CN110790839A

CN110790839A - anti-PD-1 antibody, antigen binding fragment thereof and medical application

Info

Publication number: CN110790839A
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: 2020-02-14
Anticipated expiration: 2039-08-02
Also published as: CN110790839B

Abstract

The present disclosure relates to anti-PD-1 antibodies, antigen-binding fragments thereof, and medical uses. In particular, the disclosure relates to humanized anti-PD-1 antibodies and antigen-binding fragments thereof comprising specific CDR regions, as well as pharmaceutical compositions comprising the 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 preparation of a medicament for the treatment of a tumor.

Description

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

Technical Field

The present disclosure is in the field of biotechnology, and more specifically, the present 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 hotspot in the field of tumor therapy, where tumor immunotherapy of T cells is in its central position. The tumor immunotherapy is a way for fully utilizing and mobilizing killer T cells in a tumor patient to kill the tumor, and is probably the most effective and the safest way for treating the tumor. Meanwhile, the escape of tumor cells is a great obstacle to the immunotherapy of tumors, and the tumor cells promote the rapid growth of the tumors by utilizing the inhibition effect of the tumor cells on the immune system.

There is a very complex relationship between the immune escape mechanism of tumors and the immune response of the body to the tumors. The tumor specific killer T cells in the early stage of tumor immunotherapy have the bioactivity, but lose the killing function along with the later stage of tumor growth. Therefore, the tumor immunotherapy is to improve the immune response of the patient to the tumor to the maximum extent, and it is the key of the immunotherapy for the tumor to not only activate the original immune system response in vivo, but also maintain the duration and the response intensity of the immune system response.

Two signal pathway systems are used for T cell activation in humans, and in addition to providing a first signal to T cells via MHC-antigen peptide presentation by antigen presenting cells, a second signal is provided by a series of co-stimulatory molecules, which in turn enable the T cells to generate a normal immune response. This dual signal pathway system plays a crucial role in the balance of the immune system in vivo, and it strictly regulates the body's immune response to self and non-self antigens differently. In the absence of the second signal provided by the co-stimulatory molecule, this will result in a non-responsive or sustained specific immune response by the T cell, resulting in tolerance. Thus, the second signaling pathway plays a very critical regulatory role throughout the body's immune response.

Programmed death molecule 1 (PD-l) is a protein receptor expressed on the surface of T cells discovered in 1992 and is involved in the process of apoptosis. PD-l belongs to the CD28 family, has 23% amino acid homology with cytotoxic T lymphocyte antigen4 (CTLA-4), but is expressed differently from CTLA, mainly on activated T cells, B cells and myeloid lineage cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is expressed predominantly on T cells, B cells, macrophages and Dendritic Cells (DCs), and expression on activated cells can be upregulated. While expression of PD-L2 is relatively restricted, it is predominantly expressed 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 in the microenvironment of tumor tissues express PD-L1. The new research finds that the expression of high PD-L1 protein is detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, renal 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 pathway, the blocking of the binding between PD-L1/PD-1 is an emerging target with great potential in the field of tumor immunotherapy.

At present, a plurality of pharmaceutical companies across countries develop monoclonal antibodies aiming at PD-L1/PD-1, and the monoclonal antibodies can furthest improve the immune system reaction of patients to tumors by blocking the combination between PD-L1/PD-1, thereby achieving the purpose 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 PD-L1 (Atezolizumab, Avelumab, Durvalumab), which have been approved for tumor types such as melanoma, hodgkin lymphoma patients, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck squamous carcinoma, gastric cancer, liver cancer, mercker skin cancer, and all microsatellite-unstable solid tumors. Further anti-PD-L1/PD-1 antibody patents are found in: WO 013978, WO2013173223, WO2014195852, WO2013181634, WO2015048520, WO2015036511, US2014335093, WO2014100079, WO2014055897, US6803192B1, WO2014022758, US8617546B2, WO2010089411a2, WO2015085847 and the like. 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 of single administration is only 14% in intermediate 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 been studied in a number of clinical trials and achieved good clinical results. Recent research results published at the 2018 ASCO congress show that the effective rate of treatment with anti-PD-1 antibody in combination with apatinib is 41.2% and the disease control rate is 94.1% in advanced non-squamous non-small cell carcinoma. In clinical researches on gastric cancer and liver cancer, the effective rate of the anti-PD-1 antibody and the apatinib in 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. Specifically, the disclosure provides a series of anti-PD-1 antibodies or antigen binding fragments thereof with higher affinity, longer in vivo half-life and 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 shown in SEQ ID NO: 6, as shown in SEQ ID NO: 7, and a heavy chain HCDR2 region as set forth in SEQ ID NO: a heavy chain HCDR3 region as shown in 8 or 9; and/or

(ii) Respectively shown in SEQ ID NO: 10. the light chain LCDR1, LCDR2 and LCDR3 regions shown in 11 and 12;

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

wherein: x₁Selected from D or N, X₂Selected from L, R or Q, X₃Selected from T, S, G, N or D, X₄Selected from G, A or Y, X₅Selected from T, Y, N, S or D, X₆Selected from T, N or A, X₇Selected from T, G or L, X₈Selected from T, S or H, X₉Selected from R or L, X₁₀Selected from A, E or Q, X₁₁Selected from D, Y or S, X₁₂Selected from V or A, X₁₃Selected from S or T, X₁₄Selected from V, T or I.

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

as shown in SEQ ID NO: 6 as shown in SEQ ID NO: 16 or 17, as shown in seq id NO: a heavy chain HCDR3 region as shown in 8 or 9; and/or

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

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

(b) respectively shown in SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO: 8, and heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 19. SEQ ID NO: 26. SEQ ID NO: 32, LCDR1, LCDR2, LCDR 3;

(c) respectively shown in SEQ ID NO: 6. SEQ ID NO: 17. SEQ ID NO: 8, and heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 18. SEQ ID NO: 25. SEQ ID NO: 33, LCDR1, LCDR2, LCDR 3;

(d) respectively shown in 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, LCDR1, LCDR2, LCDR 3;

(e) respectively shown in 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, LCDR1, LCDR2, LCDR 3;

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

(g) respectively shown in SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO: 8, and heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 21. SEQ ID NO: 25. SEQ ID NO: 32, LCDR1, LCDR2, LCDR 3;

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

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

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

(k) Respectively shown in SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO: 8, and heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 24. SEQ ID NO: 30. SEQ ID NO: 31, LCDR1, LCDR2, LCDR 3. 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 sequences shown as SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO: HCDR1, HCDR2 and HCDR3 as shown in fig. 8, the light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 18. SEQ ID NO: 25. SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 31; or

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

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 shown in SEQ ID NO:13 or SEQ ID NO: 14 and the heavy chain variable region as set forth in SEQ ID NO: 15, wherein SEQ ID NO 13, SEQ ID NO: 14. SEQ ID NO: 15 have the general sequences shown below:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYFAX₁SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSS SEQ ID NO：13

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYFAX₁SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS SEQ ID NO：14

DIQMTQSPSSLSASVGDX₁₅VTITCX₂ASQX₃IX₄X₅WLX₆WYQQKPGX₁₆X₁₇PKLLIX₁₈X₇ATX₈X₉X₁₀X₁ ₁GVPSRFSGSGSGTDFTLTIX₁₉SLQPEDFAX₂₀YYCQQX₁₂YX₁₃X₁₄PWTFGGGTKVEIK SEQ ID NO：15

wherein X₁Selected from D or N, X₂Selected from L, R or Q, X₃Selected from T, S, G, N or D, X₄Selected from G, A or Y, X₅Selected from T, Y, N, S or D, X₆Selected from T, N or A, X₇Selected from T, G or L, X₈Selected from T, S or H, X₉Selected from R or L, X₁₀Selected from A, E or Q, X₁₁Selected from D, Y or S, X₁₂Selected from V or A, X₁₃Selected from S or T, X₁₄Selected from V, T or I, X₁₅Selected from R or I, X₁₆Selected from K or Q, X₁₇Selected from A or P, X₁₈Selected from Y or H, X₁₉Selected from S or G, X₂₀Is selected from A or T.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises an amino acid sequence selected from the group consisting of: as shown in SEQ ID NO: 35. 36 or 37 and the heavy chain variable region 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 shown in SEQ ID NO: 36 and the heavy chain variable region as set forth in SEQ ID NO: 40;

as shown in SEQ ID NO: 37 and the heavy chain variable region as set forth in SEQ ID NO: 40;

as shown in SEQ ID NO: 36 and the heavy chain variable region as set forth in SEQ ID NO: 41; or

As shown in SEQ ID NO: 35 and the 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 shown in SEQ ID NO: 35 and the heavy chain variable region of SEQ ID NO: 38; or

As shown in SEQ ID NO: 35 and the heavy chain variable region of SEQ ID NO: 40, or a light chain variable region as shown in seq id no.

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 optimized design of human IgG1, IgG2, or IgG4 constant regions for improved function, e.g., D265A, N297A, L234A/L235A, or L234F/L235A, etc. can reduce ADCC, mutations at or near P331S can reduce CDC, further S228P, F234A, and L235A mutations, etc.; preferably comprises a human IgG1 or IgG4 heavy chain constant region, more preferably comprises an IgG1 or IgG4 heavy chain constant region incorporating F234A and L235A mutations; 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 constant region of the wild-type IgG4, or another mutation that reduces ADCC or CDC activity is introduced into the constant regions of IgG4 and IgG1, and the difference in constant regions does not substantially affect the function of the antibody of the present 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 set forth in SEQ ID NO: 48 or SEQ ID NO: 49 or at least 85% sequence identity thereto, and a light chain constant region as set forth in SEQ ID NO: 50 or has at least 85% sequence identity thereto. In some embodiments, the 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 the heavy chain as set forth in SEQ ID NO: 52, a light chain; or has the sequence as shown in SEQ ID NO: 53 and the heavy chain as shown in SEQ ID NO: 54, or a light chain as shown in figure 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 regions (diabodies), disulfide stabilized V regions (dsFv), and peptides comprising CDRs

In one aspect, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof that competes for binding 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 epitope of human PD-1 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 to 3000mg/kg of the antibody or antigen-binding fragment thereof as described above.

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

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

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

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

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

In some aspects, the present 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 agent.

In some aspects, the present disclosure provides a method of treating a tumor, the method 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: squamous cell carcinoma of head and neck, cancer of head and neck, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, kunkenberg's tumor, myeloproliferative tumor, 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, large B-cell lymphoma enriched in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia. More preferably, the tumor is melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, non-small cell lung cancer, or bladder cancer. In some embodiments, wherein the tumor is a PD-L1 positive tumor.

In some aspects, the present disclosure provides 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, for use in the preparation of a medicament for treating a tumor. In some embodiments, wherein the tumor is selected from the group consisting of: squamous cell carcinoma of head and neck, cancer of head and neck, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, kunkenberg's tumor, myeloproliferative tumor, 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, large B-cell lymphoma enriched in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia. In some embodiments, wherein the tumor is melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, non-small cell lung cancer, bladder cancer. In some embodiments, wherein the tumor is a PD-L1 positive tumor.

In some aspects, the present 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 drug.

The anti-PD-1 antibodies or antigen-binding fragments thereof of the present disclosure exhibit good results in both biochemical tests and in vitro potency experiments. In an affinity detection test, the anti-PD-1 antibodies disclosed by the invention can effectively block the binding of PD-1 and a ligand PD-L1 thereof, wherein the blocking activity of the anti-PD-1 antibodies Ab05 and Ab01 is higher than that of the parent antibody H005-1.

In an affinity detection test, the anti-PD-1 antibodies disclosed by the invention can be specifically combined with PD-1, and the affinity of the antibodies is obvious to that of the parent antibody H005-1.

In an in vitro activity detection test, the anti-PD-1 antibodies disclosed by the disclosure can effectively activate PBMC cells to secrete the cytokine IFN gamma and have a dose-dependent effect, wherein the effects of the antibodies Ab01, Ab05 and Ab09 for activating the PBMC cells to secrete the IFN gamma under a high-concentration condition are obviously higher than those of the parent antibody H005-1. Meanwhile, the influence of different constant regions on the in vitro activity of the anti-PD-1 antibody is also detected, and the result shows that the antibody of the IgG1 type still keeps higher activation effect on PBMC cells, wherein the effects of the antibodies Ab01-IgG1, Ab07-IgG1, Ab08-IgG1 and Ab09-IgG1 on activating the PBMC cells to secrete IFN gamma are obviously higher than those of the parent antibody. Differences in the constant regions do not substantially affect the function of the antibodies of the present disclosure.

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

In the transgenic PD-1 mouse colon cancer model MC38, the antibodies of the present disclosure significantly inhibited the growth of mouse colon cancer MC38 transplantable tumors, with a tumor inhibition rate of 76.4% as measured on day 20 in the Ab0530mpk group.

Drawings

FIG. 1: blocking activity of anti-PD-1 antibodies against the binding of PD-1 to PD-L1.

FIGS. 2A-2C: the PD-1 antibody promotes IFN gamma secretion of PBMC cells. Among them, FIGS. 2A and 2B show the effect of IgG4 type antibody on IFN γ secretion by PBMC cells, and FIG. 2C shows the effect of IgG1 type antibody on IFN γ secretion by PBMC cells.

FIG. 3: effect of anti-PD-1 antibodies in the transgenic PD-1 mouse colon cancer model MC 38.

Detailed Description

Term

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined 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", "PD 1", "PDCD 1", "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. The complete PD-1 sequence can be found in GenBank accession No. U64863.

The term "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2) that down-regulates T cell activation and cytokine secretion when bound to PD-1. The term "PD-L1" as used herein includes variants, isoforms, and interspecies homologs of human PD-L1(hPD-L1), hPD-L1, and 5 analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found using GenBank accession No. Q9NZQ 7.

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- γ, IL-6, TNF α, IL-17, and IL-5.

The three letter codes and the one letter codes for amino acids used in this disclosure are as described in j. diol. chem,243, p3558 (1968).

The immunoglobulin heavy chain constant region has different amino acid compositions and arrangement sequences, so that the antigenicity of the immunoglobulin heavy chain constant region is different, accordingly, the immunoglobulin can be divided into five classes, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are respectively mu chain, delta chain, gamma chain, α chain, epsilon chain, the same class of Ig can be divided into different subclasses according to the difference of the amino acid composition of the hinge region and the number and the position of the disulfide bonds of the heavy chains, for example, the IgG can be divided into IgG1, IgG2, IgG3, IgG4, the light chain is divided into kappa chain or lambda chain through the difference of the constant region.

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

In the present disclosure, the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4, or variants thereof.

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

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the 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 sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3.

Antibodies of the present disclosure include murine, chimeric, humanized 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. Preparation is performed by injecting the test subject with the PD-1 antigen and then isolating hybridomas that express 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 a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or variant thereof.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing chimeric antibody, firstly establishing hybridoma secreting mouse-derived specific monoclonal antibody, then cloning variable region gene from mouse hybridoma cell, cloning constant region gene of human antibody according to the need, connecting mouse variable region gene and human constant region gene into chimeric gene, inserting into expression vector, and finally expressing chimeric antibody molecule in eukaryotic system or 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 a variant thereof. The antibody heavy chain of the PD-1 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprises a heavy chain constant region of human IgG1, IgG2 or IgG4, or an IgG1, IgG2 or IgG4 variant using amino acid mutations (such as L234A and/or L235A mutations and/or S228P mutations).

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the heterogenous reaction induced by the chimeric antibody carrying a large amount of murine protein components. Such framework sequences can be selected from those including germline antibody gene sequencesco-DNA databases or published references. Germline DNA sequences of, for example, human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (at the Internet)www.mrccpe.com.ac.uk/vbaseAvailable), and found in Kabat, e.a. et al, 1991 Sequences of proteins of Immunological Interest, 5 th edition. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. The humanized antibodies of the present disclosure also include humanized antibodies obtained after further affinity maturation of the CDRs by yeast display.

Grafting of CDRs can result in reduced affinity of the resulting PD-1 antibody or antigen-binding fragment thereof for an antigen due to framework residues in contact with the antigen. Such interactions may be the result of a high degree of somatic mutation. Thus, there may still be a need to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues from the non-human PD-1 antibody or antigen-binding fragment thereof that are involved in antigen binding can be identified by examining the sequence and structure of the murine monoclonal antibody variable regions. Residues in the CDR donor framework that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared to a subtype consensus sequence or a consensus sequence of murine sequences with a high percentage of similarity. Rare framework residues are thought to be likely the result of somatic hypermutation and thus play an important role in binding.

"human antibodies" (HuMAb), "humanized antibodies," "fully human antibodies" are used interchangeably and can be either human derived antibodies or antibodies obtained from a transgenic organism that is "engineered" to produce specific human antibodies in response to antigenic stimuli and can 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 targeted for disruption, including targeted disruption of the endogenous heavy and light chain loci in the cell lines. The 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 can 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 can 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 an antigen (e.g., PD-1). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al, (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined 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 by a synthetic linker using recombinant methods, such that it is possible to generate a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science242: 423-. Such single chain antibodies are also intended to be encompassed within 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. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibody can be of different isotypes, e.g., IgG (e.g., IgG)E.g. IgG1, IgG2, IgG3 or IgG4 subtype), 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 peptides comprising CDRs, and the like.

Fab is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity among fragments obtained by treating an IgG antibody molecule with protease papain (which cleaves the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bonded together by a disulfide bond. In some embodiments, a 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 expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a 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 joined at the hinge position 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 can be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with pepsin. Further, the F (ab ')2 can 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 having an antigen-binding activity, which is obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F (ab') 2. In some embodiments, Fab's of the present disclosure may 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 the Fab ' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote 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) joined by a linker. Such scFv molecules can have the general structure: NH (NH)₂-VL-linker-VH-COOH or NH₂-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. USA90: 6444-. Other linkers useful in the present disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-.

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

Diabodies are antibody fragments in which the 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 antibodies and multispecific antibodies refer to antibodies that are capable of binding two or more antigens or antigenic determinants simultaneously, comprising scFv or Fab fragments.

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

The dsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues. The amino acid residue substituted with a cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (protein engineering,7,697 (1994)).

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

A CDR-containing peptide is composed of one or more regions in a CDR that contains VH or VL. Peptides comprising multiple CDRs may be linked directly or via a suitable peptide linker.

The peptides of the present disclosure comprising CDRs may be produced by: the DNA encoding the CDRs of VH and VL of the monoclonal antibody of the present disclosure, which specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, is constructed, inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or a eukaryote to express the peptide. The CDR-containing peptides can also be produced by chemical synthesis methods 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 which primarily contributes to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) per heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) per light chain variable region. The amino acid sequence boundaries of the CDRs may be determined using any of a variety of well known protocols, including the "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-, CDR amino acid numbers in VH were 26-32(HCDR1), 52-56(HCDR2) and 95-102(HCDR 3); and amino acid residues in VL are numbered 26-32(LCDR1), 50-52(LCDR2) and 91-96(LCDR 3). By combining the CDR definitions of both Kabat and Chothia, the CDRs are made up of amino acid residues 26-35(HCDR1), 50-65(HCDR2) and 95-102(HCDR3) in the human VH and amino acid residues 24-34(LCDR1), 50-56(LCDR2) and 89-97(LCDR3) in the human VL. Following the rules of IMGT, the CDR amino acid residue numbers in VH are approximately 26-35(CDR1), 51-57(CDR2) and 93-102(CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32(CDR1), 50-52(CDR2) and 89-97(CDR 3). Following the IMGT rules, the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align. .

The terms antibody "framework region", "framework region" or "FR", "FR region", 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 that variable domain. It is essentially a variable domain without 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 the PD-L1 molecule). Epitopes typically 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., epipope Mapping Protocols in methods in Molecular B biology, volume 66, g.e. morris, Ed. (1996).

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

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 less than about 10^-7M, e.g. less than about 10^-8M or 10^-9The dissociation equilibrium constant (KD) of M binds to PD-1, for example, as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR) techniques.

The term "compete" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between antigen binding proteins, as determined by the following assay: in the assay, the antigen binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., a PD-1 antigen or fragment thereof). Numerous types of competitive binding assays are available for determining whether an antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, methods Enzymology 9: 242-; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol.137: 3614-), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA with a solid phase of I-125 label (see, e.g., Morel et al, 1988, mol. 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 bound to a solid surface or cell bearing either an unlabeled test antigen binding protein or a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be detected is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds a contiguous epitope sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder binding from occurring. Additional details regarding methods for determining competitive binding are provided in the examples herein. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., decrease) the specific binding of at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of a reference antigen binding protein to a common antigen. In certain instances, 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 both 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, when the amino acid sequences are aligned and gaps are introduced, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. For purposes of determining percent amino acid sequence identity, alignments can be accomplished in a variety of ways that are within the skill in the art, e.g., 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 being 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 can 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 having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along 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 Cold spring harbor antibody protocols, chapters 5-8 and 15. For example, a mouse may be immunized with human PD-1 or a fragment thereof, and the resulting antibody can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen binding fragment of the invention is genetically engineered to add one or more human FR regions to the CDR regions of non-human origin. Human FR germline sequences can be obtained from the website http:// IMGT. cities.fr of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN012441351, by aligning the IMGT human antibody variable region germline gene database with the 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 susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

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 may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expression of antibodies that specifically bind to human PD-1. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out using an A or G Sepharose FF column containing a buffer adjusted. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting the antibody fragment by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. 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 agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administering," "administering," or "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" or "treating" also means treating, e.g., a cell, in vitro and ex vivo by an agent, a diagnostic, a 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 a therapeutic agent, e.g., a composition comprising any one of the binding compounds of the present disclosure, either internally or externally to a patient who 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 the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, to induce regression of such symptoms or to inhibit development of such symptoms to any clinically useful degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") 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 commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

"conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that changes can be made frequently without changing the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology, He Gene, The Benjamin/Cummings pub. Co., p. 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish 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, an effective amount also meaning an amount sufficient to permit or facilitate diagnosis. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening severity, or delaying onset of the condition, including biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes exhibited during development of the condition. 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-associated disorders of the disclosure, reducing the dosage of other agents required to treat the disorder, enhancing the therapeutic efficacy of another agent, and/or delaying the progression of a target antigen-associated disorder of the 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 method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance produced outside an organism, cell or human body as the case may be. "endogenous" refers to a substance produced in a cell, organism, or human body as the case may be.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., 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 matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned; two sequences are 95% homologous if there are 95 matches or homologies at 100 positions in the two sequences. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology. For example, the comparison may be performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of the respective reference sequences. The following references refer to the BLAST algorithm often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS) Altschul, S.F. et al, (1990) J.mol.biol.215: 403-; gish, W. et al, (1993) Nature Genet.3: 266-; madden, T.L. et al, (1996) meth.Enzymol.266: 131-; altschul, S.F. et al, (1997) Nucleic Acids Res.25: 3389-3402; zhang, J. et al, (1997) Genome Res.7: 649-. Other conventional BLAST algorithms, such as provided by NCBI BLAST, are also well known to those skilled in the art.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, 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 a particular portion of nucleic acid, RNA, and/or DNA are amplified as described, for example, in U.S. patent No. 4,683,195. In general, it is desirable to obtain sequence information from the ends of or beyond the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotide of the 2 primers may coincide with the end 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, and the like. See generally Mullis et al (1987) Cold Spring harborSymp. Ouant. biol.51: 263; erlich editors, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.

"isolated" refers to the 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. Generally, 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 antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a mixture containing one or more 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 facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

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

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

There is no limitation on the disease associated with PD-1 as long as it is a disease associated with PD-1, e.g., a therapeutic response induced using the molecules of the present disclosure can be achieved by binding to human PD-1 and then blocking the binding of PD-1 to its ligands PD-L1, PD-L2, or killing tumor cells that overexpress PD-1. Thus, the molecules of the present disclosure are very useful, when in preparations and formulations suitable for therapeutic applications, for those people who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, and the like.

Further, 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 a cell expressing PD-1, and a diagnostic agent for diagnosis of a disease associated with a PD-1 positive cell, which comprise, as an active ingredient, the monoclonal antibody or antibody fragment of the present disclosure that specifically recognizes human PD-1 and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof.

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

The immunoassay or measuring method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

The above-mentioned diseases associated with PD-1 positive cells can be diagnosed by detecting or assaying cells expressing PD-1 with the monoclonal antibody or antibody fragment of the present disclosure.

For detecting cells expressing the polypeptide, a known immunoassay method can be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, or the like is preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS system (applied biosystem) or the like can be used.

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

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled secondary antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance thereof, a substrate corresponding to the label, and the like.

Second, example and test

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure. The experimental methods of the present disclosure, in which specific conditions are not specified, are generally performed 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 material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1 preparation of antigen

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

Human PD-1-IgG1 Fc: (SEQ ID NO: 1)

MEFGLSWLFLVAILKGVQCPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Note that: the underlined part is the signal peptide, the positive part is the extracellular domain and the italic part is hIgG1Fc (signal peptide + extracellular domain + hIgG1 Fc).

Example 2 construction of affinity maturation Yeast library for PD-1 antibody and validation of library to obtain anti-human PD-1 antibody with better drug efficacy, affinity maturation was performed on H005-1 antibody by yeast display platform technology, and affinity maturation Yeast library against 6 CDRs of H005-1-scFv antibody was designed and prepared on the basis of H005-1 antibody, and a new human PD-1 antibody was screened therefrom. The CDR and light/heavy chain variable region sequences of the H005-1 antibody are from WO2015085847a1, with the following specific sequences:

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

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

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Note: the underlined parts are the variable region parts of the antibody, the bold italic parts are the CDR regions (amino acid residues of the CDRs are determined and annotated by the Kabat numbering system), and the other non-underlined positive parts are the constant region parts 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: the linker sequence is underlined.

Construction of the Yeast library: degenerate primers were designed and the designed mutated amino acids were introduced by PCR into libraries of H005-1-scFv antibodies, each library being 10 in size⁹And on the other hand, the diversity of the constructed yeast library is verified by a second-generation sequencing method.

Example 3 screening of antibodies

In the first round of screening, approximately 5X 10 from the H005-1 antibody library¹⁰The individual cells were incubated with 10. mu.g/ml biotinylated human PD-1-IgG1Fc protein in 50m1 buffer containing 0.1% Bovine Serum Albumin (BSA) -phosphate (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. Mu.l streptavidin microbeads (Mi1enviBiotec, Auburn, Calif.) were then added to the antibody library of PD-1 bound to biotinylated human PD-1-IgG1Fc protein and loaded onto the AutoMACS system for sorting. CollectingCells of the antibody library with high affinity for PD-1 were then cultured in SDCAA medium (20g dextrose, 6.7g Difco Yeast Nitrogen source-No amino acids, 5g Bacto Casein amino acids, 5.4g Na2HP04 and 8.56g NaH)₂PO₄·H₂O, dissolved in 1L distilled pomegranate water) at 250rpm and 30 ℃ for 24 hours. Then, the culture was cultured in SGCAA medium (20g of galactose, 6.7g of Difco yeast nitrogen source-no amino acid, 5g of Bacto casamino acid, 5.4g of Na2HP04 and 8.56g of NaH)₂PO₄·H₂O, dissolved in 1L distilled durum water) and 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 used in the second and/or third rounds of screening, 100 times the size of the library from the previous round was used as the number of cells input.

For the third and fourth rounds of screening, library cells from the previous round were incubated with 1 μ g/ml biotinylated human PD-1-IgG1Fc protein and 10 μ g/ml Mouse Anti-cMyc (9E10, sigma) antibody in 0.1% PBSA at room temperature for 1 hour, 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 Strevidin-PE (S-866, life technologies) were added and incubated at 4 ℃ for 1 hour, and the mixture was washed three times with 0.1% PBSA to remove unbound antibody fragments. Finally, high affinity antibodies were screened by FACS (BD FACSAria FUSION).

The H005-1 library utilizes biotinylated human PD-1-IgG1Fc antigen, and is subjected to two rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAria FUSION), about 400 yeast monoclonals are selected to be cultured and induced to express, FACS (FACS Canto II) is used for detecting the binding of the yeast monoclonals to the human PD-1-IgG1Fc antigen, the yeast monoclonals with higher affinity than that of the wild-type H005-1 antibody are selected for sequencing verification, the sequenced clones are subjected to alignment analysis, after redundant sequences are removed, non-redundant sequences are converted into full-length IgG (gamma 1, kappa), mammalian cell expression is carried out, and the full-length antibody after affinity purification is determined by BIAcoreTM X-100(GE Life Sciences).

1. The CDR sequences of the selected antibodies were confirmed by screening.

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

the related heavy chain variable region sequence was obtained as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVA ₁TISGGGANTYFAXSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSS(SEQ ID NO：13)

or

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVA ₁TISGGGANTYFAXSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS(SEQ ID NO：14)。

The related light chain variable region sequence was obtained as follows:

DIQMTQSPSSLSASVGDX₁₅VTITC ₂ ₃ ₄ ₅ ₆XASQXIXXWLXWYQQKPGX₁₆X₁₇PKLLIX₁₈ ₇ ₈ ₉ ₁₀ ₁XATXXXX ₁ GVPSRFSGSGSGTDFTLTIX₁₉SLQPEDFAX₂₀YYCQ ₁₂ ₁₃ ₁₄QXYXXPWTFGGGTKVEIK(SEQ ID NO：15)。

x in the above CDR and heavy and light chain variable regions₁Selected from D or N, X₂Selected from L, R or Q, X₃Selected from T, S, G, N or D, X₄Selected from G, A or Y, X₅Selected from T, Y, N, S or D, X₆Selected from T, N or A, X₇Selected from T, G or L, X₈Selected from T, S or H, X₉Selected from R or L, X₁₀Selected from A, E or Q, X₁₁Is selected from the group consisting of D, Y or S,X₁₂selected from V or A, X₁₃Selected from S or T, X₁₄Selected from V, T or I, X₁₅Selected from R or I, X₁₆Selected from K or Q, X₁₇Selected from A or P, X₁₈Selected from Y or H, X₁₉Selected from S or G, X₂₀Is selected from A or T.

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

TABLE 1 variable regions of heavy chains of antibodies obtained by affinity screening

TABLE 2 variable region sequences of antibody light chains obtained by affinity screening

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

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYFADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSS

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYFANSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSS

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYFADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS

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

DIQMTQSPSSLSASVGDIVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCRASQSIGYWLNWYQQKPGKAPKLLIYGATSLAYGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYTTPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSIPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCRASQSIGNWLAWYQQKPGKPPKLLIYLATSRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIHTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFAAYYCQQVYSIPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCQASQGIGYWLNWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYTTPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCRASQNIASWLAWYQQKPGKAPKLLIYLATTREYGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSVPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTIGSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCRASQSIYDWLAWYQQKPGQAPKLLIYLATSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

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

DIQMTQSPSSLSASVGDRVTITCLASQDIGSWLAWYQQKPGKAPKLLIYLATHRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

note: in the above light/heavy chain variable region sequences, the underlined parts are the 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 a variant thereof, to form a full length light chain of the antibody. It is well known in the art that Fc mutations of IgG1, such as D265A, N297A, L234A/L235A or L234F/L235A, etc., can reduce ADCC, and mutations at or near P331S can reduce CDC; mutations in IgG2 and Fc-hybrid antibodies of IgG2/4 also reduce ADCC and CDC; constant region ADDC activity of IgG4 is low; in addition, the introduction of F234A and L235A mutations (mAbs 4:3, 310-318; May/June 2012) in the constant region of IgG4 or IgG1 altered the CH2 domain, reduced the interaction with Fc receptors and thereby reduced ADCC activity. These well-known IgG1, IgG4, or mutants thereof can be used as constant regions of the anti-PD-1 antibodies of the present disclosure. Illustratively, antibodies of the disclosure may be selected from the group consisting of 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 H005-1 mutant antibody library obtained by the aforementioned screening is compared to the heavy chain variable region as set forth in SEQ ID NO: 48 or SEQ ID NO: 49 to form an antibody heavy chain, and a light chain variable region linked to a heavy chain constant region as set forth in SEQ ID NO: the light chain kappa constant region shown at 50 is linked to form the antibody light chain, and then the light and heavy chains are combined to form the complete antibody, and the sequences of the resulting partial antibodies are shown in table 3 below:

TABLE 3 sequences of antibodies derived from the H005-1 mutation

Remarking: for example, "Ab 01" indicates that the heavy chain variable region sequence of the antibody numbered Ab01 is as set forth in SEQ ID NO: 35, the heavy chain constant region sequence is shown as SEQ ID NO: 48 and the light chain variable region sequence is shown as SEQ ID NO: 38, and the light chain constant region sequence is shown as SEQ ID NO: shown at 50.

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYFADSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

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

DIQMTQSPSSLSASVGDIVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

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

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQAYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

note: the variable region is underlined and the CDR region is in bold.

The disclosure uses Merck's PD-1 antibody Keytruda as a positive control, and the light and heavy chain amino acid sequences of Keytruda (Merck, Pembrolizumab, see WHO Drug Information, Vol.27, No. 2, p.161-162 (2013)) are as follows:

> Keytruda heavy chain (SEQ ID NO: 55)

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVT LTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

> Keytruda light chain (SEQ ID NO: 56)

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGS GSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Remarking: the scribe line portion is a variable region.

The PD-1 antibody is obtained by purification according to a conventional method.

Example 4 antibody and ligand blocking assay

The blocking effect of the product on the combination of PD-1 and PD-L1 is deeply researched, and simultaneously, the product is compared with similar products in clinical tests. The Promega corporation constructed Jurkat cells overexpressing the PD-1 and NFAT driven luciferase reporter (CS187102, Promega) and CHO-K1 cells overexpressing PD-L1 (CS187108, Promega). The system reflects the ability of the antibody to block the binding of PD-1 to PD-L1 by detecting luminescences. The experimental procedure is briefly described as follows: CHOK1/PD-L1 cells (CS187108, Promega) were digested, added to a 96-well plate at 100. mu.L/well, and placed at 37 ℃ in a 5% CO format₂The incubator was incubated for 24 hours. The control (C25-IgG negative control (laboratory preparation)) and sample were diluted with PBS to the desired concentrations. Jurkat/PD1 cells (CS187102, Promega) were counted, inoculated at a certain ratio into a cell culture plate (90. mu.L/well) seeded with CHOK1/PD-L1 cells, diluted antibody was added at 10. mu.L/well, placed at 37 ℃ and 5% CO₂The culture was carried out in an incubator for 5 hours. The cell culture plates were removed and left at room temperature for 5 minutes, then 50. mu.l Bio-GloTMReagent (G7940, Promega) was added to each well, incubated at room temperature for 5 minutes, and the plates were read using a microplate reader (PerkinElmer Victor3, model: 1420-. The data are detailed in table 4 and figure 1.

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

The results show that the anti-PD-1 antibodies Ab05 and Ab01 exemplified in the present disclosure are able to effectively block the binding of PD-1 to PD-L1.

Example 5 BIAcore antibody affinity assay of exemplary antibodies

IgG is affinity captured by a Protein A biosensing chip (Cat. #29127556, GE), human PD-1 antigen (Cat. #10377H08H, Sino Biological) flows on the surface of the chip, and a Biacore T200 instrument detects anti-PD-1 antibody and antigen PD-1 reaction signals in real time to obtain a binding and dissociation curve. After dissociation was completed in each experimental cycle, the biosensor chip was washed and regenerated with 10mM Glycine-HClpH1.5 buffer. The experimental buffer system was 1 XHBS-EP buffer solution (Cat # BR-1001-88, GE). After the experiment, the data were fitted with the (1:1) Langmuir model using GE Biacore T200 Evaluation version3.0 software to obtain affinity values, and the experimental results of the same batch are shown in Table 5.

TABLE 5 affinity of different antibodies to human PD-L1

The results of the same batch experiment show that the anti-PD-1 antibodies screened can bind to human PD-1 with higher affinity than Keytruda and H005-1. The affinity of Ab05 and Ab01 for binding to human PD-1 was 2.8nM and 2.56nM, while the affinity of Keytruda and H005-1 was 5.81nM and 14.6nM, respectively.

Example 6 secretion of cellular IFN γ by exemplary antibodies in PBMC-T lymphocyte activation experiments

To study 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 IFN γ secretion levels were measured after 5 days of in vitro stimulation with Tuberculin (TB). The experimental procedure is briefly described as follows:

fresh blood PBMC were obtained by Ficoll-Hypaque (17-5442-02, GE), density gradient centrifugation (Stem cell technologies) and cultured in RPMI 1640(SH30809.01, GE) medium supplemented with 10% (v/v) FBS (10099-₂Culturing under the condition.

Freshly isolated and purified PBMC were adjusted to a density of 2X 10 in RPMI 1640 medium⁶40 mu/mL of cell suspension added to 20mLTuberculin (97-8800, Synbiotics), 37 deg.C, 5% CO₂The incubator is used for 5 days. On day5, the cells cultured as described above were collected, centrifuged, and resuspended in fresh RPMI 1640 medium at a density of 1.1X 10⁶One/ml, seeded into 96-well cell culture plates, 90. mu.l per well. Simultaneously, a sample of the antibody diluted in a gradient was added, and the mixture was diluted with PBS (B320, Shanghai culture Biotech Co., Ltd.) in an amount of 10. mu.l per well. The cell culture plate was placed at 37 ℃ in 5% CO₂Incubate for 3 days. The cell culture plate was removed, centrifuged (4000rpm, 10min) to collect the cell culture supernatant, and the IFN-. gamma.level was measured by ELISA (human IFN-. gamma.detection kit (EHC102g.96, Xinbo), reference reagent manual for specific procedures.

The results (see FIGS. 2A-2C) show that all antibodies can activate IFN-. gamma.secretion in the tested anti-PD-1 antibody molecules, with Ab01, Ab05, Ab09 having significantly better ability to activate IFN-. gamma.secretion at high concentrations than H005-1.

Test example 7 pharmacokinetic testing of exemplary antibodies

Animals sera were taken at different time points after administration of 1mpk of Anti-PD-1 antibody sample, standard curves for different samples were prepared using direct ELISA protocol, 100 μ L of 0.5 μ g/mL PD-1-His (Cat. #10377H08H, Nano Biological) were plated, incubated overnight at 4 ℃, blocked at 200 μ L3% mil, 100 μ L of sample was added, 50 fold plated diluted, incubated for 2H at 37 ℃, 100 μ L of adsorbed secondary antibody, goat Anti-Human IgG Fc-HRP (1:5000) was added, incubated for 1H at 37 ℃, TMB developed, 1M H₂SO₄Terminate, read plate. The obtained data were analyzed by Phoenix WinNonlin software to calculate pharmacokinetic related parameters.

Experimental cynomolgus monkeys, plain grade, male, 3-7 years old, 4-8 kg, were bred by Meidi Seipya pharmaceutical science and technology (Shanghai) Co., Ltd., from Guangxi Xiongsen Primates laboratory animal Breeding and development Co., Ltd. Animals were quarantined and acclimated before inclusion in the experiment according to SOP requirements. A breeding environment: controlled by an automatic timing controller, approximately 12 hours of light was provided per day. The dark time may be intermittently interrupted due to the need for study related activities. The environmental temperature and relative humidity of the animal room are respectively controlled within the range of 19-26 ℃ and 40-70%, and are monitored and recorded every day. The cynomolgus monkeys are grouped into 3 groups, which are respectively numbered as mon1, mon2 and mon 3. On the experimental day, each cynomolgus monkey is injected with the tested medicine intravenously, and the administration dose is 1mg/kg in a single administration, and the intravenous infusion is completed within 30 min; volume administered (2 ml/kg).

Approximately 2.0mL of each of the blood samples were collected via cephalic vein or saphenous vein puncture and placed in a blood collection tube (without anticoagulant) for serum isolation prior to administration, 5min, 15min, 0.5h, 8h, Day2(24h), Day3(48h), Day5(96h), Day8(168h), Day11(240h), Day15(336h), Day29(672h), Day43(1008h), Day57(1344h), Day71(1680h) and Day85(2016h) after intravenous infusion was initiated. After collection, the mixture was left at 4 ℃ for 30 minutes, 1000g of the mixture was centrifuged for 15 minutes, and the supernatant (serum) was placed in an EP tube and stored at-80 ℃ in 2 tubes (completed within 2 hours after serum collection).

The drug metabolism parameters of Ab05 and the parent antibody H005-1 at a dose of 1mpk are determined in a cynomolgus monkey, and the result shows that Ab05 has good in vivo drug metabolism performance of the cynomolgus monkey, the average drug half-life period t1/2 is about 6.2 days, and the following table 6 shows that the antibody has good stability in the cynomolgus monkey. While at the same dose (1mpk) the half-life t1/2 for H005-1 was found to be about 3.7 days. The results are shown in Table 6.

TABLE 6 pharmacokinetic parameters of anti-PD-1 antibodies

Note: RSD (relative standard deviation) represents a relative standard deviation.

Test example 8 Effect of anti-PD-1 antibody in transgenic PD-1 mouse Colon cancer model MC38

Transgenic PD-1 mice were derived from a fifth generation of purchased transgenic PD-1 mice (ISIS INVovation LIMITED, University Offices, Wellington Square, Oxford OX 12 JD, England) bred at Cephrim biosciences, Inc. MC38 cells were cultured at 5X 10⁵One mouse is inoculated to the subcutaneous part of the right rib of hPD-1 transgenic mice (each half of a male and a female) at a dose of 100 mu l/mouse, and when the average tumor volume of the mice reaches 80-100mm³In between, removing bodyHeavy, too large and too small tumor animals, tumor-bearing mice were randomly divided into 5 groups (8 per group) according to tumor volume: negative controls hIgG control-30mpk, H005-1-10mpk, Keytruda-30mpk, Ab05-10mpk, and Ab05-30 mpk. The group administration Day was set to Day 0. Each drug was administered in groups into the abdominal cavity for a period of 22 days, once every two days for 11 times. Tumor volumes were measured 2 times per week, body weights were weighed and data were recorded. The body weight and tumor volume of each group of animals were expressed as Mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism5 and Excel software, using student t test statistical analysis.

Tumor Volume (TV) 0.5236 XL_{Long and long}×L_{Short length} ²

Tumor proliferation rate T/C%

Tumor inhibition% TGI%

(wherein T, T0 represents the tumor volume at the end of the test and at the start of the test in the antibody-administered group, and C, C0 represents the tumor volume at the end of the test and at the start of the test in the blank control group, respectively.)

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

TABLE 7 tumor inhibition Rate Effect of anti-PD-1 antibodies on mouse Colon cancer MC38

Remarking: in the table, "IP" means intraperitoneal injection; "Q2 Dx 11" means administration 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> Hengrui pharmaceutical Co., Ltd of Jiangsu

Shanghai Hengrui pharmaceuticals, Inc

<120> anti-PD-1 antibody, antigen-binding fragment thereof, and medical use

<150>201810875621.2

<151>2018-08-03

<160>56

<170>SIPOSequenceListing 1.0

<210>1

<211>401

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<223> human PD-1-IgG1Fc

<400>1

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 105110

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

LysVal 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 general 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 general 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> general formula of heavy chain variable region sequence

<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 7580

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> general formula of heavy chain variable region sequence

<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 general 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 PheSer 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 7580

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 ProCys 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 ProGlu 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 LysThr 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

Heavy chain of <223> Ab01 antibody

<400>51

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

1 510 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

165170 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 330335

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

Heavy chain of <223> 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

225230 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

385390 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> Keytruda 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> Keytruda 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 LysSer 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, said anti-PD-1 antibody or antigen-binding fragment thereof comprising:

HCDR2TISGGGANTYFAX₁SVKG SEQ ID NO：7

LCDR1X₂ASQX₃IX₄X₅WLX₆SEQ ID NO：10

LCDR2X₇ATX₈X₉X₁₀X₁₁SEQ ID NO：11

LCDR3QQX₁₂YX₁₃X₁₄PWT SEQ ID NO：12

2. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, which comprises:

As shown in SEQ ID NO: 18. 19, 20, 21, 22, 23 or 24, as shown in SEQ id no: 25. 26, 27, 28, 29 or 30 and the light chain LCDR2 region as set forth in any one of the amino acid sequences of SEQ ID NOs: 31. 32, 33 or 34, or a light chain LCDR3 region as set forth in any one of amino acid sequences set forth herein.

3. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 2, which comprises:

(k) Respectively shown in SEQ ID NO: 6. SEQ ID NO: 16. SEQ ID NO: 8, and heavy chain HCDR1, HCDR2, HCDR3 regions as set forth in SEQ ID NOs: 24. SEQ ID NO: 30. SEQ ID NO: 31, LCDR1, LCDR2, LCDR 3.

4. The anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof includes an FR region.

5. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 4, wherein the anti-PD-1 antibody or antigen-binding fragment comprises: as shown in SEQ ID NO:13 or SEQ ID NO: 14 and the heavy chain variable region as set forth in SEQ ID NO: 15, wherein SEQ ID NO 13, SEQ ID NO: 14. SEQ ID NO: 15 are each a sequence of the general formula:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVA ₁TISGGGANTYFAXSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARTQIGFTNWGQGTTVTVSS SEQ ID NO：13

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVA ₁TISGGGANTYFAXSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS SEQIDNO：14DIQMTQSPSSLSASVGDX₁₅VTITC ₂ ₃ ₄ ₅ ₆XASQXIXXWLXWYQQKPGX₁₆X₁₇PKLLIX₁₈ ₇ ₈ ₉ ₁₀ ₁₁XATXXXXGVPSRFSGSGSGTDFTLTIX₁₉SLQPEDFAX₂₀YYCQ ₁₂ ₁₃ ₁₄QXYXXPWTFGGGTKVEIK SEQ ID NO: 15 wherein X₁Selected from D or N, X₂Selected from L, R or Q, X₃Selected from T, S, G, N or D, X₄Selected from G, A or Y, X₅Selected from T, Y, N, S or D, X₆Selected from T, N or A, X₇Selected from T, G or L, X₈Selected from T, S or H, X₉Selected from R or L, X₁₀Selected from A, E or Q, X₁₁Selected from D, Y or S, X₁₂Selected from V or A, X₁₃Selected from S or T, X₁₄Selected from V, T or I, X₁₅Selected from R or I, X₁₆Selected from K or Q, X₁₇Selected from A or P, X₁₈Selected from Y or H, X₁₉Selected from S or G, X₂₀Is selected from A or T.

6. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 5, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 35. 36 or 37 and the heavy chain variable region set forth in SEQ ID NO: 38. 39, 40, 41, 42, 43, 44, 45, 46 or 47;

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

7. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody heavy chain further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof, preferably comprises a human IgG1 or IgG4 heavy chain constant region, more preferably comprises an IgG1 or IgG4 heavy chain constant region incorporating F234A and L235A mutations; the antibody light chain further comprises a constant region of a human kappa, lambda chain or variant thereof.

8. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 7, wherein 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, and a light chain constant region as set forth in SEQ ID NO: 50 or has at least 85% sequence identity thereto.

9. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the anti-PD-1 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the heavy chain as set forth in SEQ ID NO: 52, a light chain; or comprises the amino acid sequence as set forth in SEQ ID NO: 53 and the heavy chain as set forth in SEQ ID NO: 54, or a light chain as shown in figure 54.

10. The anti-PD-1 antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), and antigen binding fragments of peptides comprising CDRs.

11. An isolated antibody or antigen-binding fragment thereof that competes for binding to human PD-1 with the antibody or antigen-binding fragment thereof of any one of claims 1-10, or binds to the same epitope of human PD-1 as the antibody or antigen-binding fragment thereof of any one of claims 1-10.

12. 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 1-11, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

13. A nucleic acid molecule encoding the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-11.

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

15. A host cell selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells, transformed with the vector according to claim 14.

16. A method of producing the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-11, comprising the steps of culturing the host cell of claim 15 in culture to form and accumulate the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-11, and recovering the anti-PD-1 antibody or antigen-binding fragment thereof from the culture.

17. A method for immunodetection or assay of PD-1, the method comprising using the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 11.

18. Use of the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1-11, in the preparation of a diagnostic agent for tumors.

19. Use of an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1-11, or a pharmaceutical composition comprising the antibody of claim 12, or the nucleic acid molecule of claim 13, in the manufacture of a medicament for the treatment of a tumor; preferably, the tumor is selected from: squamous cell carcinoma of head and neck, cancer of head and neck, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, kunkenberg's tumor, myeloproliferative tumor, 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, large B-cell lymphoma enriched in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia; most preferably, the tumor is melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, non-small cell lung cancer or bladder cancer.