for melanoma, hodgkin lymphoma patients, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck squamous carcinoma, gastric cancer, liver cancer, merck skin cancer, and all microsatellite-unstable solid tumors, and like tumor types. More anti-PD-L1/PD-1 antibodies are described in: WO0139722, WO2013173223, WO2014195852, WO2013181634, WO2015048520, WO2015036511, US2014335093, WO2014100079, WO2014055897, US6803192B1, WO2014022758, US8617546B2, WO2010089411A2, WO2015085847, etc. Although some anti-PD-1 antibodies have a better response rate (ORR) in hematological tumors, the response rate is only around 30% in advanced non-small cell lung cancer, and the total response rate alone is only 14% in mid-and advanced esophageal and gastric cancers. The applicant of the present disclosure has obtained an anti-PD-1 antibody H005-1 (disclosed in WO 2015085847) by screening, which has also entered various clinical trials and has achieved good clinical results. Recent research results published in the ASCO university in 2018 show that the effective rate of anti-PD-1 antibody in combination with apatinib in advanced non-squamous non-small cell carcinoma is 41.2% and the disease control rate is 94.1%. In clinical researches on gastric cancer and liver cancer, the effective rate of the anti-PD-1 antibody combined with the apatinib treatment is 30.6%, and the disease control rate is 83.3%. There is still a need to continue to develop more potent anti-PD-1 antibodies.

Disclosure of Invention

The present disclosure provides anti-PD-1 antibodies or antigen-binding fragments that bind to the amino acid sequence or three-dimensional structure of the extracellular region of PD-1. In particular, the present disclosure provides a series of anti-PD-1 antibodies or antigen-binding fragments thereof with higher affinity, longer in vivo half-life, higher biological activity on the basis of H005-1.

In some alternative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises:

(i) As set forth in SEQ ID NO:6, a heavy chain HCDR1 region as set forth in SEQ ID NO:7, and a heavy chain HCDR2 region as set forth in SEQ ID NO:8 or 9, and a heavy chain HCDR3 region; and/or

(ii) Respectively as SEQ ID NO: 10. 11, 12, LCDR1, LCDR2, LCDR3 region;

wherein said SEQ ID NO: 7. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 each has the general sequence shown below:

HCDR2 TISGGGANTYFAX ₁ SVKG SEQ ID NO：7

LCDR1 X ₂ ASQX ₃ IX ₄ X ₅ WLX ₆ SEQ ID NO：10

LCDR2 X ₇ ATX ₈ X ₉ X ₁₀ X ₁₁ SEQ ID NO：11

LCDR3 QQX ₁₂ YX ₁₃ X ₁₄ PWT SEQ ID NO：12

wherein: x is X ₁ Selected from D or N, X ₂ Selected from L, R or Q, X ₃ Selected from T, S, G, N or D, X ₄ Selected from G, A or Y, X ₅ Selected from T, Y, N, S or D, X ₆ Selected from T, N or A, X ₇ Selected from T, G or L, X ₈ Selected from T, S or H, X ₉ Selected from R or L, X ₁₀ Selected from A, E or Q, X ₁₁ Selected from D, Y or S, X ₁₂ Selected from V or A, X ₁₃ Selected from S or T, X ₁₄ Selected from V, T or I.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises:

As set forth in SEQ ID NO:6, and the heavy chain HCDR1 is shown as SEQ ID NO:16 or 17, and a heavy chain HCDR2 as set forth in SEQ ID NO:8 or 9, and a heavy chain HCDR3 region; and/or

As set forth in SEQ ID NO:18-24, and the light chain LCDR1 as shown in any one of the amino acid sequences of SEQ ID NO:25-30 and the light chain LCDR2 region shown in any one of amino acid sequences SEQ ID NO:31-34, and a light chain LCDR3 region represented by any one of the amino acid sequences.

(a) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 18. SEQ ID NO: 25. SEQ ID NO:31, LCDR1, LCDR2, LCDR3 regions of the light chain shown;

(b) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 19. SEQ ID NO: 26. SEQ ID NO:32, and LCDR1, LCDR2, LCDR3 regions of the light chain;

(c) Respectively as SEQ ID NO: 6. SEQ ID NO: 17. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 18. SEQ ID NO: 25. SEQ ID NO:33, light chain LCDR1, LCDR2, LCDR3 regions shown in figure 33;

(d) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:9, and HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 18. SEQ ID NO: 25. SEQ ID NO:33, light chain LCDR1, LCDR2, LCDR3 regions shown in figure 33;

(e) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 18. SEQ ID NO: 25. SEQ ID NO:33, light chain LCDR1, LCDR2, LCDR3 regions shown in figure 33;

(f) Respectively as SEQ ID NO: 6. SEQ ID NO: 17. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 20. SEQ ID NO: 27. SEQ ID NO:31, LCDR1, LCDR2, LCDR3 regions of the light chain shown;

(g) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 21. SEQ ID NO: 25. SEQ ID NO:32, and LCDR1, LCDR2, LCDR3 regions of the light chain;

(h) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 22. SEQ ID NO: 28. SEQ ID NO:34, LCDR1, LCDR2, LCDR3 region of the light chain;

(i) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 20. SEQ ID NO: 27. SEQ ID NO:31, LCDR1, LCDR2, LCDR3 regions of the light chain shown;

(j) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 23. SEQ ID NO: 29. SEQ ID NO:31, LCDR1, LCDR2, LCDR3 regions of the light chain shown; or (b)

(k) Respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 24. SEQ ID NO: 30. SEQ ID NO:31, LCDR1, LCDR2, LCDR3 regions of the light chain. Preferably, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, characterized in that:

the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 18. SEQ ID NO: 25. SEQ ID NO:31 LCDR1, LCDR2 and LCDR3; or (b)

The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 18. SEQ ID NO: 25. SEQ ID NO:33 LCDR1, LCDR2 and LCDR3.

In some embodiments, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises an FR region.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment comprises: as set forth in SEQ ID NO:13 or SEQ ID NO:14 and a heavy chain variable region as set forth in SEQ ID NO:15, wherein said light chain variable region of SEQ ID No. 13, SEQ ID NO: 14. SEQ ID NO:15 each have the general sequence shown below:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWV ATISGGGANTYFAX ₁ SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQ IGFTNWGQGTTVTVSS SEQ ID NO：13

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWV ATISGGGANTYFAX ₁ SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQL YYFDYWGQGTTVTVSS SEQ ID NO：14

DIQMTQSPSSLSASVGDX ₁₅ VTITCX ₂ ASQX ₃ IX ₄ X ₅ WLX ₆ WYQQKPGX ₁₆ X ₁₇ PKLLIX ₁₈ X ₇ ATX ₈ X ₉ X ₁₀ X ₁ ₁ GVPSRFSGSGSGTDFTLTIX ₁₉ SLQPEDFAX ₂₀ YYCQQ X ₁₂ YX ₁₃ X ₁₄ PWTFGGGTKVEIK SEQ ID NO：15

Wherein X is ₁ Selected from D or N, X ₂ Selected from L, R or Q, X ₃ Selected from T, S, G, N or D, X ₄ Selected from G, A or Y, X ₅ Selected from T, Y, N, S or D, X ₆ Selected from T, N or A, X ₇ Selected from T, G or L, X ₈ Selected from T, S or H, X ₉ Selected from R or L, X ₁₀ Selected from A, E or Q, X ₁₁ Selected from D, Y or S, X ₁₂ Selected from V or A, X ₁₃ Selected from S or T, X ₁₄ Selected from V, T or I, X ₁₅ Selected from R or I, X ₁₆ Selected from K or Q, X ₁₇ Selected from A or P, X ₁₈ Selected from Y or H, X ₁₉ Selected from S or G, X ₂₀ Selected from A or T.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises a polypeptide selected from the group consisting of: as shown in SEQ ID NO: 35. 36 or 37 and a heavy chain variable region as set forth in SEQ ID NO: 38. 39, 40, 41, 42, 43, 44, 45, 46 or 47;

preferably, the anti-PD-1 antibody or antigen-binding fragment thereof comprises:

as set forth in SEQ ID NO:36 and a heavy chain variable region as set forth in SEQ ID NO:40, and a light chain variable region shown in seq id no;

as set forth in SEQ ID NO:37 and a heavy chain variable region as set forth in SEQ ID NO:40, and a light chain variable region shown in seq id no;

as set forth in SEQ ID NO:36 and a heavy chain variable region as set forth in SEQ ID NO:41, a light chain variable region shown in seq id no; or (b)

As set forth in SEQ ID NO:35 and a heavy chain variable region as set forth in SEQ ID NO: 38. 39, 40, 41, 42, 43, 44, 45, 46 or 47;

More preferably, said anti-PD-1 antibody, or antigen-binding fragment thereof, preferably comprises:

as set forth in SEQ ID NO:35 and the heavy chain variable region shown in SEQ ID NO:38, and a light chain variable region shown in seq id no; or (b)

As set forth in SEQ ID NO:35 and the heavy chain variable region shown in SEQ ID NO: 40.

In some embodiments, the antibody is a full length antibody, wherein the antibody heavy chain further comprises a heavy chain constant region of human IgG1, igG2, igG3, or IgG4, or variants thereof, non-limiting implementations of other heavy chain constant regions include optimization of the constant region of human IgG1, igG2, igG4 for improved function, e.g., D265A, N297A, L a/L235A or L234F/L235A, etc., can reduce ADCC, P331S or nearby mutations can reduce CDC, S228P, F234A, and L235A mutations, etc.; preferably comprising a human IgG1 or IgG4 heavy chain constant region, more preferably comprising an IgG1 or IgG4 heavy chain constant region into which the F234A and L235A mutations are introduced; the antibody light chain further comprises a constant region of a human kappa, lambda chain or variant thereof.

In other embodiments, the antibody is a full length antibody, and the heavy chain constant region of the PD-1 antibody is selected from the group consisting of IgG4 wild-type constant regions, or other mutations that reduce ADCC or CDC activity are introduced into the IgG4, igG1 constant regions, without substantially affecting the function of the antibodies of the disclosure.

In some embodiments, the antibody is a full length antibody, and the heavy chain constant region sequence of the anti-PD-1 antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO:48 or SEQ ID NO:49 or at least 85% sequence identity thereto, the light chain constant region is as set forth in SEQ ID NO:50 or at least 85% sequence identity thereto. In some embodiments, the sequence having at least 85% sequence identity is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof preferably comprises an amino acid sequence as set forth in SEQ id no:51 and a heavy chain as set forth in SEQ ID NO:52, a light chain indicated by 52; or having the sequence as set forth in SEQ ID NO:53 and a heavy chain as set forth in SEQ ID NO: 54.

In some embodiments, the antigen binding fragment is an antigen binding fragment selected from the group consisting of Fab, fab ', F (ab') 2, single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), and CDR-comprising peptide

In one aspect, the disclosure also provides an isolated antibody or antigen-binding fragment thereof that competitively binds to human PD-1 with the anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding claims, or binds to the same human PD-1 epitope as the anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding claims.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-PD-1 antibody, or antigen-binding fragment thereof, according to any one of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the therapeutically effective amount of the antibody or antigen-binding fragment thereof is a unit dose of 0.1-3000mg/kg of an antibody or antigen-binding fragment thereof as described above.

In some aspects, the disclosure provides a nucleic acid molecule encoding an anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding claims.

In some aspects, the disclosure provides a vector comprising a nucleic acid molecule as described above.

In some aspects, the present disclosure provides a host cell selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell, transformed with a vector according to the foregoing.

In some aspects, the disclosure provides methods for producing an antibody or antigen-binding fragment thereof of any one of the preceding claims, the methods comprising culturing a host cell of the preceding claims in culture to form and accumulate the antibody or antigen-binding fragment thereof of any one of the preceding claims, and recovering the antibody or antigen-binding fragment thereof from the culture.

In some aspects, the disclosure provides methods for immunodetection or assay of human PD-L1, the methods comprising using an anti-PD-1 antibody, or antigen-binding fragment thereof, of any one of the foregoing.

In some aspects, the disclosure provides the use of an anti-PD-1 antibody, or antigen-binding fragment thereof, of any one of the preceding claims in the preparation of a tumor diagnostic.

In some aspects, the disclosure provides methods of treating a tumor, the methods comprising administering to a subject a pharmaceutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding claims, or a pharmaceutical composition comprising any one of the preceding claims, or a nucleic acid molecule of any one of the preceding claims. In some embodiments, wherein the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, head and neck carcinoma, brain carcinoma, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatocellular carcinoma, hepatobiliary carcinoma, pancreatic carcinoma, gastric carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, kukene's tumor, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; more preferably, the lymphoma is selected from: 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/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia. More preferably, the tumor is melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, non-small cell lung cancer, bladder cancer. In some embodiments, wherein the tumor is a PD-L1 positive tumor.

In some aspects, the disclosure provides the use of an anti-PD-1 antibody, or antigen-binding fragment thereof, as described in any one of the preceding claims, or a pharmaceutical composition comprising, or a nucleic acid molecule as described in the preceding claims, in the preparation of a tumor medicament. In some embodiments, wherein the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, head and neck carcinoma, brain carcinoma, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatocellular carcinoma, hepatobiliary carcinoma, pancreatic carcinoma, gastric carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, kukene's tumor, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; more preferably, the lymphoma is selected from: 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/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia. In some embodiments, wherein the tumor is melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, non-small cell lung cancer, bladder cancer. In some embodiments, wherein the tumor is a PD-L1 positive tumor.

In some aspects, the disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding claims for use as an anti-tumor medicament.

The anti-PD-1 antibodies or antigen-binding fragments thereof in the present disclosure exhibit good effects in biochemical tests or in vitro pharmacodynamic experiments. Wherein, in one affinity detection assay, the anti-PD-1 antibodies of the present disclosure are both effective in blocking the binding of PD-1 to its ligand PD-L1, wherein the anti-PD-1 antibodies Ab05, ab01 have a higher blocking activity than the parent antibody H005-1.

In affinity detection assays, the anti-PD-1 antibodies of the present disclosure are both capable of specifically binding PD-1, with affinity that is both pronounced to the parent antibody H005-1.

In an in vitro activity detection test, the anti-PD-1 antibodies in the disclosure can effectively activate PBMC cells to secrete cell factor IFNgamma and have a dose-dependent effect, wherein antibodies Ab01, ab05 and Ab09 can activate PBMC cells to secrete IFNgamma under the condition of high concentration, and the effect is obviously higher than that of a parent antibody H005-1. Meanwhile, the influence of different constant regions on the in vitro activity of the PD-1 antibody is detected, and as a result, the IgG1 antibody still has higher activation effect on PBMC cells, wherein the effects of antibodies Ab01-IgG1, ab07-IgG1, ab08-IgG1 and Ab09-IgG1 on activating PBMC cells to secrete IFNgamma are obviously higher than those of the parent antibody. The difference in constant regions does not substantially affect the function of the antibodies of the present disclosure.

In one in vivo pharmacokinetic test, the exemplary antibody Ab05 of the present disclosure was found to have good drug metabolism performance in cynomolgus monkeys, with an average drug half-life t1/2 of about 6.2 days, suggesting that the antibody has good stability in cynomolgus monkeys. Whereas the half-life t1/2 of the parent antibody H005-1 was found to be about 3.7 days at the same dose (1 mpk).

In experiments in transgenic PD-1 mouse colon cancer model MC38, the antibody disclosed by the invention can obviously inhibit the growth of mouse colon cancer MC38 transplanted tumor, wherein the tumor inhibition rate reaches 76.4% when measured on day 20 of Ab05 30mpk group.

Drawings

Fig. 1: anti-PD-1 antibodies block the binding activity of PD-1 to PD-L1.

Fig. 2A-2C: promotion of ifnγ secretion by PBMC cells by PD-1 antibodies. Among them, FIGS. 2A and 2B show the effect of IgG4 type antibodies on secretion of IFNγ by PBMC cells, and FIG. 2C shows the effect of IgG1 type antibodies on secretion of IFNγ by PBMC cells.

Fig. 3: role of anti-PD-1 antibodies in transgenic PD-1 mouse colon cancer model MC 38.

Detailed Description

A term of

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD1", "PDCD1", "hPD-1" and "hPD-I" are used interchangeably and include variants, isoforms, species homologs of human PD1, and analogs having at least one common epitope with PD 1. Complete PD-1 sequences can be found in GenBank accession U64863.

The term "programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which down-regulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologs of hPD-L1, as well as 5 analogs having at least one epitope in common with hPD-L1. The complete hPD-L1 sequence can be found using GenBank accession number Q9 NZQ.

The term "cytokine" is a general term for proteins released by one cell population that act 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-gamma, IL-6, TNF alpha, IL-17 and IL-5.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in J.biol. Chem,243, p3558 (1968).

The "antibody" of the present disclosure refers to an immunoglobulin that is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, and epsilon chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

In the present disclosure, the antibody light chain of the present disclosure may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin, or variants thereof.

In the present disclosure, the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4, or variants thereof, of human or murine origin.

An "affinity matured antibody" refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) that result in an improved affinity or performance of the antibody for an antigen as compared to a parent antibody that does not have the alterations.

The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each light chain variable region (LCVR or VL) and heavy chain variable region (HCVR or VH) consists of 3 CDR regions and 4 FR regions, arranged in order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, preferably humanized antibodies.

The term "murine antibody" is in the present disclosure a monoclonal antibody against human PD-1 prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with the PD-1 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the present disclosure, the murine PD-1 antibody or antigen binding fragment thereof may further comprise a light chain constant region of murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of murine IgG1, igG2, igG3 or variant thereof.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the PD-1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the PD-1 chimeric antibody further comprises a heavy chain constant region of a human IgG1, igG2, igG3, igG4, or variant thereof, preferably comprises a human IgG1, igG2, or IgG4 heavy chain constant region, or an IgG1, igG2, or IgG4 variant using an amino acid mutation (e.g., an L234A and/or L235A mutation, and/or an S228P mutation).

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available in Internet www.mrccpe.com.ac.uk/VBase) and in Kabat, E.A. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies obtained further upon affinity maturation of CDRs by yeast display.

Grafting of CDRs may result in reduced affinity of the generated PD-1 antibody or antigen-binding fragment thereof for the antigen due to framework residues that are in contact with the antigen. Such interactions may be the result of somatic hypermutations. Thus, it may still be desirable to graft such donor framework amino acids to the framework of a humanized antibody. Amino acid residues involved in antigen binding from a non-human PD-1 antibody or antigen-binding fragment thereof can be identified by examining the sequence and structure of the murine monoclonal antibody variable region. Residues in the CDR donor framework that differ from the germline can be considered relevant. If the closest germline cannot be determined, the sequences can be compared to subtype consensus sequences or consensus sequences of murine sequences with a high percentage of similarity. Rare framework residues are thought to be the result of highly mutated somatic cells, thereby playing an important role in binding.

"human antibody" (HuMAb), "human antibody", "fully human antibody" are used interchangeably and may be an antibody of human origin or an antibody obtained from a transgenic organism "engineered" to produce specific human antibodies in response to antigen stimulation and may be produced by any method known in the art. In certain techniques, the elemental elements of the human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines in which the endogenous heavy and light chain loci are disrupted by targeted disruption of the endogenous heavy and light chain loci contained in the cell lines. Transgenic organisms can synthesize human antibodies specific for human antigens, and the organisms can be used to produce human antibody-secreting hybridomas. A human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies may also be constructed by gene or chromosome transfection methods as well as phage display techniques, or from in vitro activated B cells, all of which are known in the art.

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). Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments contained in the term "antigen-binding fragment" of an antibody include (i) Fab fragmentsA monovalent segment consisting of VL, VH, CL and CH1 domains; (ii) F (ab') ₂ A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of the single arm of the antibody; (v) Single domain or dAb fragments (Ward et al, (1989) Nature 341:544-546) consisting of VH domains; 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 of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, so that they can produce a single protein chain (known as a single chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, e.g., bird et al (1988) Science242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies. In some embodiments, the antigen binding fragments of the present disclosure are Fab, F (ab ') 2, fab', single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), or CDR-containing peptides, and the like.

Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity in a fragment obtained by treating an IgG antibody molecule with protease papain (cleavage of amino acid residue at position 224 of the H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds. In some embodiments, the Fab of the present disclosure may 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 region or its three-dimensional structure with papain. In addition, the Fab may 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.

F (ab') 2 is an antibody fragment having a molecular weight of about 100,000 and having antigen binding activity and comprising two Fab regions linked at hinge positions, obtained by digestion of the lower part of the two disulfide bonds in the IgG hinge region with the enzyme pepsin. In some embodiments, the F (ab') 2 of the present disclosure may 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 region or its three-dimensional structure with pepsin. In addition, the F (ab ') 2 may be produced by linking Fab' described below with a thioether bond or a disulfide bond.

Fab 'is an antibody fragment having a molecular weight of about 50,000 and antigen binding activity obtained by cleavage of disulfide bonds in the hinge region of the above F (ab') 2. In some embodiments, fab 'of the present disclosure can be produced by treating F (ab') 2 of the present disclosure that specifically recognizes PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent, such as dithiothreitol.

In addition, the Fab ' may be produced by inserting DNA encoding a Fab ' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryotic organism to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by 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 Rooverts et al (2001), cancer immunol.

The scFv of the present disclosure can be produced by the following steps: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human PD-1 and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof of the present disclosure are obtained, DNA encoding scFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express scFv.

Diabodies are antibody fragments in which scFv or Fab is dimerized, and are antibody fragments having bivalent antigen-binding activity. In the divalent antigen binding activity, the two antigens may be the same or different. Bispecific and multispecific antibodies refer to antibodies capable of binding two or more antigens or antigenic determinants simultaneously, including scFv or Fab fragments.

Diabodies of the present disclosure may be produced by the steps of: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human PD-1 and binding to the amino acid sequence of an extracellular region or three-dimensional structure thereof of the present disclosure are obtained, DNA encoding scFv is constructed so that the amino acid sequence of a peptide linker is 8 residues or less in length, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express a diabody.

dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine residue via a disulfide bond between cysteine residues. Amino acid residues substituted with cysteine residues may be selected based on predictions of the three-dimensional structure of the antibody according to known methods (Protein Engineering,7,697 (1994)).

The dsFv of the present disclosure may be produced by the steps of: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human PD-1 and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof of the present disclosure are obtained, DNA encoding dsFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express the dsFv.

The CDR-containing peptide is constituted by one or more regions in the CDR containing VH or VL. Peptides comprising multiple CDRs may be linked directly or via suitable peptide linkers.

The CDR-containing peptides of the present disclosure can be produced by: constructing a DNA encoding CDRs of VH and VL of a monoclonal antibody specifically recognizing human PD-1 and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof of the present disclosure, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryotic organism to express the peptide. The CDR-containing peptide may also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.

The terms "CDR", "complementarity determining region", "hypervariable region" refer to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. Typically, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including "Kabat" numbering convention (see Kabat et Al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public Health Service, national Institutes of Health, bethesda, MD), "Chothia" numbering convention (see Al-Lazikani et Al, (1997) JMB 273:927-948) and ImMunoGenTics (IMGT) numbering convention (Lefranc M.P.), immunolist, 7, 132-136 (1999); lefranc, M.P. et Al, dev. Comp. Immunol.,27, 55-77 (2003) et Al, for example, for classical format, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3), the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR) and 24-34, 50-35 (VL 2) and 95 (HCDR 2) and the amino acid residues in the light chain variable domain (VL) are numbered 24-35 (HCDR 1), the amino acid residues in the light chain variable domain (VL) are numbered 26-35, VL 2-35 (VL 2) and 95-35 (HCDR 2) and the amino acid residues in the light chain variable domain (VL) are numbered 52-35 (VL 2) and 95-35 (VL 2) are numbered 52-35 and 95 (HCDR 1) and 52-52 (VL 1) are defined by the amino acid sequence of the combination of amino acid residues in the L.35-35-VL.VL.35 (VL.35 and VL.95 (2003) and 95 (2003) and so on 50-56 (LCDR 2) and 89-97 (LCDR 3). Following the IMGT rules, the CDR amino acid residues in VH are approximately 26-35 (CDR 1), 51-57 (CDR 2) and 93-102 (CDR 3), and the CDR amino acid residues in VL are approximately 27-32 (CDR 1), 50-52 (CDR 2) and 89-97 (CDR 3). Following IMGT rules, CDR regions of antibodies can be determined using the procedure IMGT/DomainGap alignment. .

The terms "framework region", "framework region" or "FR", "FR region" of an antibody as used herein refer to a portion 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 that does not have CDRs.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a PD-L1 molecule). Epitopes generally comprise at least 3,4,5,6,7,8,9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epitope Mapping Protocols in Methods in Molecular B iology, volume 66, g.e.Morris, ed. (1996).

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 ^-8 M, e.g. less than about 10 ^-9 M、10 ^- ¹⁰ M、10 ^-11 Affinity (KD) binding of M or less.

The term "KD" or "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present disclosure are administered at a ratio of less than about 10 ^-7 M, e.g. less than about 10 ^-8 M or 10 ^-9 The dissociation equilibrium constant (KD) of M binds to PD-1, e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR) techniques.

When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it is meant that competition is between antigen binding proteins, as determined by the following assay: in such assays, the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a cognate antigen (e.g., PD-1 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assay (see, e.g., stahli et al, 1983,Methodsin 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 labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual (antibodies, laboratory Manual), cold Spring Harbor Press); direct labelling of RIA with the solid phase of the I-125 label (see, e.g., morel et al, 1988, molecular. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., cheung, et al, 1990, virology 176:546-552); and directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol. 32:77-82). Typically the assay involves the use of purified antigen that binds to a solid surface or cell bearing either the unlabeled detection antigen binding protein or the 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 measured. Typically the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding protein that binds to an epitope in close proximity to the binding epitope of the reference antigen binding protein, the two epitopes spatially preventing binding from occurring. Additional details regarding methods for assaying competitive binding are provided in the examples herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more of the specific binding of the reference antigen binding protein to the cognate antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to amino acid residues in a second sequence, with gaps introduced between the aligned amino acid sequences and where necessary to achieve the maximum percentage of sequence identity, and without any conservative substitutions being considered part of the sequence identity. For the purpose of determining the percent amino acid sequence identity, the alignment may be accomplished in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the genome of a host cell upon introduction into the host cell so as to replicate with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. For example, mice may be immunized with human PD-1 or a fragment thereof, the resulting antibodies may be renatured, purified, and amino acid sequenced using conventional procedures. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions into the non-human CDR regions. Human FR germline sequences can be obtained from the website http:// IMGT. Cines. FR of ImMunoGeneTics (IMGT), or from the journal of immunoglobulins, 2001ISBN012441351 by aligning IMGT human antibody variable region germline gene databases with MOE software.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); the family of bacillus (bacillus) such as bacillus subtilis (Bacillus subtilis); pneumococci (pneumococci); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

The engineered antibodies or antigen binding fragments of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expressing antibodies that specifically bind to human PD-1. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"administering," "administering," or "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," or "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administering," or "treatment" also means in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering to a patient an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, that has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating each target disease symptom, it should be determined according to any statistical test method known in the art, such as Student t-test, chi-square test, U-test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the target disease symptom should be alleviated in a statistically significant number of patients.

"conservative modifications" or "conservative substitutions or substitutions" refer to amino acids in other amino acid substituted proteins that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. Those skilled in The art know that in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) Molecular Biology of The Gene, the Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are set forth in the following table "exemplary amino acid conservative substitutions".

Exemplary amino acid conservative substitutions

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease, and an effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening the severity, or delaying the onset of a disorder, including biochemical, histological and/or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes that are exhibited during the development of the disorder. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various target antigen-related disorders of the present disclosure, reducing the dosage of other agents required to treat a disorder, enhancing the efficacy of another agent, and/or slowing the progression of a target antigen-related disorder of the present disclosure in a patient. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the patient, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"exogenous" refers to substances produced outside of an organism, cell or human body as the case may be. "endogenous" refers to substances produced in cells, organisms or humans, as the case may be.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, when sequences are optimally aligned, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches or homologies at 100 positions in the two sequences, then the two sequences are 95% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology. For example, the comparison may be performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give a maximum match between the respective sequences over the entire length of the respective reference sequences. The following references relate to BLAST algorithms that are often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS) Altschul, S.F. et al, (1990) J.mol.biol.215:403-410; gish, W.et al, (1993) Nature Genet.3:266-272; madden, T.L. et al, (1996) meth.enzymol.266:131-141; altschul, S.F. 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 those provided by NCBI BLAST, are also known to those skilled in the art.

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell. Where different names are meant, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of nucleic acids, RNA and/or DNA of a particular moiety are amplified as described, for example, in U.S. Pat. No. 4,683,195. In general, it is necessary to obtain sequence information from the end of the target region or beyond 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 2 primers may correspond 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, phage or plasmid sequences transcribed from total cellular RNA, etc. See generally Mullis et al (1987) Cold Spring Harbor Symp. Uant. Biol.51:263; erlich editions, (1989) PCR TECHNOLOGY (stock Press, n.y.). PCR as used herein is considered an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes using known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.

"isolated" refers to a purified state and in this case means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth media. In general, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be, but need not be, present.

"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivery of an antibody or antigen-binding fragment. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antimicrobial or antifungal), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyols, sorbitol, and sodium chloride.

Furthermore, the present disclosure includes an agent for treating a disease associated with PD-1 positive cells, the agent comprising the anti-PD-1 antibody of the present disclosure or an antigen-binding fragment thereof as an active ingredient.

There is no limitation on the disease associated with PD-1, so long as it is a disease associated with PD-1, e.g., a therapeutic response induced by a molecule of the present disclosure may be produced by binding to human PD-1, then repressing the binding of PD-1 to its ligand PD-L1, PD-L2, or killing tumor cells that overexpress PD-1. Thus, the molecules of the present disclosure are very useful for those suffering from tumors or cancers, preferably melanoma, colon, breast, lung, stomach, intestinal, kidney, non-small cell lung, bladder, and the like, when in preparations and formulations suitable for therapeutic use.

Furthermore, the present disclosure relates to a method for immunodetection or assay of PD-1, a reagent for immunodetection or assay of PD-1, a method for immunodetection or assay of cells expressing PD-1, and a diagnostic agent for diagnosis of diseases associated with PD-1 positive cells, comprising as an active ingredient a monoclonal antibody or antibody fragment of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure.

In the present disclosure, the method for detecting or determining the amount of PD-1 may be any known method. For example, it includes immunological detection or assay methods.

The immunodetection or assay method is a method of detecting or assaying the amount of an antibody or an antigen using a labeled antigen or antibody. Examples of immunodetection or assay methods include radio-labeled immune antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent Immunoassays (FIA), luminescent immunoassays, western immunoblotting, physicochemical methods, and the like.

The diseases described above that are associated with PD-1 positive cells can be diagnosed by detecting or assaying PD-1 expressing cells with monoclonal antibodies or antibody fragments of the present disclosure.

For detecting the cells expressing the polypeptide, a known immunodetection method may be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, and the like are preferably used. Further, a fluorescent antibody staining method using FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, a living sample for detecting or determining PD-1 is not particularly limited as long as it has a possibility of containing cells expressing PD-1, for example, tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions, depending on the desired diagnostic method. Reagents for performing the antigen-antibody reaction include buffers, salts, and the like. Reagents for detection include reagents commonly used in immunological detection or assay methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, an antibody fragment thereof, or a conjugate thereof, a substrate corresponding to the label, and the like.

Second, examples and test cases

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure. The experimental methods without specific conditions noted in the examples of the present disclosure are generally according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1 preparation of antigen

The fusion protein of human PD-1-IgG1Fc is designed and synthesized, the N end is 150 amino acids of the extracellular region of human PD-1, and the C end is the Fc segment of human IgG 1. The recombinant PD-1-Fc Protein with high purity can be obtained by purifying the Protein A by an affinity column and is used for detecting the combination of an anti-PD-1 antibody and an antigen.

Human PD-1-IgG1Fc: (SEQ ID NO: 1)

MEFGLSWLFLVAILKGVQCPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Annotation: the underlined part is the signal peptide and the positive body part is the extracellular region.

EXAMPLE 2 construction of affinity matured Yeast library of PD-1 antibodies and validation of the library

To obtain anti-human PD-1 antibodies with better efficacy, the H005-1 antibodies are affinity-matured by yeast display platform technology, 6 CDR affinity-matured yeast libraries for H005-1-scFv antibodies are designed and prepared on the basis of the H005-1 antibodies, and new human PD-1 antibodies are screened therefrom. The CDR and light/heavy chain variable region sequences of the H005-1 antibody were derived from WO2015085847A1 and were specified as follows:

h005-1 antibody heavy chain sequence: (SEQ ID NO: 2)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWV

ATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQL

YYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS

NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV

VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW

LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL

TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW

QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

H005-1 antibody light chain sequence: (SEQ ID NO: 3)

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYT

ATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKV

EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS

FNRGEC

Note that: the underlined portion is the variable region portion of the antibody, and the other non-underlined portion is the constant region portion of the antibody.

H005-1-scFv amino acid sequence: (SEQ ID NO: 4)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

H005-1-scFv gene sequence: (SEQ ID NO: 5)

GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAGCTACATGATGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCACAATCTCTGGCGGAGGCGCCAACACCTACTACCCCGACTCTGTGAAGGGCCGGTTCACCATCTCTCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCCGGCAGCTGTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCTGGTGGAGGCGGTTCAGGCGGAGGTGGCAG CGGCGGTGGCGGGTCGGACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCTGGCCTCCCAGACCATCGGCACCTGGCTGACCTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACACCGCCACCAGTCTGGCCGATGGCGTGCCCTCTAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACTCCATCCCCTGGACCTTTGGCGGAGGCACCAAGGTGGAAATCAAG

Note that: the underlined part is the linker sequence.

Construction of a Yeast library: degenerate primers were designed and the designed mutant amino acids were introduced by PCR into libraries of H005-1-scFv antibodies, each library having a size of 10 ⁹ About, the constructed yeast library verifies the diversity of the library by a second generation sequencing method.

Example 3 screening of antibodies

In the first round of screening, about 5X 10 from the H005-1 antibody library was used ¹⁰ Individual cells were incubated with 10 μg/ml biotinylated human PD-1-IgG1Fc protein in 50m1 with 0.1% Bovine Serum Albumin (BSA) -phosphate buffer (referred to as PBSA) for 1 hour at room temperature. The mixture was then washed three times with 0.1% pbsa to remove unbound antibody fragments. 100 μl of streptavidin microbeads (Mi 1envi Biotec, auburn, calif.) were then added to the PD-1 antibody library that binds to the biotinylated human PD-1-IgG1Fc protein and loaded onto an AutoMACS system for sorting. Cells of the antibody library were collected with high affinity for PD-1 and then purified in SDCAA medium (20 g dextrose, 6.7g Difco yeast nitrogen source-no amino acids, 5g Bacto casein amino acids, 5.4g Na2HP04 and 8.56g NaH ₂ PO ₄ ·H ₂ O, dissolved in 1L of steam grenade water) was amplified at 250rpm and 30℃for 24 hours. The culture was then grown in SGCAA medium (20 g galactose, 6.7g Difco yeastNitrogen Source-amino acid free, 5g Bacto Casein amino acid, 5.4g Na2HP04 and 8.56g NaH ₂ PO ₄ ·H ₂ O, dissolved in 1L of steam grenade water) was induced at 250rpm and 20 ℃ for 18 hours. The resulting enriched library was subjected to a second round of screening for binding to biotinylated recombinant human PD-1-IgG1 Fc. To ensure sufficient diversity of the antibody libraries for the second and/or third rounds of screening, 100-fold the library size from the previous round was used as the input cell number.

For the third and fourth rounds of screening, library cells from the previous round were incubated with 1. Mu.g/ml biotinylated human PD-1-IgG1Fc protein and 10. Mu.g/ml Mouse Anti-cMyc (9E 10, sigma) antibody in 0.1% PBSA for 1 hour at room temperature, and the mixture was washed three times with 0.1% PBSA to remove unbound antibody fragments. Goat anti-mouse-Alexa488 (A-11001,life technologies) and Strepavidin-PE (S-866,Life technologies) were added and incubated for 1 hour at 4deg.C, and the mixture was washed three times with 0.1% PBSA to remove unbound antibody fragments. Finally, antibodies with high affinity were screened (BD FACSAriaTM FUSION) by FACS.

The H005-1 library was subjected to two rounds of MACS screening (streptomycin magnetic beads, invitrogen) and two rounds of FACS screening (BD FACSAriaTM FUSION), and then 400 or so yeast monoclonal clones were selected for culture and induced expression, binding of the human PD-1-IgG1Fc antigen was detected using FACS (BD FACSCanto II), sequencing was performed on the selected yeast monoclonal having a higher affinity than that of the wild-type H005-1 antibody, sequencing clones were aligned, redundant sequences were removed, non-redundant sequences were converted to full-length IgG (. Gamma.1,. Kappa.) and mammalian cell expression was performed, and full-length antibodies after affinity purification were subjected to affinity assay using BIAcore X-100 (GE Life Sciences).

1. Screening confirmed selection of CDR region sequences of the antibodies.

Clones selected from the mutant library based on the H005-1 antibody sequence were different from the H005-1 antibody in HCDR2, HCDR3, LCDR1, LCDR2, LCDR 3. The relevant CDR (amino acid residues of the CDR are determined and annotated by the Kabat numbering system) sequences or formulae, and their corresponding light/heavy chain variable regions, are described as follows:

HCDR1 SYMMS SEQ ID NO：6

HCDR2 TISGGGANTYFAX ₁ SVKG SEQ ID NO：7

HCDR3 TQIGGTN (SEQ ID NO: 8) or QLYYFDY SEQ ID NO:9

LCDR1 X ₂ ASQX ₃ IX ₄ X ₅ WLX ₆ SEQ ID NO：10

LCDR2 X ₇ ATX ₈ X ₉ X ₁₀ X ₁₁ SEQ ID NO：11

LCDR3 QQX ₁₂ YX ₁₃ X ₁₄ PWT SEQ ID NO：12

The sequence of the related heavy chain variable region is obtained as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVA ₁ TISGGGANTYFAXSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSS(SEQ ID NO：13)

Or (b)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEW VA ₁ TISGGGANTYFAXSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QLYYFDYWGQGTTVTVSS(SEQ ID NO：14)。

The sequence of the related light chain variable region is obtained as follows:

DIQMTQSPSSLSASVGDX ₁₅ VTITC ₂ ₃ ₄ ₅ ₆ XASQXIXXWLXWYQQKPGX ₁₆ X ₁₇ PKLLIX ₁₈ ₇ ₈ ₉ ₁₀ ₁ XATXXXX ₁ GVPSRFSGSGSGTDFTLTIX ₁₉ SLQPEDFAX ₂₀ YYCQ ₁₂ ₁₃ ₁₄ QXYXXPWTFGGGTKVEIK(SEQ ID NO：15)。

x in the above CDR and heavy and light chain variable regions ₁ Selected from D or N, X ₂ Selected from L, R or Q, X ₃ Selected from T, S, G, N or D, X ₄ Selected from G, A or Y, X ₅ Selected from T, Y, N, S or D, X ₆ Selected from T, N or A, X ₇ Selected from T, G or L, X ₈ Selected from T, S or H, X ₉ Selected from R or L, X ₁₀ Selected from A, E or Q, X ₁₁ Selected from D, Y or S, X ₁₂ Selected from V or A, X ₁₃ Selected from S or T, X ₁₄ Selected from V, T or I, X ₁₅ Selected from R or I, X ₁₆ Selected from K or Q, X ₁₇ Selected from A or P, X ₁₈ Selected from Y or H, X ₁₉ Selected from S or G, X ₂₀ Selected from A or T.

Specific related sequences obtained include, but are not limited to, those set forth in tables 1 and 2:

TABLE 1 affinity screening of the resulting antibody heavy chain variable regions

TABLE 2 affinity screening of the resulting antibody light chain variable region sequences

The specific sequences of the light and heavy chain variable regions of the obtained antibodies are as follows:

VH1 heavy chain variable region (SEQ ID NO: 35):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATI

SGGGANTYFADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIG

FTNWGQGTTVTVSS

VH2 heavy chain variable region (SEQ ID NO: 36):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATI

SGGGANTYFANSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIG

FTNWGQGTTVTVSS

VH3 heavy chain variable region (SEQ ID NO: 37):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATI

SGGGANTYFADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLY

YFDYWGQGTTVTVSS

VL1 light chain variable region (SEQ ID NO: 38):

DIQMTQSPSSLSASVGDIVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATS

LADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEI

K

VL2 light chain variable region (SEQ ID NO: 39):

DIQMTQSPSSLSASVGDRVTITCRASQSIGYWLNWYQQKPGKAPKLLIYGAT

SLAYGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYTTPWTFGGGTKV

EIK

VL3 light chain variable region (SEQ ID NO: 40):

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATS

LADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSIPWTFGGGTKVEI

K

VL4 light chain variable region (SEQ ID NO: 41):

DIQMTQSPSSLSASVGDRVTITCRASQSIGNWLAWYQQKPGKPPKLLIYLAT

SRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEI

K

VL5 light chain variable region (SEQ ID NO: 42):

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIHTATS

LADGVPSRFSGSGSGTDFTLTISSLQPEDFAAYYCQQVYSIPWTFGGGTKVEI

K

VL6 light chain variable region (SEQ ID NO: 43):

DIQMTQSPSSLSASVGDRVTITCQASQGIGYWLNWYQQKPGKAPKLLIYTAT

SLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYTTPWTFGGGTKV

EIK

VL7 light chain variable region (SEQ ID NO: 44):

DIQMTQSPSSLSASVGDRVTITCRASQNIASWLAWYQQKPGKAPKLLIYLAT

TREYGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSVPWTFGGGTKV

EIK

VL8 light chain variable region (SEQ ID NO: 45):

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATS

LADGVPSRFSGSGSGTDFTLTIGSLQPEDFATYYCQQVYSIPWTFGGGTKVEI

K

VL9 light chain variable region (SEQ ID NO: 46):

DIQMTQSPSSLSASVGDRVTITCRASQSIYDWLAWYQQKPGQAPKLLIYLAT

SRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEI

K

VL10 light chain variable region (SEQ ID NO: 47):

DIQMTQSPSSLSASVGDRVTITCLASQDIGSWLAWYQQKPGKAPKLLIYLAT

HRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVE

IK

note that: in the light/heavy chain variable region sequences described above, the underlined sections are CDR regions (the amino acid residues of the CDRs are determined and annotated by the Kabat numbering system).

2. Construction of full-length antibody sequences:

the antibody heavy chain variable region in the present disclosure may be linked to a heavy chain constant region selected from human IgG1, igG2, igG3, or IgG4, or variants thereof, to form an antibody full length heavy chain; the antibody light chain variable region may be linked to a light chain constant region selected from a human kappa chain or lambda chain, or variants thereof, to form an antibody full length light chain. It is well known in the art that Fc mutations such as D265A, N297A, L234A/L235A or L234F/L235A of IgG1 can reduce ADCC and P331S or nearby mutations can reduce CDC; mutations in IgG2 and Fc-hybrid antibodies to IgG2/4 can also reduce ADCC and CDC; the constant region ADDC activity of IgG4 is lower; in addition, the introduction of F234A and L235A mutations in the constant region of IgG4 or IgG1 (mAbs 4:3,310-318; may/June 2012) can alter the CH2 domain, reduce interactions with Fc receptors, and thus reduce ADCC activity. These known IgG1, igG4, or mutants thereof may all be used as constant regions of the anti-PD-1 antibodies of the present disclosure. Illustratively, the antibodies of the present disclosure may be selected from the light/heavy chain constant regions shown below:

IgG4 heavy chain constant region (SEQ ID NO: 48):

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

IgG1 heavy chain constant region (SEQ ID NO: 49):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

kappa light chain constant region (SEQ ID NO: 50):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

illustratively, the heavy chain variable region of an antibody derived from the library of H005-1 mutant antibodies obtained from the previous screen is fused to a polypeptide as set forth in SEQ ID NO:48 or SEQ ID NO:49, and a light chain variable region linked to a heavy chain constant region as set forth in SEQ ID NO:50, and then the light and heavy chains combine to form the complete antibody, the resulting partial antibodies having the sequences shown in table 3 below.

TABLE 3 sequences of antibodies derived from H005-1 mutations

Remarks: for example, "Ab01" means the heavy chain variable region sequence of the antibody numbered Ab01 as set forth in SEQ ID NO:35, the heavy chain constant region sequence is shown in SEQ ID NO:48, and the light chain variable region sequence thereof is shown in SEQ id no:38, the light chain constant region sequence is shown in SEQ ID NO: shown at 50.

Illustratively, the heavy chain of Ab01 antibody (SEQ ID NO: 51) is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYFADSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

the light chain of Ab01 antibody (SEQ ID NO: 52) is as follows:

DIQMTQSPSSLSASVGDIVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the heavy chain of Ab05 antibody (SEQ ID NO: 53) is as follows:

the light chain of Ab05 antibody (SEQ ID NO: 54) is as follows:

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQAYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

note that: the scribe line portion is a variable region.

The present disclosure uses the PD-1 antibody Keytruda of Merck as a positive control, the light and heavy chain amino acid sequences of Keytruda (Merck, pembrolizumab, see WHO Drug Information, volume 27, phase 2, pages 161-162 (2013)) are as follows:

keystuda heavy chain (SEQ ID NO: 55)

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVT LTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Key truda light chain (SEQ ID NO: 56)

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGS GSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Remarks: the scribe line portion is a variable region.

PD-1 antibodies are purified by conventional methods.

Example 4 antibody and ligand blocking assay

The blocking effect of the product on the combination of PD-1 and PD-L1 is studied deeply, and the product is compared with similar products in clinical tests. Promega company constructed Jurkat cells (CS 187102, promega) that overexpressed PD-1 and NFAT driven luciferase reporter genes and CHO-K1 cells (CS 187108, promega) that overexpressed PD-L1. This system reflects the ability of antibodies to block the binding of PD-1 to PD-L1 by detecting luminences. The experimental procedure is briefly described as follows: digestion of CHOK1/PD-L1 cells (CS 187108, promega) was added to 96-well plates at 100. Mu.L/well and placed at 37℃in 5% CO ₂ The incubator was incubated for 24 hours. The control (C25-IgG negative control (laboratory preparation)) and sample were diluted to the desired concentrations using PBS. Counting Jurkat/PD1 cells (CS 187102, promega), inoculating into a cell culture plate (90. Mu.L/well) with CHOK1/PD-L1 cells at a certain ratio, adding diluted antibody to 10. Mu.L/well, and placing at 37deg.C, 5% CO ₂ Culturing in an incubator for 5 hours. The cell culture plate was removed, left at room temperature for 5 minutes, and then 50. Mu.l of Bio-Glo was added to each well ^TM Reagent (G7940, promega), incubated for 5 minutes at room temperature, read plates using a microplate reader (PerkinelmerVictor 3, model: 1420-012), read plate channel was luminescence. The data are detailed in table 4 and fig. 1.

TABLE 4 anti-PD-1 antibodies block the binding assays of PD-1 to its ligands

The results show that exemplary anti-PD-1 antibodies Ab05 and Ab01 in the present disclosure are capable of effectively blocking the binding of PD-1 to PD-L1.

Example 5 BIAcore antibody affinity experiments for exemplary antibodies

IgG was affinity captured using a Protein A biosensing chip (Cat. # 2927556, GE), human PD-1 antigen (Cat. #10377H08H,Sino Biological) flowed across the chip surface, and the Biacore T200 instrument detected the anti-PD-1 antibody and antigen PD-1 response signals in real time to obtain binding and dissociation curves. After completion of dissociation in each experimental cycle, the biochip was washed and regenerated with 10mM Glycine-HCl buffer pH 1.5. The experimental buffer system was 1 XHBS-EP buffer (Cat#BR-1001-88, GE). After the end of the experiment, the data were fitted with the (1:1) Langmuir model using the GE Biacore T200 Evaluation version 3.0 software to give affinity values and the experimental results for the same batch are shown in table 5.

TABLE 5 affinity of different antibodies for human PD-L1

The experimental results of the same batch show that the anti-PD-1 antibodies obtained by screening can be combined with human PD-1, and the affinity is higher than that of Keystuda and H005-1. Wherein Ab05, ab01 binds to human PD-1 with an affinity of 2.8nM,2.56nM, and Keystuda, H005-1 with an affinity of 5.81nM,14.6nM, respectively.

Example 6 secretion of cell IFNγ by exemplary antibodies in PBMC-T lymphocyte activation experiments

To investigate the effect of anti-PD-1 antibodies on human primary T lymphocyte function, human Peripheral Blood Mononuclear Cells (PBMCs) were collected and purified, and cytokine ifnγ secretion levels were detected after 5 days of in vitro stimulation with Tuberculin (TB). The experimental procedure is briefly described as follows:

fresh blood was centrifuged (Stem Cell Technologies) to obtain PBMC using Ficoll-Hypaque (17-5442-02, GE) and cultured in RPMI 1640 (SH 30809.01, GE) medium supplemented with 10% (v/v) FBS (10099-141, gibco), 37℃and 5% CO ₂ Culturing under the condition.

Freshly isolated and purified PBMC were conditioned to a density of 2X 10 in RPMI 1640 medium ⁶ Mu.l tuberculin (97-8800, synbiotics), 37℃and 5% CO were added to 20mL of cell suspension at a concentration of each mL ₂ The incubator was cultured for 5 days. On day 5, the cultured cells were collected and centrifuged, resuspended in fresh RPMI 1640 medium, and the density was adjusted to 1.1X10% ⁶ Each ml was inoculated into 96-well cell culture plates at 90. Mu.l per well. Simultaneously, a gradient of diluted antibody sample was added, and diluted with PBS (B320, shanghai Biotechnology Co., ltd.) to 10. Mu.l per well. Cell culture plates were placed at 37℃with 5% CO ₂ Incubators were incubated for 3 days. The cell culture plates were removed, centrifuged (4000 rpm,10 min) to collect the cell culture supernatant, and the levels of IFN-gamma were detected by ELISA (human IFN-gamma detection kit (EHC 102g.96, xinbo) for specific procedures, see reagent instructions.

The results (see FIGS. 2A-2C) show that all antibodies can activate IFN- γ secretion in the anti-PD-1 antibody molecules tested, where Ab01, ab05, ab09 have significantly better ability to activate IFN- γ secretion at high concentrations than H005-1.

Test example 7 pharmacokinetic testing of exemplary antibodies

Animals were serum collected at various time points after dosing with 1mpk of Anti-PD-1 antibody samples, standard curves were made for the different samples using direct ELISA assays, 100. Mu.L of 0.5. Mu.g/mL PD-1-His (Cat. #10377H08H,Sino Biological) were plated, incubated overnight at 4 ℃, 200. Mu.L of 3% milk blocked, 100. Mu.L of sample was added, 50-fold plate diluted, incubated 2h at 37 ℃, 100. Mu.L of adsorbed secondary Anti-goat Anti-Human IgG Fc-HRP (1:5000) was added, incubated 1h at 37 ℃, TMB developed, 1M H ₂ SO ₄ And (5) terminating and reading the board. The resulting data was analyzed by Phoenix WinNonlin software to calculate pharmacokinetic-related parameters.

The experimental cynomolgus monkey, common grade, male, 3-7 years old, 4-8 kg, is bred by Medixic Primordia medical science and technology (Shanghai) limited company and is sourced from Guangxi androstan primate experimental animal breeding development limited company. Animals were quarantined and adapted to feed as required by SOP prior to inclusion in the experiment. Feeding environment: the illumination was provided for about 12 hours per day, controlled by an automatic timer controller. The dark time may be intermittently interrupted due to the need to study related activities. The ambient temperature and relative humidity of the animal house are controlled in the range of 19-26 ℃ and 40-70%, respectively, and monitored and recorded every day. The cynomolgus monkeys were grouped, 3 in each group, numbered mon1, mon2, mon3, respectively. On the day of the experiment, each cynomolgus monkey is respectively and intravenously injected with the tested medicine, and the single administration is carried out, the dosage of the administration is 1mg/kg, and the intravenous infusion (within 30 min) is completed; dosing volume (2 ml/kg).

Before administration, 5min,15min,0.5h,8h, day2 (24 h), day3 (48 h), day 5 (96 h), day8 (168 h), day 11 (240 h), day 15 (336 h), day 29 (672 h), day 43 (1008 h), day 57 (1344 h), day 71 (1680 h) and Day85 (2016 h) were collected by puncture of the head or saphenous vein approximately 2.0mL, respectively, and placed in a blood collection tube (without anticoagulant) for separation of serum. After collection, the mixture was left at 4℃for 30min and centrifuged at 1000g for 15min, and the supernatant (serum) was placed in an EP tube and divided into 2 tubes and stored at-80℃for 2h after serum collection.

The drug metabolism parameters of Ab05 and parent antibody H005-1 at 1mpk dose were measured in cynomolgus monkeys, and the results show that Ab05 has good in vivo drug metabolism performance of cynomolgus monkeys, the average drug half-life t1/2 is about 6.2 days, and the following table 6 indicates that the antibody has good in vivo stability in cynomolgus monkeys. Whereas the half-life t1/2 of H005-1 was found to be about 3.7 days at the same dose (1 mpk). The experimental results are shown in Table 6.

TABLE 6 pharmacokinetic parameters of anti-PD-1 antibodies

Note that: RSD (relative standard deviation) the relative standard deviation.

Test example 8 role of anti-PD-1 antibodies in transgenic PD-1 mouse colon cancer model MC38

Transgenic PD-1 mice were derived from fifth generation mice grown in commercially available transgenic PD-1 mice (ISIS INNOVATION LIMITED, university Offices, wellington Square, oxford OX1 2jd, england) at Cephrim Biosciences, inc. MC38 cells were grown at 5X 10 ⁵ Mu.l/100. Mu.l/inoculated into the rear part of right rib of hPD-1 transgenic mice (male and female half) subcutaneously until the average tumor volume of the mice reaches 80-100mm ³ Animals with body weight, oversized tumor and undersized tumor were removed, and tumor-bearing mice were randomly divided into 5 groups (8 per group) according to tumor volume: negative controls hIgG control-30mpk, H005-1-10mpk, keystuda-30 mpk, ab05-10mpk, ab05-30mpk. The group administration Day was set to Day0. Each drug was given intraperitoneally after grouping for 22 days with a total of 11 doses every two days. Tumor volumes were measured 2 times per week, weighed, and data recorded. Animals of each group Body weight, tumor volume were both expressed as Mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism 5 and Excel software, using student t test statistical analysis.

Tumor Volume (TV) = 0.5236 ×l _{Long length} ×L _{Short length} ²

Tumor proliferation rate T/C% = (T-T0)/(C-C0). Times.100%

Tumor inhibition% tgi=1-T/C%

(wherein T, T0 represents the tumor volume at the end of the test and at the beginning of the test, respectively, of the antibody administration group, C, C0 represents the tumor volume at the end of the test and at the beginning of the test, respectively, of the placebo group)

The test results are shown in the following table 7 and fig. 3, and the test results show that compared with the control group, the antibody disclosed by the invention can remarkably inhibit the growth of the mouse colon cancer MC38 transplanted tumor, wherein the tumor inhibition rate is the highest in the Ab0530mpk group, the tumor inhibition rate reaches 76.4% when measured on the 20 th day, and even in the low-dose group (10 mpk), the drug effect of the Ab05-10mpk group is better than that of the positive control H005-1-10mpk.

TABLE 7 influence of anti-PD-1 antibodies on tumor suppression Rate of mouse colon carcinoma MC38

Remarks: the "IP" in the table indicates intraperitoneal injection; "Q2Dx11" means once every two days for 11 times

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Sequence listing

<110> Jiangsu Hengrui medicine Co., ltd

Shanghai Hengrui medicine Co., ltd

<120> anti-PD-1 antibodies, antigen-binding fragments thereof, and medical uses thereof

<150> 201810875621.2

<151> 2018-08-03

<160> 56

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<223> human PD-1-IgG1Fc

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Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn

20 25 30

Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn

35 40 45

Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu

50 55 60

Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala

65 70 75 80

Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val

85 90 95

Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala

100 105 110

Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala

115 120 125

Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr

130 135 140

Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg

145 150 155 160

Pro Ala Gly Gln Phe Gln Thr Leu Val Glu Pro Lys Ser Ser Asp Lys

165 170 175

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

180 185 190

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

195 200 205

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

210 215 220

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

225 230 235 240

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

245 250 255

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

260 265 270

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

275 280 285

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

290 295 300

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

305 310 315 320

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

325 330 335

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

340 345 350

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

355 360 365

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

370 375 380

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

385 390 395 400

Lys

<210> 2

<211> 443

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> H005-1 antibody heavy chain sequence

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 3

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> H005-1 antibody light chain sequence

<400> 3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 238

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> H005-1-scFv amino acid sequence

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

130 135 140

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile

145 150 155 160

Gly Thr Trp Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

165 170 175

Leu Leu Ile Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg

180 185 190

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

195 200 205

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser

210 215 220

Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

225 230 235

<210> 5

<211> 714

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> gene

<223> H005-1-scFv Gene sequence

<400> 5

gaagtgcagc tggtggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60

tcttgtgccg cctccggctt caccttctcc agctacatga tgagctgggt gcgacaggcc 120

cctggcaagg gactggaatg ggtggccaca atctctggcg gaggcgccaa cacctactac 180

cccgactctg tgaagggccg gttcaccatc tctcgggaca acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgtgc ccggcagctg 300

tactacttcg actactgggg ccagggcacc accgtgaccg tgtcctctgg tggaggcggt 360

tcaggcggag gtggcagcgg cggtggcggg tcggacatcc agatgaccca gtccccctcc 420

agcctgtctg cctctgtggg cgacagagtg accatcacct gtctggcctc ccagaccatc 480

ggcacctggc tgacctggta tcagcagaag cctggcaagg cccccaagct gctgatctac 540

accgccacca gtctggccga tggcgtgccc tctagattct ccggctctgg ctctggcacc 600

gactttaccc tgaccatcag ctccctgcag cccgaggact tcgccaccta ctactgccag 660

caggtgtact ccatcccctg gacctttggc ggaggcacca aggtggaaat caag 714

<210> 6

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR1 sequence

<400> 6

Ser Tyr Met Met Ser

1 5

<210> 7

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR2 sequence formula

<220>

<221> DOMAIN

<222> (13)..(13)

<223> Xaa is selected from Asp or Asn.

<400> 7

Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Xaa Ser Val Lys

1 5 10 15

Gly

<210> 8

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR3

<400> 8

Thr Gln Ile Gly Phe Thr Asn

1 5

<210> 9

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR3 sequence

<400> 9

Gln Leu Tyr Tyr Phe Asp Tyr

1 5

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1 sequence formula

<220>

<221> DOMAIN

<222> (1)..(1)

<223> Xaa is selected from Leu, arg or Gln.

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Thr, ser, gly, asn or Asp.

<220>

<221> DOMAIN

<222> (7)..(7)

<223> Xaa is selected from Gly, ala or Tyr.

<220>

<221> DOMAIN

<222> (8)..(8)

<223> Xaa is selected from Thr, tyr, asn, ser or Asp.

<220>

<221> DOMAIN

<222> (11)..(11)

<223> Xaa is selected from Thr, asn or Ala.

<400> 10

Xaa Ala Ser Gln Xaa Ile Xaa Xaa Trp Leu Xaa

1 5 10

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2 sequence formula

<220>

<221> DOMAIN

<222> (1)..(1)

<223> Xaa is selected from Thr, gly or Leu.

<220>

<221> DOMAIN

<222> (4)..(4)

<223> Xaa is selected from Thr, ser or His.

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Arg or Leu.

<220>

<221> DOMAIN

<222> (6)..(6)

<223> Xaa is selected from Ala, glu or Gln.

<220>

<221> DOMAIN

<222> (7)..(7)

<223> Xaa is selected from Asp, tyr or Ser.

<400> 11

Xaa Ala Thr Xaa Xaa Xaa Xaa

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR3 general formula sequence

<220>

<221> DOMAIN

<222> (3)..(3)

<223> Xaa is selected from Val or Ala.

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Ser or Thr.

<220>

<221> DOMAIN

<222> (6)..(6)

<223> Xaa is selected from Val or Ile.

<400> 12

Gln Gln Xaa Tyr Xaa Xaa Pro Trp Thr

1 5

<210> 13

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> heavy chain variable region sequence formula

<220>

<221> DOMAIN

<222> (62)..(62)

<223> Xaa is selected from Asp or Asn.

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Xaa Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Gln Ile Gly Phe Thr Asn Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 14

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> heavy chain variable region sequence formula

<220>

<221> DOMAIN

<222> (62)..(62)

<223> Xaa is selected from Asp or Asn.

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Xaa Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 15

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> light chain variable region sequence formula

<220>

<221> DOMAIN

<222> (18)..(18)

<223> Xaa is selected from Arg or Ile.

<220>

<221> DOMAIN

<222> (24)..(24)

<223> Xaa is selected from Leu, arg or Gln.

<220>

<221> DOMAIN

<222> (28)..(28)

<223> Xaa is selected from Thr, ser, gly, asn or Asp.

<220>

<221> DOMAIN

<222> (30)..(30)

<223> Xaa is selected from Gly, ala or Tyr.

<220>

<221> DOMAIN

<222> (31)..(31)

<223> Xaa is selected from Thr, tyr, asn, ser or Asp.

<220>

<221> DOMAIN

<222> (34)..(34)

<223> Xaa is selected from Thr, asn or Ala.

<220>

<221> DOMAIN

<222> (42)..(42)

<223> Xaa is selected from Lys or Gln.

<220>

<221> DOMAIN

<222> (43)..(43)

<223> Xaa is selected from Ala or Pro.

<220>

<221> DOMAIN

<222> (49)..(49)

<223> Xaa is selected from Tyr or His.

<220>

<221> DOMAIN

<222> (50)..(50)

<223> Xaa is selected from Thr, gly or Leu.

<220>

<221> DOMAIN

<222> (53)..(53)

<223> Xaa is selected from Thr, ser or His.

<220>

<221> DOMAIN

<222> (54)..(54)

<223> Xaa is selected from Arg or Leu.

<220>

<221> DOMAIN

<222> (55)..(55)

<223> Xaa is selected from Ala, glu or Gln.

<220>

<221> DOMAIN

<222> (56)..(56)

<223> Xaa is selected from Asp, tyr or Ser.

<220>

<221> DOMAIN

<222> (76)..(76)

<223> Xaa is selected from Ser or Gly.

<220>

<221> DOMAIN

<222> (85)..(85)

<223> Xaa is selected from Ala or Thr.

<220>

<221> DOMAIN

<222> (91)..(91)

<223> Xaa is selected from Val or Ala.

<220>

<221> DOMAIN

<222> (93)..(93)

<223> Xaa is selected from Ser or Thr.

<220>

<221> DOMAIN

<222> (94)..(94)

<223> Xaa is selected from Val or Ile.

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Xaa Val Thr Ile Thr Cys Xaa Ala Ser Gln Xaa Ile Xaa Xaa Trp

20 25 30

Leu Xaa Trp Tyr Gln Gln Lys Pro Gly Xaa Xaa Pro Lys Leu Leu Ile

35 40 45

Xaa Xaa Ala Thr Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Xaa Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Xaa Tyr Tyr Cys Gln Gln Xaa Tyr Xaa Xaa Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 16

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR2

<400> 16

Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 17

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR2

<400> 17

Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Asn Ser Val Lys

1 5 10 15

Gly

<210> 18

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1

<400> 18

Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Thr

1 5 10

<210> 19

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1

<400> 19

Arg Ala Ser Gln Ser Ile Gly Tyr Trp Leu Asn

1 5 10

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1

<400> 20

Arg Ala Ser Gln Ser Ile Gly Asn Trp Leu Ala

1 5 10

<210> 21

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1

<400> 21

Gln Ala Ser Gln Gly Ile Gly Tyr Trp Leu Asn

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1

<400> 22

Arg Ala Ser Gln Asn Ile Ala Ser Trp Leu Ala

1 5 10

<210> 23

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1

<400> 23

Arg Ala Ser Gln Ser Ile Tyr Asp Trp Leu Ala

1 5 10

<210> 24

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1

<400> 24

Leu Ala Ser Gln Asp Ile Gly Ser Trp Leu Ala

1 5 10

<210> 25

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2

<400> 25

Thr Ala Thr Ser Leu Ala Asp

1 5

<210> 26

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2

<400> 26

Gly Ala Thr Ser Leu Ala Tyr

1 5

<210> 27

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2

<400> 27

Leu Ala Thr Ser Arg Glu Ser

1 5

<210> 28

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2

<400> 28

Leu Ala Thr Thr Arg Glu Tyr

1 5

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2

<400> 29

Leu Ala Thr Ser Arg Gln Ser

1 5

<210> 30

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2

<400> 30

Leu Ala Thr His Arg Gln Ser

1 5

<210> 31

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR3

<400> 31

Gln Gln Val Tyr Ser Ile Pro Trp Thr

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR3

<400> 32

Gln Gln Ala Tyr Thr Thr Pro Trp Thr

1 5

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR3

<400> 33

Gln Gln Ala Tyr Ser Ile Pro Trp Thr

1 5

<210> 34

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR3

<400> 34

Gln Gln Val Tyr Ser Val Pro Trp Thr

1 5

<210> 35

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VH1 heavy chain variable region

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Gln Ile Gly Phe Thr Asn Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 36

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VH2 heavy chain variable region

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Asn Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Gln Ile Gly Phe Thr Asn Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 37

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VH3 heavy chain variable region

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 38

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL1 light chain variable region

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ile Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL2 light chain variable region

<400> 39

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Tyr Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Thr Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL3 light chain variable region

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL4 light chain variable region

<400> 41

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Ala Thr Ser Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL5 light chain variable region

<400> 42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ala Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 43

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL6 light chain variable region

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Gly Ile Gly Tyr Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Thr Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 44

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL7 light chain variable region

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ala Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Ala Thr Thr Arg Glu Tyr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Val Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL8 light chain variable region

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Gly Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL9 light chain variable region

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Asp Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Ala Thr Ser Arg Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> VL10 light chain variable region

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Asp Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Ala Thr His Arg Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 48

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> IgG4 heavy chain constant region

<400> 48

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 49

<211> 330

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> IgG1 heavy chain constant region

<400> 49

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 50

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> kappa light chain constant region

<400> 50

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 51

<211> 443

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Ab01 antibody

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Gln Ile Gly Phe Thr Asn Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 52

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of Ab01 antibody

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ile Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 53

<211> 443

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Ab05 antibody

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Gln Ile Gly Phe Thr Asn Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 54

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of Ab05 antibody

<400> 54

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 55

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> Keystuda heavy chain

<400> 55

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 56

<211> 218

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> Keystuda light chain

<400> 56

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. An anti-PD-1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof comprising:

respectively as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO:8, and the heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 18. SEQ ID NO: 25. SEQ ID NO:31, LCDR1, LCDR2, LCDR3 regions of the light chain.

2. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises an FR region.

3. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 2, wherein the anti-PD-1 antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:13 and a heavy chain FR region in the heavy chain variable region as set forth in SEQ ID NO:15, and a light chain FR region in the light chain variable region shown in figure 15.

4. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises:

as set forth in SEQ ID NO:35 and a heavy chain variable region as set forth in SEQ ID NO:38, and a light chain variable region shown at 38.

5. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain comprises a heavy chain constant region of a human IgG1, igG2, igG3, or IgG4 or variant thereof, and the antibody light chain comprises a constant region of a human kappa, lambda chain, or variant thereof.

6. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 5, wherein the antibody heavy chain comprises a human IgG1 or IgG4 heavy chain constant region.

7. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 5, wherein the antibody heavy chain comprises an IgG1 or IgG4 heavy chain constant region into which F234A and L235A mutations are introduced.

8. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 5, which has a heavy chain constant region sequence set forth in SEQ ID NO:48 or SEQ ID NO:49 or at least 85% sequence identity thereto, the light chain constant region is as set forth in SEQ ID NO:50 or at least 85% sequence identity thereto.

9. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-PD-1 antibody comprises the amino acid sequence set forth in SEQ ID NO:51 and a heavy chain as set forth in SEQ ID NO: 52.

10. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') ₂ Single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), and antigen binding fragments of CDR-containing peptides.

11. A pharmaceutical composition comprising a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, together with one or more pharmaceutically acceptable carriers, diluents or excipients.

12. A nucleic acid molecule encoding the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

13. A vector comprising the nucleic acid molecule of claim 12.

14. A host cell transformed with the vector of claim 13, the host cell selected from the group consisting of a prokaryotic cell and a eukaryotic cell.

15. The host cell of claim 14, wherein the host cell is a eukaryotic cell.

16. The host cell of claim 14, wherein the host cell is a mammalian cell.

17. A method of making an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-10, the method comprising the steps of culturing the host cell of any one of claims 14-16 in culture to form and accumulate the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-10, and recovering the anti-PD-1 antibody or antigen-binding fragment thereof from the culture.

18. Use of an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a pharmaceutical composition comprising claim 12, or a nucleic acid molecule according to claim 12, in the manufacture of a medicament for the treatment of a PD-1 mediated tumor.

19. The use of claim 18, wherein the tumor is selected from the group consisting of: squamous cell carcinoma of head and neck, hepatocellular carcinoma, gastric cancer, intestinal cancer, clear cell renal cell carcinoma, melanoma, multiple myeloma, and myeloproliferative neoplasm.

20. The use of claim 18, wherein the tumor is selected from the group consisting of: colorectal cancer, hepatoma, hodgkin's lymphoma and non-hodgkin's lymphoma.

21. The use of claim 18, wherein the tumor is selected from the group consisting of: diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte enriched large B-cell lymphoma and lymphoplasmacytic lymphoma.

22. The use of claim 18, wherein the tumor is selected from the group consisting of: non-small cell lung cancer, lymphoblastic leukemia and myeloid leukemia, and colon cancer.

23. The use of claim 18, wherein the tumor is selected from the group consisting of: chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia.

24. The use of claim 18, wherein the tumor is selected from the group consisting of: melanoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer and bladder cancer.