CN113637075A

CN113637075A - Bispecific antigen binding molecules and medical uses thereof

Info

Publication number: CN113637075A
Application number: CN202110454892.2A
Authority: CN
Inventors: 张伟; 陈思萌; 吴建权; 王蕾蕾
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: 2020-04-27
Filing date: 2021-04-26
Publication date: 2021-11-12
Anticipated expiration: 2041-04-26
Also published as: CN113637075B

Abstract

The present disclosure relates to bispecific antigen binding molecules and their medical uses. In particular, the present disclosure relates to antigen binding molecules comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain specifically binds to PD-1 and comprises a specific CDR. The present disclosure also relates to pharmaceutical compositions comprising a first antigen-binding domain and a second antigen-binding domain, or pharmaceutical compositions comprising the antigen-binding molecules. The disclosure also relates to methods of treating and/or preventing diseases associated with PD-1 and/or PD-L1.

Description

Bispecific antigen binding molecules and medical uses thereof

Technical Field

The disclosure belongs to the field of biomedicine, and particularly relates to a bispecific antigen binding molecule and medical application thereof, and more particularly relates to an antigen binding molecule specifically binding to PD-1 and PD-L1 and medical application thereof.

Background

This section is merely to provide background information related to the present disclosure and does not necessarily constitute prior art.

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 antigen 4 (CTLA-4), but is expressed differently from CTLA, mainly on activated T cells, B cells and myeloid 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 its expression on activated cells can be up-regulated. 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 (for example, non-small cell lung cancer), gastric cancer, intestinal cancer, renal cancer, melanoma, 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.

The anti-PD-1 monoclonal antibody can improve the immune system reaction of a patient to a tumor to the maximum extent by blocking the combination between PD-1/PD-L1, thereby achieving the aim of killing tumor cells.

PD-1 and PD-L1 bispecific antibodies (BsAb) are a class of combinatorial antibodies with amphipathic properties, usually bivalent (also tetravalent and hexavalent), i.e.having two antigen binding arms, which function to bind two different specific antigens. The antibody will bind to both an Antigen Presenting Cell (APC) or tumor cell expressing PD-L1 and a T cell expressing PD-1. The bifunctional antibody has the following three functions: 1) bispecific antibodies can antagonize the PD-1/PD-L1 pathway-mediated inhibition of T cell function; 2) different from the PD-1/PD-L1 monoclonal antibody, the double antibodies promote signal synapses among T cells, DC cells and tumor cells by simultaneously combining PD-1/PD-L1, on one hand, promote the activation of the T cells by the DC cells, and on the other hand, promote the killing of the tumor cells by the T cells. 3) The double antibody can theoretically promote the infiltration of T cells to PD-L1 high-expression tumor parts by simultaneously combining the T cells and PD-L1 high-expression tumor cells. These three mechanisms ultimately promote the recognition and killing of tumor cells by the immune system. anti-PD-1/PD-L1 bispecific antibody LY3434172 (also called IBI-318) from Eli Lilly is currently in the stage I clinical stage of tumors or cancers such as non-small cell lung cancer, melanoma, squamous cell carcinoma of head and neck, urothelial cell carcinoma, renal cell carcinoma, gastric cancer, esophageal cancer, colon cancer, advanced solid tumors such as bile duct cancer, and the like, and related antibodies are described in patent application US20190010232A 1.

The disclosed study found that although the response rate (ORR) of certain anti-PD-1 antibodies in hematological tumors was as high as 90%, the response rate in advanced non-small cell lung cancer was only around 30%, and the total response rate in single administration of intermediate and advanced esophageal and gastric cancers was only 14%. Therefore, the development of new anti-PD-1 antibodies, particularly bispecific antigen-binding molecules formed from the anti-PD-1 antibody and a second antigen-binding molecule, and pharmaceutical combinations of the antibody and the second antigen-binding molecule are still needed.

Disclosure of Invention

The present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof, which is an anti-PD-1 antibody or antigen-binding fragment thereof selected from any one of i) to iii) below:

i) an anti-PD-1 antibody or antigen-binding fragment thereof, whose heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 or HCDR1 having up to 3, 2 or 1 amino acid mutations thereto, the sequence being as set forth in SEQ ID NO: 9 or having at most 3, 2 or 1 amino acid mutations thereto, and a HCDR2 having a sequence as set forth in SEQ ID NO: 10 or HCDR3 having up to 8, 3, 2 or 1 amino acid mutations therefrom; the light chain variable region comprises a sequence as set forth in SEQ ID NO: 11 or LCDR1 having up to 4, 3, 2 or 1 amino acid mutations thereto, the sequence set forth in SEQ ID NO: 12 or with at most 3, 2 or 1 amino acid mutations thereof, and an LCDR2 having a sequence as set forth in SEQ ID NO: 13 or LCDR3 having up to 3, 2 or 1 amino acid mutations therefrom;

ii) an anti-PD-1 antibody or antigen-binding fragment thereof, whose heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14 or HCDR1 having at most 3, 2 or 1 amino acid mutations thereto, the sequence being as set forth in SEQ ID NO: 15 or having at most 3, 2 or 1 amino acid mutations thereto, and a HCDR2 having a sequence as set forth in SEQ ID NO: 16 or HCDR3 having up to 8, 3, 2 or 1 amino acid mutations therefrom; the light chain variable region comprises a sequence as set forth in SEQ ID NO: 17 or LCDR1 having up to 4, 3, 2 or 1 amino acid mutations thereto, the sequence set forth in SEQ ID NO: 12 or with at most 3, 2 or 1 amino acid mutations thereof, and an LCDR2 having a sequence as set forth in SEQ ID NO: 18 or LCDR3 having at most 3, 2 or 1 amino acid mutations therefrom; and

iii) an anti-PD-1 antibody or antigen-binding fragment thereof whose heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21 or HCDR1 having at most 3, 2 or 1 amino acid mutations thereto, the sequence being as set forth in SEQ ID NO: 22 or having at most 3, 2 or 1 amino acid mutations thereto, and a HCDR2 having a sequence as set forth in SEQ ID NO: 23 or HCDR3 having up to 3, 2 or 1 amino acid mutations therefrom; the light chain variable region comprises a sequence shown as SEQ ID NO: 24 or LCDR1 having up to 3, 2 or 1 amino acid mutations thereto, the sequence set forth in SEQ ID NO: 25 or LCDR2 having at most 3, 2 or 1 amino acid mutations thereto, and a polypeptide having the sequence set forth in SEQ ID NO: 26 or LCDR3 having at most 3, 2 or 1 amino acid mutations therefrom.

In some embodiments, the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof of the present disclosure are administered at 10 or less^-7M dissociation equilibrium constant associated with human PD-1And (6) mixing. In some embodiments, 10 or less^-8M、 10^-9M、10^-10M or 10^-11The M dissociation equilibrium constant binds to human PD-1.

In some alternative embodiments, the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof, whose heavy chain variable region comprises: the sequence is shown as SEQ ID NO: 65, and the sequence is shown in SEQ ID NO: 66, and HCDR2 having the sequence set forth in SEQ ID NO: 67 HCDR 3; the light chain variable region comprises: the sequence is shown as SEQ ID NO: 68, and the sequence is shown as SEQ ID NO: 12, and LCDR2 having the sequence set forth in SEQ ID NO: LCDR3 shown at 69; the sequence is shown in the following table 1:

TABLE 1

In some alternative embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises a light chain variable region having a sequence set forth in SEQ ID NO: 8, and the sequence is shown as SEQ ID NO: 9 and HCDR2 having the sequence shown in SEQ ID NO: HCDR3 shown at 10; the light chain variable region comprises a sequence shown as SEQ ID NO: 12, and the sequence is shown as SEQ ID NO: 13, and the sequence is shown as the general formula RSSQSX₁₃VHSX₁₄X₁₅X₁₆TYLE (SEQ ID NO: 68), and wherein X₁₃Is selected from L, X₁₄Selected from N, Q, L, T or D, X₁₅Selected from G, A or V, X₁₆LCDR1 selected from N.

In some alternative embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof selected from any one of (a) to (e) below:

(a) an anti-PD-1 antibody or antigen-binding fragment thereof, comprising the amino acid sequences set forth in SEQ ID NOs: 8. SEQ ID NO: 9 and SEQ ID NO: 10, HCDR1, HCDR2 and HCDR3, the sequences of which are set forth in SEQ ID NOs: 12 and SEQ ID NO: 13, and LCDR2 and LCDR3 having sequences set forth in SEQ ID NO: 11. LCDR1 shown at 47, 48, 49, 50, 51 or 52;

(b) an anti-PD-1 antibody or antigen-binding fragment thereof, comprising the amino acid sequences set forth in SEQ ID NOs: 14. SEQ ID NO: 15 and SEQ ID NO: 16, and HCDR1, HCDR2 and HCDR3 having sequences set forth in SEQ ID NOs: 17. SEQ ID NO: 12 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 18;

(c) an anti-PD-1 antibody or antigen-binding fragment thereof, comprising the amino acid sequences set forth in SEQ ID NOs: 21. SEQ ID NO: 22 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 23, and sequences set forth in SEQ ID NOs: 24. SEQ ID NO: 25 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 26;

(d) an anti-PD-1 antibody or antigen-binding fragment thereof, comprising the amino acid sequences set forth in SEQ ID NOs: 14. SEQ ID NO: 15 and SEQ ID NO: 16, and HCDR1, HCDR2 and HCDR3 having sequences set forth in SEQ ID NOs: 12 and SEQ ID NO: 13, and LCDR2 and LCDR3 as set forth in SEQ ID NO: 11. LCDR1 shown at 47, 48, 49, 50, 51 or 52; and

(e) the heavy chain variable region comprises sequences respectively shown as SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10, and HCDR1, HCDR2 and HCDR3, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 17. SEQ ID NO: 12 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 18.

In some alternative embodiments, the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof selected from any one of the following iv) to vi):

iv) an anti-PD-1 antibody or antigen-binding fragment thereof, whose heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:4 having the same sequence as HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 5 light chain variable regions with the same sequences of LCDR1, LCDR2 and LCDR 3;

v) an anti-PD-1 antibody or antigen-binding fragment thereof, whose heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 6 having the same sequence as HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: the light chain variable region shown in the sequence 7 has LCDR1, LCDR2 and LCDR3 with the same sequence; and

vi) an anti-PD-1 antibody or antigen-binding fragment thereof, whose heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:19 having the same sequence as HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: the light chain variable region shown by the 20 sequence has LCDR1, LCDR2 and LCDR3 with the same sequence.

In some embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, or a humanized antibody.

In some embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment is a humanized antibody. In some embodiments, the humanized antibody comprises a framework region derived from a human antibody or a variant of a framework region thereof. In some embodiments, the framework region variant is a back-mutation having up to 11 amino acids on the basis of the light chain framework region and/or the heavy chain framework region of a human antibody, respectively. In some embodiments, the framework region variant comprises a mutation selected from any one of (f) to (h) below:

(f) a 2G amino acid back mutation in the light chain variable region, and/or one or more amino acid back mutations selected from 27Y, 48I, 67T, 69L, 82F and 93T in the heavy chain variable region;

(g) a 2V amino acid back mutation in the light chain variable region, and/or one or more amino acid back mutations selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T in the heavy chain variable region; and

(h) the light chain variable region comprises one or more amino acid back mutations selected from 42G, 44V and 71Y, and/or the heavy chain variable region comprises 1K and/or 94S amino acid back mutations.

In some embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises an antibody variable region selected from any one of (i) to (o) below;

(i) the sequence is shown as SEQ ID NO:4 and/or the sequence of the heavy chain variable region shown in SEQ ID NO: 5, a light chain variable region;

(j) the sequence is shown as SEQ ID NO: 6 and/or the sequence of the heavy chain variable region shown in SEQ ID NO: 7;

(k) the heavy chain variable region with the sequence shown as SEQ ID NO 19 and/or the heavy chain variable region with the sequence shown as SEQ ID NO: 20, a light chain variable region;

(l) The sequence is shown as SEQ ID NO: 27. 30, 31 or 32 and/or the heavy chain variable region and/or the sequence shown in SEQ ID NO: 28. 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64;

(m) a heavy chain variable region having the sequence shown in SEQ ID NO:33, 36, 37, 38, 39 or 40 and/or a light chain variable region having the sequence shown in SEQ ID NO: 34. 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64;

(n) a heavy chain variable region having a sequence as set forth in SEQ ID NO:41, 45 or 46 and/or a heavy chain variable region having a sequence as set forth in SEQ ID NO: 42. 43 or 44;

(o) a heavy chain variable region having the sequence shown in SEQ ID NO:70 and/or a light chain variable region having the sequence shown in 71; and

(p) the sequence is as shown in SEQ ID NO: 27. 30, 31 or 32 and/or the heavy chain variable region and/or the sequence shown in SEQ ID NO: 34 or 35.

Wherein, the sequence SEQ ID NO:70 and SEQ ID NO: 71 is a sequence of the general formula:

TABLE 2

In still other embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antibody further comprises an antibody constant region; in still other embodiments, the heavy chain constant region of the antibody constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region of the antibody constant region is selected from the group consisting of human kappa and lambda chain constant regions and conventional variants thereof; in still other embodiments, the antibody constant region comprises an IgG4 heavy chain constant region comprising one or more mutations introduced in S228P, F234A, and L235A, e.g., three amino acid mutations comprising S228P, F234A, and L235A; in some other embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 72 or as set forth in SEQ ID NO: 79 as shown in SEQ ID NO: 73, or a light chain constant region.

In some embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 78 and a light chain as set forth in SEQ ID NO: 77 or 82; or comprises the amino acid sequence as set forth in SEQ ID NO: 75 and the light chain as set forth in SEQ ID NO: 74. 76, 80 or 81.

In some embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, 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.

In some embodiments, also disclosed is an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes with the anti-PD-1 antibody, or antigen-binding fragment thereof, of any one of the preceding claims for binding to human PD-1.

In some embodiments, the present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof of any one of the foregoing, or a therapeutically effective amount of the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

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

In some embodiments, the present disclosure also provides a host cell comprising the aforementioned nucleic acid molecule.

In some embodiments, the present disclosure also provides a method for the immunodetection or assay of PD-1, the method comprising the step of using the anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding, or the step of using the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof.

In some embodiments, the present disclosure also provides a kit comprising the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof or the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof. In some embodiments, there is also provided the use of the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof or the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof in the preparation of a diagnostic agent for a disease associated with PD-1.

In some embodiments, the present disclosure also provides a method of treating a disease associated with PD-1, the method comprising administering to a subject a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding, or the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition, or the aforementioned nucleic acid molecule; in some embodiments, the present disclosure also provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof as described above, or an isolated monoclonal antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition as described above, or a nucleic acid molecule as described above, in the preparation or prevention of a disease associated with PD-1; in some embodiments, the present disclosure also provides an anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding, or an isolated monoclonal antibody or antigen-binding fragment thereof of the foregoing, or a nucleic acid molecule of the foregoing, or a pharmaceutical composition of the foregoing, for use as a medicament; in some of these embodiments, the medicament is for treating or preventing a disease associated with PD-1.

In some embodiments, the disease is a tumor; in other embodiments, the disease 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, adenosquamous carcinoma, 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, myeloproliferative tumors, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and meikel cell carcinoma; in some of these embodiments, 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 other embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, renal cancer, bladder cancer, intestinal cancer and colon cancer. In some embodiments, the disease can be associated with PD-1 and/or PD-L1.

The present disclosure also provides an antigen binding molecule comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain specifically binds PD-1, including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 of an anti-PD-1 antibody or antigen-binding fragment thereof of the present disclosure as described previously.

In some embodiments, the first antigen-binding domain that specifically binds PD-1 described above comprises any one selected from the group consisting of i) to vi) as follows:

i) the sequences are respectively shown as SEQ ID NO: 8. 9 and 10, HCDR1, HCDR2 and HCDR3, and/or the sequences are respectively shown in SEQ ID NO: 11. LCDR1, LCDR2, LCDR3 shown in fig. 12 and 13;

ii) the sequences are respectively shown in SEQ ID NO: 14. 15, 16, and/or sequences are respectively shown as SEQ ID NO: 17. LCDR1, LCDR2, LCDR3 shown in fig. 12 and 18;

iii) the sequences are respectively shown in SEQ ID NO: 21. 22 and 23, HCDR1, HCDR2 and HCDR3, and/or the sequences are respectively shown in SEQ ID NO: 24. LCDR1, LCDR2, LCDR3 shown at 25, 26;

iv) is a variant of i) to iii) having 1, 2, 3 or more amino acid differences at HCDR1, and/or 1, 2, 3 or more amino acid differences at HCDR2, and/or 1, 2, 3 or more amino acid differences (e.g., 8) at HCDR3, 1, 2, 3 or more amino acid differences (e.g., 4) at LCDR1, and/or 1, 2, 3 or more amino acid differences at LCDR2, and/or 1, 2, 3 or more amino acid differences at LCDR3, e.g., such that the variant still has substantially the same function as i) to iii);

v) sequences of the general formula DYEX₁H (SEQ ID NO: 65) as shown in HCDR1, with the sequence shown in general formula LX₂DPETGGX₃VYNQKFKX₄(SEQ ID NO: 66) HCDR2 having the sequence of general formula EX₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂HCDR3 as shown in DWYFDV (SEQ ID NO: 67); wherein, X₁Is I or M; x₂Is F or I; x₃Is I or T; x₄Is G or D; x₅Is G or R; x₆Is F or a vacancy; x₇Is S or a vacancy; x₈Is Y or a vacancy; x₉Is G or a vacancy; x₁₀Is S or a vacancy; x₁₁Is N or T; x₁₂Is R or S; and

the sequence is shown as the general formula RSSQSX₁₃VHSX₁₄X₁₅X₁₆LCDR1 shown by TYLE (SEQ ID NO: 68), LCDR2 with sequence KVSNRFS (SEQ ID NO: 12), and sequence shown as general formula FQGSHVPYX₁₇LCDR3 as shown (SEQ ID NO: 69); wherein, X₁₃Is I or L; x₁₄Selected from N, Q, L, T or D; x₁₅Selected from G, A or V; x₁₆Is N or K; x₁₇Is A or T; or

The sequences are respectively shown as SEQ ID NO: 24. LCDR1, LCDR2 and LCDR3 shown at 25, 26;

vi) the sequences are respectively shown in SEQ ID NO: 21. HCDR1, HCDR2 and HCDR3 shown at 22, 23; and/or

The sequence is shown as the general formula RSSQSX₁₃VHSX₁₄X₁₅X₁₆LCDR1 shown by TYLE (SEQ ID NO: 68) having the sequence of SEQ ID NO: 12, the sequence of LCDR2 is shown as the general formula FQGSHVPYX₁₇LCDR3 as shown (SEQ ID NO: 69); wherein, X₁₃Is I or L; x₁₄Selected from N, Q, L, T or D; x₁₅Selected from G, A or V; x₁₆Is N or K; x₁₇Is A or T;

vii) the sequences are respectively shown as SEQ ID NO: 8. HCDR1, HCDR2, HCDR3 shown in fig. 9 and 10; and/or

The sequence is shown as the general formula RSSQSX₁₃VHSX₁₄X₁₅X₁₆LCDR1 shown by TYLE (SEQ ID NO: 68), and wherein X₁₃Is L, X₁₄Selected from N, Q, L, T or D, X₁₅Selected from G, A or V, X₁₆Is N; the sequences are respectively shown as SEQ ID NO: 12. LCDR2, LCDR3 shown at 13;

viii) the sequences are as shown in SEQ ID NO: 8. HCDR1, HCDR2 and HCDR3 shown at 9, 10; and/or

The sequence is shown as SEQ ID NO: 11. 47-52, the sequences are respectively shown in SEQ ID NO: 12. LCDR2, LCDR3 shown at 13;

ix) sequences are respectively shown in SEQ ID NO: 14. HCDR1, HCDR2 and HCDR3 shown at 15, 16; and/or

The sequence is shown as SEQ ID NO: 11. 47-52, the sequences are respectively shown in SEQ ID NO: 12. LCDR2 and LCDR3 shown in FIG. 13.

In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH) and a first light chain variable region (VL), the first VH and first VL comprising a framework region or framework region variant derived from a human antibody, wherein the framework region variant is a back mutation having up to 11 amino acids in the heavy chain framework region and/or light chain framework region, respectively, of a human antibody.

In some specific embodiments, the first VH and the first VL are selected from a) -k) as follows:

a) one or more amino acid back-mutations selected from 27Y, 48I, 67T, 69L, 82F and 93T in the first VH, and/or 2G amino acid back-mutation in the first VL;

b) one or more amino acid back-mutations selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T in the first VH, and/or a 2V amino acid back-mutation in the first VL;

c) the first VH comprises 1K and/or 94S amino acid back mutations, and/or the first VL comprises one or more amino acid back mutations selected from 42G, 44V and 71Y.

d) The sequence is shown as SEQ ID NO:4 and/or the sequence of the first VH shown in SEQ ID NO: 5;

e) the sequence is shown as SEQ ID NO: 6 and/or the sequence of the first VH shown in SEQ ID NO: 7;

f) the sequence is shown as SEQ ID NO:19 and/or the sequence of the first VH shown in SEQ ID NO: a first VL shown at 20;

h) the sequence is shown as SEQ ID NO: 27. 30, 31 or 32 and/or a first VH having a sequence as set forth in SEQ ID NO: 28. 29, 34, 35, 53-64;

i) the sequence is shown as SEQ ID NO: 33. 36-40 and/or the sequence of the first VH as shown in one of SEQ ID NOs: 34. 35, 28, 29, 53-64;

j) the sequence is shown as SEQ ID NO: 41. 45 or 46 and/or the first VH and/or the sequence shown in SEQ ID NO: 42. 43 or 44; and

k) the sequence is shown as SEQ ID NO:70 and/or the sequence is shown in SEQ ID NO: 71, respectively.

In some embodiments, the first antigen binding domain comprises:

(s) a first scFv comprising a first VH and a first VL covalently linked via a first peptide linker;

(t) a first scFv-hinge region-CH 2-CH3, wherein the first scFv comprises a first VH and a first VL covalently linked via a first peptide linker;

(u) a first heavy chain and a first light chain, wherein the first heavy chain comprises the first VH and a first heavy chain constant region comprising a CH 1-hinge region-CH 2-CH 3; (e.g., the sequences shown in SEQ ID NO: 72 or 79); wherein the second light chain comprises a second VL and a second light chain constant region, wherein the second light chain constant region is derived from a human antibody kappa chain constant region, lambda chain constant region, and conventional variants thereof (e.g., the sequence set forth in SEQ ID NO: 73);

wherein the first peptide linker is GS, GAP, ASGS, G₄S、(G₄S)₂、(G₄S)₃、(G₄S) ₄、(G₄S)₅、(G₄S)₆、YGNGT、(YGNGT)₂、(YGNGT)₃、(YGNGT)₄、(YGNGT₎₅、 (YGNGT₎₆(ii) a For example, the first peptide linker is (G)₄S)₃；

The hinge region-CH 2-CH3 or CH 1-hinge region-CH 2-CH3 may be derived from human IgG1, IgG2, IgG3 or IgG4, for example from human IgG 1; may have mutations, for example, human IgG1 with at least one of L234A, L235A, T366S or T366W, L368A, Y407V and K447A, human IgG1 with L234A, L235A, T366S or T366W, L368A and Y407V mutations, or human IgG4 with at least one of S228P, F234A and L235A mutations.

In some embodiments, the first antigen-binding domain comprises a combination of a first heavy chain and a first light chain selected from any one of (u1) - (u 4):

(u1) the sequence is as shown in SEQ ID NO: 77 or 82; and the sequence is as shown in SEQ ID NO: 78 with a first light chain;

(u2) the sequence is as shown in SEQ ID NO: 74. 76, 80 or 81; and the sequence is as shown in SEQ ID NO: 75 a first light chain; and

(u3) the sequence is as shown in SEQ ID NO: 144. 145 or 146; and the sequence is as shown in SEQ ID NO: 75, or a second light chain.

(u4) is a variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the heavy and/or light chain of (bn) or (bo).

In some embodiments, the second antigen-binding domain specifically binds PD-L1.

In some embodiments, the second antigen binding domain comprises:

HCDR1 having a sequence selected from: NDYWZ₁(SEQ ID NO: 83), SYWMH (SEQ ID NO: 89), DGSAYWS (SEQ ID NO: 95) and Z₁₁Z₁₂WMZ₁₃(SEQ ID NO: 96), wherein Z₁Is N or T, Z₁₁Is S or D, Z₁₂Is Y or K, Z₁₃Is H or M;

HCDR2 having a sequence selected from: YISYTGSTYYNPSLKS (SEQ ID NO: 84), RIZ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀KN (SEQ ID NO: 90) and Z₁₄ISZ₁₅AGSTYZ₁₆TPSLKG (SEQ ID NO: 97), wherein Z₄Selected from T, S, H and G, Z₅Selected from S, N and G, Z₆Selected from S, L and G, Z₇Selected from G, F, L, W and M, Z₈Selected from A, P and T, Z₉Selected from M, V, L and S, Z₁₀Is F or Y, Z₁₄Is F or M, Z₁₅Is R or V, Z₁₆Is N or H;

HCDR3 having a sequence of SGGWLAPFDY (SEQ ID NO: 85) or GGSSYDYFDY (SEQ ID NO: 91); and

LCDR1 with the sequence shown in general formula KSSQSLFYZ₂SNQKZ₃SLA (SEQ ID NO: 86) or RASESVSIHGTHLMH (SEQ ID NO: 92), wherein Z₂Is R or H, Z₃Is N or H;

LCDR2 with the sequence GASTRES (SEQ ID NO: 87) or the general formula Z₁₇ASZ₁₈Z₁₉Z₂₀S (SEQ ID NO: 93), wherein Z₁₇Is V or A, Z₁₈Is Y, K or N, Z₁₉Selected from A, L and V, Z₂₀Selected from E, F, Y and A;

LCDR3, LCDR3 with sequence QQYYGYPYT (SEQ ID NO: 88) or QQSFEDPLT (SEQ ID NO: 94).

In some embodiments, the second antigen binding domain comprises a combination of CDRs selected from any one of (aa) - (aj):

(aa) sequences of the general formula NDYWZ₁(SEQ ID NO: 83) HCDR1 wherein Z₁HCDR2 of sequence YISYTGSTYYNPSLKS (SEQ ID NO: 84), and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85), being N or T; and/or

The sequence is shown as the general formula KSSQSLFYZ₂SNQKZ₃LCDR1 according to SLA (SEQ ID NO: 86), wherein Z₂Is R or H, Z₃LCDR2 of sequence GASTRES (SEQ ID NO: 87) and LCDR3 of sequence QQYYGYPYT (SEQ ID NO: 88) being N or H;

(ab) HCDR1 with sequence SYWMH (SEQ ID NO: 89), the sequence is shown as general formula RIZ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀HCDR2 of KN (SEQ ID NO: 90), wherein Z₄Selected from T, S, H and G, Z₅Selected from S, N and G, Z₆Selected from S, L and G, Z₇Selected from G, F, L, W and M, Z₈Selected from A, P and T, Z₉Selected from M, V, L and S, Z₁₀HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91) and F or Y; and/or

LCDR1 of sequence RASESVSIHGTHLMH (SEQ ID NO: 92) and sequence Z₁₇ASZ₁₈Z₁₉Z₂₀LCDR2 of S (SEQ ID NO: 93), wherein Z₁₇Is V or A, Z₁₈Is Y, K or N, Z₁₉Selected from A, L and V, Z₂₀Selected from E, F and A, and LCDR3 of sequence QQSFEDPLT (SEQ ID NO: 94);

(ac) sequences of the general formula NDYWZ₁(SEQ ID NO: 83) HCDR1 wherein Z₁HCDR2 of sequence YISYTGSTYYNPSLKS (SEQ ID NO: 84), and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85), being N or T; and/or

LCDR1 of sequence RASESVSIHGTHLMH (SEQ ID NO: 92) and sequence Z₁₇ASZ₁₈Z₁₉Z₂₀S(SEQ ID NO：93) LCDR2 of (1), wherein Z₁₇Is V or A, Z₁₈Is Y, K or N, Z₁₉Selected from A, L and V, Z₂₀Selected from E, F and A, and LCDR3 of sequence QQSFEDPLT (SEQ ID NO: 94);

(ad) HCDR1 with sequence SYWMH (SEQ ID NO: 89), the sequence is shown as general formula RIZ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀HCDR2 of KN (SEQ ID NO: 90), wherein Z₄Selected from T, S, H and G, Z₅Selected from S, N and G, Z₆Selected from S, L and G, Z₇Selected from G, F, L, W and M, Z₈Selected from A, P and T, Z₉Selected from M, V, L and S, Z₁₀HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91) and F or Y; and/or

(ae) the sequence is DGSAYWS (SEQ ID NO: 95) or NDYWZ₁(SEQ ID NO: 83) HCDR1 with a sequence of formula Z₁₄ISZ₁₅AGSTYZ₁₆HCDR2 as shown in TPSLKG (SEQ ID NO: 97), and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85), wherein Z₁Is N or T, Z₁₄Is F or M, Z₁₅Is R or V, Z₁₆Is N or H; and/or

(af) sequences of the general formula Z₁₁Z₁₂WMZ₁₃(SEQ ID NO: 96) HCDR1 wherein Z₁₁Is S or D, Z₁₂Is Y or K, Z₁₃Is H or M; the sequence is shown as the general formula RIZ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀HCDR2 of KN (SEQ ID NO: 90), wherein Z₄Selected from T, S, H andG，Z₅selected from S, N and G, Z₆Selected from S, L and G, Z₇Selected from G, F, L, W and M, Z₈Selected from A, P and T, Z₉Selected from M, V, L and S, Z₁₀Is F or Y; the sequence is SEQ ID NO: HCDR3 of 91; and/or

The sequence is SEQ ID NO: 92 LCDR 1; the sequence is shown as the general formula Z₁₇ASZ₁₈Z₁₉Z₂₀LCDR2 as shown by S (SEQ ID NO: 93), wherein Z₁₇Is V or A, Z₁₈Is Y, K or N, Z₁₉Selected from A, L and V, Z₂₀Selected from E, F, Y and A; the sequence is SEQ ID NO: LCDR3 of 94;

(ag) HCDR1 of sequence DGSAYWS (SEQ ID NO: 95), HCDR2 of sequence FISRAGSTYNTPSLKG (SEQ ID NO: 98) or MISVAGSTYHTPSLKG (SEQ ID NO: 99), and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85); and/or

(ah) sequences of the general formula NDYWZ₁(SEQ ID NO: 83) HCDR1 wherein Z₁HCDR2 of sequence FISRAGSTYNTPSLKG (SEQ ID NO: 98) or MISVAGSTYHTPSLKG (SEQ ID NO: 99) and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85) being N or T; and/or

(ai) HCDR1 with sequence SYWMH (SEQ ID NO: 89), and the sequence is shown in SEQ ID NO: 100-106, and HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

LCDR1 with sequence RASESVSIHGTHLMH (SEQ ID NO: 92), having the sequence shown in SEQ ID NO: LCDR2 as shown in any of items 107-109, 161-163 and LCDR3 having sequence QQSFEDPLT (SEQ ID NO: 94);

and

(aj) HCDR1 with sequence DKWMM (SEQ ID NO: 110), sequence as shown in SEQ ID NO: 100-106, and HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

LCDR1 with sequence RASESVSIHGTHLMH (SEQ ID NO: 92), having the sequence shown in SEQ ID NO: LCDR2 as shown in any of items 107-109, 161-163, and LCDR3 with sequence QQSFEDPLT (SEQ ID NO: 94).

SEQ ID NO: the sequences of 100-:

RITPSSGFAMYNEKFKN(SEQ ID NO：100)；

RISPSLGLAVYNEKFKN(SEQ ID NO：101)；

RIHPSLGLPLYNEKFKN(SEQ ID NO：102)；

RIGPNLGWAMYNEKYKN(SEQ ID NO：103)；

RISPSSGMAVYNEKFKN(SEQ ID NO：104)；

RISPGGGFTLYNEKFKN(SEQ ID NO：105)；

RIGPNSGFTSYNEKFKN(SEQ ID NO：106)；

VASYAAS(SEQ ID NO：107)；

AASNLES(SEQ ID NO：108)；

VASNVFS(SEQ ID NO：109)；

AASKLES(SEQ ID NO：142)；

VASNVES(SEQ ID NO：161)；

VASNVWS(SEQ ID NO：162)；

VASNVYS(SEQ ID NO：163)。

in some embodiments, the second antigen binding domain comprises an antigen selected from (ak) - (ap) as follows:

(ak) as NDYWN (SEQ ID NO: 164), SEQ ID NO: 84. SEQ ID NO: 85, and/or a HCDR1, HCDR2, HCDR3 as set forth in KSSQSLFYRSNQKNSLA (SEQ ID NO: 165), SEQ ID NO: 87. SEQ ID NO: LCDR1, LCDR2, LCDR3 shown at 88;

(al) as shown in SEQ ID NO: 89. RIHPNSGGTSYNEKFKN (SEQ ID NO: 166), SEQ ID NO: 91, HCDR1, HCDR2, HCDR3, and/or a nucleic acid sequence as set forth in SEQ ID NO: 92. SEQ ID NO: 108 or 142, SEQ ID NO: LCDR1, LCDR2, LCDR3 shown at 95;

(am) as set forth in SEQ ID NO: 95. 98, 85, HCDR1, HCDR2, HCDR3, or a nucleic acid sequence as set forth in SEQ ID NO: 83. 98, 85, HCDR1, HCDR2, HCDR3, or a nucleic acid sequence as set forth in SEQ ID NO: 83. HCDR1, HCDR2, HCDR3 shown at 99, 85; and/or as shown in SEQ ID NO: 86. LCDR1, LCDR2, LCDR3 shown at 97, 88;

(an) as set forth in SEQ ID NO: 89. HCDR1, HCDR2, HCDR3 shown at 100, 91; and/or as shown in SEQ ID NO: 92. LCDR1, LCDR2, LCDR3 shown at 107, 94;

(ao) as shown in SEQ ID NO: 89 as shown in SEQ ID NO: 100-106, the HCDR2 shown in one of SEQ ID NOs: HCDR3 shown at 91; and/or

As shown in SEQ ID NO: 92. LCDR1, LCDR2, LCDR3 shown at 108 or 142, 94;

(ap) as set forth in SEQ ID NO: 110. HCDR1, HCDR2, HCDR3 shown at 100, 91; and/or as shown in SEQ ID NO: 92, as shown in SEQ ID NO: 108, 109, 142, 161, 163, an LCDR2 shown in SEQ ID NO: LCDR3 shown at 94.

In some embodiments, the second antigen-binding domain comprises a second heavy chain variable region (VH) and a second light chain variable region (VL), the second VH and second VL comprising a framework region or a framework region variant derived from a human antibody, wherein the framework region variant is a back mutation having up to 10 amino acids in the heavy chain framework region and/or the light chain framework region, respectively, of the human antibody.

In some embodiments, the heavy chain FR sequence of the second heavy chain variable region is derived from the combined sequence of human germline heavy chain IGHV4-30-4 a 01 and hjh2, which comprises the FR1, FR2, FR3 regions of human germline heavy chain IGHV4-30-4 a 01 and the FR4 region of hjh 2; or a combined sequence derived from human germline heavy chain IGHV1-46 a 01 and hjh 6.1.1 comprising the FR1, FR2, FR3 region of human germline heavy chain IGHV1-46 a 01 and the FR4 region of hjh 6.1.1;

the light chain FR sequence of the second light chain variable region is derived from the combined sequences of human germline light chains IGKV4-1 × 01 and hjk4.1, comprising the FR1, FR2, FR3 regions of human germline light chain IGKV4-1 × 01 and FR4 regions of hjk 4.1; alternatively, a combined sequence derived from human germline light chains IGKV7-3 x 01 and hjk2.1 comprising the FR1, FR2, FR3 regions of human germline light chains IGKV7-3 x 01 and the FR4 region of hjk 2.1.

In some embodiments, in the second antigen-binding domain:

(ba) one or more amino acid mutations selected from W47Y, V71R, G27Y, I48M, V67L, F78Y, S30T, Q39K and P49S are comprised in the second VH, for example one or more amino acid mutations selected from W47Y, V71R and P49S are comprised;

(bb) one or more amino acid mutations in the second VH selected from T74K, R72V, M48I, M70L, R38Q, L83F, V68A, V79A, Y91F, T22S and G72E, for example, one or more amino acid back mutations selected from Y91F, T22S and G72E; or a deglycosylation mutation comprising N85E.

In some embodiments, the second antigen-binding domain comprises a second VH and/or a second VL selected from any one of (bc) to (bj):

(bc) is as shown in SEQ ID NO: 111 and/or the sequence of SEQ ID NO: 112, a second VL;

(bd) sequence as shown in SEQ ID NO: 113 and/or the sequence of SEQ ID NO: 114 or 169;

(be) has a sequence shown in SEQ ID NO: 115 and/or a second VH having the sequence set forth in SEQ ID NO: 116, a second VL;

(bf) has the sequence shown in SEQ ID NO: 117 and/or the sequence shown in SEQ ID NO: 118 or 170;

(bg) the sequence is as shown in SEQ ID NO: 119. 123, 124, 128, 129, 130 and/or a second VH having a sequence as set forth in one of SEQ ID NOs: a second VL, shown at 120;

(bh) has the sequence shown in SEQ ID NO: 121 and/or a second VH having the sequence set forth in SEQ ID NO: a second VL shown as 122 or 171;

(bi) has a sequence shown in SEQ ID NO: 126 and/or SEQ ID NO: 127, a second VL;

(bj) the sequence is as shown in SEQ ID NO: 131. 132-140 and/or the second VH and/or SEQ ID NO: 141 and one of 143, 125, 167, 168.

In some embodiments, the second antigen binding domain comprises:

(bk) a second scFv comprising a second VH and a second VL covalently linked via a first peptide linker;

(bl) a second scFv-hinge region-CH 2-CH3, wherein the second scFv comprises a second VH and a second VL covalently linked via a first peptide linker;

(bm) a second heavy chain and a second light chain, wherein the second heavy chain comprises the second VH and a second heavy chain constant region comprising CH 1-hinge region-CH 2-CH3 (e.g., the sequence shown in SEQ ID NOs: 72 or 79); wherein the second light chain comprises a second VL and a second light chain constant region, wherein the second light chain constant region is derived from a human antibody kappa chain constant region, lambda chain constant region, and conventional variants thereof (e.g., the sequence set forth in SEQ ID NO: 73);

In some embodiments, the second antigen binding domain comprises a combination of a second heavy chain and a second light chain selected from any one of (bn) - (bp):

(bn) the sequence is as shown in SEQ ID NO: 147 and a second heavy chain as set forth in SEQ ID NO: 148 a second light chain;

(bo) is as shown in SEQ ID NO: 149 or 151 and a second heavy chain as set forth in one of SEQ ID NOs: 150 or 172 from a second light chain;

(bp) is a variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the heavy and/or light chain of (bn) or (bo).

In some embodiments, the antigen binding molecule comprises a combination of polypeptides selected from any one of (aaa) - (iii):

(aaa) a first polypeptide comprising a first heavy chain, a second scFv, and optionally a second peptide linker covalently linking the first heavy chain to the second scFv; and

a second polypeptide comprising a first light chain;

wherein the second scFv is linked to the N-terminus or C-terminus of the first heavy chain;

(bbb) a first polypeptide comprising a second heavy chain, a first scFv, and optionally a second peptide linker covalently linking the second heavy chain to the first scFv; and

a second polypeptide comprising a second light chain;

wherein the first scFv is linked to the N-terminus or C-terminus of the second heavy chain;

(ccc) a first polypeptide comprising a first heavy chain variable region, CH1, an optional second peptide linker, and a second heavy chain connected in that order from N-terminus to C-terminus; and

a second polypeptide comprising a first light chain, optionally a second peptide linker, and a second light chain connected in sequence from N-terminus to C-terminus;

(ddd) a first polypeptide comprising, connected in order from N-terminus to C-terminus, a second heavy chain variable region, CH1, an optional second peptide linker, and a first heavy chain; and

a second polypeptide comprising a second light chain, optionally a second peptide linker, and a first light chain connected in sequence from N-terminus to C-terminus;

(eee) a first polypeptide comprising a first heavy chain;

a second polypeptide comprising a second scFv-hinge region-CH 2-CH 3; and

a third polypeptide comprising a first light chain;

wherein the hinge region-CH 2-CH3 may be derived from human IgG1 with mutations introduced in L234A, L235A, and T366W;

(fff) a first polypeptide comprising a second heavy chain;

a second polypeptide comprising a first scFv-hinge region-CH 2-CH 3; and

a third polypeptide comprising a second light chain;

wherein the hinge region-CH 2-CH3 may be derived from human IgG1 introduced with L234A, L235A and T366W mutations;

(ggg) a first polypeptide comprising a first scFv-hinge region-CH 2-CH 3; and

a second polypeptide comprising a second scFv-hinge region-CH 2-CH 3;

(hhh) a first polypeptide comprising, connected in order from N-terminus to C-terminus, a first heavy chain variable region, CH1, a hinge region, CH2, CH3, optionally a second peptide linker, and a second heavy chain variable region;

a second polypeptide comprising a first light chain; and

a third polypeptide comprising a second light chain;

and

(iii) a first polypeptide comprising a second heavy chain variable region, CH1, a hinge region, CH2, CH3, an optional second peptide linker, and a first heavy chain variable region connected in that order from N-terminus to C-terminus;

a second polypeptide comprising a first light chain; and

a third polypeptide comprising a second light chain;

wherein the second peptide linker may be a GS linker, for example, a linker having the following amino acid sequence: GGGGSGGGGSGGGGSGGGGS.

In some embodiments, the antigen binding molecule comprises a combination of polypeptides selected from any one of (jjj) - (mmm):

(jjj) as set forth in SEQ ID NO: 152. 157, 158, 159, 160; and as shown in SEQ ID NO: 75 or a second polypeptide;

(kkk) as shown in SEQ ID NO: 153; as shown in SEQ ID NO: 75 or a second polypeptide; and as shown in SEQ ID NO: 154;

(lll) as shown in SEQ ID NO: 155; as shown in SEQ ID NO: 145; and as shown in SEQ ID NO: 156;

(mmm) is a variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the first polypeptide, the second polypeptide and/or the third polypeptide in (jjj), (kk), (lll).

In an alternative embodiment, the antigen binding molecule is an anti-PD-1/PD-L1 bispecific antibody.

In an alternative embodiment, the first antigen-binding domain is an anti-PD-1 antibody and the second antigen-binding domain is an anti-PD-L1 antibody.

In an alternative embodiment, the anti-PD-1/PD-L1 bispecific antibody, anti-PD-1 antibody, and/or anti-PD-L1 antibody is a murine, chimeric, or humanized antibody.

The present disclosure also provides a pharmaceutical composition comprising the above antigen binding molecule and at least one pharmaceutically acceptable carrier, diluent, buffer or excipient. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent, radiation, and/or other agent for cancer immunotherapy.

The present disclosure also provides a pharmaceutical composition comprising a first antigen-binding domain as defined above and a second antigen-binding domain as defined above. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent, radiation, and/or other agent for cancer immunotherapy.

The present disclosure also provides an intermediate product of a recombinant DNA technique, which is:

(1) a nucleic acid composition comprising at least one nucleic acid molecule encoding an antigen binding molecule according to the present disclosure;

(2) an expression vector composition comprising at least one expression vector comprising at least one nucleic acid molecule as defined in (1) above; or

(3) A host cell comprising the vector composition according to (2) above, or transformed or transfected with the vector composition according to (2) above; in some embodiments, the host cell is selected from the group consisting of bacteria such as enterobacter, yeast such as pichia, insect cells, and mammalian cells such as CHO cells, NSO cells, HEK293 cells, or COS cells.

The present disclosure also provides a method of making the aforementioned antigen binding molecule, comprising:

culturing a host cell as defined above under conditions such that the antigen binding molecule is expressed; and

recovering the antigen binding molecule.

The present disclosure also provides a kit comprising the above antigen binding molecule or pharmaceutical composition.

The present disclosure also provides a method for diagnosing a PD-1 and/or PD-L1-associated disease, comprising the step of using the above antigen binding molecule, pharmaceutical composition or kit.

The present disclosure also provides the use of the above antigen binding molecule, pharmaceutical composition or kit in the preparation of a reagent for diagnosing a disease associated with PD-1 and/or PD-L1.

Embodiments of the present disclosure also provide a method of treating and/or preventing a disease associated with PD-1 and/or PD-L1, the method comprising administering to a subject in need thereof an effective amount of the above-described antigen binding molecule, the above-described pharmaceutical composition, the nucleic acid molecule or the expression vector as defined above; embodiments of the present disclosure also provide the use of the above-described antigen binding molecule, the above-described pharmaceutical composition, the nucleic acid molecule as defined above or the expression vector for the preparation of a medicament for the treatment and/or prevention of a disease associated with PD-1 and/or PD-L1; embodiments of the present disclosure also provide the above antigen binding molecules, the above pharmaceutical compositions, the nucleic acid molecules or expression vectors as defined above for use as a medicament; embodiments of the present disclosure also provide the above antigen binding molecules, the above pharmaceutical compositions, the nucleic acid molecules or expression vectors as defined above for use in the treatment and/or prevention of a disease associated with PD-1 and/or PD-L1. In some embodiments, the disease may be a tumor or cancer; in some embodiments, the disease 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, myeloproliferative tumors, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some specific embodiments, 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 more specific embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, renal cancer, bladder cancer, and intestinal cancer and colon cancer.

Drawings

FIG. 1: anti-PD-1 antibodies block the results of the binding assay of PD-1 to its ligand.

FIG. 2: effect of anti-PD-1 antibodies on IFN γ secretion by PBMC cells.

FIG. 3: therapeutic efficacy of anti-PD-1 antibodies against mouse colon carcinoma MC38 transplantable tumors.

FIG. 4: effect of anti-PD-1 antibody on mouse colon cancer MC38 tumor volume.

FIG. 5: FIG. 5A is a schematic structural diagram of an anti-PD-1/PD-L1 bispecific antibody TJ021-PR 0001; FIG. 5B is a schematic structural diagram of an anti-PD-1/PD-L1 bispecific antibody TJ021-PR 0002; FIG. 5C is a schematic structural diagram of an anti-PD-1/PD-L1 bispecific antibody TJ021-PR 0003; FIG. 5D is a schematic structural diagram of an anti-PD-1/PD-L1 bispecific antibody TJ021-PR 0004.

FIG. 6: SDS-PAGE electrophorograms of anti-PD-1/PD-L1 bispecific antibodies TJ021-PR0001, TJ021-PR 0004.

FIG. 7A: binding of an anti-PD-1/PD-L1 bispecific antibody to a PD-1 protein; FIG. 7B: binding of an anti-PD-1/PD-L1 bispecific antibody to PD-L1 protein.

FIG. 8A: determining the affinity of the anti-PD-1/PD-L1 bispecific antibody TJ021-PR0001 and PD-1 protein; FIG. 8B: determination of the affinity of the anti-PD-1/PD-L1 bispecific antibody TJ021-PR0001 to PD-L1 protein.

FIG. 9A: cell bridging experiments with anti-PD-1/PD-L1 bispecific antibodies; FIG. 9B: cell bridging experiments against the PD-1/PD-L1 bispecific antibody.

FIG. 10A: the anti-PD-1/PD-L1 bispecific antibody blocks a PD-1/PD-L1 signal channel experiment; FIG. 10B: the anti-PD-1/PD-L1 bispecific antibody blocks a PD-1/PD-L1 signal pathway experiment.

FIG. 11A: t lymphocyte activation assay (SEB); FIG. 11B: t lymphocyte activation assay (SEB).

FIG. 12A: mixed lymphocyte reaction experiments (MLR); FIG. 12B: mixed lymphocyte reaction experiments (MLR).

Detailed Description

In order that the disclosure may be more readily understood, some technical and scientific terms are specifically defined below. Unless otherwise defined explicitly 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 factor 1", "programmed death 1", "protein PD-1", "PDCD 1", and "hPD-1" are used interchangeably and include variants, isoforms, species homologs of human PD-1, 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 homologues of human PD-L1(hPD-L1), hPD-L1, and 5 analogues 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-gamma, IL-6, TNF alpha, 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 "antibody" as described in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in amino acid composition and arrangement, and therefore, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as immunoglobulin isotypes, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

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 light chain variable region (VL) and heavy chain variable region (VH) consists of 4 FRs of 3 CDR regions, in order from N-terminus to C-terminus: 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.

The heavy chain constant region (CH) of the present disclosure is a CH 1-hinge region-CH 2-CH3 or, for some heavy chain classes, CH 1-hinge region-CH 2-CH3-CH4, connected sequentially from amino terminus to carboxy terminus. The "hinge region" connects the CH1 domain to CH2-CH3 of the Fc portion.

In the present disclosure, Fc typically includes at least CH 2. For example, the Fc may comprise the entire immunoglobulin heavy chain constant region (CH 1-hinge region-CH 2-CH 3). Alternatively, the Fc may comprise all or part of the hinge region, CH2 domain, and CH3 domain. The constant region of an immunoglobulin is responsible for many important antibody effector functions, including those mediated by Fc receptor (FcR) binding and by complement binding.

"hinge region-CH 2-CH 3" of the present disclosure denotes a hinge region-CH 2-CH3 connected in sequence from the N-terminus to the C-terminus; "scFv-hinge region-CH 2-CH 3" each denote an scFv-hinge region-CH 2-CH3 connected in succession from the N-terminus to the C-terminus;

antibodies of the present disclosure include murine, chimeric, humanized and fully human antibodies, which may be humanized, for example.

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 specific embodiment of the present disclosure, the murine anti-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 one embodiment of the disclosure, the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the PD-1 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, e.g., comprises a human IgG1, IgG2, or IgG4 heavy chain constant region, or an IgG1, IgG2, or IgG4 variant using amino acid mutations (e.g., L234A and/or L235A mutations, and/or S228P mutations).

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), 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 obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of genes such as the human heavy and light chain variable regions can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/VBase), as well as in Kabat, E.A. et al, 1991Sequences 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 after further affinity maturation mutagenesis of the CDRs by yeast display.

Because of the contacting residues of the antigen, grafting of the CDRs can result in a reduction in the affinity of the resulting antibody or antigen-binding fragment thereof for the antigen due to the framework residues that are contacted 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 antibody or antigen-binding fragment thereof that are involved in antigen binding can be identified by examining the sequence and structure of the variable region of an animal monoclonal antibody. Various 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 subclass consensus sequence or a consensus sequence of animal antibody 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.

In one embodiment of the present disclosure, the antibody or antigen binding fragment thereof may further comprise a light chain constant region of a kappa, lambda chain of human or murine origin or a variant thereof, or further comprise a heavy chain constant region of IgG1, IgG2, IgG3, IgG4 or a variant thereof of human or murine origin; for example comprising a human IgG1, IgG2, or IgG4 heavy chain constant region, or an IgG1, IgG2, or IgG4 variant using amino acid mutations (e.g., L234A and/or L235A mutations, and/or S228P mutations).

The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region as described in the present disclosure refer to variants of the heavy chain constant region or light chain constant region of human origin that have been disclosed in the prior art without altering the structure and function of the antibody variable region, and exemplary variants include variants of the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 heavy chain constant region with site-directed modification and amino acid substitution, specifically substitution of YTE mutations as known in the prior art, L234A and/or L235A mutations, S228P mutations, and/or mutations that achieve a knob-in-hole structure (such that the antibody heavy chain has a knob-Fc and hole-Fc combination), which have been confirmed to give the antibody new properties without altering the function of the antibody variable region.

"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 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 such 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. The human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by gene or chromosome transfection methods as well as phage display techniques, or by in vitro activated B cells, all of which are known in the art.

The terms "full-length antibody," "intact antibody," "complete antibody," and "whole antibody" are used interchangeably herein to refer to a substantially intact form of an antibody, as distinguished from antigen-binding fragments defined below. The term particularly refers to heavy chain antibodies comprising an Fc region.

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) which consist of a VH domain; and (vi) separation(viii) 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 they can be generated as 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) Science 242: 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 functionality 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 may be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

Antigen binding fragments of the present disclosure include Fab, F (ab ')2, Fab', single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), 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.

For example, a Fab of the present disclosure may be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human PD-1 or PD-L1 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')₂By digesting the lower part of the two disulfide bonds in the IgG hinge region with the enzyme pepsinThe resulting antibody fragment has a molecular weight of about 100,000 and has antigen binding activity and comprises two Fab regions linked at a hinge position.

For example, F (ab')₂The monoclonal antibody of the present disclosure that specifically recognizes human PD-1 or PD-L1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure can be produced by treating with pepsin. Further, the F (ab ') can be produced by linking Fab' described below with a thioether bond or a disulfide bond₂。

Fab 'is obtained by cleaving the above-mentioned F (ab')₂Has a molecular weight of about 50,000 and has an antigen binding activity. The Fab 'of the present disclosure may be produced by treating the F (ab')2 of the present disclosure, which specifically recognizes PD-1 and is bound 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 eukaryotic expression vector and introducing the vector into a prokaryote or 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 this disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-: 94-106, Hu et al (1996), Cancer Res.56:3055-3061, Kipriyanov et al (1999), J.mol.biol.293: 41-56 and Rovers et al (2001), Cancer Immunol.

For example, the scFv of the present disclosure can be produced by: obtaining the encoding cdnas of VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 or PD-L1 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 an scFv 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.

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 or PD-L1 and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof, constructing DNA encoding scFv such 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)).

For example, the dsFv of the present disclosure can be produced by: obtaining cDNA encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 or PD-L1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing DNA encoding dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express dsFv.

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

For example, the peptides of the present disclosure comprising CDRs can 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 or PD-L1 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 term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide as compared to the original protein or polypeptide, including insertions, deletions or substitutions of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

The term "antibody framework" or "FR" refers 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 "complementarity determining region", "CDR" or "hypervariable region" refers 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: 927-948) and Munogenetics IMGT (Lefranc M.P., Immunogest, 7, 132-136 (1999); Leanc M.P., Dev.Comp., 27, 55-77, et Al, for example, for the Kabat numbering convention, the amino acid sequence boundaries in the variable domain (VH) are amino acid residues 31-35 (VH 35), the variable domain (VH 35-34) are amino acid residues 31-34 (VH-35), the variable domain (VH-11) are amino acid residues 102-34 (HCDR 35) and the variable domain (HCDR 3-34) are amino acid residues 35(HCDR 35) (HCDR 3-11) and the variable domain (HCDR 6335) (HCDR3) are amino acid residues 35) (HCDR 3-11) for the Kabat numbering convention, 50-56(LCDR2) and 89-97(LCDR 3). CDR amino acid numbers in VH were 26-32(HCDR1), 52-56(HCDR2) and 95-102(HCDR3) following the Chothia rule; 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 CDRs of an antibody can be determined using the program IMGT/DomainGap Align.

The "PD-1/PD-L1 bispecific antibody", "anti-PD-1/PD-L1 bispecific antibody", "anti-PD-1/anti-PD-L1 antibody", "bispecific PD-1/PD-L1 antibody" are used interchangeably in this disclosure. The anti-PD-1/PD-L1 bispecific antibodies provided by the present disclosure specifically bind to human PD-1 and human PD-L1. The present disclosure also provides an antigen binding molecule comprising 12 CDRs, wherein 6 CDRs specifically bind to PD-1 and 6 CDRs specifically bind to PD-L1.

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 "linker", "linker" and "linker" refer to a peptide linker used in the context of an scFv, bispecific antibody, or bispecific antigen binding molecule as described herein. The peptide linker should be of sufficient length to link the two molecules to each other in the correct configuration to maintain the desired activity. In one embodiment, the linker is about 1 to about 50 amino acids in length, more specifically, about 1 to about 30 amino acids, and for example, may be 1 to 20 amino acids. Useful linkers include glycine-serine polymers such as (GS) n, (GSGGS) n, (GGGGS) n, and (GGGS) n, where n is an integer of at least 1, typically 3 to 4; glycine-alanine polymer; alanine-serine polymers; and other flexible joints.

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to 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 Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, methods in Enzymology 9: 242-; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol.137: 3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA with a solid phase of I-125 marker (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 ADCC, namely, the antibody-dependent cell-mediated cytotoxicity, of the present disclosure means that cells expressing Fc receptors directly kill target cells coated with antibodies by recognizing the Fc fragment of the antibodies. The ADCC effector function of an antibody may be reduced or eliminated by modification of the Fc-fragment of the IgG. The modification refers to a mutation in the heavy chain constant region of an antibody, such as N297A, L234A, L235A selected from IgG 1; IgG2/4chimera, F235E of IgG4, or L234A/E235A mutations.

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, and may be, for example, double-stranded DNA or single-stranded mRNA or modified mRNA. 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.

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 Experimental technical guide, chapters 5-8 and 15. For example, a mouse can 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 antibodies or antigen-binding fragments are disclosed as being genetically engineered to incorporate one or more FRs of human origin in a CDR 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. Easily transformed bacteria include members of the family Enterobacteriaceae (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 expressing antibodies that bind specifically to human PD-1, or antibodies that bind to both PD-1 and PD-L1. 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 product obtained is immediately frozen, e.g. -70 ℃, or lyophilized.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "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" and "treating" also mean treating, for example, 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 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 each of the symptoms of the 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 altering 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 of The 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".

TABLE 3 exemplary amino acid conservative substitutions

An "effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. 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.

"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.

In the present disclosure, the terms "homology" and "identity" are used interchangeably to refer 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. Typically, a comparison is made when aligning two sequences to give the maximum percent homology. For example, the comparison may be performed by a 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: 131-141; altschul, s.f. et al, (1997) Nucleic Acids res.25: 3389 and 3402; zhang, j, et al, (1997) Genome res.7: 649-656. 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 Harbor Symp. 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 that 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 mean 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 active ingredients and further 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., an 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.

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

The disease associated with PD-1 in the present disclosure is not limited 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 for those who have tumors or cancers, such as melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, non-small cell lung cancer, bladder cancer, and the like, when in preparations and formulations suitable for therapeutic use.

Further, the present disclosure relates to a method for immunodetection or assay of a target antigen (e.g., PD-1), a reagent for immunodetection or assay of a target antigen (e.g., PD-1), a method for immunodetection or assay of a cell expressing a target antigen (e.g., PD-1), and a diagnostic agent for diagnosing a disease associated with a target antigen (e.g., PD-1) -positive cell, which comprise, as an active ingredient, an antibody or an antibody fragment of the present disclosure that specifically recognizes a target antigen (e.g., 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 a target antigen (e.g., 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 protein 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 PD-1 expressing cells with the antibodies or antibody fragments of the present disclosure.

For detecting cells expressing the polypeptide, known immunoassay methods, for example, immunoprecipitation, fluorescent cell staining, immunohistological staining, and the like, can be 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 a target antigen (e.g., PD-1) is not particularly limited as long as it has a possibility of containing cells expressing the target antigen (e.g., PD-1), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may 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 an immunoassay or measuring method, 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.

Examples

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 technology 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 anti-human PD-1 murine antibody acquisition

Preparation of antigens

1. PD-1 antigen construction and screening:

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 the Fc segment of human IgG1 (hIgG1 Fc). 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-IgG1Fc (SEQ ID NO: 1):

note that: the underlined part is the signal peptide, the positive part is the extracellular domain of human PD-1, and the italic part is hIgG1Fc (signal peptide + extracellular domain + hIgG1 Fc).

Human PD-1-his (SEQ ID NO: 2):

MEFGLSWLFLVAILKGVQCPGWFLDSPDRPWNPPTFSPALLVVTEGDNAT FTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRD FHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPS PRPAGQFQTLVGSSDYKDDDDKHHHHHH。

PD-1 antigen encoded by transfected cell nucleic acid (SEQ ID NO: 3):

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNG RDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPS PSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLK EDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPAR RGSADGPRSAQPLRPEDGHCSWPL。

preparation of anti-human PD-1 antibody

The anti-human PD-1 antibody can be produced by immunizing a mouse, and can also be obtained by immunizing a library with an anti-human PD-1 phage mouse.

The method for preparing the anti-human PD-1 antibody by immunizing a mouse is as follows:

1. immunization: the experimental study was performed on SJL white mice, female, 6-8 weeks old and Balb/c white mice, female, 6-8 weeks old. A breeding environment: SPF grade. After the mice are purchased, the mice are raised in a laboratory environment for 1 week, and the light/dark period is regulated for 12/12 hours at the temperature of 20-25 ℃; the humidity is 40-60%. Mice adapted to the environment were immunized according to different protocols, 6-10 mice per group. The immunizing antigen may be purified recombinant protein PD-1-IgG1Fc (shown in SEQ ID NO: 1), PD-1-his (shown in SEQ ID NO: 2), or Jurkat/CHO-PD-1 cells can be cross-immunized by a single agent with different immunological adjuvants or different types of immunogens. The immunization part can be abdominal cavity or back subcutaneous, or the two positions are alternatively immunized. Immunologic adjuvant

Gold Adjuvant (hereinafter Titermax, from Sigma Cat. No. T2684) was cross-immunized with Imject Alum Adjuvant (hereinafter Alum, from Pierce Cat. No. 77161). The ratio of antigen to adjuvant (Titermax) is 1:1, the ratio of antigen to adjuvant (Alum) is 3:1, 25-50. mu.g/body (prime), 50. mu.g/body (boost), or 1X 10⁷Jurkat/CHO-PD-1 cells/cell. On day0, intraperitoneal injections of 25-50. mu.g/mouse of emulsified antigen were given, either weekly or biweekly after first immunization, and Titermax and Alum were used alternately for 5-8 times.

2. Cell fusion: selecting mice with high antibody titer in serum for spleen cell fusion, discharging blood from eyeballs of the mice after the thorns are immunized for 72 hours, pulling the necks for killing, and putting the mice into 75% ethanol for disinfection. Splenic lymphocytes were fused with myeloma cells Sp2/0 cells (Chinese academy of sciences) using an optimized PEG-mediated fusion procedure to give hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHAT and 1 XPPI), and distributed in 96-well cell culture plates (1X 10)⁵150. mu.l/well), 37 ℃ 5% CO₂Incubate, about 10-30 plates. HAT complete medium was added at day 5 after fusion, 50. mu.l/well, 37 ℃ C, 5% CO₂And (4) incubating. Day 7 to 8 after fusion, according to cell growth density, total fluid change, 200. mu.l/well, 37 ℃, 5% CO₂And (4) incubating.

3. Screening hybridoma cells: 7-9 days after fusion, performing ELISA method detection of the combination of the antibody and PD-1 according to cell growth density, performing blocking ELISA detection of the combination of PD-1/PDL1 on the detected positive well cells, changing the liquid of the positive well, and expanding the positive well cells into a 24-well plate in time according to cell density. The cell lines transferred into the 24-well plate were retested and first subcloned. And (4) performing seed preservation on the positive subclone selected for the first time, and performing secondary or third subcloning until a single cell clone is obtained. Multiple fusions resulted in hybridoma cells that blocked the binding of PD-1 to PDL 1.

The method for obtaining the anti-human PD-1 antibody through the anti-human PD-1 phage mouse immune library comprises the following steps:

1. constructing an anti-human PD-1 phage mouse immune library: the spleen of a mouse with a high antibody titer in serum was selected, and total RNA from the tissue was extracted using Trizol (Invitrogen Cat No. 15596-018). Using PrimeScript^TMII 1st Strand cDNA Synthesis Kit (Takara Cat No.6210A) was used to obtain cDNA by reverse transcription. Primers for constructing the library were designed and synthesized according to the IMGT database. Through three rounds of PCR reaction, single-chain antibody fragments were obtained. The single-chain antibody fragment and the modified library construction vector pCantab5E (Amersham Biosciences/GE Cat No.27-9400-01) are subjected to enzyme digestion by Sfi1(NEB Cat No. # R0123L), and after electrophoresis, the single-chain antibody fragment is subjected to enzyme digestion by using a kit

The Gel Extraction Kit (Omega Cat No. D2500-02) was purified and recovered. Then, the DNA fragments were ligated with T4 DNA ligase (NEB Cat No. # M0202L) at 16 ℃ for 16 to 18 hours, purified and recovered by the kit, and finally eluted with deionized water. Mu.g of the ligation product was mixed with 1 branch of electrotransformation competent TG1(Lucigen Cat No.60502-2), and the parameters of an electrotransformation apparatus (Bio Rad Micropulser) were set to 2.5kV, 200. omega., 25uF for electrotransformation. The transformation was repeated 10 times, plated and cultured in an inverted medium at 37 ℃ for 16 to 18 hours. All colonies were scraped off and mixed together, glycerol was added to a final concentration of 15%, and stored at-80 ℃ for future use.

2. Screening of anti-human PD-1 phage mouse immune library: packaging anti-human PD-1 phage immune library (1X 10)¹²-1×10¹³) Mu.l of streptavidin beads (Mi1envi Biotec, Auburn, Calif.) were incubated with 1m1 buffer containing 2% skim milk-phosphate (abbreviated MPBS) at room temperature for 1 hour, placed on a magnetic frame, and the supernatant was collected. The supernatant was incubated at room temperature for 1 hour with 10. mu.g/ml biotinylated human PD-1-ECD-his protein (purchased from Nano Biological), and then 100. mu.l streptavidin-coated magnetic beads (1ml MPBS pre-incubation) were added and incubated at room temperature for 1 hourThen (c) is performed. And loaded on a magnetic rack system for sorting, and the supernatant is aspirated. 1ml of PBST (phosphate buffered saline containing 0.1% Tween-20) was added, the inversion was repeated several times, after exhaustion, fresh wash was added, and the process was repeated 11 times to remove unbound antibody fragments, and 0.5ml of eluent (50. mu.l of 10mg/ml tryptsin stock solution in 450. mu.l of PBS) was added. Shaking at room temperature for 15 min. Place on magnetic stand and aspirate supernatant into a new EP tube. TG1 was inoculated into 2YT medium and amplified until the density of cultured bacteria OD600 became 0.4. 1.75ml of TG1(OD600 ═ 0.4) was added to each tube, and 250. mu.l of eluted phase (phage) was added, incubated in a 37 ℃ water bath for 30min, and plates were diluted in a gradient to test titer. The remaining TG1 solution was centrifuged, plated and incubated overnight at 37 ℃.

The phage mouse immune library utilizes biotinylated human PD-1-ECD-his antigen, and is subjected to 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen), so that monoclonal with binding PD-1 and blocking the binding of PD-1 and PD-L1 is finally obtained, and sequencing verification is carried out to obtain the variable region sequence of the antibody.

Purification of recombinant antigenic proteins/antibodies

1. Hybridoma supernatant isolation purification/ProteinG affinity chromatography:

for purification of mouse hybridoma supernatant, preferably, protein G is subjected to affinity chromatography, cultured hybridoma is centrifuged to obtain supernatant, and 10-15% by volume of 1M Tris-HCl (pH8.0-8.5) is added to adjust the pH of the supernatant according to the volume of the supernatant. Washing the ProteinG column by using 6M guanidine hydrochloride for 3-5 times of column volume, and then washing the ProteinG column by using pure water for 3-5 times of column volume; equilibrating the column for 3-5 column volumes using, for example, 1 XPBS (pH7.4) buffer system as equilibration buffer; the cell supernatant is combined by low flow rate sample loading, and the flow rate is controlled to keep the retention time for about 1min or more; washing the column with 1 × PBS (pH7.4) for 3-5 times of column volume until the UV absorption falls to baseline; eluting with 0.1M acetic acid/sodium acetate (pH3.0) buffer solution, collecting eluate peak according to ultraviolet detection, and rapidly adjusting pH of the eluate product to 5-6 with 1M Tris-HCl (pH8.0) for temporary storage. The eluted product may be subjected to solution displacement by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to a desired buffer system, or may be replaced with a desired buffer system by size exclusion such as G-25 desalting, or may be subjected to removal of the polymer component from the eluted product using a high resolution size exclusion column such as Superdex 200 to increase the purity of the sample.

Protein a affinity chromatography purification of Protein or antibody:

firstly, cell culture supernatant expressing the antigen protein or the antibody is subjected to high-speed centrifugation to collect the supernatant. The ProteinA affinity column was washed 3-5 column volumes with 6M guanidine hydrochloride and then 3-5 column volumes with pure water. The column is equilibrated 3-5 column volumes using, for example, a1 XPBS (pH7.4) buffer system as the equilibration buffer. The cell supernatant was combined by low flow loading, the flow rate was controlled to allow retention time of about 1min or more, and after the combination was completed, the column was washed 3-5 column volumes with 1 × PBS (ph7.4) until the uv absorbance fell back to baseline. Eluting with 0.1M acetic acid/sodium acetate (pH3.0-3.5) buffer solution, collecting eluate peak according to ultraviolet detection, and rapidly adjusting pH of the eluate to 5-6 with 1M Tris-HCl (pH8.0) for temporary storage. The eluted product may be subjected to solution displacement by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to a desired buffer system, or by size exclusion such as desalting with G-25 to replace the desired buffer system, or by removing the polymer component from the eluted product using a high resolution size exclusion column such as Superdex 200 to increase the purity of the sample.

PD-L1 antibody

Methods for the preparation and purification of PD-L1 antigen and anti-PD-L1 antibodies may be conventional methods known in the art, and may be, for example, the methods described in ZL201680027181.4 and WO2019137397a1, which are incorporated by reference in their entirety into the present disclosure. PD-L1 antibodies prepared in ZL201680027181.4 and WO2019137397a1 may also be used in the present disclosure.

The murine anti-variable region sequence of hybridoma clone 9-2 is as follows:

>9-2 mVH: 9-2 murine heavy chain variable region sequence (SEQ ID NO: 111)

>9-2 mVL: 9-2 murine light chain variable region sequence (SEQ ID NO: 112)

Note: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the FR sequences are in italics and the CDR sequences are underlined.

The murine variable region sequence of hybridoma clone 24D5 is as follows:

24D 5-VH: 24D5 murine heavy chain variable region sequence (SEQ ID NO: 113)

24D 5-VL: 24D5 murine light chain variable region sequence (SEQ ID NO: 114)

The humanized light chain templates of the murine antibody 9-2 are IGKV4-1 × 01 and hjk4.1, the humanized heavy chain templates are IGHV4-30-4 × 01 and hjh2, and the humanized variable region sequences are as follows:

>9-2hVH-CDR graft(SEQ ID NO：115)

>9-2hVL CDR graft(SEQ ID NO：116)

The humanized light chain templates of the murine antibody 24D5 are IGKV7-3 x 01 and hjk2.1, the humanized heavy chain templates are IGHV1-46 x 01 and hjh 6.1.1, and the humanized variable region sequences are as follows:

VH.1 humanized heavy chain variable region of >24D5 (SEQ ID NO: 117)

24D5 humanized light chain variable region VL.1(SEQ ID NO: 118)

The variable region sequences of antibodies 9-2 and 24D5 obtained after affinity maturation were as follows:

>9-2hVH(T)(SEQ ID NO：119)

>9-2hVL(H)(SEQ ID NO：120)

>24-D5 hVH(GF)(SEQ ID NO：121)

>24-D5 hVL(SEQ ID NO：122)

note: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the sequence is in italics as the FR sequence; the CDR sequences are underlined, with double underlined sites being sites obtained after affinity maturation screening.

The present disclosure also constructs the nucleic acid sequences as set forth in SEQ ID NO: 169-VL shown in 171:

the sequence of the heavy chain variable region of the anti-PD-L1 antibody 24D 5H 21 provided by the disclosure is shown as SEQ ID NO: 131 shows:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDKWMMWVRQAPGQGLEW MGRITPSSGFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGS SYDYFDYWGQGTTVTVSS(SEQ ID NO：131)

the variable region sequence of the anti-PD-L1 antibody 24D 5L 67 light chain provided by the disclosure is shown as SEQ ID NO: 125, as shown:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI YVASNVESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTK LEIK(SEQ ID NO：125)

the variable region sequence of the anti-PD-L1 antibody 24D 5L 68 light chain provided by the disclosure is shown as SEQ ID NO: 167 of the formula:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI YVASNVWSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGT KLEIK(SEQ ID NO：167)

the variable region sequence of the anti-PD-L1 antibody 24D 5L 69 light chain provided by the disclosure is shown as SEQ ID NO: 168, as shown:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI YVASNVYSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTK LEIK(SEQ ID NO：168)

heavy and light chain variable regions of antibodies from HRP00049 mutation library:

the related heavy chain variable region sequence is obtained by the following general formula:

QVQLQESGPGLVKPSQTLSLTCTVSGGSISDGSAYWSWIRQHPGKGLEYIG ₁₄ ₁₅ ₁₆ZISZAGSTYZTP SLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS(SEQ ID NO：123)

or

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIG ₁₄ ₁₅ ₁₆ZISZAGSTYZTPSL KGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS (SEQ ID NO: 124). Z in the above-mentioned heavy chain variable region₁₄Is F or M, Z₁₅Is R or V, Z₁₆Is N or H.

The related light chain variable region sequence is shown as SEQ ID NO: shown at 120.

Further, the related heavy chain variable region sequence is obtained by the following general formula:

QVQLVQSGAEVKKPGASVKVSCKASGYTFT ₁₁ ₁₂ ₁₃ZZWMZWVRQAPGQGL EWMG ₄ ₅ ₆ ₇ ₈ ₉RIZPZZGZZZ ₁₀YNEKZKNRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARGGSSYDYFDYWGQGTTVTVSS (SEQ ID NO: 126), the above CDRs and Z in the heavy chain variable region₁₁Is S or D, Z₁₂Is Y or K, Z₁₃Is H or M, Z₄Selected from T, S, H and G, Z₅Selected from S, N and G, Z₆Selected from S, L and G, Z₇Selected from F, L, W and M, Z₈Selected from A, P and T, Z₉Selected from M, V, L and S, Z₁₀Is F or Y.

Further, the related light chain variable region sequence is obtained by the following general formula:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI Y ₁₇ ₁₈ ₁₉ ₂₀ZASZZZSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFG QGTKLEIK (SEQ ID NO: 127), wherein Z is₁₇Is V or A, Z₁₈Is Y, K or N, Z₁₉Selected from A, L and V, Z₂₀Selected from E, F, Y and A.

Thereby obtaining the following heavy and light chain variable regions:

9-2H5 heavy chain variable region (SEQ ID NO: 128)

QVQLQESGPGLVKPSQTLSLTCTVSGGSISDGSAYWSWIRQHPGKGLEYIGFISRAGSTYNTPSLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDY WGRGTLVTVSS

9-2H6 heavy chain variable region (SEQ ID NO: 129)

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGFISRAGSTYNTPSLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWG RGTLVTVSS

9-2H7 heavy chain variable region (SEQ ID NO: 130)

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGMISVAGSTYHTPSLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYW GRGTLVTVSS

The variable region of the 9-2L11 light chain is shown as SEQ ID NO: shown at 120.

Heavy and light chain variable regions of antibodies from HRP00052 mutation library:

24D 5H 12 heavy chain variable region (SEQ ID NO: 132)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRITPSSGFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 13 heavy chain variable region (SEQ ID NO: 133)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPSLGLAVYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 14 heavy chain variable region (SEQ ID NO: 134)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIHPSLGLPLYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 15 heavy chain variable region (SEQ ID NO: 135)

QVQLVQSGAEVKKPGASVKVSCKANGYTFTDKWMMWVRQAPGQGLEWMGRITPSSGFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 16 heavy chain variable region (SEQ ID NO: 136)

QVQLVQSGAEVKKPGASMKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPSLGLAVYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 17 heavy chain variable region (SEQ ID NO: 137)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNLGWAMYNEKYKNRVTMTRDTSTSTVYMELSSLGSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 18 heavy chain variable region (SEQ ID NO: 138)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPSSGMAVYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 19 heavy chain variable region (SEQ ID NO: 139)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPGGGFTLYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5H 20 heavy chain variable region (SEQ ID NO: 140)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D 5L 64 light chain variable region (SEQ ID NO: 141)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYVASYAASGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEI K

24D 5L 61 light chain variable region (SEQ ID NO: 122)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

24D 5L 66 light chain variable region (SEQ ID NO: 143)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYVASNVFSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

HRP 00049: 9-2(H2/L10) IgG4(AA) heavy chain: SEQ ID NO: 147

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGYISYTGSTYYNPSLKSRVT ISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK

HRP00049 antibody light chain: SEQ ID NO: 148

DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLAWYQQKPGQPPKLLIYGASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

HRP 00052: 24D5(GF) IGG4(AA) antibody heavy chain: SEQ ID NO: 149

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRV TMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK

HRP00052 antibody light chain sequence SEQ ID NO: 150

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG SGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The present disclosure also constructs the nucleic acid sequences as set forth in SEQ ID NO: 172 of an antibody light chain sequence:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASKLESGVPARFSG SGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the full-length sequence of the constructed PD-L1 antibody in the form of IgG 1is exemplified as follows:

heavy chain: SEQ ID NO: 151

The light chain is shown as SEQ ID NO: 150, respectively.

The anti-human PD-1 murine antibody obtained by the method is subjected to an antigen binding experiment, and 3 clones with good activity are obtained by screening: m23, M32 and M33, and performing cloning, amplification and culture on single cells, extracting RNA, and performing reverse transcription amplification (RT-PCR) by using degenerate primers of mouse-Ig to obtain the variable region sequence of the antibody. Connecting the variable region sequence of the mouse antibody with the constant region sequence of the human antibody, cloning and recombining to express the chimeric antibody of the mouse monoclonal antibody, and carrying out in vitro activity experiments to confirm that the variable region sequence of the obtained monoclonal antibody is correct.

The variable region sequences of murine antibodies M23, M32 and M33 were determined as follows:

heavy chain variable region of murine antibody M23 (SEQ ID NO: 4):

QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPIHGLEWIGLIDPETGGTVYNQKFKDKTILTADKSSSTAYMEFRSLTSEDSAVYHCTRERFSYYGSTSDWYFDVWGTGTTVTVSS。

the variable region in the light chain of murine antibody M23 (SEQ ID NO: 5):

DGLMTQTPLSLPVSLGDHASISCRSSQSLVHSNGNTYLEWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYTFGGG TKLEIK。

heavy chain variable region of murine antibody M32 (SEQ ID NO: 6):

QVQLQQSGAELVRPGASVTLSCKASDFTFTDYEIHWVKQTPVHGLEWIGLFDPETGGIVYNQKFKGKAILTADKSSNTAYMEFRSLTSEDSAVYYCTREGYNRDWYFDVWGTGTTVTVSS。

the variable region in the light chain of murine antibody M32 (SEQ ID NO: 7):

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYAFGGG TKLEIK。

heavy chain variable region of murine antibody M33: (SEQ ID NO: 19)

KVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVA TISGGGVDTYYQDNVQGRFTISRDNAKNTLYLQMSSLRSEDTALYYCASPYGHGYFDVWGTGTTVTVSS。

Light chain variable region of murine antibody M33: (SEQ ID NO: 20)

DIQMTQTTSSLSASLGDRVTISCRASQDINNFLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEI K。

Remarking: in the heavy chain variable region and light chain variable region sequences of the above antibody, the CDR sequences determined by the Kabat numbering system are underlined, and FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are in order.

TABLE 4 murine antibodies M23, M32, and M33 heavy and light chain CDR region sequences

Remarking: the antibody CDR sequences in the tables are determined according to the Kabat numbering system.

Example 2 humanization of anti-human PD-1 monoclonal antibodies

By comparing an IMGT human antibody heavy-light chain variable region germline gene database with MOE software, heavy-light chain variable region germline genes with high light-heavy chain sequence identity with M23, M32 and M33 are respectively selected as templates, CDRs of the 3 murine antibodies are respectively transplanted into corresponding human antibody templates, and corresponding humanized antibodies are respectively constructed.

1. Humanization of murine antibody M23

1.1 murine antibody M23 humanized framework selection

The humanized light chain templates of murine antibody M23 were IGKV2-40 × 01 and IGKJ4 × 01, and the humanized heavy chain templates were IGHV1-69 × 02 and IGHJ6 × 01, and the variable region sequences after humanization were as follows (underlined as CDR sequences):

hu23VH-CDR grafting (grafted): (SEQ ID NO: 27)

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWM GLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSS。

Hu23VL-CDR grafting: (SEQ ID NO: 28)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLEWYLQKPGQSPQLLI YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGT KVEIK。

1.2 humanized template selection and Back-mutation design of murine antibody M23

TABLE 5 humanized template selection and Back-mutation design of murine antibody M23

Note: grafting (Grafted) represents the implantation of murine antibody CDRs into human germline FR sequences with amino acid residues determined and annotated by the Kabat numbering system, as I2G represents the mutation of position 2I of Kabat numbering back to G according to the Kabat numbering system.

The humanized antibody light/heavy chain variable region sequence of M23 is as follows:

hu23VL1 (grafted with Hu23 VL-CDR): (SEQ ID NO: 28)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGG TKVEIK。

>Hu23VL2(SEQ ID NO：29)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGG GTKVEIK。

Hu23VH1 (grafted with Hu23 VH-CDR): (SEQ ID NO: 27)

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWM GLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFS YYGSTSDWYFDVWGQGTTVTVSS。

>Hu23VH2(SEQ ID NO：30)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWM GLIDPETGGTVYNQKFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARERFS YYGSTSDWYFDVWGQGTTVTVSS。

>Hu23VH3(SEQ ID NO：31)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWIG LIDPETGGTVYNQKFKDRTTLTADKSTSTAYMEFSSLRSEDTAVYYCARERFSY YGSTSDWYFDVWGQGTTVTVSS。

>Hu23VH4(SEQ ID NO：32)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWIG LIDPETGGTVYNQKFKDRTTLTADKSTSTAYMEFSSLRSEDTAVYYCTRERFSY YGSTSDWYFDVWGQGTTVTVSS。

1.3 humanized sequence combinations of murine antibody M23

The humanized template of murine antibody M23 was designed for back mutations, and different variable region back mutations were combined into different humanized antibody variable regions, as shown in the following Table.

TABLE 6 humanized antibody variable region combinations of Hu23

Remarking: in the tables, for example, "Hu 23-1" refers to an antibody light/heavy chain variable region combination having an antibody light chain variable region of Hu23VL1 and a heavy chain variable region of Hu23VH1, and so on.

The antibody light/heavy chain variable region combinations referred to in the above table (e.g., Hu23-1) can be linked to the antibody light/heavy chain constant regions, respectively, to form full-length antibodies; as not explicitly stated in this disclosure, the light chain variable region when forming a full-length antibody is identical to SEQ ID NO: 73 to form an antibody light chain, and a heavy chain variable region linked to the Kappa chain constant region of SEQ ID NO: 72 or the constant region of the heavy chain of IgG4-AA shown in SEQ ID NO: 79 to form an antibody heavy chain, and are referred to in the table as the designation antibody light/heavy chain variable region combination (e.g., Hu23-1) followed byThe suffix ". IgG4 AA" indicates the full length antibody formed by the linkage to the IgG4-AA heavy chain constant region, and the suffix ". IgG 4P" indicates the full length antibody formed by the linkage to the IgG4-P heavy chain constant region, e.g., "Hu 23-1.IgG4 AA" indicates the full length antibody formed by the linkage to the IgG4-P heavy chain constant regionHu23VH1A heavy chain variable region and a heavy chain variable region as set forth in SEQ ID NO: 72 and an IgG4-AA heavy chain constant region linked theretoHu23VL1A light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73 (the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region), and "Hu 23-1.IgG 4P" is represented byHu23VH1A heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 79 IgG4-P heavy chain constant region andHu23VL1a light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73 to the constant region of the Kappa chain (in which the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region).

2. Humanization of murine antibody M32

2.1 murine antibody M32 humanized framework selection

The humanized light chain templates of murine antibody M32 were IGKV2-40 × 01 and IGKJ4 × 01, the humanized heavy chain templates were IGHV1-69 × 02 and IGHJ6 × 01, and the humanized variable region sequences were as follows (underlined CDR sequences):

hu32VH-CDR grafting: (SEQ ID NO: 33) IGHV1-69 x 02 and IGHJ6 x 01

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEIHWVRQAPGQGLEWMG LFDPETGGIVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGYNRDWYFDVWGQGTTVTVSS。

Hu32VL-CDR grafting: (SEQ ID NO: 34)

DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLI YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGGT KVEIK。

2.2 humanized template selection and Back-mutation design of murine antibody M32

TABLE 7 humanized template selection and Back-mutation design of murine antibody M32

Note: grafting (Grafted) represents the grafting of murine antibody CDRs into human germline FR sequences. Amino acid residues are determined and annotated by the Kabat numbering system, as I2V indicates that position 2I of Kabat numbering is mutated back to V according to the Kabat numbering system.

The sequence of the humanized antibody light-heavy chain variable region of the murine antibody M32 is as follows:

hu32VL1 (grafted with Hu32 VL-CDR): (SEQ ID NO: 34)

>Hu32VL2(SEQ ID NO：35)

DVVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGG TKVEIK。

Hu32VH1 (grafted with Hu32 VH-CDR): (SEQ ID NO: 33)

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEIHWVRQAPGQGLEWMG LFDPETGGIVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGYNR DWYFDVWGQGTTVTVSS。

>Hu32VH2(SEQ ID NO：36)

EVQLVQSGAEVKKPGSSVKVSCKASGFTFSDYEIHWVRQAPGQGLEWMG LFDPETGGIVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCTREGYN RDWYFDVWGQGTTVTVSS。

>Hu32VH3(SEQ ID NO：37)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFSDYEIHWVRQAPGQGLEWMG LFDPETGGIVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCTREGYN RDWYFDVWGQGTTVTVSS。

>Hu32VH4(SEQ ID NO：38)

EVQLVQSGAEVKKPGSSVKVSCKASGFTFSDYEIHWVRQAPGQGLEWIGL FDPETGGIVYNQKFKGRATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNR DWYFDVWGQGTTVTVSS。

>Hu32VH5(SEQ ID NO：39)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVRQAPGQGLEWIGL FDPETGGIVYNQKFKGRATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNR DWYFDVWGQGTTVTVSS。

>Hu32VH6(SEQ ID NO：40)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGL FDPETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNR DWYFDVWGQGTTVTVSS。

2.3 humanized sequence combinations of murine antibody M32

The humanized template of murine antibody M32 was designed for back mutations, and different back mutations were combined into different humanized antibodies, as shown in the following Table.

TABLE 8 humanized antibody light/heavy chain variable region combinations

Remarking: in the tables, for example, "Hu 32-1" refers to an antibody light/heavy chain variable region combination having an antibody light chain variable region of Hu32VL1 and a heavy chain variable region of Hu32VH1, and so on.

The antibody light/heavy chain variable region combinations referred to in the above table (e.g., Hu32-1) can be linked to the antibody light/heavy chain constant regions, respectively, to form full-length antibodies; as not explicitly stated in this disclosure, the light chain variable region when forming a full-length antibody is identical to SEQ ID NO: 73 to form an antibody light chain, and a heavy chain variable region linked to the Kappa chain constant region of SEQ ID NO: 72 or the constant region of the heavy chain of IgG4-AA shown in SEQ ID NO: 79 to form an antibody heavy chain, and the full length antibody formed by association with the IgG4-AA heavy chain constant region is indicated by the designation indicating the light/heavy chain variable region combination of the antibody in the table (e.g., Hu32-1) suffixed ". IgG4 AA" to form a full length antibody formed by association with the IgG4-AA heavy chain constant region, and suffixed ". IgG 4P" to form a full length antibody formed by association with the IgG4-P heavy chain constant region, e.g., "Hu 32-1.IgG4 AA" to form a full length antibody formed by association with the IgG4-P heavy chain constant regionHu32VH1A heavy chain variable region and a heavy chain variable region as set forth in SEQ ID NO: 72 and an IgG4-AA heavy chain constant region linked theretoHu32VL1A light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73 (the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region), and "Hu 32-1.IgG 4P" is represented byHu32VH1A heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 79 IgG4-P heavy chain constant region ligationTo a heavy chain ofHu32VL1A light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73 to the constant region of the Kappa chain (in which the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region).

3. Humanization of murine antibody M33

3.1 murine antibody M33 humanized framework selection

The humanized light chain templates of the murine antibody M33 are IGKV1-39 and IGKJ4 and 01, the humanized heavy chain templates are IGHV3-7 and IGHJ6 and 01, and the humanized variable region sequences are as follows:

hu33VH-CDR grafting (SEQ ID NO: 41):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVA TISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYGHGYFDVWGQGTTVTVSS。

Hu33VL-CDRgrafted(SEQ ID NO：42)：

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEI K。

3.2 humanized template selection and Back-mutation design of murine antibody M33

TABLE 9 murine antibody M33 humanized antibody light/heavy chain variable region sequences

Note: grafting (Grafted) represents the grafting of murine antibody CDRs into human germline FR sequences. Amino acid residues are determined and annotated by the Kabat numbering system, as F71Y indicates that position 71F of Kabat numbering is mutated back to Y according to the Kabat numbering system.

The sequences of the humanized antibody light chain variable region and heavy chain variable region of murine antibody M33 are as follows:

hu33VL1 (grafted with Hu33 VL-CDR): (SEQ ID NO: 42)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYT SSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEI K。

>Hu33VL2(SEQ ID NO：43)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYT SSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEI K。

>Hu33VL3(SEQ ID NO：44)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGGAVKLLIYYT SSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEI K。

Hu33VH1 (grafted with Hu33 VH-CDR): (SEQ ID NO: 41)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVA TISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYG HGYFDVWGQGTTVTVSS。

>Hu33VH2(SEQ ID NO：45)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVA TISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGH GYFDVWGQGTTVTVSS。

>Hu33VH3(SEQ ID NO：46)

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVA TISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGH GYFDVWGQGTTVTVSS。

3.3 humanized sequence combinations of murine antibody M33

TABLE 10 humanized antibody light and heavy chain variable region combinations

Remarking: in the tables, for example, "Hu 33-6" refers to an antibody light/heavy chain variable region combination having an antibody light chain variable region of Hu33VL2 and a heavy chain variable region of Hu33VH3, and so on.

The antibody light/heavy chain variable region combinations referred to in the above table (e.g., Hu33-6) can be linked to the antibody light/heavy chain constant regions, respectively, to form full-length antibodies; as not explicitly stated in this disclosure, the light chain variable region when forming a full-length antibody is identical to SEQ ID NO: 73 to form an antibody light chain, and a heavy chain variable region linked to the Kappa chain constant region of SEQ ID NO: 72 or the constant region of the heavy chain of IgG4-AA shown in SEQ ID NO: 79 IgG4-P heavy chain constant region ligationAntibody heavy chains are formed and are suffixed with the designation indicating the antibody light/heavy chain variable region combination in the table (e.g., Hu 33-6). IgG4AA "indicates full length antibodies formed by association with the IgG4-AA heavy chain constant region and suffixed with the designation IgG 4P" indicates full length antibodies formed by association with the IgG4-P heavy chain constant region, e.g., "Hu 33-6.IgG4 AA" indicates antibodies formed by association with the IgG4-P heavy chain constant regionHu33VH3A heavy chain variable region and a heavy chain variable region as set forth in SEQ ID NO: 72 and an IgG4-AA heavy chain constant region linked theretoHu33VL2A light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73 (the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region) and "Hu 33-6.IgG 4P" represents a full-length antibody composed of a light chain in which Kappa chain constant regions are linked togetherHu33VH3A heavy chain variable region and a heavy chain variable region as set forth in SEQ ID NO: 79 IgG4-P heavy chain constant region andHu33VL2a light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73 to the constant region of the Kappa chain (in which the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region).

4. Mutants of humanized antibodies

4.1 mutant antibodies to the Hu23 humanized antibody

Site-specific mutagenesis was performed in silico on amino acids at specific positions of LCDR1(SEQ ID NO: 11) of the light chain of the Hu23 humanized antibody, and the specific mutagenesis is shown in Table 11:

table 11. mutant sequences of Hu23 light chain LCDR 1:

note: hu23LCDR1(N28Q) represents the LCDR1 mutant sequence in which N at position 28 is mutated to Q according to the Kabat numbering convention for the Hu23 humanized antibody light chain variable region Hu23VL1 or Hu23VL2, and Hu23LCDR1(G29A) represents the LCDR1 mutant sequence in which G at position 29 is mutated to A according to the Kabat numbering convention for the Hu23 humanized antibody light chain variable region Hu23VL1 or Hu23VL 2(CDR is determined by the Kabat numbering system).

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

the sequence of > Hu23VL1(N28Q) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGG TKVEIK(SEQ ID NO：53)

the sequence of > Hu23VL1(N28L) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGG TKVEIK(SEQ ID NO：54)

the sequence of > Hu23VL1(N28T) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGG TKVEIK(SEQ ID NO：55)

the sequence of > Hu23VL1(N28D) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGG TKVEIK(SEQ ID NO：56)

the sequence of > Hu23VL1(G29A) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGG TKVEIK(SEQ ID NO：57)

the sequence of > Hu23VL1(G29V) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGG TKVEIK(SEQ ID NO：58)

the sequence of > Hu23VL2(N28Q) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGG GTKVEIK(SEQ ID NO：59)

the sequence of > Hu23VL2(N28L) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGG GTKVEIK(SEQ ID NO：60)

the sequence of > Hu23VL2(N28T) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGG GTKVEIK(SEQ ID NO：61)

the sequence of > Hu23VL2(N28D) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGG GTKVEIK(SEQ ID NO：62)

the sequence of > Hu23VL2(G29A) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGG GTKVEIK(SEQ ID NO：63)

the sequence of > Hu23VL2(G29V) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGG GTKVEIK(SEQ ID NO：64)

TABLE 12 Hu23 humanized antibody light/heavy chain variable region combinations

Remarking: in the tables, for example, "Hu 23-11" refers to an antibody light/heavy chain variable region combination with an antibody light chain variable region of Hu23VL1(N28T) and a heavy chain variable region of Hu23VH1, and so on.

The antibody light/heavy chain variable region combinations referred to in the above table (e.g., Hu23-11) can be linked to the antibody light/heavy chain constant regions, respectively, to form full-length antibodies; as not explicitly stated in this disclosure, the light chain variable region when forming a full-length antibody is identical to SEQ ID NO: 73 to form an antibody light chain, and a heavy chain variable region linked to the Kappa chain constant region of SEQ ID NO: 72 or the constant region of the heavy chain of IgG4-AA shown in SEQ ID NO: 79 to form an antibody heavy chain, and the full length antibody formed by association with the IgG4-AA heavy chain constant region is indicated by the designation indicating the light/heavy chain variable region combination of the antibody in the table (e.g., Hu23-11) suffixed ". IgG4 AA", and the full length antibody formed by association with the IgG4-P heavy chain constant region is indicated by the suffix ". IgG 4P", e.g., "Hu 23-11.IgG4 AA", indicating the full length antibody formed by association with the IgG4-P heavy chain constant regionHu23VH1A heavy chain variable region and a heavy chain variable region as set forth in SEQ ID NO: 72 and an IgG4-AA heavy chain constant region linked theretoHu23VL1(N28T)A light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73The full-length antibody comprising a light chain comprising linked Kappa chain constant regions as shown (the variable regions are linked to the constant regions such that the variable regions are located at the amino terminal ends of the constant regions) is represented by "Hu 23-11.IgG 4P" which is represented byHu23VH1A heavy chain variable region and a heavy chain variable region as set forth in SEQ ID NO: 79 IgG4-P heavy chain constant region andHu23VL1(N28T)a light chain variable region and a light chain variable region as set forth in SEQ ID NO: 73 to the constant region of the Kappa chain (in which the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region).

The experimental results show that the humanized antibodies mutated at the Hu23LCDR1(N28Q), Hu23LCDR1(N28L), Hu23LCDR 1(N28T), Hu23LCDR1(N28D), Hu23LCDR1(G29A) and Hu23LCDR1(G29V) sites all maintain the binding capacity with PD-1 (Table 16).

4.2 mutant antibodies to the Hu32 humanized antibody

Through sequence analysis, the sequence identity of serial humanized antibodies Hu23 and Hu32 from M23 and M32 is high, and a Hu23 light chain variable region and a Hu32 heavy chain variable region are combined into a novel light chain variable region combination. The results of the experiments show that the humanized antibodies comprising the newly combined light and heavy chain variable regions all retain the binding ability to the PD-1 antigen (table 16).

TABLE 13 Hu32 and Hu23 antibody variable region consensus sequences general formulas

TABLE 14 combination of Hu32 heavy chain variable region with Hu23 light chain variable region

Remarking: in the tables, for example, "Hu 32 a-85" refers to an antibody light/heavy chain variable region combination with an antibody light chain variable region of Hu23VL1(N28T) and a heavy chain variable region of Hu32VH6, and so on.

The antibody light/heavy chain variable region combinations referred to in the above table (e.g., Hu32a-85) can be linked to the antibody light/heavy chain constant regions, respectively, to form full-length antibodies; as not explicitly stated in this disclosure, the light chain variable region when forming a full-length antibody is identical to SEQ ID NO: 73 to form an antibody light chain, and a heavy chain variable region linked to the Kappa chain constant region of SEQ ID NO: 72 or the constant region of the heavy chain of IgG4-AA shown in SEQ ID NO: 79 and the full length antibody formed by association with the IgG4-AA heavy chain constant region is designated by the designation indicating the light/heavy chain variable region combination of the antibody in the table (e.g., Hu32a-85) with the suffix "IgG 4 AA" for the full length antibody formed by association with the IgG 3663-P heavy chain constant region, e.g., "Hu 32a-85.IgG4 AA" for the full length antibody formed by association with the IgG4-P heavy chain constant region, and the heavy chain variable region of Hu32VH6 with the amino acid sequence as set forth in SEQ ID NO: 72, to the light chain variable region of Hu23VL1(N28T) and the heavy chain variable region as set forth in SEQ ID NO: 73 (the variable regions are linked to the constant regions at the amino terminus of the constant regions) and "Hu 32a-85.IgG 4P" represents a full-length antibody comprising the heavy chain variable region of Hu32VH6 and the amino acid sequence as set forth in SEQ ID NO: 79 to the heavy chain consisting of the constant region of the heavy chain of Hu23VL1(N28T) and the heavy chain consisting of the heavy chain of IgG4-P linked to the variable region of the light chain as shown in SEQ ID NO: 73 to the constant region of the Kappa chain (in which the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region).

TABLE 15 combination of Hu23 heavy chain variable region and Hu32 light chain variable region

Remarking: in the tables, for example, "Hu 23 a-57" refers to an antibody light/heavy chain variable region combination having an antibody light chain variable region of Hu32VL1 and a heavy chain variable region of Hu23VH1, and so on.

The antibody light/heavy chain variable region combinations referred to in the above table (e.g., Hu23a-57) can be linked to the antibody light/heavy chain constant regions, respectively, to form full-length antibodies; as not explicitly stated in this disclosure, the light chain variable region when forming a full-length antibody is identical to SEQ ID NO: 73 to form an antibody light chain, and a heavy chain variable region linked to the Kappa chain constant region of SEQ ID NO: 72 or the constant region of the heavy chain of IgG4-AA shown in SEQ ID NO: 79 and the full length antibody formed by association with the IgG4-AA heavy chain constant region is designated by the designation indicating the antibody light/heavy chain variable region combination in the table (e.g., Hu32a-85) with the suffix ". IgG4 AA" and the full length antibody formed by association with the IgG4-P heavy chain constant region is designated by the suffix ". IgG 4P", e.g., "Hu 23a-57.IgG4 AA", indicating the variable region formed by the Hu23VH1 heavy chain variable region and the amino acid sequence as set forth in SEQ ID NO: 72 to the heavy chain consisting of the constant region of the heavy chain of Hu32VL1 and the heavy chain of IgG4-AA shown in SEQ ID NO: 73 (the variable regions are linked to the constant regions at the amino terminus of the constant regions) and "Hu 23a-57.IgG 4P", which represents a full-length antibody comprising the heavy chain variable region of Hu23VH1 and the amino acid sequence as set forth in SEQ ID NO: 79, and a heavy chain consisting of the variable region of the Hu32VL1 light chain and the variable region of the IgG4-P heavy chain constant region as shown in SEQ ID NO: 73 to the constant region of the Kappa chain (in which the variable region and the constant region are linked such that the variable region is located at the amino terminus of the constant region).

5. Screening of humanized antibody

Affinity detection of different humanized antibodies was performed by Biacore (see test example 3 for the method), and the results are shown in table 16, which shows that different humanized antibodies retain the binding ability to PD-1, and the affinity of partially humanized antibody is even substantially similar to that of its murine antibody.

TABLE 16 affinity of Hu23 humanized antibody to human PD-1

Example 3 construction and expression of a humanized antibody for PD-1

Designing primers, carrying out PCR (polymerase chain reaction) to build each humanized antibody VH/VK gene fragment, carrying out homologous recombination with an expression vector pHr (a fragment with a signal peptide and a constant region gene (CH 1-Fc/CL)), and constructing an antibody full-length expression vector VH-CH 1-Fc-pHr/VK-CL-pHr. IgG4-P represents the mutation S228P (corresponding to position 108 of the sequences SEQ ID NO: 72 or SEQ ID NO: 79), IgG4-AA represents the mutations F234A (corresponding to position 114 of the sequences SEQ ID NO: 72 or SEQ ID NO: 79), L235A (corresponding to position 115 of the sequences SEQ ID NO: 72 or SEQ ID NO: 79) and S228P (corresponding to position 108 of the sequences SEQ ID NO: 72 or SEQ ID NO: 79), and the IgG4-AA and IgG4-P antibody formats can be obtained by simple point mutations of the IgG4 antibody format.

The IgG4-AA heavy chain constant region sequence is as follows (SEQ ID NO: 72):

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK。

the light chain (Kappa chain) constant region sequence of the antibody is as follows (SEQ ID NO: 73):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC。

an exemplary list of full-length antibody sequences constructed for the IgG4AA format is as follows:

hu23-11, IgG4AA antibody heavy chain (SEQ ID NO: 74):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLI DPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYY GSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK。

hu23-11.IgG4AA light chain (SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC。

hu32a-85.IgG4AA heavy chain (SEQ ID NO: 76):

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDP ETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDW YFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK。

light chain of Hu32a-85.IgG4AA (same as Hu23-11. light chain of IgG4AA, SEQ ID NO: 75):

hu33-6.IgG4AA heavy chain (SEQ ID NO: 77):

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISG GGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYF DVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK。

hu33-6.IgG4AA light chain (SEQ ID NO: 78):

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLH SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

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

LSLGK。

an exemplary list of full-length antibody sequences constructed for the IgG4-P format is as follows:

hu23-11, IgG4P antibody heavy chain (SEQ ID NO: 80):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLI DPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYY GSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK。

hu23-11.IgG4P light chain (identical to Hu23-11.IgG4AA light chain, SEQ ID NO: 75):

hu32a-85.IgG4P heavy chain (SEQ ID NO: 81):

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDP ETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDW YFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK。

light chain of Hu32a-85.IgG4P (same as Hu23-11. light chain of IgG4AA, SEQ ID NO: 75):

hu33-6.IgG4P heavy chain (SEQ ID NO: 82):

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISG GGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYF DVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK。

hu33-6.IgG4P light chain (as Hu33-6.IgG4AA light chain, SEQ ID NO: 78):

the full-length sequence of the constructed PD-1 antibody in the form of IgG 1is exemplified as follows:

(1) heavy chain amino acid sequence: SEQ ID NO: 144

Light chain amino acid sequence: SEQ ID NO: 75

(2) Heavy chain amino acid sequence: SEQ ID NO: 145

Light chain amino acid sequence: SEQ ID NO: 75

(3) Heavy chain amino acid sequence: SEQ ID NO: 146

Light chain amino acid sequence: SEQ ID NO: 75

Test example:

test example 1 anti-PD-1 antibody in vitro PD-1 ligand binding and binding blockade ELISA experiments

PD-L1 on the surface of the tumor cell binds to PD-1 on the surface of the T cell, thereby inhibiting the proliferation of the T cell. The antibody of PD-1 can block the signal path of PD-1/PD-L1 through the combination with PD-1, thereby stimulating the proliferation of T cells. The binding blocking assay for PD-1/PD-L1 was used to detect the blocking activity of anti-PD-1 antibodies on the signaling pathway.

In this experiment, the PD-1 protein (PD-1-Fc, SEQ ID NO: 1) in which the extracellular region and Fc were fused was coated on a 96-well plateThen adding anti-PD-1 antibodies (comprising antibodies: Hu23-11, IgG4AA, Hu32a-85., IgG4AA and Hu33-6, IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO 2015085847) to be tested respectively, carrying out incubation reaction, later adding biotin-labeled PD-L1/PD-L2, carrying out incubation reaction, washing the plate, detecting the binding amount of the biotin-labeled PD-L1/PD-L2, and calculating the IC of the anti-PD-1 antibody for blocking the binding of the ligand PD-L1/PD-L2₅₀The value is obtained.

With pH 9.6CB buffer (1.59g Na)₂CO₃And 2.93g NaHCO₃Dissolved in 1L of distilled water) PD-1-Fc was diluted to 1. mu.g/ml, added to a 96-well plate in a volume of 100. mu.l/well and left at 4 ℃ for 16h to 20 h. The 96-well plate was aspirated off PBS buffer, and after washing the plate 1 time with PBST (pH7.4 PBS containing 0.05% tween20), 120. mu.l/well of PBST/1% mil was added and incubated at room temperature for 1h for blocking. After removing the blocking solution and washing the plate 1 time with PBST buffer, 90. mu.l of the anti-PD-1 antibody to be tested diluted to the appropriate concentration with a sample diluent (5% BSA in PBS pH7.4, 0.05% Tween20) was added and preincubated at 4 ℃ for 1 h. 10 Xconcentration of biotin-labeled PD-L1/PD-L2 (Beijing Yi Qian Shenzhou Biotechnology Co., Ltd.) (10. mu.g/ml) was added to a volume of 10. mu.l/well, and the mixture was shaken and mixed in a shaker, and then incubated at 37 ℃ for 1 hour. The reaction was removed and after washing the plate 6 times with PBST, 100. mu.l/well of Streptavidin-Peroxidase Polymer (Streptavidin-Peroxidase Polymer) diluted 1: 400 in PBST buffer was added and incubated for 50 minutes at room temperature with shaking. After washing the plates 6 times with PBST, 100. mu.l/well of TMB was added and incubated for 5-10min at room temperature. Add 100. mu.l/well 1M H₂SO₄The reaction was terminated. The absorbance was read at 450nm with a microplate reader and the IC of the anti-PD-1 antibody for the binding blockade of ligand PD-L1/PD-L2 was calculated₅₀The value is obtained. The data are detailed in Table 17 below.

TABLE 17 anti-PD-1 antibodies of the present disclosure bind to PD-1, block ELISA for ligand PD-L1/PD-L2 binding

The anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA exemplified by the present disclosure were all able to effectively block the binding of PD-1 to PD-L1/PD-L2 with blocking activity similar to the positive control antibody.

Test example 2 exemplary antibody and ligand blocking assays

The blocking effect of the antibodies on the binding of PD-1 to PD-L1 was investigated. The experimental procedure is briefly described as follows: CHOK1/PD-L1 cells (Promega) were digested, added to a 96-well plate at 100. mu.L/well, and placed at 37 ℃ in 5% CO₂The incubator was incubated for 24 hours. The control and sample were diluted to the desired concentration using PBS. Counting Jurkat/PD-1 cells (Jurkat cells stably transferring PD-1), adding 10 μ L/well diluted antibodies (antibodies: Hu23-11, IgG4AA, Hu32a-85.IgG4AA and Hu33-6, IgG4AA, positive control antibody: H005-1), negative control IgG4 protein, antibody gradient dilution concentration of 0.3mg/ml, 3mg/ml and 30mg/ml) into a cell culture plate (90 μ L/well) containing CHOK1/PD-L1 cells according to a certain proportion, placing at 37 ℃, and placing at 5% CO 2₂The incubator was incubated for 5 hours. The cell culture plate was removed, left at room temperature for 5 minutes, and 50. mu.l Bio-Glo was added to each well^TMReagent, incubate for 5 minutes at room temperature and read the plate. The results are shown in figure 1 and demonstrate that the anti-PD-1 antibodies Hu23-11, IgG4AA, Hu32a-85., IgG4AA and Hu33-6, IgG4AA exemplified in the present disclosure are able to effectively block the binding of PD-1 to PD-L1, with blocking activity similar to that of the positive control antibody H005-1.

Test example 3 BIAcore antibody affinity assay for exemplary antibodies

IgG is affinity captured by a Protein A biosensing chip (Cat. #29127556, GE), human PD-1 antigen (Cat. #10377H08H, Sino Biological), and Cyno PD-1 antigen (purchased from Sino Biological) flow through the surface of the chip, and a Biacore T200 instrument detects PD-1 antibody and antigen PD-1 reaction signals in real time to obtain a binding and dissociation curve. After dissociation was complete for each experimental cycle, the biosensor chip was washed and regenerated with 10mM Glycine-HCl buffer pH 1.5. The experimental buffer system was 1 XHBS-EP buffer solution (Cat # BR-1001-88, GE). After the experiment was completed, the data were fitted with (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software to obtain affinity values, and the results are shown in Table 18.

TABLE 18 affinity of anti-PD-1 antibodies to human PD-1 and monkey PD-1

The results show that the anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA exemplified by the present disclosure are all able to bind to human PD-1 and monkey PD-1.

Test example 4 secretion of cellular IFN γ by 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 medium⁶Each 20mL cell suspension was added with 40. mu.l tuberculin (97-8800, Synbiotics) at 37 deg.C and 5% CO₂The incubator is used for 5 days. On day 5, 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. Antibody samples (including antibodies of the present disclosure: Hu23-11, IgG4AA, Hu32a-85.IgG4AA and Hu33-6, IgG4AA, positive control antibody H005-1, and negative control IgG4 protein, antibody gradient dilution concentrations of 0.3mg/ml, 3mg/ml, 30mg/ml) were added at the same time in a gradient dilution of 10. mu.l per well with PBS (B320, Shanghai-culture Biotech Ltd.). The cell culture plate was placed at 37 ℃ in 5% CO₂Incubate for 3 days. Taking out the cell culture plate, centrifuging (4000rpm, 10min), collecting cell culture supernatant, and detecting IFN-gamma level by ELISA (human IFN-gamma detection kit (EHC102g.96, Xinbo Sheng) methodReference is made to the reagent specification.

The results are shown in fig. 2, and indicate that the anti-PD-1 antibodies Hu23-11, IgG4AA, Hu32a-85., IgG4AA, and Hu33-6, IgG4AA of the present disclosure are all effective in activating IFN- γ secretion, and their ability to activate IFN- γ secretion is similar to that of the positive H005-1 control.

Test example 5 Effect of anti-PD-1 antibody in transgenic PD-1 mouse Colon cancer model MC38

MC38 cells 5x10 ^5 cells/mouse/100 μ l were inoculated subcutaneously under the right flank of 90 hPD-1TG mice (Pogostemon), 10 days later the animals with too large tumor volume were removed, and the mice were randomized to an average tumor volume of about 120mm ^ 3: 7 groups of blank control Vehicle (PBS), positive control H005-13 mpk, Hu32a-85.IgG4AA 1mpk, Hu32a-85.IgG4AA 3mpk, Hu23-11.IgG4AA 1mpk, Hu23-11.IgG4AA 3mpk and Hu33-6.IgG4AA 3mpk, and 8 animals in each group. Each group of antibodies was intraperitoneally injected three times a week from Day0 (Day 0), and after the end of the first week of administration, tumors were found to be significantly inhibited, and the frequency of administration was adjusted once a week for 5 times in the second and third weeks. Tumor volume, animal weight were monitored 2 times per week and data were recorded. The tumor-bearing animals were euthanized as experimental end-points when tumor volumes exceeded 2000mm ^3 or most tumors appeared ulcerated or body weight decreased 20%.

Tumor Volume (TV) 1/2 XL_{Long and long}×L_{Short length} ²

Tumor proliferation rate (T/C%) (T-T0)/(C-C0) × 100%

Tumor inhibition rate (TGI%) < 1-T/C%

(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 experimental results are shown in table 19 and figure 3, and the experimental results show that compared with a blank control, the antibodies disclosed by the invention can obviously inhibit the growth of mouse colon cancer MC38 transplanted tumors, wherein the tumor inhibition rate is the highest in Hu32a-85.IgG4AA-3mpk group, and the tumor inhibition rate is 77.64% in the last measurement. When the administration frequency is 3 times of three times of administration in one week, the result shows that the tumor inhibition rate of the antibody of the disclosure is obviously superior to that of the positive control antibody H005-1; thereafter the frequency of dosing decreased to once a week, 2 times after dosing (Day21), the potency of the antibodies of the present disclosure was progressively spread apart and showed dose-dependence, with Hu32a-85.IgG4AA being significantly better than H005-1 at equivalent doses (p < 0.05). And the tumor-bearing mice can well tolerate the anti-PD-1 antibody, the body weight stably rises in the whole administration process, and no obvious symptoms such as weight loss caused by medicaments occur.

TABLE 19 tumor inhibition Rate Effect of anti-PD-1 antibodies on mouse Colon cancer MC38

Test example 6 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) grown in Cephrim Biosciences, Inc. MC38 cells were plated at 5X10⁵Each 100. mu.l of the mice were inoculated subcutaneously into the posterior part of the right rib of hPD-1 transgenic mice (male and female halves), when the average tumor volume of the mice reached 80-100mm3, the animals with heavy weight, over-large tumor and under-small tumor were removed, and the tumor-bearing mice were randomly divided into 5 groups (8 mice per group) according to the tumor volume: negative controls hIgG control 30mpk, H005-110 mpk, H005-130 mpk, Hu33-6.IgG4AA 10mpk, Hu33-6.IgG4AA 30 mpk. The group administration date is 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 Prism 5 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%

The experimental results are shown in table 20 and fig. 4, and show that compared with the 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 Hu33-6.IgG4AA 30mpk group, and the tumor inhibition rate is 80.4% when measured on day 20. In the low dose group (10mpk), Hu33-6.IgG4AA-10mpk had better efficacy than the positive control H005-1-10 mpk.

TABLE 20 Effect of anti-PD-1 antibodies on mouse Colon cancer MC38 tumor volume

Remarking: the mean tumor volume in each group is given in the table: mm is³。

Example 4: preparation of anti-PD-1/PD-L1 bispecific antibody

1. Structure of anti-PD-1/PD-L1 bispecific antibody

Seven anti-PD-1/PD-L1 bispecific antibodies were designed and prepared as follows, wherein the structures of TJ021-PR0001, TJ021-PR0004, TJ021-PR0005, TJ021-PR0006 and TJ021-PR0007 are all IgG-scFv. The structures of TJ021-PR0002 and TJ021-PR0003 are scFv-Fab IgG. The Fc portion of all antibodies was mutated at L234A, L235A to eliminate Fc-mediated ADCC.

The schematic structure diagram of TJ021-PR0001 is shown in FIG. 5A, wherein the IgG part is anti-human PD-1 antibody (anti-PD-1 IgG), the scFv part is composed of heavy chain variable region (VH) and light chain variable region (VL) of anti-human PD-L1 antibody (anti-PD-L1 scFv), and the heavy chain variable region (VH) and the light chain variable region (VL) are connected by first peptide linker (GGGGS GGGGS GGGGS). Anti-PD-L1 scFv was linked at the C-terminus of Anti-PD-1IgG by a second peptide linker (GGGGSGGGGSGGSGGGGS).

The sequence of TJ021-PR0001 is as follows:

amino acid sequence of TJ021-PR0001 heavy chain (PD-1 heavy chain-second peptide linker-PD-L1 scFv) (SEQ ID NO: 152)

Amino acid sequence of TJ021-PR0001 light chain (PD-1 light chain) (SEQ ID NO: 75)

The schematic structure diagram of TJ021-PR0002 is shown in FIG. 5B, wherein the Fab IgG part is anti-human PD-1 antibody (anti-PD-1 Fab), the scFv part is composed of heavy chain variable region (VH) and light chain variable region (VL) of anti-human PD-L1 antibody (anti-PD-L1 scFv), and the heavy chain variable region (VH) and the light chain variable region (VL) are connected by a first peptide linker. Anti-PD-L1 scFv was attached to the Fc-terminus of IgG by a second peptide linker.

The sequence of TJ021-PR0002 is as follows:

amino acid sequence of TJ021-PR0002 heavy chain (PD-1 heavy chain) (SEQ ID NO: 153)

Amino acid sequence of TJ021-PR0002 light chain (PD-1 light chain) (SEQ ID NO: 75)

Amino acid sequence of TJ021-PR0002 heavy chain (PD-L1 scFv-hinge region-CH 2-CH3) (SEQ ID NO: 154)

The structural diagram of TJ021-PR0003 is shown in FIG. 5C, which is different from TJ021-PR0002 in that the Fab IgG part is anti-human PD-L1 antibody (anti-PD-L1 Fab) and the scFv part is anti-human PD-1 antibody.

The sequence of TJ021-PR0003 is as follows:

amino acid sequence of TJ021-PR0003 heavy chain (PD-L1 heavy chain) (SEQ ID NO: 155)

Amino acid sequence of TJ021-PR0003 light chain (PD-L1 light chain) (SEQ ID NO: 145)

Amino acid sequence of TJ021-PR0003 heavy chain (PD-1 scFv-hinge region-CH 2-CH3) (SEQ ID NO: 156)

The structural diagram of TJ021-PR0004 is shown in FIG. 5D, which is different from TJ021-PR0001 in that Anti-PD-L1 scFv is linked to the N-terminus of Anti-PD-1IgG by a second peptide linker.

The sequence of TJ021-PR0004 is as follows:

amino acid sequence of TJ021-PR0004 heavy chain (PD-L1 scFv-PD-1 heavy chain) (SEQ ID NO: 157)

Amino acid sequence of TJ021-PR0004 light chain (PD-1 light chain) (SEQ ID NO: 75)

The schematic structure diagram of TJ021-PR0005 is shown in FIG. 5A, and the difference from TJ021-PR0001 lies in one amino acid mutation at the Fc C terminal, from K mutation in TJ021-PR0001 to A mutation.

The sequence of TJ021-PR0005 is as follows:

amino acid sequence of TJ021-PR0005 heavy chain (PD-1 heavy chain-PD-L1 scFv) (SEQ ID NO: 158)

Amino acid sequence of TJ021-PR0005 light chain (PD-1 light chain) (SEQ ID NO: 75)

The structural diagram of TJ021-PR0006 is shown in FIG. 5D, which is different from TJ021-PR0004 in the sequence of PD-L1 binding domain.

The sequence of TJ021-PR0006 is as follows:

TJ021-PR0006 heavy chain (PD-L1 scFv-PD-1 heavy chain) amino acid sequence (SEQ ID NO: 159)

TJ021-PR0006 light chain (PD-1 light chain) amino acid sequence (SEQ ID NO: 75)

The structural diagram of TJ021-PR0007 is shown in FIG. 5A, and the difference from TJ021-PR0001 lies in the sequence difference of the PD-L1 binding domain.

The sequence of TJ021-PR0007 is as follows:

TJ021-PR0007 heavy chain (PD-1 heavy chain-PD-L1 scFv) amino acid sequence (SEQ ID NO: 160)

TJ021-PR0007 light chain (PD-1 light chain) amino acid sequence (SEQ ID NO: 75)

2. Preparation of anti-PD-1/PD-L1 bispecific antibody

CHO-S cells (purchased from Thermo, Inc., Cat. A29133) which grew well in a logarithmic growth phase were used, centrifuged and grown at 6X 10⁶Cells/ml were seeded at 250 ml. Add solution 2 (diluted 800. mu.l transfection reagent with 9.2ml culture medium, mix) to solution 1 (diluted 250. mu.g plasmid with 10ml culture medium, mix) for a total volume of 20ml, incubate gently and mix for 1-5 minutes at room temperature for no more than 5 minutes, add mixed transfection solution drop by drop to the cell sap, shake and add. The flasks were then placed in 5% CO₂Cultured at 32 ℃ and 123rpm in a shaker, and 16ml of an adjuvant Feed (purchased from Thermo, cat # A29133) and 0.6ml of an Enhancer Enhancer (purchased from Thermo, cat # A29133) were added to the mixture for 18 to 22 hours. On the fifth day, 16ml adjuvant Feed (available from Thermo company, Cat. A29133), 5% CO were added₂Shaking at 32 ℃ and 123rpm, and collecting the supernatant after centrifugation on days 12-14. Purification of recombinant antibodies was performed by affinity chromatography (Protein a) and ion exchange two-step process. The media used for the purification were MabSelect SuRe column (GE, 17-5438) and 1ml CM pre-packed column (GE, 17505601), respectively, from GE. The seven anti-PD-1/PD-L1 bispecific antibodies were prepared, wherein the results of detecting TJ021-PR0001 and TJ021-PR0004 by 10% SDS-PAGE gel electrophoresis are shown in FIG. 6.

The results show that the theoretical molecular weights of the two diabodies are 200kDa, the heavy chain is 75kDa, the light chain is 25kDa, and the sizes of the bands are judged by the size of the non-reducing band and the size of the reducing band of SDS-PAGE gel electrophoresis, and the sizes of the bands are consistent with expectations, which indicates that the recombinant bispecific antibody can be correctly assembled and expressed without obvious aggregation degradation.

Example 5 detection of antigen binding Activity of anti-PD-1/PD-L1 bispecific antibody

The ELISA method is used for detecting the binding activity of anti-PD-1/PD-L1 bispecific antibodies TJ021-PR0001, TJ021-PR0002 and TJ021-PR0003 with human PD-L1 protein and human PD-1 protein respectively. The 96-well plates were labeled accordingly before the start of the experiment and coated with 1. mu.g/ml antigen concentration, 50. mu.l per well, overnight in a refrigerator at 4 ℃. The next day, the coated antigen plates were removed and the plate washer was washed once with 1 × PBST wash. After washing, the cells were blocked with 1% BSA blocking solution prepared in 1 XPBST at 37 ℃ for 1 hour. After washing the plate 3 times with 1 XPBST, the double antibody to be detected was added at different dilution concentrations (0.01nM, 0.1nM, 1nM, 10nM, 100nM) and incubated for 1h at 37 ℃ in an incubator. After washing the plate 3 times with 1 XPBST, 100. mu.l of goat anti-human IgG secondary antibody diluted 1: 5000 was added and incubated at 37 ℃ for 0.5 hour in an incubator. After washing the plate, taking a TMB developing solution A and a liquid B, and mixing the TMB developing solution A and the liquid B in a ratio of 1: the color was developed after mixing at a ratio of 1. The color reaction was terminated with 1M hydrochloric acid for 15 minutes. Absorbance at 450nm was measured on a Spectra Max M5 multifunctional plate reader. The results are shown in FIGS. 7A and 7B.

The results show that the binding activities of the anti-PD-1/PD-L1 bispecific antibodies TJ021-PR0001, TJ021-PR0002 and TJ021-PR0003 with the human PD-L1 protein and the human PD-1 protein respectively keep the similar binding activity with the original parent monoclonal antibody.

Example 6 detection of antigen binding affinity of anti-PD-1/PD-L1 bispecific antibody

The Surface Plasmon Resonance (SPR) technology is adopted to detect the affinity between the anti-PD-1/PD-L1 bispecific antibody TJ021-PR0001 and the antigen human PD-L1 protein and human PD-1 protein thereof respectively. Antigen human PD-L1 protein and human PD-1 protein were immobilized to a CM5 chip, respectively, at a coupling level set at 100RU, and detected using Biacore (Biacore T200, GE Healthcare, BIAC-B20-03). The running buffer was HBS-EP + (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). The diluted antibodies (concentrations of 6.25nM, 12.5nM, 25nM, 50nM, 100nM, respectively) were flowed through the experimental and control channels (100nM antigen human PD-L1 protein/antigen human PD-1) at a flow rate of 30. mu.l/min for 3 min and dissociated for 5 min. The regeneration buffer 10mM Glycine pH1.5 (GE Healthcare, BR-1003-54) was then run at a flow rate of 30. mu.l/min for 30 seconds. Data were analyzed using Biacore 8K evaluation software. The results are shown in FIGS. 8A and 8B.

The results showed that the affinity KD value of the bispecific antibody TJ021-PR0001 with human PD-1 protein was 3.18nM, and the affinity KD value with human PD-L1 protein was 0.75 nM. TJ021-PR0001 has about 4 times greater affinity for human PD-L1 protein than for human PD-1 protein.

Example 7 anti-PD-1/PD-L1 bispecific antibody promotes cell bridging experiments

The human PD-1 and PD-L1 plasmids were transferred into CHOK1 cells (ATCC, CCL-61) to construct stably expressed cell lines. CHOK1-hPD-L1 and CHOK1-hPD-1 were resuspended in PBS and cell density was adjusted to 2X 10⁶and/mL. Respectively with 4M CFSE (Invitrogen), 10M Cell promotion Dye eFluor^TM670(eBioscience) were mixed in equal volumes, stained at 37 ℃ for 10 minutes in the absence of light and then washed free of fluorescent dye. Adjusting cell density to 2X 10⁶The concentration of the antibody is/mL, the diluted antibody to be detected is added into a 96-hole round bottom plate, the mixture is incubated for 60 minutes at 4 ℃ in a dark place, and then the proportion of double positive cells is detected by a flow cytometer, and the result is shown in FIGS. 9A and 9B.

FIGS. 9A and 9B show that bispecific antibodies TJ021-PR0001, TJ021-PR0002, TJ021-PR0003, TJ021-PR0006, TJ021-PR007, and positive control LY3434172 were able to dose-dependently promote bridging of CHOK1-hPD-L1 cells and CHOK1-hPD-1 cells, while PD-1 control mab and PD-L1 control mab blocked PD-1/PD-L1 mediated bridging between the two cells by epitope-occupancy.

The VH sequence of the PD-1 control monoclonal antibody in the disclosed embodiment is shown in SEQ ID NO: 27, and the VL sequence is shown in SEQ ID NO: as shown at 55.

The VH sequence of the PD-L1 control monoclonal antibody in the disclosed embodiment is shown in SEQ ID NO: 121, and a VL sequence is shown as SEQ ID NO: 122, respectively.

Example 8 anti-PD-1/PD-L1 bispecific antibody blocks PD-1/PD-L1 Signaling pathway experiments

PD-1 and NFAT luciferase reporter plasmids (Promega, pGL4.30[ luc2P/NFAT-RE/Hygro ] Vector) were stably transfected into Jurkat (ATCC) cells to obtain a stable cell line Jurkat-NFAT-PD-1 with PD-1 expression and NFAT luciferase reporter. Cell line Hep3B-PD-L1, which stably highly expressed PD-L1, was seeded in 96-well plates (Perkin Elmer, Cat #6005181) the day before the experiment. Adding different concentrations of antibodies to be tested (0.04nM, 0.12nM, 0.37nM, 1.11nM, 3.33nM, 10nM) and incubating at 37 deg.C for 30 min; then adding Jurkat-NFAT-PD-1 cells, placing the cells in a 5% carbon dioxide incubator at 37 ℃ for culturing for 6 hours; the plate was removed and allowed to equilibrate at room temperature for 10 minutes, then the luciferase assay reagent ONE-GLUTM (Promega, Cat # E6120) was added and the fluorescence read using Envision (PerkinElmer, 2150) and the results are shown in FIGS. 10A and 10B. It can be seen that TJ021-PR0001, TJ021-PR0002, TJ021-PR0006, TJ021-PR0007 and positive control LY3434172 can release dose-dependently the inhibition of the signal pathway of PD-1/PD-L1 on NFAT fluorescence signal, and the inhibitory ability of bispecific antibody is stronger than that of monoclonal antibody PD-1 control monoclonal antibody and PD-L1 control monoclonal antibody.

Example 9 anti-PD-1/PD-L1 bispecific antibody enhances T cell activation experiments under SEB-stimulated PBMC

Will be 1 × 10⁵100ng/mL staphylococcal enterotoxin B (SEB, combobenine) was added to/mL human peripheral blood mononuclear cells (Stemexpress), while seven test antibodies and positive controls LY3434172 were added at a range of concentrations (0.01nM, 0.1nM, 1nM, 10nM, 100nM), respectively, and non-specific IgG controls and no SEB controls were set. CO at 37 deg.C₂After 72 hours of incubator culture, the secretion of IL-2(Invitrogen, human IL-2kit) in the supernatant was measured by ELISA. The results are shown in FIG. 7, in which the bispecific antibodies TJ021-PR0001, TJ021-PR0002, TJ021-PR0003, TJ021-PR0006 and TJ021-PR007 and the positive control LY3434172 can dose-dependently promote IL-2 secretion from T cells. In FIG. 11A, TJ021-PR0001 activated T cells more strongly than the combination of PD-1 control mab and PD-L1 control mab, andTJ021-PR0001 is stronger than TJ021-PR 0002. TJ021-PR0006 in FIG. 11B activated T cells more strongly than the combination of PD-1mAb and PD-L1 mAb.

Example 10 Mixed lymphocyte reaction experiments against the bispecific antibody PD-1/PD-L1

Resuscitating cryopreserved human peripheral blood mononuclear cells (StemExpress) and adjusting cell density to 2 × 10⁶Perml, transferred into 10cm cell culture dishes, 10mL per dish, and placed in CO at 37 ℃₂Incubating the culture box for 2 hours to make the monocyte adhere to the wall, washing the monocyte with serum-free cell culture medium once, adding fresh culture medium containing 50ng/mL GM-CSF (Peprotech, Cat #300-03) and 50ng/mL hIL-4(Peprotech, Cat #200-04) for culturing for five days, changing the medium every 2-3 days, and adjusting the cell density to 2 x10⁵and/mL. Isolation of CD4+ T cells from human peripheral blood mononuclear cells of different individuals (STEMCELL, EasySep)^TMHuman CD4+ T Cell Isolation Kit), which regulates CD4+ T Cell density to 1 × 10⁶and/mL. Dendritic cells and CD4+ T cells were added to a 96-well U-plate (Corning, Cat #3799) at a volume of 1:1, and a gradient of diluted antibody (concentration 0.01nM, 0.1nM, 1nM, 10nM, 100nM) was added to the well, which was placed in a CO chamber at 37 deg.C₂Incubate 72 hours, take the supernatant TR-FRET method to detect the IL-2 secretion (Cisbio, human IL-2kit), the result is shown in FIG. 12; the results show that in FIG. 12, TJ021-PR0006 and TJ021-PR0007 activated mixed lymphocytes more strongly than the combination of PD-1 control mAb and PD-L1 control mAb.

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 to limit 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 PHARMACEUTICAL Co.,Ltd.

<120> bispecific antigen-binding molecules and medical uses thereof

<150> CN202010344729.6

<151> 2020-04-27

<160> 172

<170> PatentIn version 3.5

<210> 1

<211> 401

<212> PRT

<213> Artificial Sequence

<220>

<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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Lys

<210> 2

<211> 186

<212> PRT

<213> Artificial Sequence

<220>

<223> human PD-1-his

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Asp Asp Asp Lys His His His His His His

180 185

<210> 3

<211> 288

<212> PRT

<213> Artificial Sequence

<220>

<223> PD-1 antigen encoded by nucleic acid of transfected cells

<400> 3

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

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

195 200 205

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

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 4

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain variable region of murine antibody M23

<400> 4

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

1 5 10 15

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

20 25 30

Glu Met His Trp Val Lys Gln Thr Pro Ile His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Thr Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr His Cys

85 90 95

Thr Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> variable region of light chain of murine antibody M23

<400> 5

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

1 5 10 15

Asp His Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 6

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain variable region of murine antibody M32

<400> 6

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

1 5 10 15

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

20 25 30

Glu Ile His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Thr

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 7

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> variable region of light chain of murine antibody M32

<400> 7

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 8

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> M23 HCDR1

<400> 8

Asp Tyr Glu Met His

1 5

<210> 9

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> M23 HCDR2

<400> 9

Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 10

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> M23 HCDR3

<400> 10

Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe Asp Val

1 5 10 15

<210> 11

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> M23 LCDR1

<400> 11

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

1 5 10 15

<210> 12

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> M23 LCDR2

<400> 12

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> M23 LCDR3

<400> 13

Phe Gln Gly Ser His Val Pro Tyr Thr

1 5

<210> 14

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> M32 HCDR1

<400> 14

Asp Tyr Glu Ile His

1 5

<210> 15

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> M32 HCDR2

<400> 15

Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> M32 HCDR3

<400> 16

Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val

1 5 10

<210> 17

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> M32 LCDR1

<400> 17

Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 18

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> M32 LCDR3

<400> 18

Phe Gln Gly Ser His Val Pro Tyr Ala

1 5

<210> 19

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain variable region of murine antibody M33

<400> 19

Lys Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Thr Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> variable region of light chain of murine antibody M33

<400> 20

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> M33 HCDR1

<400> 21

Ser Tyr Ala Met Ser

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> M33 HCDR2

<400> 22

Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val Gln

1 5 10 15

Gly

<210> 23

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> M33 HCDR3

<400> 23

Pro Tyr Gly His Gly Tyr Phe Asp Val

1 5

<210> 24

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> M33 LCDR1

<400> 24

Arg Ala Ser Gln Asp Ile Asn Asn Phe Leu Asn

1 5 10

<210> 25

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> M33 LCDR2

<400> 25

Tyr Thr Ser Ser Leu His Ser

1 5

<210> 26

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> M33 LCDR3

<400> 26

Gln Gln Gly Asn Thr Leu Pro Trp Thr

1 5

<210> 27

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VH-CDR grafted (grafting)

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 28

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL-CDR grafted

<400> 28

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 29

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL2

<400> 29

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 30

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VH2

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 31

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VH3

<400> 31

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

1 5 10 15

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

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 32

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VH4

<400> 32

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

1 5 10 15

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

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 33

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VH-CDR grafted: IGHV1-69 x 02 and IGHJ6 x 01

<400> 33

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

1 5 10 15

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

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 34

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VL-CDR grafted

<400> 34

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 35

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VL2

<400> 35

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 36

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VH2

<400> 36

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

1 5 10 15

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

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 37

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VH3

<400> 37

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Ser Asp Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 38

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VH4

<400> 38

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

1 5 10 15

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

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 39

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VH5

<400> 39

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 40

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32VH6

<400> 40

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 41

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu33VH-CDR grafted

<400> 41

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

Ala 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 Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln 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 Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 42

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu33VL-CDRgrafted

<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 Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Ser Leu His 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 Gly Asn Thr Leu 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

<220>

<223> Hu33VL2

<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 Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu 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

<220>

<223> Hu33VL3

<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 Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu33VH2

<400> 45

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

Ala 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 Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln 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 Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 46

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu33VH3

<400> 46

Lys 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

Ala 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 Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln 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 Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 47

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23LCDR1（N28Q）

<400> 47

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

1 5 10 15

<210> 48

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23LCDR1（N28L）

<400> 48

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

1 5 10 15

<210> 49

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23LCDR1（N28T）

<400> 49

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

1 5 10 15

<210> 50

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23LCDR1（N28D）

<400> 50

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

1 5 10 15

<210> 51

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23LCDR1（G29A）

<400> 51

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

1 5 10 15

<210> 52

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23LCDR1（G29V）

<400> 52

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

1 5 10 15

<210> 53

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL1(N28Q)

<400> 53

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 54

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL1(N28L)

<400> 54

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 55

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL1(N28T)

<400> 55

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 56

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL1(N28D)

<400> 56

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 57

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL1(G29A)

<400> 57

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Ala Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 58

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL1(G29V)

<400> 58

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 59

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL2(N28Q)

<400> 59

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 60

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL2(N28L)

<400> 60

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 61

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL2(N28T)

<400> 61

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 62

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL2(N28D)

<400> 62

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 63

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL2(G29A)

<400> 63

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Ala Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 64

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23VL2(G29V)

<400> 64

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 65

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32 and Hu23 antibody variable region consensus sequence general formula HCDR1, wherein, X1 is selected from I or M

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa can be Ile or Met.

<400> 65

Asp Tyr Glu Xaa His

1 5

<210> 66

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32 and Hu23 antibody variable region consensus sequence general formula HCDR2

Wherein X2 is selected from F or I, X3 is selected from I/T, wherein X4 is selected from G or D

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be Phe or Ile.

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa can be Ile or Thr.

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa can be Gly or Asp.

<400> 66

Leu Xaa Asp Pro Glu Thr Gly Gly Xaa Val Tyr Asn Gln Lys Phe Lys

1 5 10 15

Xaa

<210> 67

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32 and Hu23 antibody variable region consensus sequence general formula HCDR3

Wherein X5 is selected from G or R, X6 is selected from F or a vacancy, X7 is selected from S or a vacancy, X8 is selected from Y or a vacancy,

x9 is selected from G or vacancy, X10 is selected from S or vacancy, X11 is selected from N or T, X12 is selected from R or S

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be Gly or Arg.

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa can be Phe or delete.

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa can be Ser or delete.

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa can be Tyr or delete.

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be Gly or delete.

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa can be Ser or delete.

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa can be Asn or Thr.

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa can be Arg or Ser.

<400> 67

Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe Asp Val

1 5 10 15

<210> 68

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32 and Hu23 antibody variable region consensus sequence general formula LCDR1

Wherein X13 is selected from I or L, X14 is selected from N, Q, L, T or D

X15 is selected from G, A or V, X16 is selected from N or K

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa can be Ile or Leu.

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa can be Asn, Gln, Leu, Thr or Asp.

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa can be Gly, Ala or Val.

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa can be Asn or Lys.

<400> 68

Arg Ser Ser Gln Ser Xaa Val His Ser Xaa Xaa Xaa Thr Tyr Leu Glu

1 5 10 15

<210> 69

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32 and Hu23 antibody variable region consensus sequence general formula LCDR3 wherein X17 is selected from A or T

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa can be Ala or Thr.

<400> 69

Phe Gln Gly Ser His Val Pro Tyr Xaa

1 5

<210> 70

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32 and Hu23 antibody variable region consensus sequence general formula VH

<220>

<221> misc_feature

<222> (26)..(26)

<223> Xaa can be Gly or Asp.

<220>

<221> misc_feature

<222> (27)..(27)

<223> Xaa can be Gly, Phe or Tyr.

<220>

<221> misc_feature

<222> (30)..(30)

<223> Xaa can be Ser or Thr.

<220>

<221> misc_feature

<222> (34)..(34)

<223> Xaa can be Ile or Met.

<220>

<221> misc_feature

<222> (38)..(38)

<223> Xaa can be Arg or Lys.

<220>

<221> misc_feature

<222> (43)..(43)

<223> Xaa can be Gln or His.

<220>

<221> misc_feature

<222> (48)..(48)

<223> Xaa can be Met or Ile.

<220>

<221> misc_feature

<222> (51)..(51)

<223> Xaa can be Phe or Ile.

<220>

<221> misc_feature

<222> (58)..(58)

<223> Xaa can be Ile or Thr.

<220>

<221> misc_feature

<222> (66)..(66)

<223> Xaa can be Gly or Asp.

<220>

<221> misc_feature

<222> (67)..(67)

<223> Xaa can be Arg or Lys.

<220>

<221> misc_feature

<222> (68)..(68)

<223> Xaa can be Val, Ala or Thr.

<220>

<221> misc_feature

<222> (70)..(70)

<223> Xaa can be Arg or Lys.

<220>

<221> misc_feature

<222> (83)..(83)

<223> Xaa can be Leu or Phe.

<220>

<221> misc_feature

<222> (97)..(97)

<223> Xaa can be Ala or Thr.

<220>

<221> misc_feature

<222> (100)..(100)

<223> Xaa can be Gly or Arg.

<220>

<221> misc_feature

<222> (101)..(101)

<223> Xaa can be Phe or delete.

<220>

<221> misc_feature

<222> (102)..(102)

<223> Xaa can be Ser or delete.

<220>

<221> misc_feature

<222> (103)..(103)

<223> Xaa can be Tyr or delete.

<220>

<221> misc_feature

<222> (105)..(105)

<223> Xaa can be Gly or delete.

<220>

<221> misc_feature

<222> (106)..(106)

<223> Xaa can be Ser or delete.

<220>

<221> misc_feature

<222> (107)..(107)

<223> Xaa can be Asn or Thr.

<220>

<221> misc_feature

<222> (108)..(108)

<223> Xaa can be Arg or Ser.

<400> 70

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Xaa Xaa Thr Phe Xaa Asp Tyr

20 25 30

Glu Xaa His Trp Val Xaa Gln Ala Pro Gly Xaa Gly Leu Glu Trp Xaa

35 40 45

Gly Leu Xaa Asp Pro Glu Thr Gly Gly Xaa Val Tyr Asn Gln Lys Phe

50 55 60

Lys Xaa Xaa Xaa Thr Xaa Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Xaa Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Xaa Arg Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 71

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32 and Hu23 antibody variable region consensus sequence general formula LCDR3

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be Ile, Val or Gly.

<220>

<221> misc_feature

<222> (29)..(29)

<223> Xaa can be Ile or Leu.

<220>

<221> misc_feature

<222> (33)..(33)

<223> Xaa can be Asn, Gln, Leu, Thr or Asp.

<220>

<221> misc_feature

<222> (34)..(34)

<223> Xaa can be Gly, Ala or Val.

<220>

<221> misc_feature

<222> (35)..(35)

<223> Xaa can be Asn or Lys.

<220>

<221> misc_feature

<222> (102)..(102)

<223> Xaa can be Ala or Thr.

<400> 71

Asp Xaa Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Xaa Val His Ser

20 25 30

Xaa Xaa Xaa Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Xaa Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 72

<211> 327

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG4-AA heavy chain constant region sequence

<400> 72

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 73

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> light chain (Kappa chain) constant region sequence of antibody

<400> 73

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> 74

<211> 452

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23-11 IgG4AA antibody heavy chain

<400> 74

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Leu Gly Lys

450

<210> 75

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23-11.IgG4AA light chain

<400> 75

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 76

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32a-85.IgG4AA heavy chain

<400> 76

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val 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 Ala Ala 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> 77

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu33-6.IgG4AA heavy chain

<400> 77

Lys 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

Ala 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 Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln 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 Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

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

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 78

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu33-6.IgG4AA light chain

<400> 78

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 Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu 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> 79

<211> 327

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain constant region sequence of IgG4-P

<400> 79

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 80

<211> 452

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu23-11 IgG4P antibody heavy chain

<400> 80

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Leu Gly Lys

450

<210> 81

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu32a-85 IgG4P heavy chain

<400> 81

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val 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> 82

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu33-6 IgG4P heavy chain

<400> 82

Lys 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

Ala 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 Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln 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 Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

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

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 83

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> formula HCDR 1Z 1is N or T

<220>

<221> misc_feature

<222> (5)..(5)

<223> Znn can be Asn or Thr.

<400> 83

Asn Asp Tyr Trp Znn

1 5

<210> 84

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 84

Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 85

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 85

Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr

1 5 10

<210> 86

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1 wherein Z2 is R or H, Z3 is N or H

<220>

<221> misc_feature

<222> (9)..(9)

<223> Znn can be Arg or His.

<220>

<221> misc_feature

<222> (14)..(14)

<223> Znn can be Asn or His.

<400> 86

Lys Ser Ser Gln Ser Leu Phe Tyr Znn Ser Asn Gln Lys Znn Ser Leu

1 5 10 15

Ala

<210> 87

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 87

Gly Ala Ser Thr Arg Glu Ser

1 5

<210> 88

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 88

Gln Gln Tyr Tyr Gly Tyr Pro Tyr Thr

1 5

<210> 89

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 89

Ser Tyr Trp Met His

1 5

<210> 90

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2 general formula

Wherein Z4 is selected from T, S, H and G, Z5 is selected from S, N and G, Z6 is selected from S, L and G, Z7 is selected from G, F, L

W and M, Z8 selected from A, P and T, Z9 selected from M, V, L and S

<220>

<221> misc_feature

<222> (3)..(3)

<223> Znn can be Thr, Ser, His or Gly.

<220>

<221> misc_feature

<222> (5)..(5)

<223> Znn can be Ser, Asn or Gly.

<220>

<221> misc_feature

<222> (6)..(6)

<223> Znn can be Ser, Leu or Gly.

<220>

<221> misc_feature

<222> (8)..(8)

<223> Znn can be Gly, Phe, Leu, Trp or Met.

<220>

<221> misc_feature

<222> (9)..(9)

<223> Znn can be Ala, Pro or Thr.

<220>

<221> misc_feature

<222> (10)..(10)

<223> Znn can be Met, Val, Leu or Ser.

<220>

<221> misc_feature

<222> (15)..(15)

<223> Znn can be Phe or Tyr.

<400> 90

Arg Ile Znn Pro Znn Znn Gly Znn Znn Znn Tyr Asn Glu Lys Znn Lys

1 5 10 15

Asn

<210> 91

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 91

Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210> 92

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 92

Arg Ala Ser Glu Ser Val Ser Ile His Gly Thr His Leu Met His

1 5 10 15

<210> 93

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2 general formula

Wherein Z17 is V or A, Z18 is Y, K or N, Z19 is selected from A, L and V, Z20 is selected from E, F, Y and A

<220>

<221> misc_feature

<222> (1)..(1)

<223> Znn can be Val or Ala.

<220>

<221> misc_feature

<222> (4)..(4)

<223> Znn can be Tyr, Lys or Asn.

<220>

<221> misc_feature

<222> (5)..(5)

<223> Znn can be Ala, Leu or Val.

<220>

<221> misc_feature

<222> (6)..(6)

<223> Znn can be Glu, Phe, Tyr or Ala.

<400> 93

Znn Ala Ser Znn Znn Znn Ser

1 5

<210> 94

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 94

Gln Gln Ser Phe Glu Asp Pro Leu Thr

1 5

<210> 95

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 95

Asp Gly Ser Ala Tyr Trp Ser

1 5

<210> 96

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1 general formula wherein Z1 is N or T, Z11 is S or D, Z12 is Y or K, Z13 is H or M

<220>

<221> misc_feature

<222> (1)..(1)

<223> Znn can be Ser or Asp.

<220>

<221> misc_feature

<222> (2)..(2)

<223> Znn can be Tyr or Lys.

<220>

<221> misc_feature

<222> (5)..(5)

<223> Znn can be His or Met.

<400> 96

Znn Znn Trp Met Znn

1 5

<210> 97

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2 general formula wherein Z14 is F or M, Z15 is R or V, Z16 is N or H

<220>

<221> misc_feature

<222> (1)..(1)

<223> Znn can be Phe or Met.

<220>

<221> misc_feature

<222> (4)..(4)

<223> Znn can be Arg or Val.

<220>

<221> misc_feature

<222> (10)..(10)

<223> Znn can be Asn or His.

<400> 97

Znn Ile Ser Znn Ala Gly Ser Thr Tyr Znn Thr Pro Ser Leu Lys Gly

1 5 10 15

<210> 98

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 98

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

1 5 10 15

<210> 99

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 99

Met Ile Ser Val Ala Gly Ser Thr Tyr His Thr Pro Ser Leu Lys Gly

1 5 10 15

<210> 100

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 100

Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 101

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 101

Arg Ile Ser Pro Ser Leu Gly Leu Ala Val Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 102

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 102

Arg Ile His Pro Ser Leu Gly Leu Pro Leu Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 103

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 103

Arg Ile Gly Pro Asn Leu Gly Trp Ala Met Tyr Asn Glu Lys Tyr Lys

1 5 10 15

Asn

<210> 104

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 104

Arg Ile Ser Pro Ser Ser Gly Met Ala Val Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 105

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 105

Arg Ile Ser Pro Gly Gly Gly Phe Thr Leu Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 106

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 106

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

1 5 10 15

Asn

<210> 107

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 107

Val Ala Ser Tyr Ala Ala Ser

1 5

<210> 108

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 108

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 109

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 109

Val Ala Ser Asn Val Phe Ser

1 5

<210> 110

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 110

Asp Lys Trp Met Met

1 5

<210> 111

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2 mVH: 9-2 murine heavy chain variable region sequences

<400> 111

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

1 5 10 15

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

20 25 30

Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 112

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2 mVL: 9-2 murine light chain variable region sequences

<400> 112

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

1 5 10 15

Glu Lys Val Ile Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Tyr Arg

20 25 30

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

35 40 45

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

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Val Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 113

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5-VH: 24D5 murine heavy chain variable region sequence

<400> 113

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Gln Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile His Pro Asn Ser Gly Gly Thr Ser Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 114

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5-VL: 24D5 murine light chain variable region sequence

<400> 114

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 115

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2 hVH- CDR graft

<400> 115

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 116

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2 hVL CDR graft

<400> 116

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr Arg

20 25 30

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

35 40 45

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

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

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

100 105 110

Lys

<210> 117

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D5 humanized heavy chain variable region VH.1

<400> 117

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile His Pro Asn Ser Gly Gly Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 118

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D5 humanized light chain variable region VL.1

<400> 118

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn 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 Asn

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 119

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2 hVH(T)

<400> 119

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 120

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2 hVL(H)

<400> 120

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr His

20 25 30

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

35 40 45

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

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

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

100 105 110

Lys

<210> 121

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24-D5 hVH(GF)

<400> 121

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 122

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> 24-D5 hVL

<400> 122

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn 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 Asn

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 123

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain variable region of antibody from HRP00049 mutation library, Z14 is F or M, Z15 is R or V, Z16

Is N or H

<220>

<221> misc_feature

<222> (52)..(52)

<223> Znn can be Phe or Met.

<220>

<221> misc_feature

<222> (55)..(55)

<223> Znn can be Arg or Val.

<220>

<221> misc_feature

<222> (61)..(61)

<223> Znn can be Asn or His.

<400> 123

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asp Gly

20 25 30

Ser Ala Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Tyr Ile Gly Znn Ile Ser Znn Ala Gly Ser Thr Tyr Znn Thr Pro Ser

50 55 60

Leu Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 124

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

Is N or H

<220>

<221> misc_feature

<222> (52)..(52)

<223> Znn can be Phe or Met.

<220>

<221> misc_feature

<222> (55)..(55)

<223> Znn can be Arg or Val.

<220>

<221> misc_feature

<222> (61)..(61)

<223> Znn can be Asn or His.

<400> 124

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Znn Ile Ser Znn Ala Gly Ser Thr Tyr Znn Thr Pro Ser Leu Lys

50 55 60

Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 125

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-PD-L1 antibody 24D 5L 67 light chain variable region sequence

<400> 125

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 126

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> general formula of heavy chain variable region sequence

Z11 is S or D, Z12 is Y or K, Z13 is H or M, Z4 is selected from T, S, H and G, Z5 is selected from S, N and G,

z6 is selected from S, L and G, Z'7 is selected from F, L, W and M, Z8 is selected from A, P and T, Z9 is selected from M, V, L and S

Z10 is F or Y

<220>

<221> misc_feature

<222> (31)..(31)

<223> Znn can be Ser or Asp.

<220>

<221> misc_feature

<222> (32)..(32)

<223> Znn can be Tyr or Lys.

<220>

<221> misc_feature

<222> (35)..(35)

<223> Znn can be His or Met.

<220>

<221> misc_feature

<222> (52)..(52)

<223> Znn can be Thr, Ser, His or Gly.

<220>

<221> misc_feature

<222> (54)..(54)

<223> Znn can be Ser, Asn or Gly.

<220>

<221> misc_feature

<222> (55)..(55)

<223> Znn can be Ser, Leu or Gly.

<220>

<221> misc_feature

<222> (57)..(57)

<223> Znn can be Phe, Leu, Trp or Met.

<220>

<221> misc_feature

<222> (58)..(58)

<223> Znn can be Ala, Pro or Thr.

<220>

<221> misc_feature

<222> (59)..(59)

<223> Znn can be Met, Val, Leu or Ser.

<220>

<221> misc_feature

<222> (64)..(64)

<223> Znn can be Phe or Tyr.

<400> 126

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Znn Znn

20 25 30

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

35 40 45

Gly Arg Ile Znn Pro Znn Znn Gly Znn Znn Glx Tyr Asn Glu Lys Znn

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 127

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> light chain variable region sequence general formula

Z17 is V or A, Z18 is Y, K or N, Z19 is selected from A, L and V, Z20 is selected from E, F, Y and A

<220>

<221> misc_feature

<222> (54)..(54)

<223> Znn can be Val or Ala.

<220>

<221> misc_feature

<222> (57)..(57)

<223> Znn can be Tyr, Lys or Asn.

<220>

<221> misc_feature

<222> (58)..(58)

<223> Znn can be Ala, Leu or Val.

<220>

<221> misc_feature

<222> (59)..(59)

<223> Znn can be Glu, Phe, Tyr or Ala.

<400> 127

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Znn Ala Ser Znn Znn Znn Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 128

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2H5 heavy chain variable region

<400> 128

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asp Gly

20 25 30

Ser Ala Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Tyr Ile Gly Phe Ile Ser Arg Ala Gly Ser Thr Tyr Asn Thr Pro Ser

50 55 60

Leu Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 129

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2H6 heavy chain variable region

<400> 129

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

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

50 55 60

Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 130

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> 9-2H7 heavy chain variable region

<400> 130

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Met Ile Ser Val Ala Gly Ser Thr Tyr His Thr Pro Ser Leu Lys

50 55 60

Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 131

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-PD-L1 antibody 24D 5H 21 heavy chain variable region sequence

<400> 131

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Asp Lys

20 25 30

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

35 40 45

Gly Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 132

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 12 heavy chain variable region

<400> 132

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 133

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 13 heavy chain variable region

<400> 133

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile Ser Pro Ser Leu Gly Leu Ala Val Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 134

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 14 heavy chain variable region

<400> 134

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile His Pro Ser Leu Gly Leu Pro Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 135

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 15 heavy chain variable region

<400> 135

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 136

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 16 heavy chain variable region

<400> 136

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ser Pro Ser Leu Gly Leu Ala Val Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 137

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 17 heavy chain variable region

<400> 137

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile Gly Pro Asn Leu Gly Trp Ala Met Tyr Asn Glu Lys Tyr

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 138

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 18 heavy chain variable region

<400> 138

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile Ser Pro Ser Ser Gly Met Ala Val Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 139

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 19 heavy chain variable region

<400> 139

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile Ser Pro Gly Gly Gly Phe Thr Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 140

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5H 20 heavy chain variable region

<400> 140

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 141

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5L 64 light chain variable region

<400> 141

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Val Ala Ser Tyr Ala Ala Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 142

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 142

Ala Ala Ser Lys Leu Glu Ser

1 5

<210> 143

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> 24D 5L 66 light chain variable region

<400> 143

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Phe Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 144

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> PD-1 antibody heavy chain amino acid sequence in form of IgG1

<400> 144

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 145

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> constructed PD-1 antibody heavy chain amino acid sequence in the form of IgG1

<400> 145

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 146

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<400> 146

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro Gly Ala

450 455

<210> 147

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> HRP 00049: 9-2(H2/L10) IgG4(AA) heavy chain

<400> 147

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 148

<211> 220

<212> PRT

<213> Artificial Sequence

<220>

<223> HRP00049 antibody light chain

<400> 148

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr His

20 25 30

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

35 40 45

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

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 149

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> HRP 00052: 24D5(GF) IGG4(AA) antibody heavy chain

<400> 149

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 150

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> HRP00052 antibody light chain sequence

<400> 150

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn 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 Asn

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 151

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> construction of heavy chain of PD-L1 antibody in IgG1 form

<400> 151

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 152

<211> 720

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of TJ021-PR0001 heavy chain (PD-1 heavy chain-second peptide linker-PD-L1 scFv)

<400> 152

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

465 470 475 480

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

485 490 495

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val

500 505 510

Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Gly Pro

515 520 525

Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe Lys Asn Arg Val Thr

530 535 540

Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser

545 550 555 560

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

565 570 575

Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

580 585 590

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

595 600 605

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

610 615 620

Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile

625 630 635 640

His Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro

645 650 655

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

660 665 670

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

675 680 685

Asn Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser

690 695 700

Phe Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

705 710 715 720

<210> 153

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of TJ021-PR0002 heavy chain (PD-1 heavy chain)

<400> 153

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 154

<211> 477

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of TJ021-PR0002 heavy chain (PD-L1 scFv-hinge region-CH 2-CH3)

<400> 154

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Val Ser Ile His Gly Thr His Leu Met His Trp Tyr Gln Gln

165 170 175

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

180 185 190

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

195 200 205

Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr

210 215 220

Tyr Cys Gln Gln Ser Phe Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr

225 230 235 240

Lys Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475

<210> 155

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of heavy chain of TJ021-PR0003 (PD-L1 heavy chain)

<400> 155

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 156

<211> 484

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of TJ021-PR0003 heavy chain (PD-1 scFv-hinge region-CH 2-CH3)

<400> 156

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

130 135 140

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

145 150 155 160

Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Thr Gly Asn Thr

165 170 175

Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu

180 185 190

Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser

195 200 205

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

210 215 220

Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro

225 230 235 240

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Glu Pro Lys Ser

245 250 255

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

420 425 430

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

435 440 445

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

450 455 460

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

465 470 475 480

Ser Pro Gly Lys

<210> 157

<211> 720

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of TJ021-PR0004 heavy chain (PD-L1 scFv-PD-1 heavy chain)

<400> 157

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Val Ser Ile His Gly Thr His Leu Met His Trp Tyr Gln Gln

165 170 175

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

180 185 190

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

195 200 205

Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr

210 215 220

Tyr Cys Gln Gln Ser Phe Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr

225 230 235 240

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

245 250 255

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

260 265 270

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

275 280 285

Ala Ser Gly Gly Thr Phe Ser Asp Tyr Glu Met His Trp Val Arg Gln

290 295 300

Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile Asp Pro Glu Thr

305 310 315 320

Gly Gly Thr Val Tyr Asn Gln Lys Phe Lys Asp Arg Val Thr Ile Thr

325 330 335

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

340 345 350

Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Arg Phe Ser Tyr

355 360 365

Tyr Gly Ser Thr Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr

370 375 380

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

385 390 395 400

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

405 410 415

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

420 425 430

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

435 440 445

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

450 455 460

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

465 470 475 480

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

485 490 495

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

<210> 158

<211> 720

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of TJ021-PR0005 heavy chain (PD-1 heavy chain-PD-L1 scFv)

<400> 158

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

465 470 475 480

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

485 490 495

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val

500 505 510

Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Gly Pro

515 520 525

Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe Lys Asn Arg Val Thr

530 535 540

Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser

545 550 555 560

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

565 570 575

Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

580 585 590

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

595 600 605

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

610 615 620

Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile

625 630 635 640

His Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro

645 650 655

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

660 665 670

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

675 680 685

Asn Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser

690 695 700

Phe Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

705 710 715 720

<210> 159

<211> 720

<212> PRT

<213> Artificial Sequence

<220>

<223> TJ021-PR0006 heavy chain (PD-L1 scFv-PD-1 heavy chain) amino acid sequence

<400> 159

Gln Val Gln Leu Val Gln Ser Gly Ala 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 Ser Tyr

20 25 30

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

35 40 45

Gly Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Val Ser Ile His Gly Thr His Leu Met His Trp Tyr Gln Gln

165 170 175

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

180 185 190

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

195 200 205

Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr

210 215 220

Tyr Cys Gln Gln Ser Phe Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr

225 230 235 240

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

245 250 255

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

260 265 270

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

275 280 285

Ala Ser Gly Gly Thr Phe Ser Asp Tyr Glu Met His Trp Val Arg Gln

290 295 300

Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile Asp Pro Glu Thr

305 310 315 320

Gly Gly Thr Val Tyr Asn Gln Lys Phe Lys Asp Arg Val Thr Ile Thr

325 330 335

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

340 345 350

Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Arg Phe Ser Tyr

355 360 365

Tyr Gly Ser Thr Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr

370 375 380

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

385 390 395 400

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

405 410 415

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

420 425 430

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

435 440 445

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

450 455 460

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

465 470 475 480

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

485 490 495

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

<210> 160

<211> 720

<212> PRT

<213> Artificial Sequence

<220>

<223> TJ021-PR0007 heavy chain (PD-1 heavy chain-PD-L1 scFv) amino acid sequence

<400> 160

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

465 470 475 480

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

485 490 495

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val

500 505 510

Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Thr Pro

515 520 525

Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe Lys Asn Arg Val Thr

530 535 540

Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser

545 550 555 560

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

565 570 575

Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

580 585 590

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

595 600 605

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

610 615 620

Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile

625 630 635 640

His Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro

645 650 655

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

660 665 670

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

675 680 685

Asn Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser

690 695 700

Phe Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

705 710 715 720

<210> 161

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 161

Val Ala Ser Asn Val Glu Ser

1 5

<210> 162

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 162

Val Ala Ser Asn Val Trp Ser

1 5

<210> 163

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 163

Val Ala Ser Asn Val Tyr Ser

1 5

<210> 164

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> second antigen-binding domain HCDR1

<400> 164

Asn Asp Tyr Trp Asn

1 5

<210> 165

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> second antigen binding domain LCDR1

<400> 165

Lys Ser Ser Gln Ser Leu Phe Tyr Arg Ser Asn Gln Lys Asn Ser Leu

1 5 10 15

Ala

<210> 166

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> second antigen-binding domain HCDR2

<400> 166

Arg Ile His Pro Asn Ser Gly Gly Thr Ser Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 167

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-PD-L1 antibody 24D 5L 68 light chain variable region sequence

<400> 167

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Trp Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 168

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-PD-L1 antibody 24D 5L 69 light chain variable region sequence

<400> 168

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Tyr Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 169

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 169

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 170

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 170

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn 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 Asn

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 171

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 171

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Lys 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 Asn

65 70 75 80

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

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 172

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 172

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Lys 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 Asn

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. An antigen binding molecule comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain specifically binds PD-1 and comprises a combination of CDRs selected from any one of (a) - (d):

(a) the sequence is shown as general formula DYEX₁H (SEQ ID NO: 65) as shown in HCDR1, with the sequence shown in general formula LX₂DPETGGX₃VYNQKFKX₄(SEQ ID NO: 66) HCDR2 having the sequence of general formula EX₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂HCDR3 as shown in DWYFDV (SEQ ID NO: 67); wherein, X₁Is I or M; x₂Is F or I; x₃Is I or T; x₄Is G or D; x₅Is G or R; x₆Is F or a vacancy; x₇Is S or a vacancy; x₈Is Y or a vacancy; x₉Is G or a vacancy; x₁₀Is S or nullLacking; x₁₁Is N or T; x₁₂Is R or S; and

the sequence is shown as the general formula RSSQSX₁₃VHSX₁₄X₁₅X₁₆LCDR1 shown by TYLE (SEQ ID NO: 68), LCDR2 with sequence KVSNRFS (SEQ ID NO: 12), and sequence shown as general formula FQGSHVPYX₁₇LCDR3 as shown (SEQ ID NO: 69); wherein, X₁₃Is I or L; x₁₄Selected from N, Q, L, T or D; x₁₅Selected from G, A or V; x₁₆Is N or K; x₁₇Is A or T;

(b) HCDR1, HCDR2 and HCDR3 having sequences SYAMS (SEQ ID NO: 21), TISGGGVDTYYQDNVQG (SEQ ID NO: 22) and PYGHGYFDV (SEQ ID NO: 23), respectively, and

LCDR1, LCDR2 and LCDR3 having sequences of RASQDINNFLN (SEQ ID NO: 24), YTSSLHS (SEQ ID NO: 25) and QQGNTLPWT (SEQ ID NO: 26), respectively;

(c) the sequence is shown as general formula DYEX₁H (SEQ ID NO: 65) as shown in HCDR1, with the sequence shown in general formula LX₂DPETGGX₃VYNQKFKX₄(SEQ ID NO: 66) HCDR2 having the sequence of general formula EX₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂HCDR3 as shown in DWYFDV (SEQ ID NO: 67); wherein, X₁Is I or M; x₂Is F or I; x₃Is I or T; x₄Is G or D; x₅Is G or R; x₆Is F or a vacancy; x₇Is S or a vacancy; x₈Is Y or a vacancy; x₉Is G or a vacancy; x₁₀Is S or a vacancy; x₁₁Is N or T; x₁₂Is R or S; and

LCDR1, LCDR2 and LCDR3 having sequences of RASQDINNFLN (SEQ ID NO: 24), YTSSLHS (SEQ ID NO: 25) and QQGNTLPWT (SEQ ID NO: 26), respectively; and

(d) HCDR1, HCDR2 and HCDR3 having sequences SYAMS (SEQ ID NO: 21), TISGGGVDTYYQDNVQG (SEQ ID NO: 22) and PYGHGYFDV (SEQ ID NO: 23), respectively, and

the sequence is shown as the general formula RSSQSX₁₃VHSX₁₄X₁₅X₁₆LCDR1 shown by TYLE (SEQ ID NO: 68), LCDR2 with sequence KVSNRFS (SEQ ID NO: 12), and sequence shown as general formula FQGSHVPYX₁₇LCDR3 as shown (SEQ ID NO: 69); wherein, X₁₃Is I or L; x₁₄Selected from N, Q, L, T or D; x₁₅Selected from G, A or V; x₁₆Is N or K; x₁₇Is A or T.

2. The antigen binding molecule of claim 1, the first antigen binding domain comprising:

HCDR1 of sequence DYEMH (SEQ ID NO: 8), HCDR2 of sequence LIDPETGGTVYNQKFKD (SEQ ID NO: 9), HCDR3 of sequence ERFSYYGSTSDWYFDV (SEQ ID NO: 10); and

LCDR2 with sequence KVSNRFS (SEQ ID NO: 12), LCDR3 with sequence FQGSHVPYT (SEQ ID NO: 13); the sequence is shown as the general formula RSSQSX₁₃VHSX₁₄X₁₅X₁₆TYLE (SEQ ID NO: 68), and wherein X₁₃Is L, X₁₄Selected from N, Q, L, T or D, X₁₅Selected from G, A or V, X₁₆LCDR1 of N.

3. The antigen binding molecule of claim 1 or 2, wherein the first antigen binding domain comprises a combination of CDRs selected from any one of (e) to (h):

(e) HCDR1, HCDR2 and HCDR3 having sequences of DYEMH (SEQ ID NO: 8), LIDPETGGTVYNQKFKD (SEQ ID NO: 9) and ERFSYYGSTSDWYFDV (SEQ ID NO: 10), respectively; and/or

LCDR2 with sequence KVSNRFS (SEQ ID NO: 12), LCDR3 with sequence FQGSHVPYT (SEQ ID NO: 13); LCDR1 having a sequence selected from RSSQSLVHSNGNTYLE (SEQ ID NO: 11), RSSQSLVHSQGNTYLE (SEQ ID NO: 47), RSSQSLVHSLGNTYLE (SEQ ID NO: 48), RSSQSLVHSTGNTYLE (SEQ ID NO: 49), RSSQSLVHSDGNTYLE (SEQ ID NO: 50), RSSQSLVHSNANTYLE (SEQ ID NO: 51) or RSSQSLVHSNVNTYLE (SEQ ID NO: 52);

(f) HCDR1, HCDR2 and HCDR3 having sequences of DYEIH (SEQ ID NO: 14), LFDPETGGIVYNQKFKG (SEQ ID NO: 15) and EGYNRDWYFDV (SEQ ID NO: 16), respectively, and/or

LCDR1, LCDR2 and LCDR3 having sequences of RSSQSIVHSNGNTYLE (SEQ ID NO: 17), KVSNRFS (SEQ ID NO: 12) and FQGSHVPYA (SEQ ID NO: 18), respectively;

(j) HCDR1, HCDR2 and HCDR3 and/or of sequences DYEIH (SEQ ID NO: 14), LFDPETGGIVYNQKFKG (SEQ ID NO: 15) and EGYNRDWYFDV (SEQ ID NO: 16), respectively

and

(h) HCDR1, HCDR2 and HCDR3 having sequences of DYEMH (SEQ ID NO: 8), LIDPETGGTVYNQKFKD (SEQ ID NO: 9) and ERFSYYGSTSDWYFDV (SEQ ID NO: 10), respectively; and/or

LCDR1, LCDR2 and LCDR3 having sequences of RSSQSIVHSNGNTYLE (SEQ ID NO: 17), KVSNRFS (SEQ ID NO: 12) and FQGSHVPYA (SEQ ID NO: 18), respectively.

4. The antigen binding molecule of any one of claims 1 to 3, the first antigen binding domain comprising a first heavy chain variable region and a first light chain variable region comprising a framework region or a framework region variant derived from a human antibody; the framework region variant is a back mutation having at most 11 amino acids in the heavy chain framework region and/or the light chain framework region of a human antibody, respectively,

preferably, the first and second electrodes are formed of a metal,

(i) the first light chain variable region comprises a 2G amino acid back mutation, and/or the first heavy chain variable region comprises one or more amino acid back mutations selected from 27Y, 48I, 67T, 69L, 82F and 93T;

(j) a 2V amino acid back mutation in the first light chain variable region, and/or one or more amino acid back mutations selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F, and 93T in the first heavy chain variable region; or

(k) The first light chain variable region comprises one or more amino acid back mutations selected from 42G, 44V and 71Y, and/or the first heavy chain variable region comprises 1K and/or 94S amino acid back mutations.

5. The antigen binding molecule of any one of claims 1 to 4, wherein the first antigen binding domain comprises a first heavy chain variable region and/or a first light chain variable region selected from any one of (l) to (r):

(l) The sequence is shown as SEQ ID NO:4 and/or the sequence of the first heavy chain variable region shown in SEQ ID NO: 5;

(m) has the sequence shown in SEQ ID NO: 6 and/or the sequence of the first heavy chain variable region shown in SEQ ID NO: 7;

(n) the sequence is as shown in SEQ ID NO:19 and/or the sequence of the first heavy chain variable region shown in SEQ ID NO: 20, a first light chain variable region;

(o) the sequence is as shown in SEQ ID NO: 27. 30, 31 or 32 and/or the sequence of the first heavy chain variable region is as shown in SEQ ID NO: 28. 29, 34, 35, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64;

(p) the sequence is as shown in SEQ ID NO: 33. 36, 37, 38, 39 or 40 and/or the first heavy chain variable region as set forth in SEQ ID NO: 34. 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64;

(q) the sequence is as shown in SEQ ID NO: 41. 45 or 46 and/or the sequence of the first heavy chain variable region shown in SEQ ID NO: 42. 43 or 44; and

(r) has the sequence shown in SEQ ID NO:70 and/or the sequence of the first heavy chain variable region shown in SEQ ID NO: 71.

6. The antigen binding molecule of claim 4 or 5, wherein the first antigen binding domain comprises:

(s) a first scFv comprising a first heavy chain variable region and a first light chain variable region covalently linked via a first peptide linker; preferably, the first peptide linker is a GS linker; more preferably, the first peptide linker has the following amino acid sequence: GGGGSGGGGSGGS;

(t) a first scFv-hinge region-CH 2-CH3, wherein the first scFv comprises a first heavy chain variable region and a first light chain variable region covalently linked via a first peptide linker; preferably, the first peptide linker is a GS linker; more preferably, the first peptide linker has the following amino acid sequence: GGGGSGGGGSGGS; also preferably, the hinge region-CH 2-CH3 is derived from human IgG1, IgG2, IgG3 or IgG4, more preferably from human IgG1, especially preferably from human IgG1 to which at least one mutation of L234A, L235A, T366S, L368A, Y407V and K447A is introduced, human IgG1 to which L234A, L235A, T366S, L368A and Y407V mutations is introduced, or human IgG4 to which at least one mutation of S228P, F234A and L235A is introduced; or

(u) a first heavy chain and a first light chain, wherein the first heavy chain comprises the first heavy chain variable region and a first heavy chain constant region comprising a CH 1-hinge region-CH 2-CH 3; preferably, CH 1-hinge region-CH 2-CH3 is derived from human IgG1, IgG2, IgG3, IgG4 or a conventional variant thereof, more preferably from human IgG1 or a conventional variant thereof, especially preferably from human IgG1 introduced with at least one mutation of L234A, L235A, T366S, L368A, Y407V and K447A, human IgG1 introduced with L234A, L235A, T366S, L368A and Y407V mutations, or human IgG4 introduced with at least one mutation of S228P, F234A and L235A; also preferably, the first heavy chain constant region has the amino acid sequence as set forth in SEQ ID NO: 72 or 79; wherein the first light chain comprises a first light chain variable region and a first light chain constant region, wherein the first light chain constant region is derived from a human antibody kappa chain constant region, lambda chain constant region, and conventional variants thereof; more preferably, the first light chain constant region has the amino acid sequence as set forth in SEQ ID NO: 73;

preferably, the first antigen-binding domain comprises a combination of a first heavy chain and a first light chain selected from any one of (u1) - (u 3):

(u1) and SEQ ID NO: 77 or 82, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and

and SEQ ID NO: 78, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity;

(u2) and SEQ ID NO: 74. 76, 80 or 82, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and

and SEQ ID NO: 75, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and

(u3) and SEQ ID NO: 144. 145 or 146, having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; and

and SEQ ID NO: 75, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in seq id no.

7. The antigen binding molecule of any one of claims 1 to 6, the second antigen binding domain specifically binds PD-L1;

preferably, the second antigen-binding domain comprises:

LCDR2 with the sequence GASTRES (SEQ ID NO: 87) or the general formula Z₁₇ASZ₁₈Z₁₉Z₂₀S (SEQ ID NO: 93), wherein Z₁₇Is V or A, Z₁₈Is Y, K or N, Z₁₉Selected from A, L and V, Z₂₀Selected from E, F and A;

8. The antigen binding molecule of any one of claims 1 to 7, wherein the second antigen binding domain comprises a combination of CDRs selected from any one of (aa) - (ah):

(ab) HCDR1 with sequence SYWMH (SEQ ID NO: 89), the sequence is shown as general formula RIZ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀KN(SEQ IDNO: 90) shown is HCDR2 wherein Z₄Selected from T, S, H and G, Z₅Selected from S, N and G, Z₆Selected from S, L and G, Z₇Selected from G, F, L, W and M, Z₈Selected from A, P and T, Z₉Selected from M, V, L and S, Z₁₀HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91) and F or Y; and/or

(ae) HCDR1 having the sequence DGSAYWS (SEQ ID NO: 95), HCDR2 having the sequence FISRAGSTYNTPSLKG (SEQ ID NO: 98) or MISVAGSTYHTPSLKG (SEQ ID NO: 99), and HCDR3 having the sequence SGGWLAPFDY (SEQ ID NO: 85); and/or

(af) sequences of the general formula NDYWZ₁(SEQ ID NO: 83) HCDR1 wherein Z₁HCDR2 of sequence FISRAGSTYNTPSLKG (SEQ ID NO: 98) or MISVAGSTYHTPSLKG (SEQ ID NO: 99) and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85) being N or T; and/or

(ag) HCDR1 with sequence SYWMH (SEQ ID NO: 89), and the sequence is shown in SEQ ID NO: 100-106, and HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

LCDR1 with sequence RASESVSIHGTHLMH (SEQ ID NO: 92), having the sequence shown in SEQ ID NO: LCDR2 as shown in any one of items 107-109 and LCDR3 having sequence QQSFEDPLT (SEQ ID NO: 94);

and

(ah) HCDR1 with sequence DKWMM (SEQ ID NO: 110), sequence as shown in SEQ ID NO: 100-106, and HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

LCDR1 with sequence RASESVSIHGTHLMH (SEQ ID NO: 92), having the sequence shown in SEQ ID NO: LCDR2 as shown in any one of items 107-109 and LCDR3 having sequence QQSFEDPLT (SEQ ID NO: 94).

9. The antigen binding molecule of any one of claims 1 to 8, the second antigen binding domain comprising:

HCDR1 of sequence SYWMH (SEQ ID NO: 89), HCDR2 of sequence RIGPNSGFTSYNEKFKN (SEQ ID NO: 106), and HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91); and LCDR1 of sequence RASESVSIHGTHLMH (SEQ ID NO: 92), LCDR2 of sequence AASKLES (SEQ ID NO: 142), and LCDR3 of sequence QQSFEDPLT (SEQ ID NO: 94);

or

HCDR1 of sequence SYWMH (SEQ ID NO: 89), HCDR2 of sequence RITPSSGFAMYNEKFKN (SEQ ID NO: 100), and HCDR3 of sequence GGSSYDYFDY (SEQ ID NO: 91); and LCDR1 of sequence RASESVSIHGTHLMH (SEQ ID NO: 92), LCDR2 of sequence AASKLES (SEQ ID NO: 142), and LCDR3 of sequence QQSFEDPLT (SEQ ID NO: 94).

10. The antigen binding molecule of any one of claims 1 to 9, wherein the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region comprising a framework region or a framework region variant derived from a human antibody,

preferably, the heavy chain FR sequence of the second heavy chain variable region is derived from the combined sequences of human germline heavy chain IGHV4-30-4 x 01 and hjh2, comprising the FR1, FR2, FR3 regions and the FR4 region of hjh2 of human germline heavy chain IGHV4-30-4 x 01; or a combined sequence derived from human germline heavy chain IGHV1-46 a 01 and hjh 6.1.1 comprising the FR1, FR2, FR3 region of human germline heavy chain IGHV1-46 a 01 and the FR4 region of hjh 6.1.1;

the light chain FR sequence of the second light chain variable region is derived from the combined sequences of human germline light chains IGKV4-1 × 01 and hjk4.1, comprising the FR1, FR2, FR3 regions of human germline light chain IGKV4-1 × 01 and FR4 regions of hjk 4.1; alternatively, a combined sequence derived from human germline light chains IGKV7-3 x 01 and hjk2.1 comprising the FR1, FR2, FR3 regions of human germline light chain IGKV7-3 x 01 and the FR4 region of hjk 2.1; more preferably, wherein the framework region variant is a back mutation having at most 10 amino acids in the heavy chain framework region and/or the light chain framework region, respectively, of a human antibody, such as:

(ai) one or more amino acid back-mutations selected from W47Y, V71R, G27Y, I48M, V67L, F78Y, S30T, Q39K and P49S, more preferably one or more amino acid back-mutations selected from W47Y, V71R and P49S, in the second heavy chain variable region;

(aj) one or more amino acid back-mutations selected from T74K, R72V, M48I, M70L, R38Q, L83F, V68A, V79A, Y91F, T22S and G72E in the second heavy chain variable region; or

(ak) the second heavy chain variable region comprises an N85E deglycosylation mutation.

11. The antigen binding molecule according to any one of claims 1 to 10, the second antigen binding domain comprising a second heavy chain variable region and/or a second light chain variable region selected from any one of (al) to (bc):

(al) the sequence is shown in SEQ ID NO: 111 and/or the sequence of the second heavy chain variable region shown in SEQ ID NO: 112, a second light chain variable region;

(am) sequence as set forth in SEQ ID NO: 113 and/or the sequence of the second heavy chain variable region shown in SEQ ID NO: a second light chain variable region as shown at 181;

(an) has the sequence shown in SEQ ID NO: 115 and/or the sequence of the second heavy chain variable region shown in SEQ ID NO: 116, a second light chain variable region;

(ao) has a sequence shown in SEQ ID NO: 117 is as set forth in SEQ ID NO: 170;

(ap) sequence as shown in SEQ ID NO: 119 and/or a second heavy chain variable region and/or a sequence as set forth in SEQ ID NO: 120, a second light chain variable region;

(aq) sequence as shown in SEQ ID NO: 121 and/or a second heavy chain variable region and/or a sequence as set forth in SEQ ID NO: 183;

(ar) the sequence is as shown in SEQ ID NO: 123 and/or the second heavy chain variable region of SEQ ID NO: 120, a second light chain variable region;

(as) the sequence is as shown in SEQ ID NO: 124 and/or the second heavy chain variable region of SEQ ID NO: 120, a second light chain variable region;

(at) the sequence is as shown in SEQ ID NO: 126 and/or the second heavy chain variable region of SEQ ID NO: 127;

(au) the sequence is as shown in SEQ ID NO: 128 and/or the second heavy chain variable region of SEQ ID NO: 120, a second light chain variable region;

(av) is as shown in SEQ ID NO: 129 and/or the second heavy chain variable region of SEQ ID NO: 120, a second light chain variable region;

(aw) the sequence is as shown in SEQ ID NO: 130 and/or the second heavy chain variable region of SEQ ID NO: 120, a second light chain variable region;

(ax) the sequence is as shown in SEQ ID NO: 132 and/or the second heavy chain variable region of SEQ ID NO: 141;

(ay) the sequence as set forth in SEQ ID NO: 132 and/or the second heavy chain variable region of SEQ ID NO: 122;

(az) has a sequence shown in SEQ ID NO: 132 and/or the second heavy chain variable region of SEQ ID NO: 143;

(ba) sequences as shown in SEQ ID NO: 133 and/or the second heavy chain variable region of SEQ ID NO: 141 and 143;

(bb) the sequence is shown in SEQ ID NO: 134 and/or the second heavy chain variable region of SEQ ID NO: 141 and 143; and

(bc) is as shown in SEQ ID NO: 135-140 and/or the second heavy chain variable region as set forth in any one of SEQ ID NOs: 141 and 143.

12. The antigen binding molecule of claim 10 or 11, the second antigen binding domain comprising:

(bd) a second scFv comprising a second heavy chain variable region and a second light chain variable region covalently linked via a first peptide linker; preferably, the first peptide linker is a GS linker; more preferably, the first peptide linker has the following amino acid sequence: GGGGSGGGGSGGS;

(be) a second scFv-hinge region-CH 2-CH3, wherein the second scFv comprises a second heavy chain variable region and a second light chain variable region covalently linked via a first peptide linker; preferably, the first peptide linker is a GS linker; more preferably, the first peptide linker has the following amino acid sequence: GGGGSGGGGSGGS; also preferably, the hinge region-CH 2-CH3 is derived from human IgG1, IgG2, IgG3 or IgG4, more preferably from human IgG1, especially preferably from human IgG1 introduced with at least one mutation of L234A, L235A, T366S or T366W, L368A, Y407V and K447A, human IgG1 introduced with L234A, L235A, T366S or T366W, L368A and Y407V mutations, or human IgG4 introduced with at least one mutation of S228P, F234A and L235A; or

(bf) a second heavy chain and a second light chain, wherein the second heavy chain comprises the second heavy chain variable region and a second heavy chain constant region comprising CH 1-hinge region-CH 2-CH 3; preferably, CH 1-hinge region-CH 2-CH3 is derived from human IgG1, IgG2, IgG3, IgG4 or a conventional variant thereof, more preferably from human IgG1 or a conventional variant thereof, especially preferably from human IgG1 introduced with at least one mutation of L234A, L235A, T366S, L368A, Y407V and K447A, human IgG1 introduced with L234A, L235A, T366S, L368A and Y407V mutations, or human IgG4 introduced with at least one mutation of S228P, F234A and L235A; also preferably, the second heavy chain constant region has the sequence as set forth in SEQ ID NO: 72 or 79; wherein the second light chain comprises a second light chain variable region and a second light chain constant region, wherein the second light chain constant region is derived from a human antibody kappa chain constant region, lambda chain constant region, and conventional variants thereof; more preferably, the second light chain constant region has the sequence as set forth in SEQ ID NO: 73;

preferably, the second antigen-binding domain comprises a combination of a second heavy chain and a second light chain selected from any one of (bf1) - (bf 3):

(bf1) and SEQ ID NO: 147, having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; and

and SEQ ID NO: 148, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical;

(bf2) and SEQ ID NO: 149, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; and

and SEQ ID NO: 172, a second light chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in seq id no;

and

(bf3) and SEQ ID NO: 151, having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; and

and SEQ ID NO: 172, a second light chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

13. The antigen binding molecule of claim 6 or 12, comprising a combination of polypeptides selected from any one of (aaa) - (iii):

a second polypeptide comprising a first light chain;

a second polypeptide comprising a second light chain;

(eee) a first polypeptide comprising a first heavy chain;

a second polypeptide comprising a second scFv-hinge region-CH 2-CH 3; and

a third polypeptide comprising a first light chain;

preferably, the hinge region-CH 2-CH3 is derived from human IgG1 introduced with L234A, L235A and T366W mutations;

(fff) a first polypeptide comprising a second heavy chain;

a second polypeptide comprising a first scFv-hinge region-CH 2-CH 3; and

a third polypeptide comprising a second light chain;

(ggg) a first polypeptide comprising a first scFv-hinge region-CH 2-CH 3; and

a second polypeptide comprising a second scFv-hinge region-CH 2-CH 3;

and

a second polypeptide comprising a first light chain; and

a third polypeptide comprising a second light chain;

and

a second polypeptide comprising a first light chain; and

a third polypeptide comprising a second light chain;

preferably, the second peptide linker is a GS linker; more preferably, the second peptide linker has the following amino acid sequence: GGGGSGGGGSGGGGSGGGGS.

14. The antigen binding molecule of claim 13, comprising a combination of polypeptides selected from any one of (jjj) - (ppp):

(jjjj) and SEQ ID NO: 152, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; and

and SEQ ID NO: 75, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical;

(kkk) and SEQ ID NO: 153, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical;

and SEQ ID NO: 154, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity;

(lll) and SEQ ID NO: 155, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical;

and SEQ ID NO: 145, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and

and SEQ ID NO: 156, is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical;

(mmm) and SEQ ID NO: 157, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and

(nnn) and SEQ ID NO: 158, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and

(ooo) and SEQ ID NO: 159, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; and

and

(ppp) comparison to SEQ ID NO: 160, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and

and SEQ ID NO: 75, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

15. A pharmaceutical composition comprising an antigen binding molecule according to any one of claims 1 to 14 and at least one pharmaceutically acceptable carrier, diluent, buffer or excipient; alternatively, the first and second electrodes may be,

the pharmaceutical composition comprises a first antibody comprising a first antigen-binding domain as defined in any one of claims 1 to 14 and a second antibody comprising a second antigen-binding domain as defined in any one of claims 1 to 14;

preferably, the pharmaceutical composition further comprises a chemotherapeutic agent, a radiopharmaceutical agent and/or other agent for cancer immunotherapy.

16. A nucleic acid composition comprising at least one nucleic acid molecule encoding the antigen binding molecule of any one of claims 1-14.

17. An expression vector composition comprising at least one expression vector comprising at least one nucleic acid molecule as defined in claim 16.

18. A host cell comprising the vector composition of claim 17, or transformed or transfected with the vector composition of claim 17; preferably, the host cell is selected from bacteria such as e.coli, yeast such as pichia, insect cells, and mammalian cells such as CHO cells, NSO cells, HEK293 cells or COS cells.

19. A method of making an antigen binding molecule according to any one of claims 1 to 14, comprising:

culturing a host cell as defined in claim 16 under conditions such that the antigen binding molecule is expressed; and

recovering the antigen binding molecule;

preferably, the method comprises a purification step of the antigen binding molecule.

20. A method of treating and/or preventing a disease, the method comprising administering to a subject in need thereof an effective amount of the antigen binding molecule of any one of claims 1 to 14, the pharmaceutical composition of claim 15, the nucleic acid composition of claim 16, or the expression vector composition of claim 17;

wherein the disease is preferably a proliferative disease, more preferably a tumor or cancer; more preferably the tumor or cancer is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative tumors, 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 disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, renal cancer, bladder cancer, and intestinal and colon cancer;

preferably, the disease is associated with PD-1 and/or PD-L1.