CN113637075B

CN113637075B - Bispecific antigen binding molecules and medical uses thereof

Info

Publication number: CN113637075B
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: 2024-04-16
Anticipated expiration: 2041-04-26
Also published as: CN113637075A

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 specific CDRs. The disclosure also relates to pharmaceutical compositions comprising a first antigen binding domain and a second antigen binding domain, or pharmaceutical compositions comprising said antigen binding molecules. The present 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 biological medicine, and in particular 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 merely provides 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 found in 1992 and is involved in the apoptosis process of cells. PD-l belongs to the CD28 family, has 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (cytotoxic T Iymphocyte antigen, CTLA-4), but 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, respectively. PD-L1 is expressed predominantly on T cells, B cells, macrophages and dendritic cells (DENDRITIC CELL, DC) and expression on activated cells can be up-regulated. Whereas PD-L2 expression is relatively limited, primarily on antigen presenting cells, such as activated macrophages and dendritic cells.

PD-L1 inhibits the immune system by binding to PD-1 and B7-1, and many tumor cells and immune cells of the tumor tissue microenvironment express PD-L1. New studies have found that high PD-L1 protein expression is detected in human tumor tissues such as breast cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, intestinal cancer, kidney cancer, melanoma, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, liver cancer, and the like, and that the expression level of PD-L1 is closely correlated with the clinical and prognostic properties of patients.

The anti-PD-1 monoclonal antibody can furthest improve the immune system response of a patient to tumors by blocking the combination of PD-1/PD-L1, thereby achieving the purpose of killing tumor cells.

PD-1 and PD-L1 bispecific antibodies (BsAb) are a class of combinatorial antibodies with amphiphilicity, usually bivalent (also tetravalent and hexavalent), i.e.having two antigen binding arms, with the function of binding two different specific antigens. The antibody will bind both to an Antigen Presenting Cell (APC) or tumor cell expressing PD-L1 and to a T cell expressing PD-1. The bifunctional antibody has the following three functions: 1) Bispecific antibodies can antagonize PD-1/PD-L1 pathway-mediated T cell function inhibition; 2) Different from PD-1/PD-L1 monoclonal antibody, the double antibody promotes T cells, DC cells and tumor cells to form signal synapses by combining with PD-1/PD-L1, so that on one hand, activation of the T cells by the DC cells is promoted, and on the other hand, killing of the tumor cells by the T cells is promoted. 3) The double antibody can promote infiltration of T cells to PD-L1 high-expression tumor parts theoretically by combining the T cells and the PD-L1 high-expression tumor cells. The mechanisms of these three aspects ultimately promote recognition and killing of tumor cells by the immune system. The anti-PD-1/PD-L1 bispecific antibody LY3434172 (also known as IBI-318) of Eli Lilly corporation is currently in stage I clinical stage of tumors or cancers such as non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, urothelial cell carcinoma, renal cell carcinoma, gastric cancer, esophageal cancer, colon cancer, cholangiocarcinoma, and other advanced solid tumors, and related antibodies are described in patent application US20190010232 A1.

The present disclosure found that although some anti-PD-1 antibodies responded at rates (ORR) as high as 90% in hematological tumors, the response rate was only around 30% in advanced non-small cell lung cancer and only 14% in medium and advanced esophageal and gastric cancers alone. Thus, there is still a need to develop new anti-PD-1 antibodies, in particular bispecific antigen binding molecules formed from such anti-PD-1 antibodies and a second antigen binding molecule, and pharmaceutical combinations of such antibodies and second antigen binding molecules.

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 the following i) to iii):

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

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

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

In some embodiments, the foregoing anti-PD-1 antibodies of the present disclosure, or antigen-binding fragments thereof, bind to human PD-1 with a dissociation equilibrium constant of equal to or less than 10 ^-7 M. In some embodiments, the binding to human PD-1 occurs with a dissociation equilibrium constant equal to or less than 10 ^-8M、 10^-9M、10^-10 M or 10 ^-11 M.

In some alternative embodiments, the present disclosure provides an anti-PD-1 antibody, or antigen-binding fragment thereof, the heavy chain variable region of which comprises: the sequence is shown in SEQ ID NO:65, and the sequence of the HCDR1 is shown as SEQ ID NO: 66, and HCDR2 having the sequence set forth in SEQ ID NO: HCDR3 as shown at 67; the light chain variable region comprises: the sequence is shown in SEQ ID NO:68, and the sequence of LCDR1 is shown as SEQ ID NO:12, and LCDR2 having the sequence set forth in SEQ ID NO: LCDR3 as indicated at 69; the sequences are shown in Table 1 below:

TABLE 1

In some alternative embodiments, the aforementioned anti-PD-1 antibody, or antigen-binding fragment thereof, the heavy chain variable region comprises a sequence as set forth in SEQ ID NO:8, and the sequence of the HCDR1 is shown as SEQ ID NO:9, and HCDR2 having the sequence shown in SEQ ID NO: HCDR3 as shown in fig. 10; the light chain variable region comprises a sequence as set forth in SEQ ID NO:12, and the sequence of LCDR2 is shown as SEQ ID NO:13, and the sequence is represented by the general formula RSSQSX ₁₃VHSX₁₄X₁₅X₁₆ TYLE (SEQ ID NO: 68), and wherein X ₁₃ is selected from L, X ₁₄ is selected from N, Q, L, T or D, X ₁₅ is selected from G, A or V, and X ₁₆ is selected from N LCDR1.

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 sequences set forth in SEQ ID NOs: 8. SEQ ID NO:9 and SEQ ID NO:10, and the sequences of HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NOs: 12 and SEQ ID NO:13, and LCDR2 and LCDR3 as set forth in SEQ ID NO: 11. 47, 48, 49, 50, 51 or 52;

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

(c) An anti-PD-1 antibody or antigen-binding fragment thereof comprising the sequences set forth in SEQ ID NOs: 21. SEQ ID NO:22 and SEQ ID NO:23, and HCDR1, HCDR2, and HCDR3, and sequences are 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 sequences set forth in SEQ ID NOs: 14. SEQ ID NO:15 and SEQ ID NO:16, and HCDR1, HCDR2, and HCDR3, and sequences are set forth in SEQ ID NOs: 12 and SEQ ID NO:13 and LCDR2 and LCDR3 as set forth in SEQ ID NO: 11. 47, 48, 49, 50, 51 or 52; and

(E) The heavy chain variable region comprises the sequences as set forth in SEQ ID NOs: 8. SEQ ID NO:9 and SEQ ID NO:10, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the sequences as set forth in SEQ ID NOs: 17. SEQ ID NO:12 and SEQ ID NO:18 LCDR1, LCDR2 and LCDR3.

In some alternative embodiments, 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 iv) to vi) below:

iv) an anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region of which comprises a sequence identical to the sequence set forth in SEQ ID NO: 4, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO:5 has the same sequence of LCDR1, LCDR2 and LCDR3;

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

Vi) an anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region of which comprises a sequence identical to the sequence set forth in SEQ ID NO: 19, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO:20 have the same sequence of LCDR1, LCDR2 and LCDR3.

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

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

(g) The light chain variable region comprises a 2V amino acid back mutation, and/or the heavy chain variable region comprises one or more amino acid back mutations selected from the group consisting of 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F, and 93T; 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 anti-PD-1 antibody or antigen-binding fragment thereof described above, 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 in SEQ ID NO:4 and/or the heavy chain variable region shown in SEQ ID NO:5, a light chain variable region shown in seq id no;

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

(k) The heavy chain variable region shown in SEQ ID NO. 19 and/or the heavy chain variable region shown in SEQ ID NO: 20. the light chain variable region shown;

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

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

(n) the heavy chain variable region shown in SEQ ID NO 41, 45 or 46 and/or the heavy chain variable region shown 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 SEQ ID NO. 71; and

(P) the sequence is as shown in SEQ ID NO: 27. 30, 31 or 32 and/or the heavy chain variable region 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 antibodies, or antigen-binding fragments thereof, wherein the antibodies further comprise 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 antibody kappa and lambda chain constant regions and conventional variants thereof; in other embodiments, the antibody constant region comprises an IgG4 heavy chain constant region that introduces one or more mutations in S228P, F a and L235A, e.g., three amino acid mutations of S228P, F234A and L235A; in other embodiments, the antibody comprises a sequence as set forth in SEQ ID NO:72 or as set forth in SEQ ID NO:79 and a heavy chain constant region of SEQ ID NO: 73.

In some embodiments, the foregoing anti-PD-1 antibodies, or antigen-binding fragments thereof, wherein the anti-PD-1 antibodies comprise the 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 a light chain as set forth in SEQ ID NO: 74. 76, 80 or 81.

In some embodiments, the foregoing 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') ₂, single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), and antigen-binding fragment of a CDR-comprising peptide.

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

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

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

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

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

In some embodiments, the 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 foregoing anti-PD-1 antibody or antigen-binding fragment thereof or the foregoing isolated monoclonal antibody or antigen-binding fragment thereof in the manufacture of a diagnostic agent for a PD-1 related disease.

In some embodiments, the present disclosure also provides a method of treating a PD-1-related disease, the method comprising administering to a subject a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of the preceding claims, 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 disclosure also provides the use of the foregoing anti-PD-1 antibody or antigen-binding fragment thereof, or the foregoing isolated monoclonal antibody or antigen-binding fragment thereof, or the foregoing pharmaceutical composition, or the foregoing nucleic acid molecule, for the preparation or prevention of a PD-1 related disease; in some embodiments, the disclosure further provides an anti-PD-1 antibody or antigen-binding fragment thereof, or an isolated monoclonal antibody or antigen-binding fragment thereof, or a nucleic acid molecule, or a pharmaceutical composition, as described in any of the preceding claims for use as a medicament; in some 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 the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, parathyroid 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 neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and mecell 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, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia; in other embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, kidney cancer, bladder cancer, bowel cancer and colon cancer. In some embodiments, the disease may be PD-1 and/or PD-L1 related.

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 to PD-1, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3 of an anti-PD-1 antibody of the disclosure or antigen binding fragment thereof as previously described.

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) below:

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

ii) sequences are shown in SEQ ID NO: 14. 15, 16, and/or HCDR1, HCDR2, HCDR3, and/or HCDR3 having the sequence shown in SEQ ID NO: 17. LCDR1, LCDR2, LCDR3 shown in fig. 12, 18;

iii) The sequences are shown in SEQ ID NO: 21. 22, 23, and/or HCDR1, HCDR2, HCDR3, and/or HCDR3 having the sequence of 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 amino acid differences such that the variant is still substantially the same as i) to iii);

v) the sequence is of the general formula DYEX ₁ H (SEQ ID NO:65 HCDR1 of the general formula LX ₂DPETGGX₃VYNQKFKX₄ (SEQ ID NO:66 HCDR2 represented by the general formula EX₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂DWYFDV(SEQ ID NO：67), HCDR3 represented by the sequence; wherein X ₁ is I or M; x ₂ is F or I; x ₃ is I or T; x ₄ is G or D; x ₅ is G or R; x ₆ is F or a vacancy; x ₇ is S or a vacancy; x ₈ is Y or a void; x ₉ is G or a void; x ₁₀ is S or a vacancy; x ₁₁ is N or T; x ₁₂ is R or S; and

LCDR1 of the sequence formula RSSQSX ₁₃VHSX₁₄X₁₅X₁₆ TYLE (SEQ ID NO: 68), LCDR2 of the sequence KVSNSRFS (SEQ ID NO: 12), and LCDR3 of the sequence formula FQGSHVPYX ₁₇ (SEQ ID NO: 69); wherein X ₁₃ is I or L; x ₁₄ is selected from N, Q, L, T or D; x ₁₅ is selected from G, A or V; x ₁₆ is N or K; x ₁₇ is A or T; or (b)

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

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

LCDR1 of the general formula RSSQSX ₁₃VHSX₁₄X₁₅X₁₆ TYLE (SEQ ID NO: 68) has the sequence SEQ ID NO:12, LCDR3 having a sequence according to formula FQGSHVPYX ₁₇ (SEQ ID NO: 69); wherein X ₁₃ is I or L; x ₁₄ is selected from N, Q, L, T or D; x ₁₅ is selected from G, A or V; x ₁₆ is N or K; x ₁₇ is A or T;

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

LCDR1 of the general formula RSSQSX ₁₃VHSX₁₄X₁₅X₁₆ TYLE (SEQ ID NO: 68), and wherein X ₁₃ is L, X ₁₄ is selected from N, Q, L, T or D, X ₁₅ is selected from G, A or V, and X ₁₆ is N; the sequences are shown in SEQ ID NO: 12. LCDR2, LCDR3 shown in fig. 13;

viii) sequences are set forth in SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown in figures 9 and 10; and/or

The sequence is shown in SEQ ID NO: 11. 47-52, the sequence of which is shown in SEQ ID NO: 12. LCDR2, LCDR3 shown in fig. 13;

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

The sequence is shown in SEQ ID NO: 11. 47-52, the sequence of which is shown in SEQ ID NO: 12. LCDR2, 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 framework regions or framework region variants derived from a human antibody, wherein the framework region variants are back mutations having up to 11 amino acids in the heavy chain framework region and/or the light chain framework region, respectively, of the human antibody.

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

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

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

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 in SEQ ID NO:4 and/or the first VH and/or the sequence shown in SEQ ID NO:5, a first VL;

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

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

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

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

j) The sequence is shown in 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 in SEQ ID NO:70 and/or the first VH and/or the sequence shown in SEQ ID NO:71, a first VL.

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-CH3; (e.g., the sequence 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;, 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 IgG1; may have a mutation, for example, human IgG1 with at least one mutation of L234A, L235A, T366S or T366W, L368A, Y V and K447A, human IgG1 with a mutation of L234A, L235A, T366S or T366W, L a and Y407V, or human IgG4 with at least one mutation of S228P, F234A and L235A.

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 (u 1) - (u 4):

(u 1) the sequence of which is shown in SEQ ID NO:77 or 82; the sequence is shown as SEQ ID NO: 78, a first light chain indicated by 78;

(u 2) the sequence of which is shown in SEQ ID NO: 74. 76, 80 or 81; the sequence is shown as SEQ ID NO: 75. And

(U 3) the sequence of which is shown in SEQ ID NO: 144. 145 or 146; the sequence is shown as SEQ ID NO: 75.

(U 4) 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 the group consisting of: NDYWZ ₁ (SEQ ID NO: 83), SYWMH (SEQ ID NO: 89), DGSAYWS (SEQ ID NO: 95) and Z ₁₁Z₁₂WMZ₁₃ (SEQ ID NO: 96), wherein Z ₁ is N or T, Z ₁₁ is S or D, Z ₁₂ is Y or K, Z ₁₃ is H or M;

HCDR2 having a sequence ：YISYTGSTYYNPSLKS(SEQ ID NO：84), RIZ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀KN(SEQ ID NO：90) and Z ₁₄ISZ₁₅AGSTYZ₁₆ TPSLKG (SEQ ID NO: 97) selected from wherein Z ₄ is selected from T, S, H and G, Z ₅ is selected from S, N and G, Z ₆ is selected from S, L and G, Z ₇ is selected from G, F, L, W and M, Z ₈ is selected from A, P and T, Z ₉ is selected from M, V, L and S, Z ₁₀ is F or Y, Z ₁₄ is F or M, Z ₁₅ is R or V, Z ₁₆ is N or H;

HCDR3, which has the sequence SGGWLAPFDY (SEQ ID NO: 85) or GGSSYDYFDY (SEQ ID NO: 91); and

LCDR1, the sequence of which is shown in the general formula KSSQSLFYZ ₂SNQKZ₃ SLA (SEQ ID NO: 86) or RASESVSIHGTHLMH (SEQ ID NO: 92), wherein Z ₂ is R or H and Z ₃ is N or H;

LCDR2, having a sequence GASTRES (SEQ ID NO: 87) or as shown in general formula Z ₁₇ASZ₁₈Z₁₉Z₂₀ S (SEQ ID NO: 93), wherein Z ₁₇ is V or A, Z ₁₈ is Y, K or N, Z ₁₉ is selected from A, L and V, and Z ₂₀ is selected 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) HCDR1 of sequence formula NDYWZ ₁ (SEQ ID NO: 83), wherein Z ₁ is N or T, HCDR2 of sequence YISYTGSTYYNPSLKS (SEQ ID NO: 84), and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85); and/or

LCDR1 of the sequence shown in formula KSSQSLFYZ ₂SNQKZ₃ SLA (SEQ ID NO: 86), wherein Z ₂ is R or H, Z ₃ is N or H, LCDR2 of the sequence GASTRES (SEQ ID NO: 87), and LCDR3 of the sequence QQYYGYPYT (SEQ ID NO: 88);

(ab) HCDR1 having the sequence SYWMH (SEQ ID NO: 89), HCDR2 having the sequence shown in general formula RIZ ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀ KN (SEQ ID NO: 90), wherein Z ₄ is selected from T, S, H and G, Z ₅ is selected from S, N and G, Z ₆ is selected from S, L and G, Z ₇ is selected from G, F, L, W and M, Z ₈ is selected from A, P and T, Z ₉ is selected from M, V, L and S, Z ₁₀ is F or Y, and HCDR3 having the sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

LCDR1 of sequence RASESVSIHGTHLMH (SEQ ID NO: 92), LCDR2 of sequence Z ₁₇ASZ₁₈Z₁₉Z₂₀ S (SEQ ID NO: 93), wherein Z ₁₇ is V or a, Z ₁₈ is Y, K or N, Z ₁₉ is selected from A, L and V, Z ₂₀ is selected from E, F and a, and LCDR3 of sequence QQSFEDPLT (SEQ ID NO: 94);

(ac) HCDR1 of sequence formula NDYWZ ₁ (SEQ ID NO: 83), wherein Z ₁ is N or T, HCDR2 of sequence YISYTGSTYYNPSLKS (SEQ ID NO: 84), and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85); and/or

(ad) HCDR1 having the sequence SYWMH (SEQ ID NO: 89), HCDR2 having the sequence shown in general formula RIZ ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀ KN (SEQ ID NO: 90), wherein Z ₄ is selected from T, S, H and G, Z ₅ is selected from S, N and G, Z ₆ is selected from S, L and G, Z ₇ is selected from G, F, L, W and M, Z ₈ is selected from A, P and T, Z ₉ is selected from M, V, L and S, Z ₁₀ is F or Y, and HCDR3 having the sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

(ae) HCDR1 of sequence DGSAYWS (SEQ ID NO: 95) or NDYWZ ₁ (SEQ ID NO: 83), HCDR2 of sequence formula Z ₁₄ISZ₁₅AGSTYZ₁₆ TPSLKG (SEQ ID NO: 97), and HCDR3 of sequence SGGWLAPFDY (SEQ ID NO: 85), wherein Z ₁ is N or T, Z ₁₄ is F or M, Z ₁₅ is R or V, and Z ₁₆ is N or H; and/or

(af) a HCDR1 of the general formula Z ₁₁Z₁₂WMZ₁₃ (SEQ ID NO: 96), wherein Z ₁₁ is S or D, Z ₁₂ is Y or K, and Z ₁₃ is H or M; HCDR2 of sequence shown in general formula RIZ ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀ KN (SEQ ID NO: 90), wherein Z ₄ is selected from T, S, H and G, Z ₅ is selected from S, N and G, Z ₆ is selected from S, L and G, Z ₇ is selected from G, F, L, W and M, Z ₈ is selected from A, P and T, Z ₉ is selected from M, V, L and S, Z ₁₀ is F or Y; the sequence is SEQ ID NO: HCDR3 of 91; and/or

The sequence is SEQ ID NO: LCDR1 of 92; LCDR2 having a sequence of formula Z ₁₇ASZ₁₈Z₁₉Z₂₀ S (SEQ ID NO: 93), wherein Z ₁₇ is V or A, Z ₁₈ is Y, K or N, Z ₁₉ is selected from A, L and V, and Z ₂₀ is selected from E, F, Y and A; the sequence is SEQ ID NO:94 LCDR3;

(ag) 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

(ah) HCDR1 of sequence formula NDYWZ ₁ (SEQ ID NO: 83), wherein Z ₁ is N or T, 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

(ai) HCDR1 having the sequence SYWMH (SEQ ID NO: 89) as set forth in SEQ ID NO: 100-106, and HCDR3 having the sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

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

And

(Aj) HCDR1 having the sequence DKWMM (SEQ ID NO: 110), having the sequence set forth in SEQ ID NO: 100-106, and HCDR3 having the sequence GGSSYDYFDY (SEQ ID NO: 91); and/or

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

SEQ ID NO:100-109, 161-163 are shown below:

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 (ak) - (ap) selected from the group consisting of:

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

(al) SEQ ID NO: 89. RIHPNSGGTSYNEKFKN (SEQ ID NO: 166), SEQ ID NO:91, HCDR1, HCDR2, HCDR3, and/or an amino 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) the sequence set forth in SEQ ID NO: 95. 98, 85, or HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 83. 98, 85, or HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 83. HCDR1, HCDR2, HCDR3 shown as 99, 85; and/or as set forth in SEQ ID NO: 86. LCDR1, LCDR2, LCDR3 shown at 97, 88;

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

(ao) is as set forth in SEQ ID NO:89, HCDR1 as shown in SEQ ID NO:100-106, HCDR2 shown as one of SEQ ID NOs: HCDR3 as shown at 91; and/or

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

(ap) as set forth in SEQ ID NO: 110. HCDR1, HCDR2, HCDR3 shown in 100, 91; and/or as set forth in SEQ ID NO:92, LCDR1 as set forth in SEQ ID NO:108-109, 142, 161-163, LCDR2 shown in SEQ ID NO: LCDR3 as 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 framework regions or framework region variants derived from a human antibody, wherein the framework region variants are back mutations having up to 10 amino acids on 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 sequences of human germline heavy chain IGHV4-30-4 x 01 and hjh2, comprising the FR1, FR2, FR3 region of human germline heavy chain IGHV4-30-4 x 01 and FR4 region of hjh; or a combination sequence derived from human germline heavy chain IGHV1-46 x 01 and hjh 6.1.1 comprising the FR1, FR2, FR3 region and FR4 region of hjh 6.1.1 of human germline heavy chain IGHV1-46 x 01;

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

In some embodiments, the second antigen binding domain:

(ba) one or more amino acid mutations in the second VH selected from W47Y, V R, G27Y, I5348M, V67L, F Y, S3530T, Q39K and P49S, e.g., one or more amino acid mutations selected from W47Y, V71R and P49S;

(bb) the second VH comprises one or more amino acid mutations selected from T74K, R72V, M48I, M L, R38Q, L83F, V68A, V79A, Y91F, T S and G72E, e.g., comprises one or more amino acid back mutations selected from Y91F, T22S and G72E; or comprises a deglycosylation mutation of 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) sequence as set forth in SEQ ID NO: second VH and 111 and/or the sequence is shown in SEQ ID NO: 112. a second VL shown;

(bd) sequence as set forth in SEQ ID NO:113 and/or a second VH and a second SEQ ID NO: 114. or 169;

(be) sequence as set forth in SEQ ID NO:115 and a second VH and and/or the sequence is shown in SEQ ID NO: 116. a second VL shown;

(bf) has the sequence shown in SEQ ID NO:117 and/or a second VH and a second SEQ ID NO: 118. or a second VL shown at 170;

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

(bh) has the sequence shown in SEQ ID NO: second VH and 121 and/or the sequence is shown in SEQ ID NO: 122. or a second VL shown at 171;

(bi) sequence as set forth in SEQ ID NO:126 and/or the second VH and/or SEQ ID NO: 127.

(Bj) has the sequence shown in SEQ ID NO: 131. 132-140 and/or the second VH and/or SEQ ID NO: 141-143, 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 a CH 1-hinge region-CH 2-CH3 (e.g., the sequence set forth 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);

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 of which is shown in SEQ ID NO:147 and a second heavy chain as set forth in SEQ ID NO:148, a second light chain shown at 148;

(bo) sequence as set forth in SEQ ID NO:149 or 151 and a second heavy chain as set forth in SEQ ID NO: 150 or 172;

(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 the 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 the C-terminus of the second heavy chain;

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

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

(ddd) a first polypeptide comprising a second heavy chain variable region, CH1, optionally a second peptide linker, and a first heavy chain, linked in sequence from N-terminus to C-terminus; and

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

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

a second polypeptide comprising a second scFv-hinge region-CH 2-CH3; and

A third polypeptide comprising a first light chain;

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

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

A second polypeptide comprising a first scFv-hinge region-CH 2-CH3; and

A third polypeptide comprising a second light chain;

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

(ggg) a first polypeptide comprising a first scFv-hinge region-CH 2-CH3; and

A second polypeptide comprising a second scFv-hinge region-CH 2-CH3;

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

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, optionally a second peptide linker, and a first heavy chain variable region, connected in sequence 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 amino acid sequence: GGGGSGGGGSGGGGSGGGGS.

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

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

(kkk) is as set forth in SEQ ID NO:153, a first polypeptide as set forth in seq id no; as set forth in SEQ ID NO:75, a second polypeptide shown in seq id no; and as set forth in SEQ ID NO:154, a third polypeptide as set forth in seq id no;

(lll) as set forth in SEQ ID NO:155, a first polypeptide as set forth in seq id no; as set forth in SEQ ID NO:145, a second polypeptide shown in figure 145; and as set forth in SEQ ID NO:156, a third polypeptide shown in seq id no;

(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 antibody, a chimeric antibody, or a 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 agents 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 agents for cancer immunotherapy.

The present disclosure also provides an intermediate product of recombinant DNA technology, 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 (b)

(3) A host cell comprising the vector composition of (2) above, or transformed or transfected with the vector composition of (2) above; in some embodiments, the host cell is selected from bacteria such as E.coli, yeast such as Pichia pastoris, insect cells, and mammalian cells such as CHO cells, NSO cells, HEK293 cells, or COS cells.

The present disclosure also provides a method of preparing 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 PD-1 and/or PD-L1-related diseases, comprising the step of using the above-described antigen binding molecule, pharmaceutical composition, or kit.

The present disclosure also provides the use of the above antigen binding molecules, pharmaceutical compositions or kits for the preparation of a reagent for diagnosing PD-1 and/or PD-L1 related diseases.

Embodiments of the present disclosure also provide a method of treating and/or preventing a PD-1 and/or PD-L1-related disease, the method comprising administering to a subject in need thereof an effective amount of an antigen binding molecule as described above, a pharmaceutical composition as described above, a nucleic acid molecule as defined above, or an expression vector; embodiments of the present disclosure also provide the use of the above antigen binding molecule, the above 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 PD-1 and/or PD-L1 related disease; embodiments of the present disclosure also provide the above antigen binding molecule for use as a medicament, the above pharmaceutical composition, a nucleic acid molecule as defined above or an expression vector; embodiments of the present disclosure also provide the above antigen binding molecules, the above pharmaceutical compositions, the nucleic acid molecules as defined above or expression vectors for use in the treatment and/or prevention of diseases 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: head and neck squamous cell carcinoma, head and neck carcinoma, brain carcinoma, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatocellular carcinoma, hepatobiliary carcinoma, pancreatic carcinoma, gastric carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some specific embodiments, the lymphoma is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia; in some more specific embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, kidney cancer, bladder cancer and bowel cancer and colon cancer.

Drawings

Fig. 1: anti-PD-1 antibodies block the binding test results of PD-1 to its ligand.

Fig. 2: effect of anti-PD-1 antibodies on ifnγ secretion by PBMC cells.

Fig. 3: therapeutic effects of anti-PD-1 antibodies on mouse colon cancer MC38 transplantation tumor.

Fig. 4: effect of anti-PD-1 antibodies on tumor volume of mouse colon carcinoma MC 38.

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 anti-PD-1/PD-L1 bispecific antibody TJ021-PR 0002; FIG. 5C is a schematic structural diagram of anti-PD-1/PD-L1 bispecific antibody TJ021-PR 0003; FIG. 5D is a schematic diagram of the structure of anti-PD-1/PD-L1 bispecific antibody TJ021-PR 0004.

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

Fig. 7A: binding of anti-PD-1/PD-L1 bispecific antibodies to PD-1 proteins; fig. 7B: binding of anti-PD-1/PD-L1 bispecific antibodies to PD-L1 proteins.

Fig. 8A: affinity determination of anti-PD-1/PD-L1 bispecific antibody TJ021-PR0001 to PD-1 protein; fig. 8B: affinity determination of anti-PD-1/PD-L1 bispecific antibody TJ021-PR0001 with PD-L1 protein.

Fig. 9A: cell bridging experiments with anti-PD-1/PD-L1 bispecific antibodies; fig. 9B: cell bridging experiments against PD-1/PD-L1 bispecific antibodies.

Fig. 10A: anti-PD-1/PD-L1 bispecific antibodies block PD-1/PD-L1 signaling pathway experiments; fig. 10B: anti-PD-1/PD-L1 bispecific antibodies block PD-1/PD-L1 signaling pathway experiments.

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

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

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

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

The term "cytokine" is a general term for proteins released by one cell population that act on other cells as intercellular mediators. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IFN-gamma, IL-6, TNF alpha, IL-17 and IL-5.

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

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

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

The heavy chain constant region (CH) of the present disclosure is CH 1-hinge region-CH 2-CH3 or, for some heavy chain classes, CH 1-hinge region-CH 2-CH3-CH4, connected in sequence 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 CH2. For example, fc can include the complete 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 immunoglobulins is responsible for many important antibody effector functions including those mediated by Fc receptor (FcR) binding and by complement fixation.

"Hinge region-CH 2-CH3" of the present disclosure means a hinge region-CH 2-CH3 that is connected sequentially from the N-terminus to the C-terminus; "scFv-hinge region-CH 2-CH3" means scFv-hinge region-CH 2-CH3 linked sequentially from N-terminus to C-terminus;

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

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

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce the immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In one embodiment of the present disclosure, the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the PD-1 chimeric antibody further comprises a heavy chain constant region of a human IgG1, igG2, igG3, igG4 or variant thereof, 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 (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., into framework sequences of different types of human germline antibodies. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/VBase) and in Kabat, E.A. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation mutations of the CDRs by yeast display.

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

In one embodiment of the present disclosure, the antibody or antigen binding fragment thereof may further comprise a light chain constant region of human or murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of human or murine IgG1, igG2, igG3, igG4 or variant thereof; such as an IgG1, igG2 or IgG4 variant comprising a human IgG1, igG2 or IgG4 heavy chain constant region, or using amino acid mutations (e.g., L234A and/or L235A mutations, and/or S228P mutations).

"Conventional variants" of human antibody heavy chain constant regions and human antibody light chain constant regions as described in this disclosure refer to variants of human derived heavy chain constant regions or light chain constant regions that have been disclosed in the prior art that do not alter the structure and function of the antibody variable regions, exemplary variants include IgG1, igG2, igG3 or IgG4 heavy chain constant region variants that are site-directed engineering and amino acid substitutions of the heavy chain constant regions, specific substitutions such as YTE mutations, L234A and/or L235A mutations, S228P mutations, and/or mutations that result in a knob-into-hole structure (such that the antibody heavy chain has a knob-Fc and hole-Fc combination), which have been demonstrated to confer new properties to the antibody, but do not alter the function of the antibody variable regions, as known in the prior art.

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

The terms "full length antibody", "whole antibody", "complete antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in substantially complete form, as distinguished from antigen binding fragments as defined below. The term particularly refers to antibodies whose heavy chain comprises 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 to an antigen (e.g., PD-1). Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments comprised in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') ₂ fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of the single arm of the antibody; (v) Single domain or dAb fragments (Ward et al, (1989) Nature 341:544-546) consisting of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, so that they can produce a single protein chain (known as a single chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, e.g., bird et al (1988) Science242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

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), CDR-containing peptides, and the like.

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

For example, the Fab of the present disclosure can 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 or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab.

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

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

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

In addition, the Fab ' may be produced by inserting DNA encoding a Fab ' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or 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) connected by a linker. Such scFv molecules may have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other joints useful in the present disclosure are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immuno l.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol. Biol.293:41-56 and Roovers et al (2001), cancer immunol.

For example, the scFv of the present disclosure can be produced by the following steps: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human PD-1 or PD-L1 of the present disclosure and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof are obtained, DNA encoding scFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express scFv.

Diabodies are antibody fragments in which scFv 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 may be produced by the steps of: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human PD-1 or PD-L1 of the present disclosure and binding to the amino acid sequence of an extracellular region or three-dimensional structure thereof are obtained, DNA encoding scFv is constructed so that the amino acid sequence of a peptide linker is 8 residues or less in length, the DNA is inserted into a prokaryotic expression vector or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express diabodies.

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

For example, the dsFv of the present disclosure may be produced by the following steps: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human PD-1 or PD-L1 of the present disclosure and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof are obtained, DNA encoding dsFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express dsFv.

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

For example, a CDR-containing peptide of the present disclosure can be produced by: constructing a DNA encoding CDRs of VH and VL of a monoclonal antibody specifically recognizing human PD-1 or PD-L1 and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof of the present disclosure, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the peptide. The CDR-containing peptide may also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.

The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of an amino acid in a variant protein or polypeptide as compared to the original protein or polypeptide, including an insertion, deletion or substitution of 1,2, 3 or more amino acids on the basis of 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 the variable domain. Essentially, it is a variable domain that does not have 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 that contribute primarily to antigen binding. Typically, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known protocols, including "Kabat" numbering convention (see Kabat et Al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD), "Chothia" numbering convention (see Al-Lazikani et Al, (1997) JMB 273:927-948) and ImMunoGenTics (IMGT) numbering convention (Lefranc M.P., immunologist,7, 132-136 (1999); lefranc, M.P., et Al, dev. Comp. Immunol.,27, 55-77 (2003) et Al for example, for classical formats, CDR amino acid residues numbering 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3) in the heavy chain variable domain (VL), lightchain amino acid residues numbering 24, LC34-34 (LC35), 50-65 (HCDR 2) and 95-102 (HCDR 3) in the light chain variable domain (VL) are defined as amino acid residues 31-35 (HCDR 1), LCDR 2-35 (LCDR 2), LC35 (LCDR 2) and amino acid residues 26-35 (LC32) and 96-52 (HCDR 2) are defined as amino acid residues (16-35, LC32) and 96-96 (VH) in the amino acid sequence numbering convention (VH 2) for the classical format, for example 50-65 (HCDR 2) and 95-102 (HCDR 3) and amino acid residues 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately 26-35 (CDR 1), 51-57 (CDR 2) and 93-102 (CDR 3), and the CDR amino acid residues in VL are approximately 27-32 (CDR 1), 50-52 (CDR 2) and 89-97 (CDR 3). Following the IMGT rules, CDRs for antibodies can be determined using the program IMGT/DomainGap Align.

"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 a PD-L1 molecule). Epitopes generally comprise at least 3,4,5, 6,7,8,9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epitope Mapping Protocols in Methods in Molecular B iology, volume 66, g.e.Morris, ed. (1996).

The term "linker" refers 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 have a length sufficient 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, for example, 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 binding" and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of about less than 10 ^-8 M, e.g., about less than 10 ^-9M、10^-10M、10^-11 M or less.

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

When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it is meant that competition is between antigen binding proteins, as determined by the following assay: in such assays, the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a cognate antigen (e.g., PD-1 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assay (see, e.g., stahli et al, 1983,Methodsin Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J.Immunol.137:3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual (antibody, laboratory Manual), cold Spring Harbor Press); direct labelling of RIA with the solid phase of the I-125 label (see, e.g., morel et al, 1988, molecular. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., cheung, et al, 1990, virology 176:546-552); and a directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol. 32:77-82). Typically the assay involves the use of purified antigen that binds to a solid surface or cell bearing either the unlabeled detection antigen binding protein or the labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding protein that binds to an epitope in close proximity to the binding epitope of the reference antigen binding protein, the two epitopes spatially preventing binding from occurring. Additional details regarding the method for determining competitive binding are provided in the embodiments herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more of the specific binding of the reference antigen binding protein to the cognate antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

ADCC of the present disclosure, namely anti-body-DEPENDENT CELL-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity refers to direct killing of target cells coated with an antibody by recognizing the Fc segment of the antibody by cells expressing the Fc receptor. ADCC effector function of antibodies may be reduced or eliminated by modification of the Fc fragment on IgG. The modification refers to mutation in the heavy chain constant region of the antibody, such as N297A, L234A, L a selected from IgG 1; igG2/4chimera, F235E of IgG4, or L234A/E235A mutation.

The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, for example, may be 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 and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the genome of a host cell upon introduction into the host cell so as to replicate with the host genome (e.g., non-episomal mammalian vectors).

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

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

The engineered antibodies or antigen binding fragments of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expressing antibodies that specifically bind to human PD-1, or antibodies that bind to both PD-1 and PD-L1. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either frozen immediately, e.g., at-70 ℃, or lyophilized.

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

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

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

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 the severity, or delaying the onset of a disorder, including biochemical, histological and/or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes that are exhibited during the development of the disorder. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various target antigen-related disorders of the present disclosure, reducing the dosage of other agents required to treat a disorder, enhancing the efficacy of another agent, and/or slowing the progression of a target antigen-related disorder of the present disclosure in a patient.

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

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 comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, when sequences are optimally aligned, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches or homologies at 100 positions in the two sequences, then the two sequences are 95% homologous. Typically, the comparison is made when two sequences are aligned to give the greatest percent homology. For example, the comparison may be performed by a BLAST algorithm, wherein the parameters of the algorithm are selected to give a maximum match between the individual sequences over the entire length of the individual reference sequences. The following references relate to BLAST algorithms that are often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): altschul, S.F. et al, (1990) J.mol.biol.215:403-410; gish, W.et al, (1993) Nature Genet.3:266-272; madden, t.l. et al, (1996) meth.enzymol.266:131-141; altschul, S.F. et al, (1997) Nucleic Acids Res.25:3389-3402; zhang, j et al, (1997) Genome res.7:649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also known to those skilled in the art.

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

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a particular portion of nucleic acid, RNA, and/or DNA are amplified as described, for example, in U.S. patent No. 4,683,195. Generally, it is necessary to obtain sequence information from the end of the target region or beyond so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' -terminal nucleotides of the 2 primers may correspond to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, etc. 1. See generally Mullis et al (1987) Cold Spring Harbor Symp. Uant. Biol.51:263; erlich editions, (1989) PCR TECHNOLOGY (stock Press, n.y.). PCR as used herein is considered one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes using known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.

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

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

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

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

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

The PD-1-associated disease in the present disclosure is not limited as long as it is a PD-1-associated disease, e.g., a therapeutic response induced by a molecule utilizing the present disclosure may be produced by binding to human PD-1, then repressing the binding of PD-1 to its ligand PD-L1, PD-L2, or killing tumor cells that overexpress PD-1. Thus, the molecules of the present disclosure are very useful for those suffering from tumors or cancers, such as melanoma, colon, breast, lung, stomach, bowel, kidney, non-small cell lung, bladder, etc., when in preparations and formulations suitable for therapeutic applications.

Furthermore, the present disclosure relates to methods for immunodetection or assay of an antigen of interest (e.g., PD-1), reagents for immunodetection or assay of an antigen of interest (e.g., PD-1), methods for immunodetection or assay of cells expressing an antigen of interest (e.g., PD-1), and diagnostic agents for diagnosis of diseases associated with positive cells of an antigen of interest (e.g., PD-1), comprising as an active ingredient an antibody or antibody fragment of the present disclosure that specifically recognizes an antigen of interest (e.g., human PD-1) and binds to the amino acid sequence of the extracellular region or three-dimensional structure thereof.

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

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

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

For detecting the cells expressing the polypeptide, known immunodetection methods, for example, immunoprecipitation, fluorescent cell staining, immunohistological staining, etc., can be used. Furthermore, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, a living sample for detecting or assaying an antigen of interest (e.g., PD-1) is not particularly limited as long as it has a possibility of containing cells expressing the antigen of interest (e.g., PD-1), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue juice, or culture solution.

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

Examples

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure. Experimental methods not specifying specific conditions in the examples of the present disclosure are generally conducted according to conventional conditions such as a laboratory manual of antibody technology in Cold spring harbor, a manual of molecular cloning; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1 obtaining anti-human PD-1 murine antibody

Preparation of antigens

1. PD-1 antigen construction and screening:

The fusion protein of human PD-1-IgG1Fc is designed and synthesized, wherein the N end is 150 amino acids of the extracellular region of human PD-1, and the C end is the Fc segment of human IgG1 (hIgG 1 Fc). The recombinant PD-1-Fc Protein with high purity can be obtained by purifying the Protein A by an affinity column and is used for detecting the combination of an anti-PD-1 antibody and an antigen.

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

Annotation: the underlined part is the signal peptide, the orthotopic part is the human PD-1 extracellular domain, and the italic part is hIgG1Fc (signal peptide+extracellular domain+hIgG 1 Fc).

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

MEFGLSWLFLVAILKGVQCPGWFLDSPDRPWNPPTFSPALLVVTEGDNAT FTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPS PRPAGQFQTLVGSSDYKDDDDKHHHHHH.

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

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPS PSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPAR RGSADGPRSAQPLRPEDGHCSWPL.

Preparation of anti-human PD-1 antibodies

Anti-human PD-1 antibodies can be produced by immunizing mice, or can be obtained by immunizing human PD-1 phage mice against the Website.

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

1. Immunization: the experimental SJL white mice, females, 6-8 weeks old and Balb/c white mice, females, 6-8 weeks old. Feeding environment: SPF stage. After the mice are purchased, the mice are fed in a laboratory environment for 1 week, the light/dark period is regulated for 12/12 hours, and the temperature is 20-25 ℃; humidity is 40-60%. The mice which are adapted to the environment are immunized according to different schemes, and 6-10 mice are used in each group. The immune antigen can be purified recombinant protein PD-1-IgG1Fc (see SEQ ID NO: 1), PD-1-his (see SEQ ID NO: 2), or PD-1 as antigen (see SEQ ID NO: 3) transfected Jurkat/CHO-PD-1 cells, and can be matched with different immune adjuvants or different types of immunogens by a single reagent for cross immunization. The immune site can be the abdominal cavity or the subcutaneous of the back, or the two sites are alternatively immunized. The immunological adjuvants gold adjuvant (hereinafter Titermax, available from Sigma cat# T2684) were cross-immunized with Imject Alum Adjuvant (hereinafter Alum, available from Pierce cat# 77161). Antigen to adjuvant (Titermax) ratio of 1:1, antigen to adjuvant (Alum) ratio of 3:1, 25-50 μg/per (priming), 50 μg/per (boost) or 1X 10 ⁷ Jurkat/CHO-PD-1 cells/per. Day 0 intraperitoneal injection of 25-50 μg/dose of emulsified antigen, once weekly or every two weeks after priming, alternating with Titermax and Alum for 5-8 times.

2. Cell fusion: mice with high antibody titer in serum are selected for spleen cell fusion, and the eyeballs of the mice after 72 hours of sprint immunization are exsanguinated, and the mice are killed by neck pulling and put into 75% ethanol for disinfection. The spleen lymphocytes and myeloma cells Sp2/0 cells (China academy of sciences) are fused by adopting an optimized PEG-mediated fusion step to obtain the hybrid tumor cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHAT and 1 XOPI), and plated in 96-well cell culture plates (1X 10 ⁵/150. Mu.l/well), incubated at 37℃with 5% CO ₂, and approximately 10-30 plates. HAT complete medium was added at day 5 after fusion, 50 μl/well, incubated at 37deg.C with 5% CO ₂. From day 7 to day 8 after fusion, according to cell growth density, the whole exchange was performed with 200 μl/well, 37℃and 5% CO ₂.

3. Hybridoma cell selection: and on the 7 th to 9 th days after fusion, detecting by an ELISA method for combining the antibody and the PD-1 according to the cell growth density, detecting the detected positive hole cells by a blocking ELISA for combining the PD-1/PDL1, replacing the positive hole liquid, and expanding the positive hole cells into a 24-hole plate in time according to the cell density. The cell lines engrafted into 24-well plates were retested for seed protection and first subcloning. Seed conservation is carried out when the first subclone screening is positive, and the second or third subclone is carried out until single cell clone is obtained. And obtaining the hybridoma cells with the effect of blocking the combination of PD-1 and PDL1 through multiple times of fusion.

The method for obtaining anti-human PD-1 antibodies from an anti-human PD-1 phage mouse immune library is as follows:

1. Construction of an anti-human PD-1 phage mouse immune library: spleen of mice with high antibody titer in serum was selected, and total RNA of tissues was extracted with Trizol (Invitrogen Cat No. 15596-018). cDNA was obtained by reverse transcription using PRIMESCRIPT ^TM II 1st Strand cDNA Synthesis Kit kit (TAKARA CAT No. 6210A). Primers for constructing the library were designed and synthesized according to IMGT database. The single-chain antibody fragment is obtained through three rounds of PCR reaction. Single-chain antibody fragments and engineered pool-building vector pCantab E (Amersham Biosciences/GE Cat No. 27-9400-01) were digested with Sfi1 (NEB Cat No. #R0123L), electrophoresed, and purified and recovered by Gel Extraction Kit (omega Cat No. D2500-02). Then, the mixture was ligated with T4 DNA ligase (NEB Cat No. # M0202L) at 16℃for 16-18 hours, and then purified and recovered by the above-mentioned kit, and finally eluted with deionized water. 1. Mu.g of the ligation product was mixed with 1 branch of electrotransformation competent TG1 (Lucigen Cat No. 60502-2), and the parameters of the electrotransformation apparatus (Bio Rad Micropulser) were set to 2.5kV,200Ω,25uF for electrotransformation. The transformation was repeated 10 times, plated, and incubated at 37℃for 16-18 hours in an inverted state. All colonies were scraped off and mixed together, glycerol was added at a final concentration of 15%, and stored at-80℃for later use.

2. Screening of anti-human PD-1 phage mouse immune library: the packaged anti-human PD-1 phage immune library (1X 10 ¹²-1×10¹³) was incubated with 100. Mu.l of streptavidin microbeads (Mi 1envi Biotec, auburn, calif.) in 1m1 of 2% nonfat milk-phosphate buffer (abbreviated MPBS) for 1 hour at room temperature, placed on a magnetic rack, and the supernatant was taken. The supernatant was incubated with 10. Mu.g/ml biotinylated human PD-1-ECD-his protein (purchased from Sino Biological) for 1 hour at room temperature, and 100. Mu.l of streptavidin-coated magnetic beads (1 ml of MPBS pre-incubation) was added and incubated for 1 hour at room temperature. And loaded on a magnetic rack system for sorting, and the supernatant is sucked. 1ml of PBST (phosphate buffer containing 0.1% Tween-20) was added, turned over multiple times, and after blotting, fresh wash was added, and repeated 11 times to remove unbound antibody fragments, 0.5ml of eluent (50. Mu.l of 10mg/ML TRYPSIN stock solution) was added to 450. Mu.l of PBS. Shake for 15min at room temperature. Placed on a magnetic rack and the supernatant aspirated into a new EP tube. TG1 was expanded to a bacterial density od600=0.4 in 2YT medium. 1.75ml of TG1 (OD 600 = 0.4) was added to each tube, and 250. Mu.l of post-elution phase (phage) was added, incubated for 30min in a 37℃water bath, and the plates were serially diluted for testing titers. The remaining TG1 solution was centrifuged, plated, and incubated overnight at 37 ℃.

Phage mouse immune library utilizes biotinylated human PD-1-ECD-his antigen, and through 2-3 rounds of MACS screening (streptomycin magnetic beads, invitrogen), monoclonal antibodies with the functions of binding PD-1 and blocking the binding of PD-1 and PD-L1 are finally obtained, and sequencing verification is carried out, so that the variable region sequence of the antibodies is obtained.

Purification of recombinant antigen proteins/antibodies

1. Hybridoma supernatant separation and purification/ProteinG affinity chromatography:

the method is characterized in that ProteinG is first selected for purification of mouse hybridoma supernatant, the supernatant is obtained by taking the hybridoma obtained by culture through centrifugation, and the pH of the supernatant is adjusted by adding 10-15% by volume of 1M Tris-HCl (pH 8.0-8.5) according to the volume of the supernatant. The ProteinG column is washed by 6M guanidine hydrochloride for 3 to 5 times of column volume, and then is washed by pure water for 3 to 5 times of column volume; the column is equilibrated for 3-5 column volumes using, for example, a1 XPBS (pH 7.4) buffer system as an equilibration buffer; cell supernatants were combined using low flow loading, and the flow was controlled to leave the retention time for about 1min or longer; washing the column with 1 XPBS (pH 7.4) for 3-5 times the column volume until the UV absorption falls back to baseline; sample elution was performed using 0.1M acetic acid/sodium acetate (pH 3.0) buffer, elution peaks were collected according to UV detection, and the eluted product was rapidly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0) for buffer 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 desalting using a molecular exclusion column such as G-25 to replace the desired buffer system, or removing the polymer component in the eluted product using a high resolution molecular exclusion column such as Superdex 200 to increase the sample purity.

Protein a affinity chromatography purification of proteins or antibodies:

The supernatant is first harvested by high-speed centrifugation of cell culture supernatants expressing the antigen proteins or antibodies. 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 for 3-5 column volumes using, for example, a1 XPBS (pH 7.4) buffer system as an equilibration buffer. Cell supernatants were loaded and bound using a low flow rate, the flow rate was controlled to allow a retention time of about 1min or longer, and after binding was completed, the column was washed 3-5 column volumes with 1 XPBS (pH 7.4) until UV absorption fell back to baseline. Sample elution is carried out by using 0.1M acetic acid/sodium acetate (pH 3.0-3.5) buffer solution, elution peaks are collected according to ultraviolet detection, and the eluting product is rapidly adjusted to pH 5-6 by using 1M Tris-HCl (pH 8.0) for temporary storage. For the eluted product, solution displacement can be performed 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 desalting using a molecular exclusion column such as G-25 to a desired buffer system, or removing the polymer component in the eluted product using a high resolution molecular exclusion column such as Superdex 200 to increase the sample purity.

PD-L1 antibodies

Methods for the preparation and purification of PD-L1 antigens and anti-PD-L1 antibodies may be conventional methods known in the art, as well as methods such as those 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:

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

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

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

The murine anti-variable region sequence of hybridoma clone 24D5 was as follows:

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

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

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

>9-2hVH-CDR graft(SEQ ID NO：115)

>9-2hVL CDR graft(SEQ ID NO：116)

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

24D5 humanized heavy chain variable region VH.1 (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 that: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the sequence is italic FR sequence; the CDR sequences are underlined, wherein the double underlined sites are the sites obtained after affinity maturation screening.

The present disclosure also constructs a polypeptide as set forth in SEQ ID NO: VL shown in 169-171:

the variable region sequence of the anti-PD-L1 antibody 24D5H21 heavy chain provided by the disclosure is shown in SEQ ID NO: 131:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDKWMMWVRQAPGQGLEW MGRITPSSGFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGS SYDYFDYWGQGTTVTVSS(SEQ ID NO：131)

the variable region sequence of the anti-PD-L1 antibody 24D5L 67 light chain provided by the disclosure is shown in SEQ ID NO:125, shown in:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI YVASNVESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK(SEQ ID NO：125)

the variable region sequence of the anti-PD-L1 antibody 24D5L 68 provided by the disclosure is shown in SEQ ID NO:167, as shown in:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI YVASNVWSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK(SEQ ID NO：167)

The variable region sequence of the anti-PD-L1 antibody 24D5L 69 provided by the disclosure is shown in SEQ ID NO:168, shown in:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI YVASNVYSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK(SEQ ID NO：168)

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

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

QVQLQESGPGLVKPSQTLSLTCTVSGGSISDGSAYWSWIRQHPGKGLEYIGZ₁₄ISZ₁₅AGSTYZ₁₆TPSLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS(SEQ ID NO：123)

Or (b)

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGZ₁₄ISZ₁₅AGSTYZ₁₆TPSLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS(SEQ ID NO：124). Z ₁₄ in the heavy chain variable region is F or M, Z ₁₅ is R or V, and Z ₁₆ is N or H.

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

Further, the related heavy chain variable region sequence general formula is obtained as follows:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTZ₁₁Z₁₂WMZ₁₃WVRQAPGQGL EWMGRIZ₄PZ₅Z₆GZ₇Z₈Z₉YNEKZ₁₀KNRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARGGSSYDYFDYWGQGTTVTVSS(SEQ ID NO：126), Z ₁₁ in the CDR and heavy chain variable region is S or D, Z ₁₂ is Y or K, Z ₁₃ is H or M, Z ₄ is selected from T, S, H and G, Z ₅ is selected from S, N and G, Z ₆ is selected from S, L and G, Z ₇ is selected from F, L, W and M, Z ₈ is selected from A, P and T, Z ₉ is selected from M, V, L and S, and Z ₁₀ is F or Y.

Further, the sequence formula of the related light chain variable region is obtained as follows:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLI YZ₁₇ASZ₁₈Z₁₉Z₂₀SGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFG QGTKLEIK(SEQ ID NO：127), Wherein Z ₁₇ is V or A, Z ₁₈ is Y, K or N, Z ₁₉ is selected from A, L and V, and Z ₂₀ is selected from E, F, Y and A.

The following heavy and light chain variable regions were obtained:

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

QVQLQESGPGLVKPSQTLSLTCTVSGGSISDGSAYWSWIRQHPGKGLEYIGFISRAGSTYNTPSLKGRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS

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 9-2L11 light chain variable region is shown in SEQ ID NO: 120.

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

24D5H 12 heavy chain variable region (SEQ ID NO: 132)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRITPSSGFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 13 heavy chain variable region (SEQ ID NO: 133)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPSLGLAVYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 14 heavy chain variable region (SEQ ID NO: 134)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIHPSLGLPLYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 15 heavy chain variable region (SEQ ID NO: 135)

QVQLVQSGAEVKKPGASVKVSCKANGYTFTDKWMMWVRQAPGQGLEWMGRITPSSGFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 16 heavy chain variable region (SEQ ID NO: 136)

QVQLVQSGAEVKKPGASMKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPSLGLAVYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 17 heavy chain variable region (SEQ ID NO: 137)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNLGWAMYNEKYKNRVTMTRDTSTSTVYMELSSLGSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 18 heavy chain variable region (SEQ ID NO: 138)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPSSGMAVYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 19 heavy chain variable region (SEQ ID NO: 139)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRISPGGGFTLYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5H 20 heavy chain variable region (SEQ ID NO: 140)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

24D5L 64 light chain variable region (SEQ ID NO: 141)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYVASYAASGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEI K

24D5L 61 light chain variable region (SEQ ID NO: 122)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

24D5L 66 light chain variable region (SEQ ID NO: 143)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYVASNVFSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

HRP00049:9-2 (H2/L10) IgG4 (AA) heavy chain: SEQ ID NO:147

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGYISYTGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK

HRP00049 antibody light chain: SEQ ID NO:148

DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

HRP00052:24D5 (GF) IGG4 (AA) antibody heavy chain: SEQ ID NO:149

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK

HRP00052 antibody light chain sequence SEQ ID NO:150

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The present disclosure also constructs a polypeptide as set forth in SEQ ID NO:172, an antibody light chain sequence shown in seq id no:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASKLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The full length sequence of the constructed PD-L1 antibody in IgG1 form is exemplified as follows:

Heavy chain: SEQ ID NO:151

The light chain is shown in SEQ ID NO: 150.

Carrying out antigen binding experiments on the anti-human PD-1 murine antibody obtained by the method, and screening to obtain 3 clones with good activity: m23, M32 and M33, and performing reverse transcription amplification (RT-PCR) by using degenerate primers of mouse-Ig to obtain variable region sequences of the antibody. The variable region sequence of the mouse antibody is connected with the constant region sequence of the human antibody, the chimeric antibody of the mouse monoclonal antibody is cloned and expressed in a recombination way, and an in vitro activity experiment is carried out 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 light chain variable region 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 light chain variable region of murine antibody M32 (SEQ ID NO: 7):

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYAFGGG TKLEIK.

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

KVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNTLYLQMSSLRSEDTALYYCASPYGHGYFDVWGTGTTVTVSS.

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

DIQMTQTTSSLSASLGDRVTISCRASQDINNFLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEI K.

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

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

Remarks: the antibody CDR sequences in the table are determined according to the Kabat numbering system.

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

And (3) respectively selecting heavy and light chain variable region germline genes with high sequence identity with the light and heavy chains of M23, M32 and M33 as templates by comparing an IMGT human antibody heavy and light chain variable region germline gene database and MOE software, respectively transplanting the CDRs of the 3 murine antibodies into corresponding humanized antibody templates, and respectively constructing the corresponding humanized antibodies.

1. Humanized murine antibody M23

1.1 Murine antibody M23 humanized framework selection

Humanized light chain templates of the murine antibody M23 are IGKV2-40 x 01 and IGKJ4 x 01, humanized heavy chain templates are IGHV1-69 x 02 and IGHJ6 x 01, and the sequence of a humanized modified variable region is as follows (underlined as a CDR sequence):

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 reverse mutation design of murine antibody M23

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

Note that: grafting (Grafted) represents the implantation of murine antibody CDRs into human germline FR sequences, amino acid residues are determined and annotated by the Kabat numbering system, as I2G represents the mutation of Kabat numbering at position 2I 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 (grafting onto 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 the murine antibody M23 is subjected to back mutation design, and different variable regions are combined into different humanized antibody variable regions through back mutation, and the specific table is shown below.

TABLE 6 Hu23 humanized antibody variable region combinations

Remarks: in the table, "Hu23-1" refers to, for example, the antibody light chain variable region being Hu23VL1 and the heavy chain variable region being Hu23VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 23-1) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu 23-1) as indicated in the table with the suffix ". IgG4AA" indicates a full length antibody formed by the attachment of an IgG4-AA heavy chain constant region, and with the suffix ". IgG4P" indicates a full length antibody formed by the attachment of an IgG4-P heavy chain constant region, e.g., "Hu23-1.Igg4aa" indicates a variable region of Hu23VH1 and a variable region of a heavy chain as set forth in SEQ ID NO:72, and a heavy chain comprising a Hu23VL1 light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region), "Hu23-1.Igg4p" means a full length antibody formed by the Hu23VH1 heavy chain variable region and the amino acid sequence as set forth in SEQ ID NO:79 and a heavy chain comprising an IgG4-P heavy chain constant region linked to a light chain variable region comprising Hu23VL1 and a light chain variable region as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region).

2. Humanized murine antibody M32

2.1 Murine antibody M32 humanized framework selection

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

Hu32VH-CDR grafting: (SEQ ID NO: 33) IGHV 1-69.times.02 and IGHJ 6.times.01

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEIHWVRQAPGQGLEWMGLFDPETGGIVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGYNRDWYFDVWGQGTTVTVSS.

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

DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLI YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGGT KVEIK.

2.2 Humanized template selection and reverse mutation design of murine antibody M32

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

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

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

Hu32VL1 (grafting onto Hu32 VL-CDR): (SEQ ID NO: 34)

>Hu32VL2(SEQ ID NO：35)

DVVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGG TKVEIK.

Hu32VH1 (grafting onto 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 the murine antibody M32 is subjected to back mutation design, and different back mutations are combined into different humanized antibodies, and the specific details are shown in the following table.

TABLE 8 humanized antibody light/heavy chain variable region regions

Remarks: in the table, "Hu32-1" for example refers to the antibody light chain variable region of Hu32VL1 and the heavy chain variable region of Hu32VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 32-1) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu 32-1) as indicated in the table with the suffix ". IgG4AA" indicates a full length antibody formed by the attachment of an IgG4-AA heavy chain constant region, and with the suffix ". IgG4P" indicates a full length antibody formed by the attachment of an IgG4-P heavy chain constant region, e.g., "Hu32-1.Igg4aa" indicates a variable region of Hu32VH1 heavy chain and a variable region of SEQ ID NO:72, and a heavy chain comprising a Hu32VL1 light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region), "Hu32-1.Igg4p" means a full length antibody formed by the Hu32VH1 heavy chain variable region and the amino acid sequence as set forth in SEQ ID NO:79 and a heavy chain comprising an IgG4-P heavy chain constant region linked to a light chain variable region comprising Hu32VL1 and a heavy chain variable region as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region).

3. Humanized murine antibody M33

3.1 Murine antibody M33 humanized framework selection

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

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYGHGYFDVWGQGTTVTVSS.

Hu33VL-CDRgrafted(SEQ ID NO：42)：

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEI K.

3.2 humanized template selection and reverse mutation design of murine antibody M33

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

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

The humanized antibody light chain variable region and heavy chain variable region sequences 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 regions

Remarks: in the table, "Hu33-6" refers to, for example, the antibody light chain variable region being Hu33VL2 and the heavy chain variable region being Hu33VH3, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 33-6) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu 33-6) as indicated in the table with the suffix ". IgG4AA" indicates a full length antibody formed by the attachment of an IgG4-AA heavy chain constant region, and with the suffix ". IgG4P" indicates a full length antibody formed by the attachment of an IgG4-P heavy chain constant region, e.g., "Hu33-6.Igg4aa" indicates a variable region of Hu33VH3 and a variable region of a heavy chain as set forth in SEQ ID NO:72, and a heavy chain comprising a Hu33VL2 light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked by the amino terminus of the constant region), "Hu33-6.Igg4p", representing a full length antibody formed by the Hu33VH3 heavy chain variable region and the amino terminus of the constant region as set forth in SEQ ID NO:79, and a heavy chain comprising a Hu33VL2 light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region).

4. Mutants of humanized antibodies

4.1 Mutant antibodies of humanized Hu23 antibodies

Site-directed mutagenesis of amino acids at specific sites of the light chain LCDR1 (SEQ ID NO: 11) of Hu23 humanized antibodies was performed by computer simulation and the specific mutations are shown in Table 11:

table 11. Mutant sequences of hu23 light chain LCDR 1:

Note that: hu23LCDR1 (N28Q) represents an LCDR1 mutant sequence in which the N at position 28 of Kabat numbering convention on Hu23VL1 or Hu23VL2 of the light chain variable region of Hu23 is mutated to Q, hu23LCDR1 (G29A) represents an LCDR1 mutant sequence in which the G at position 29 of Kabat numbering convention on Hu23VL1 or Hu23VL2 of the light chain variable region of Hu23 is mutated to A (the CDR is determined by the Kabat numbering convention).

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

the > Hu23VL1 (N28Q) sequence is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：53)

the > Hu23VL1 (N28L) sequence:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：54)

the > Hu23VL1 (N28T) sequence is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：55)

the > Hu23VL1 (N28D) sequence is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：56)

the > Hu23VL1 (G29A) sequence:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：57)

the > Hu23VL1 (G29V) sequence:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：58)

the > Hu23VL2 (N28Q) sequence is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：59)

the > Hu23VL2 (N28L) sequence is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：60)

the > Hu23VL2 (N28T) sequence is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：61)

the > Hu23VL2 (N28D) sequence is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：62)

The > Hu23VL2 (G29A) sequence:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：63)

The > Hu23VL2 (G29V) sequence:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：64)

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

Remarks: in the table, "Hu23-11" refers to, for example, the region of the antibody light chain variable region being Hu23VL1 (N28T) and the heavy chain variable region being Hu23VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 23-11) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu 23-11) as indicated in the table with the suffix ". IgG4AA" indicates a full length antibody formed by the attachment of an IgG4-AA heavy chain constant region, and with the suffix ". IgG4P" indicates a full length antibody formed by the attachment of an IgG4-P heavy chain constant region, e.g., "Hu23-11.Igg4aa" indicates a variable region of Hu23VH1 and a variable region of SEQ ID NO:72 to a heavy chain comprising an IgG4-AA heavy chain constant region linked to a light chain variable region consisting of Hu23VL1 (N28T) and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked by the amino terminus of the constant region), "Hu23-11.Igg4p", representing a full length antibody formed from the Hu23VH1 heavy chain variable region and the amino terminus of the constant region as set forth in SEQ ID NO:79, and a heavy chain comprising a Hu23VL1 (N28T) light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region).

Experimental results show that humanized antibodies after site mutation of Hu23LCDR1 (N28Q), hu23LCDR1 (N28L), hu23LCDR1 (N28T), hu23LCDR1 (N28D), hu23LCDR1 (G29A), hu23LCDR1 (G29V) all maintain the binding capacity to PD-1 (Table 16).

4.2 Mutant antibodies of Hu32 humanized antibodies

Through sequence analysis, the sequence identity of the series humanized antibodies Hu23 and Hu32 from M23 is higher, and the Hu23 light chain variable region and the Hu32 heavy chain variable region are combined into a new light and heavy chain variable region. The experimental results show that humanized antibodies comprising the newly combined light and heavy chain variable regions all retain binding capacity to PD-1 antigen (table 16).

TABLE 13 general formula of variable region consensus sequences for Hu32 and Hu23 antibodies

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

Remarks: in the table, "Hu32a-85" for example refers to the region of the antibody light/heavy chain variable region where the antibody light chain variable region is Hu23VL1 (N28T) and the heavy chain variable region is Hu32VH6, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu32 a-85) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu32 a-85) with the suffix ". Lg4aa" indicates a full length antibody formed by the linkage to an lg4-AA heavy chain constant region, and ". Lg4p" indicates a full length antibody formed by the linkage to an lg4-P heavy chain constant region, e.g., "Hu32a-85.Igg4aa" indicates a variable region of Hu32VH6 and a variable region of SEQ ID NO:72 to a heavy chain comprising an IgG4-AA heavy chain constant region linked to a light chain variable region consisting of Hu23VL1 (N28T) and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region is linked to the constant region in such a way that the variable region is at the amino terminus of the constant region), "Hu32a-85.Igg4p", which means a full length antibody formed by the light chain linked to the Kappa chain constant region as shown in SEQ ID NO:79, and a heavy chain comprising a Hu23VL1 (N28T) light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region).

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

Remarks: in the table, "Hu23a-57" refers to, for example, the antibody light chain variable region being Hu32VL1 and the heavy chain variable region being Hu23VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu23 a-57) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu32 a-85) with the suffix ". Lg4aa" indicates a full length antibody formed by the linkage to an lg4-AA heavy chain constant region, and ". Lg4p" indicates a full length antibody formed by the linkage to an lg4-P heavy chain constant region, e.g., "Hu23a-57.Igg4aa" indicates a variable region of Hu23VH1 and a variable region of SEQ ID NO:72, and a heavy chain comprising a Hu32VL1 light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73 (the variable region is linked to the constant region in such a way that the variable region is at the amino terminus of the constant region), "Hu23a-57.Igg4p", which means a full length antibody formed by the light chain linked to the Kappa chain constant region as shown in SEQ ID NO:79 and a heavy chain comprising an IgG4-P heavy chain constant region linked to a light chain variable region comprising Hu32VL1 and a heavy chain variable region as set forth in SEQ ID NO:73 (the variable region and the constant region are both linked at the amino terminus of the constant region).

5. Screening of humanized antibodies

The affinity assays of the different humanized antibodies were performed by Biacore (see test example 3 for methods), and the results are shown in table 16, which show that the different humanized antibodies retain binding capacity for PD-1, and the affinity of the partially humanized antibodies is even substantially similar to that of their murine antibodies.

TABLE 16 affinity of Hu23 humanized antibodies for human PD-1

EXAMPLE 3 construction and expression of PD-1 humanized antibodies

Designing primer PCR to build each humanized antibody VH/VK gene fragment, and then carrying out homologous recombination with an expression vector pHr (with signal peptide and constant region gene (CH 1-Fc/CL) fragment) to construct an antibody full-length expression vector VH-CH1-Fc-pHr/VK-CL-pHr. IgG4-P represents the S228P (corresponding to position 108 of the sequence SEQ ID NO:72 or SEQ ID NO: 79) mutation, igG4-AA represents the F234A (corresponding to position 114 of the sequence SEQ ID NO:72 or SEQ ID NO: 79), L235A (corresponding to position 115 of the sequence SEQ ID NO:72 or SEQ ID NO: 79) and S228P (corresponding to position 108 of the sequence SEQ ID NO:72 or SEQ ID NO: 79) mutation, and IgG4-AA and IgG4-P antibody forms can be obtained by simple point mutation of IgG4 antibody forms.

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

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK.

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

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC.

the sequence of the constructed full-length antibody of IgG4AA format is exemplified as follows:

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

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLI DPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK.

Hu23-11.Igg4aa light chain (SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC.

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

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDP ETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK.

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

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

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISG GGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK.

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

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLH SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

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

LSLGK。

The sequence of the constructed IgG4-P version of the full-length antibody is exemplified as follows:

Hu23-11.IgG4P antibody heavy chain (SEQ ID NO: 80):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLI DPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK.

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

Hu32a-85.Igg4p heavy chain (SEQ ID NO: 81):

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDP ETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDW YFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Light chain of Hu32a-85.IgG4P (light chain identical to Hu23-11.IgG4AA, SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC.

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

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISG GGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK.

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

The full length sequence of the constructed PD-1 antibody in IgG1 format is 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 in vitro PD-1 ligand binding and binding blocking ELISA experiments with anti-PD-1 antibodies

PD-L1 on the surface of tumor cells can inhibit proliferation of T cells by combining with PD-1 on the surface of T cells. Antibodies to PD-1 block the signaling pathway of PD-1/PD-L1 by binding to PD-1, thereby stimulating proliferation of T cells. The binding blocking assay of PD-1/PD-L1 was used to detect the blocking activity of anti-PD-1 antibodies on signal pathways.

In the experiment, after the extracellular region and Fc fused PD-1 protein (PD-1-Fc, the sequence is shown in SEQ ID NO: 1) are coated on a 96-well plate, anti-PD-1 antibodies (comprising antibodies: hu23-11.IgG4AA, hu32a-85.IgG4AA and Hu33-6.IgG4 AA) to be detected respectively, a positive control antibody: H005-1 (see H005-1 antibody in WO 2015085847) is subjected to incubation reaction, biotin-labeled PD-L1/PD-L2 is added later, the incubation reaction is carried out, after washing the plate, the binding amount of the biotin-labeled PD-L1/PD-L2 is detected, and the IC ₅₀ value of the anti-PD-1 antibody for ligand PD-L1/PD-L2 binding blocking is calculated.

PD-1-Fc was diluted to 1. Mu.g/ml with pH 9.6CB buffer (1.59 g Na ₂CO₃ and 2.93g NaHCO ₃ in 1L distilled water), added to 96-well plates at a volume of 100. Mu.l/well and left to stand at 4℃for 16-20 h. After washing the plates 1 time with PBST (pH 7.4 PBS containing 0.05% tween 20) buffer, 120 μl/well PBST/1% mill was added and incubated for 1h at room temperature for blocking. After removing the blocking solution and washing the plate 1 time with PBST buffer, 90. Mu.l of anti-PD-1 antibody to be tested diluted to an appropriate concentration with sample dilution (pH 7.4 PBS containing 5% BSA,0.05% Tween 20) was added, and pre-incubated at 4℃for 1 hour. 10 Xconcentration of biotin-labeled PD-L1/PD-L2 (10. Mu.g/ml, biotechnology Co., ltd. In Beijing Yiqiao) was added in a volume of 10. Mu.l/well, and after shaking on a shaker, the mixture was incubated at 37℃for 1 hour. After the reaction system was removed and the plate was washed 6 times with PBST, 100. Mu.l/well of strepitavidin-Peroxidase Polymer (streptavidin-peroxidase polymer) diluted 1:400 with 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 TMB was added and incubated for 5-10min at room temperature. The reaction was terminated by adding 100. Mu.l/well 1M H ₂SO₄. The absorbance was read at 450nm using a microplate reader and the IC ₅₀ value of the anti-PD-1 antibody for ligand PD-L1/PD-L2 binding blockade was calculated. The data are detailed in table 17 below.

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

The exemplary anti-PD-1 antibodies of the present disclosure, hu23-11.IgG4AA, hu32a-85.IgG4AA, and Hu33-6.IgG4AA, are all capable of effectively blocking the binding of PD-1 to PD-L1/PD-L2, with blocking activity similar to that of the positive control antibody.

Test example 2 exemplary antibody and ligand blocking assays

The blocking effect of 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 and added to 96-well plates at 100. Mu.L/well, and incubated in a 5% CO ₂ incubator at 37℃for 24 hours. The control and samples were diluted to the desired concentration using PBS. Jurkat/PD-1 cells (Jurkat cells stably transferring PD-1) were counted, 10. Mu.L/well of diluted antibodies (antibodies: hu23-11.IgG4AA, hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1) were added simultaneously to a cell culture plate (90. Mu.L/well) for the CHOK1/PD-L1 cells in a certain ratio, and the negative control IgG4 protein was incubated in a 5% CO ₂ incubator at 37℃for 5 hours at a gradient dilution concentration of 0.3mg/ml, 3mg/ml, 30 mg/ml. The cell culture plate was removed, left to stand at room temperature for 5 minutes, then 50. Mu.l of Bio-Glo ^TM Reagent was added to each well, incubated at room temperature for 5 minutes, and the plate was read. The experimental results are shown in the attached figure 1, and the results show that the anti-PD-1 antibodies Hu23-11.IgG4AA, hu32a-85.IgG4AA and Hu33-6.IgG4AA in the disclosure can effectively block the combination of PD-1 and PD-L1, and the blocking activity of the anti-PD-1 antibodies is similar to that of a positive control antibody H005-1.

Test example 3 BIAcore antibody affinity experiments for exemplary antibodies

IgG was affinity captured using A protein A biosensing chip (Cat. # 2927556, GE), human PD-1 antigen (Cat. #10377H08H,Sino Biological), cyno PD-1 antigen (purchased from Sino Biological) flowed across the chip surface, and the Biacore T200 instrument detected the PD-1 antibody and antigen PD-1 reaction signal in real time to obtain binding and dissociation curves. After completion of dissociation in each experimental cycle, the biochip was washed and regenerated with 10mM Glycine-HCl buffer pH 1.5. The experimental buffer system was 1 XHBS-EP buffer (Cat#BR-1001-88, GE). The data were fitted with the (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0.0 software after the end of the experiment to give affinity values, 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 exemplary anti-PD-1 antibodies of the present disclosure, hu23-11.IgG4AA, hu32a-85.IgG4AA, and Hu33-6.IgG4AA, are each capable of binding to human PD-1 and monkey PD-1.

Test example 4 secretion of cell IFNγ by antibodies in PBMC-T lymphocyte activation experiments

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

Fresh blood was subjected to Ficoll-Hypaque (17-5442-02, GE), density gradient centrifugation (Stem Cell Technologies) to obtain PBMC, and cultured in RPMI 1640 (SH 30809.01, GE) medium supplemented with 10% (v/v) FBS (10099-141, gibco) at 37℃under 5% CO ₂.

Fresh isolated and purified PBMC were conditioned to a density of 2X 10 ⁶/mL in RPMI 1640 medium and 40. Mu.l tuberculin (97-8800, synbiotics) was added to 20mL cell suspension and incubated in a 5% CO ₂ incubator at 37℃for 5 days. On day 5, the above cultured cells were collected, centrifuged, resuspended in fresh RPMI 1640 medium, and plated at a density of 1.1X10 ⁶/ml in 96-well cell culture plates at 90. Mu.l per well. Simultaneously, gradient diluted 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) were added, and the antibody gradient diluted to a concentration of 0.3mg/ml, 3mg/ml, 30mg/ml, and diluted with PBS (B320, shanghai source biosciences Co., ltd.) at 10. Mu.l per well. The cell culture plates were incubated at 37℃in a 5% CO ₂ incubator for 3 days. The cell culture plates were removed, centrifuged (4000 rpm,10 min) to collect the cell culture supernatant, and the levels of IFN-gamma were detected by ELISA (human IFN-gamma detection kit (EHC 102g.96, xinbo) for specific procedures, see reagent instructions.

The experimental results are shown in FIG. 2, and the results show that the anti-PD-1 antibodies Hu23-11.IgG4AA, hu32a-85.IgG4AA and Hu33-6.IgG4AA can effectively activate IFN-gamma secretion, and the capacity of activating IFN-gamma secretion is similar to that of positive H005-1 control.

Test example 5 effect of anti-PD-1 antibody in transgenic PD-1 mouse colon cancer model MC38

MC38 cells 5×10≡5 cells/mouse/100 μl were inoculated subcutaneously in right rib of 90 hPD-1TG mice (Baiocigram), and after 10 days animals with excessively large and small tumor volumes were removed, and the mice were randomly divided into: blank control Vehicle (PBS), positive control H005-1 3mpk、 Hu32a-85.IgG4AA 1mpk、Hu32a-85.IgG4AA 3mpk、Hu23-11.IgG4AA 1mpk、 Hu23-11.IgG4AA 3mpk、Hu33-6.IgG4AA 3mpk, 7 groups of 8 each. Day0 (Day 0) was given three times per week by intraperitoneal injections of each group of antibodies, and after the end of the first week, the tumor was found to be significantly inhibited, and the frequency of administration was adjusted once per week for 5 times in the second and third weeks. Tumor volume, animal weight were monitored 2 times per week and data recorded. Euthanasia of tumor-bearing animals was taken as the end point of the experiment when tumor volumes exceeded 2000mm 3 or most tumors had crumbed or lost 20% of body weight.

Tumor Volume (TV) =1/2×l _{Long length}×L_{Short length} ²

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

Tumor inhibition (TGI%) =1-T/C%

(Wherein T, T0 represents the tumor volume at the end of the test and at the start of the test, respectively, of the antibody administration group, C, C represents the tumor volume at the end of the test and at the start of the test, respectively, of the placebo group.)

The experimental results are shown in table 19 and fig. 3, and the experimental results show that compared with a blank control, the antibody disclosed by the invention can obviously inhibit the growth of the mouse colon cancer MC38 transplanted tumor, wherein the tumor inhibition rate is 77.64% in the last measurement of the Hu32a-85.IgG4AA-3mpk group. When the administration frequency is 3 times of three times a week, and the detection is carried out on the seventh day, the results show that the tumor inhibition rate of the antibody disclosed by the invention is obviously superior to that of a positive control antibody H005-1; the frequency of dosing was then reduced once a week and 2 times after dosing (Day 21), the efficacy of the antibodies of the present disclosure gradually pulled apart and showed a dose dependency, with Hu32a-85.Igg4aa being significantly better than the equivalent dose of H005-1 (p < 0.05). Moreover, the tumor-bearing mice can well tolerate the anti-PD-1 antibody, the weight of the tumor-bearing mice steadily rises in the whole administration process, and no obvious symptoms such as weight loss caused by medicines occur.

TABLE 19 influence of anti-PD-1 antibodies on tumor suppression Rate of mouse colon carcinoma MC38

Test example 6 role of anti-PD-1 antibodies in transgenic PD-1 mouse colon cancer model MC38

Transgenic PD-1 mice were derived from fifth generation mice grown in commercially available transgenic PD-1 mice (ISIS INNOVATION LIMITED, university Offices, wellington Square, oxford OX 12 jd, england) at Cephrim Biosciences, inc. MC38 cells were inoculated subcutaneously into the rear part of right ribs of hPD-1 transgenic mice (male and female halves) at a ratio of 5X10 ⁵/100. Mu.l, animals with body weight, excessive and excessively small tumors were removed when the average tumor volume of the mice reached 80-100mm3, and tumor-bearing mice were randomly divided into 5 groups (8 per group) according to tumor volume size: negative controls hIgG control 30mpk, H005-1 10mpk, H005-1 30mpk, hu33-6.IgG4AA 10mpk, hu33-6.IgG4AA 30mpk. The group administration date is set to Day 0. Each drug was given intraperitoneally after grouping for 22 days with a total of 11 doses every two days. Tumor volumes were measured 2 times per week, weighed, and data recorded. Body weight and tumor volume of each group were expressed as Mean ± standard deviation (Mean ± SEM) and plotted using GRAPHPAD PRISM and Excel software using student t test statistical analysis.

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

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

Tumor inhibition% tgi=1-T/C%

The experimental results are shown in table 20 and fig. 4, and compared with the control group, the experimental results show that the antibody can obviously inhibit the growth of the mouse colon cancer MC38 transplanted tumor, wherein the tumor inhibition rate is the highest in the Hu33-6.IgG4AA 30mpk group, and the tumor inhibition rate is 80.4% when measured on the 20 th day. In the low dose group (10 mpk), hu33-6.IgG4AA-10mpk was better than the positive control H005-1-10mpk.

TABLE 20 influence of anti-PD-1 antibodies on mouse colon carcinoma MC38 tumor volume

Remarks: the average tumor volume units for each group in the table are: mm ³.

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

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 were all IgG-scFv. TJ021-PR0002 and TJ021-PR0003 have the structure of scFv-Fab IgG. All antibodies were subjected to the L234A, L a mutation in the Fc portion to eliminate Fc-mediated ADCC.

The structure of TJ021-PR0001 is schematically shown in FIG. 5A, wherein the IgG moiety is an anti-human PD-1 antibody (anti-PD-1 IgG), the scFv moiety consists of a heavy chain variable region (VH) and a light chain variable region (VL) of the 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 (GGGGS GGGGS GGGGS). The Anti-PD-L1 scFv is linked to the C-terminus of Anti-PD-1IgG by a second peptide linker (GGGGSGGGGSGGGGSGGGGS).

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 structure of TJ021-PR0002 is schematically shown in FIG. 5B, wherein the Fab IgG fraction is anti-human PD-1 antibody (anti-PD-1 Fab), the scFv fraction consists 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. The Anti-PD-L1 scFv is linked 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-CH 3) (SEQ ID NO: 154)

The structure of TJ021-PR0003 is shown in FIG. 5C, and differs from TJ021-PR0002 in that the Fab IgG moiety is an anti-human PD-L1 antibody (anti-PD-L1 Fab) and the scFv moiety is an 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)

The amino acid sequence of the heavy chain (PD-1 scFv-hinge region-CH 2-CH 3) of TJ021-PR0003 (SEQ ID NO: 156)

The structural schematic of TJ021-PR0004 is shown in FIG. 5D, and differs from TJ021-PR0001 in that the Anti-PD-L1 scFv is linked to the N-terminus of Anti-PD-1 IgG 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 structure of TJ021-PR0005 is shown in FIG. 5A, and differs from TJ021-PR0001 in that one amino acid at the C-terminal end of Fc is mutated from K in TJ021-PR0001 to A.

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 schematic of TJ021-PR0006 is shown in FIG. 5D, and differs from TJ021-PR0004 in the sequence of the 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 schematic of TJ021-PR0007 is shown in FIG. 5A, and differs from TJ021-PR0001 in the sequence 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 antibodies

CHO-S cells (available from Thermo, cat. No. A29133) well grown in the logarithmic growth phase were centrifuged and inoculated at 250ml per ml of 6X 10 ⁶ cells. Solution 2 (9.2 ml of culture solution was used to dilute 800. Mu.l of transfection reagent, and mixed well) was added to solution 1 (10 ml of culture solution was used to dilute 250. Mu.g of plasmid, mixed well), the total volume was 20ml, and after gentle mixing, incubation was performed at room temperature for 1-5 minutes, not more than 5 minutes, the mixed transfection solution was added dropwise to the cell solution, and shaking was performed. The flask was then placed in 5% CO ₂, at 32℃and 123rpm, and incubated with shaking for 18-22 hours and 16ml of adjuvant Feed (available from Thermo company under the trade designation A29133) and 0.6ml of Enhancer (available from Thermo company under the trade designation A29133). On the fifth day 16ml of adjuvant Feed (available from Thermo company under the trade designation A29133), 5% CO ₂, 32℃at 123rpm, shaking culture and centrifugation of the supernatant for 12-14 days. Purification of the recombinant antibodies was performed by affinity chromatography (Protein A) and ion exchange two-step method. The media used in the purification were MabSelect SuRe column (GE, 17-5438) and 1ml CM pre-packed column (GE, 17505601) produced by GE company, respectively. The above seven anti-PD-1/PD-L1 bispecific antibodies were prepared, and 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 weight of the two double antibodies is 200kDa, the heavy chain is 75kDa, the light chain is 25kDa, and the double antibodies are judged by the non-reducing band size and the reducing band size of SDS-PAGE gel electrophoresis, the band size accords with expectations, and the recombinant double-specificity antibody can be assembled and expressed correctly without obvious aggregation degradation.

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

ELISA was used to detect the binding activity of anti-PD-1/PD-L1 bispecific antibodies TJ021-PR0001, TJ021-PR0002 and TJ021-PR0003 to human PD-L1 protein and human PD-1 protein, respectively. The 96-well plate was labeled before the start of the experiment, and coated overnight at a concentration of 1. Mu.g/ml antigen, 50. Mu.l per well, at 4 ℃. The next day, the coated antigen plate was removed and the plate washer was washed once with 1 XPBST. After washing, 1% BSA blocking solution prepared in 1 XPBST was blocked at 37℃for 1 hour. After washing the plates 3 times with 1 XPBST, the diabodies to be examined were added at different dilution concentrations (0.01 nM, 0.1nM, 1nM, 10nM, 100 nM) and incubated in an incubator at 37℃for 1 hour. After 3 washes of 1 XPBST, 100. Mu.l of goat anti-human IgG secondary antibody at 1:5000 dilution was added and incubated for 0.5 hours at 37 ℃. After washing the plate, TMB developing solution A and TMB developing solution B are taken according to the proportion of 1: mixing at a ratio of 1, and developing. The color reaction was stopped with 1M hydrochloric acid for 15 minutes. The 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 respectively with the human PD-L1 protein and the human PD-1 protein are kept similar to the binding activities of the original parent monoclonal antibodies.

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

The affinity of anti-PD-1/PD-L1 bispecific antibody TJ021-PR0001 with its antigens human PD-L1 protein and human PD-1 protein, respectively, was detected using surface plasmon resonance (surface plasmon resonance, SPR). Antigens human PD-L1 protein and human PD-1 protein were immobilized on CM5 chip, respectively, and the coupling level was set at 100RU, and detection was performed 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). Diluted antibodies (concentrations 6.25nM, 12.5nM, 25nM, 50nM, 100nM, respectively) were flowed through the experimental and control channels (100 nM antigen human PD-L1 protein/antigen human PD-1) for 3 min at a flow rate of 30. Mu.l/min and dissociated for 5 min. Regeneration buffer 10mM Glycine pH 1.5 (GE HEALTHCARE, BR-1003-54) was then run at a flow rate of 30 μl/min for 30 seconds. The data were analyzed using biacore 8K evaluation software software. The results are shown in FIGS. 8A and 8B.

The results show that the bispecific antibody TJ021-PR0001 has an affinity KD value of 3.18nM for the human PD-1 protein and 0.75nM for the human PD-L1 protein. TJ021-PR0001 has about 4 times higher affinity for human PD-L1 protein than for human PD-1 protein.

Example 7 anti-PD-1/PD-L1 bispecific antibodies promote cell bridging experiments

The human PD-1 and human PD-L1 plasmids were transferred into CHOK1 cells (ATCC, CCL-61), respectively, to construct a stably expressed cell line. CHOK1-hPD-L1 and CHOK1-hPD-1 were resuspended in PBS and the cell density was adjusted to 2X 10 ⁶/mL. Mix with 4M CFSE (Invitrogen), 10M Cell Proliferation Dye eFluor ^TM 670 (ebioscience) and wash off the fluorochromes after 10 minutes of shading at 37 ℃. The cell density was adjusted to 2X 10 ⁶/mL, and diluted antibody to be tested was added to a 96-well round bottom plate, incubated at 4℃for 60 minutes in the absence of light, and then the proportion of double positive cells was measured by flow cytometry, and the results are 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 are able to promote bridging of CHOK1-hPD-L1 cells and CHOK1-hPD-1 cells dose-dependently, whereas PD-1 control mab and PD-L1 control mab block PD-1/PD-L1 mediated bridging between the two cells by epitope-binding.

In the embodiment of the disclosure, the VH sequence of the PD-1 control monoclonal antibody is shown as SEQ ID NO:27, the VL sequence is set forth in SEQ ID NO: shown at 55.

In the embodiment of the disclosure, the VH sequence of the PD-L1 control monoclonal antibody is shown as SEQ ID NO:121, the VL sequence is set forth in SEQ ID NO: 122.

Example 8 anti-PD-1/PD-L1 bispecific antibody blocking PD-1/PD-L1 Signal pathway experiments

PD-1 and NFAT luciferase reporter plasmids (Promega, pGL4.30[ luc2P/NFAT-RE/Hygro ] Vector) were stably transfected in Jurkat (ATCC) cells to obtain a stable cell line Jurkat-NFAT-PD-1 with PD-1 expression and NFAT luciferase reporter. The day prior to the experiment, cell lines Hep3B-PD-L1 stably expressing high levels of PD-L1 were seeded in 96-well plates (PERKIN ELMER, cat# 6005181). After adding different concentrations of the antibody to be tested (0.04 nM, 0.12nM, 0.37nM, 1.11nM, 3.33nM, 10 nM), incubation was carried out for 30 min at 37 ℃; then Jurkat-NFAT-PD-1 cells were added and incubated at 37℃in a 5% carbon dioxide incubator for 6 hours; the test plate was taken out and equilibrated at room temperature for 10 minutes, then luciferase assay reagent ONE-GLUTM (Promega, cat#E6120) was added and fluorescence values were read using Envision (Perkinelmer, 2150), see FIGS. 10A and 10B for results. It can be seen that TJ021-PR0001, TJ021-PR0002, TJ021-PR0006, TJ021-PR0007 and positive control LY3434172 can release the inhibition of NFAT fluorescent signal mediated by PD-1/PD-L1 signal pathway in a dose-dependent manner, and the bispecific antibody inhibition ability is stronger than that of monoclonal antibody PD-1 control mab and PD-L1 control mab.

Example 9 anti-PD-1/PD-L1 bispecific antibodies enhance T cell activation experiments in SEB stimulated PBMC

1X 10 ⁵/mL human peripheral blood mononuclear cells (StemExpress) were added with 100ng/mL staphylococcal enterotoxin B (SEB, conbaberin) while a series of concentrations (0.01 nM, 0.1nM, 1nM, 10nM, 100 nM) of seven antibodies to be tested and positive control LY3434172 were added, respectively, and a nonspecific IgG control and SEB-free control were set. After incubation in a CO ₂ incubator at 37℃for 72 hours, the supernatant was assayed for IL-2 (Invitrogen, human IL-2 kit) secretion by ELISA. The results are shown in FIG. 7 for bispecific antibodies TJ021-PR0001, TJ021-PR0002, TJ021-PR0003, TJ021-PR0006, TJ021-PR007 and positive control LY3434172 that are dose dependent to promote IL-2 secretion by T cells. In FIG. 11A TJ021-PR0001 activated T cells more than the combination of PD-1 control mab and PD-L1 control mab, and TJ021-PR0001 was stronger than TJ021-PR0002. In FIG. 11B TJ021-PR0006 is more activated for T cells than the combination of PD-1mAb and PD-L1 mAb.

Example 10 Mixed lymphocyte reaction experiments with anti-PD-1/PD-L1 bispecific antibodies

The frozen human peripheral blood mononuclear cells (StemExpress) were resuscitated and the cell density was adjusted to 2X 10 ⁶/mL, transferred to 10cm cell culture dishes, 10mL each, incubated in a CO ₂ incubator at 37℃for 2 hours to allow the mononuclear cells to adhere, washed once with serum-free cell culture medium, and then cultured for five days with fresh medium containing 50ng/mL GM-CSF (Peprotech, cat # 300-03) and 50ng/mL hIL-4 (Peprotech, cat # 200-04) for 2 to 3 days with liquid changes and the cell density adjusted to 2X 10 ⁵/mL. CD4+ T cells (STEMCELL, easySep ^TM human CD4+ T Cell Isolation Kit) were isolated from human peripheral blood mononuclear cells of different individuals and the CD4+ T cell density was adjusted to 1X 10 ⁶/mL. Dendritic cells and CD4+ T cells were added 1:1 to 96-well U-shaped plates (Corning, cat#3799) and gradient diluted antibodies (0.01 nM, 0.1nM, 1nM, 10nM, 100 nM) were added and incubated in a CO ₂ incubator at 37℃for 72 hours, and the supernatant TR-FRET method was used to detect IL-2 secretion (Cisbio, human IL-2 kit) as shown in FIG. 12; the results show that TJ021-PR0006 and TJ021-PR0007 in FIG. 12 activate more mixed lymphocytes than do 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 as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

SEQUENCE LISTING

<110> Jiangsu Hengrui medicine Co., ltd

Shanghai Hengrui medicine Co., ltd

<120> 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> Transfected cell nucleic acid encoding PD-1 antigen

<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> Light chain variable region 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> Light chain variable region 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> Light chain variable region 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: IGHV 1-69.times.02 and IGHJ 6.times.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 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 general formula HCDR2

Wherein X2 is selected from F or I and 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 general formula HCDR3

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

X9 is selected from G or a vacancy, X10 is selected from S or a vacancy, X11 is selected from N or T, and 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> General formula LCDR1 of the variable region consensus sequence of Hu32 and Hu23 antibodies

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

X15 is G, A or V, X16 is 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 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 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> General formula LCDR3 of the variable region consensus sequence of Hu32 and Hu23 antibodies

<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.IgG 4P 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.IgG 4P 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> General formula HCDR 1Z 1 is 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> General formula LCDR1 wherein Z2 is R or H and 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, and Z7 is selected from G, F, L

W and M, Z8 is selected from A, P and T, Z9 is 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, and 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 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 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> 24D5-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> 24D5-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 mutant 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 24D5L 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> Heavy chain variable region sequence formula

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, and 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 formula

Z17 is V or A, Z18 is Y, K or N, Z19 is selected from A, L and V, and 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> 9-2 H5 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> 9-2 H6 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> 9-2 H7 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 24D5H21 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> Heavy chain amino acid sequence of PD-1 antibody in IgG1 form

<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> Construction of heavy chain amino acid sequence of IgG 1-form PD-1 antibody

<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> HRP00049:9-2 (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> HRP00052:24D5 (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> Constructed heavy chain of IgG 1-form PD-L1 antibody

<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 heavy chain (PD-L1 scFv-hinge region-CH 2-CH 3) TJ021-PR0002

<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 TJ021-PR0003 heavy chain (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 heavy chain (PD-1 scFv-hinge region-CH 2-CH 3) TJ021-PR0003

<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 24D5L 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 24D5L 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. A bispecific antibody 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 as follows:

(e) HCDR1, HCDR2 and HCDR3 having sequences DYEMH (SEQ ID NO: 8), LIDPETGGTVYNQKFKD (SEQ ID NO: 9) and ERFSYYGSTSDWYFDV (SEQ ID NO: 10), respectively; and

LCDR2 of KVSNSRFS (SEQ ID NO: 12) and LCDR3 of FQGSHVPYT (SEQ ID NO: 13); LCDR1 is 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).

2. The bispecific antibody of claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region and a first light chain variable region comprising a framework region or framework region variant derived from a human antibody; the back mutations of the framework region variants on the heavy and/or light chain framework regions of the human antibody are shown below:

(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 the group consisting of 27Y, 48I, 67T, 69L, 82F, and 93T;

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

(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.

3. The bispecific antibody of claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region and a first light chain variable region selected from any one of (l) and (o):

(l) The sequence is shown in SEQ ID NO:4 and the first heavy chain variable region shown in SEQ ID NO:5, a first light chain variable region shown in figure 5;

(o) the sequence of SEQ ID NO: 27. 30, 31 or 32 and a first heavy chain variable region having the sequence set forth in SEQ ID NO: 28. 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64.

4. The bispecific antibody of claim 2, 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;

(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; or alternatively

(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-CH3; wherein the first light chain comprises a first light chain variable region and a first light chain constant region derived from a human antibody kappa chain constant region, lambda chain constant region and conventional variants thereof.

5. The bispecific antibody of claim 4, wherein the first peptide linker is a GS linker.

6. The bispecific antibody of claim 5, wherein the amino acid sequence of the first peptide linker is: GGGGSGGGGSGGGGS.

7. The bispecific antibody of claim 4, wherein the hinge region-CH 2-CH3 is derived from human IgG1, igG2, igG3 or IgG4.

8. The bispecific antibody of claim 7, wherein the hinge region-CH 2-CH3 is derived from human IgG1.

9. The bispecific antibody of claim 8, wherein the hinge region-CH 2-CH3 is derived from human IgG1 introduced with at least one mutation of L234A, L235A, T366S, L368A, Y V and K447A, human IgG1 introduced with mutations of L234A, L235A, T366S, L368A and Y407V, or human IgG4 introduced with at least one mutation of S228P, F234A and L235A.

10. The bispecific antibody of claim 4, wherein the CH 1-hinge region-CH 2-CH3 is derived from human IgG1, igG2, igG3, igG4 or conventional variants thereof.

11. The bispecific antibody of claim 10, wherein the CH 1-hinge region-CH 2-CH3 is derived from human IgG1 or a conventional variant thereof.

12. The bispecific antibody of claim 11, wherein the CH 1-hinge region-CH 2-CH3 is derived from human IgG1 introduced with at least one mutation in L234A, L235A, T366S, L368A, Y V and K447A, human IgG1 introduced with mutations in L234A, L235A, T366S, L368A and Y407V, or human IgG4 introduced with at least one mutation in S228P, F234A and L235A.

13. The bispecific antibody of claim 4, wherein the first heavy chain constant region has a sequence as set forth in SEQ ID NO:72 or 79.

14. The bispecific antibody of claim 4, wherein the first light chain constant region has a sequence as set forth in SEQ ID NO: shown at 73.

15. The bispecific antibody of claim 4, wherein the first antigen-binding domain comprises a combination of a first heavy chain and a first light chain selected from any one of (u 2) - (u 3):

(u 2) and SEQ ID NO:74 or 80, a first heavy chain having at least 80% identity to the amino acid sequence shown in seq id no; and

And SEQ ID NO:75, a first light chain having at least 80% identity to the amino acid sequence set forth in seq id no; and

(U 3) and SEQ ID NO: 144. 145 or 146, and a first heavy chain having at least 80% identity to the amino acid sequence shown in seq id no; and

And SEQ ID NO:75, and a first light chain having at least 80% identity to the amino acid sequence set forth in seq id no.

16. The bispecific antibody of any one of claims 1-15, wherein the second antigen-binding domain specifically binds PD-L1.

17. The bispecific antibody of claim 16, wherein the second antigen binding domain comprises:

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 alternatively

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).

18. The bispecific antibody of claim 1, 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 framework region variant derived from a human antibody.

19. The bispecific antibody of claim 18, wherein 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 x 01 and hjh2, comprising the FR1, FR2, FR3 region of human germline heavy chain IGHV4-30-4 x 01 and FR4 region of hjh; or a combination sequence derived from human germline heavy chain IGHV1-46 x 01 and hjh 6.1.1 comprising the FR1, FR2, FR3 region and FR4 region of hjh 6.1.1 of human germline heavy chain IGHV1-46 x 01;

20. The bispecific antibody of claim 18, wherein the back mutation of the framework region variant in the heavy chain framework region and/or the light chain framework region of a human antibody is as follows:

(ai) one or more amino acid back mutations in the second heavy chain variable region selected from W47Y, V71R, G Y, I M, V67L, F78Y, S30T, Q K and P49S;

(aj) one or more amino acid back mutations in the second heavy chain variable region selected from T74K, R72, 72V, M48I, M L, R Q, L83F, V68A, V79A, Y F, T S and G72E; or alternatively

(Ak) the second heavy chain variable region comprises an N85E deglycosylation mutation.

21. The bispecific antibody of claim 20, wherein the second heavy chain variable region comprises one or more amino acid back mutations selected from W47Y, V R and P49S.

22. The bispecific antibody of claim 1, wherein the second antigen-binding domain comprises a second heavy chain variable region and a second light chain variable region as shown below:

(aq) sequence as set forth in SEQ ID NO:121 and 132, and a second heavy chain variable region having a sequence as set forth in any one of SEQ ID NOs: 171, and a second light chain variable region shown at 171.

23. The bispecific antibody of claim 18, wherein the second antigen binding domain comprises:

(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;

(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; or alternatively

(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 a CH 1-hinge region-CH 2-CH3; 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.

24. The bispecific antibody of claim 23, wherein the first peptide linker is a GS linker.

25. The bispecific antibody of claim 24, wherein the amino acid sequence of the first peptide linker is: GGGGSGGGGSGGGGS.

26. The bispecific antibody of claim 23, wherein the hinge region-CH 2-CH3 is derived from human IgG1, igG2, igG3 or IgG4.

27. The bispecific antibody of claim 26, wherein the hinge region-CH 2-CH3 is derived from human IgG1.

28. The bispecific antibody of claim 27, wherein the hinge region-CH 2-CH3 is derived from human IgG1 introduced with at least one mutation in L234A, L235A, T S or T366W, L368A, Y407V and K447A, human IgG1 introduced with a mutation in L234A, L235A, T366S or T366W, L368A and Y407V, or human IgG4 introduced with at least one mutation in S228P, F234A and L235A.

29. The bispecific antibody of claim 23, wherein the CH 1-hinge region-CH 2-CH3 is derived from human IgG1, igG2, igG3, igG4, or conventional variants thereof.

30. The bispecific antibody of claim 29, wherein the CH 1-hinge region-CH 2-CH3 is derived from human IgG1 or a conventional variant thereof.

31. The bispecific antibody of claim 30, wherein the CH 1-hinge region-CH 2-CH3 is derived from human IgG1 introduced with at least one mutation in L234A, L235A, T366S, L368A, Y V and K447A, human IgG1 introduced with mutations in L234A, L235A, T366S, L368A and Y407V, or human IgG4 introduced with at least one mutation in S228P, F234A and L235A.

32. The bispecific antibody of claim 23, wherein the second heavy chain constant region has a sequence as set forth in SEQ ID NO:72 or 79, and/or the sequence of the second light chain constant region is set forth in SEQ ID NO: shown at 73.

33. The bispecific antibody of claim 23, wherein the second antigen binding domain comprises a combination of a second heavy chain and a second light chain selected from any one of (bf 2) - (bf 3):

(bf 2) and SEQ ID NO:149, a second heavy chain having at least 80% identity to the amino acid sequence shown in seq id no; and

And SEQ ID NO:172, a second light chain having at least 80% identity to the amino acid sequence set forth herein; and

(Bf 3) and SEQ ID NO:151, a second heavy chain having at least 80% identity to the amino acid sequence set forth in seq id no; and

And SEQ ID NO:172 has a second light chain having at least 80% identity.

34. The bispecific antibody of claim 4 or 23, 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;

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

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

a second polypeptide comprising a second scFv-hinge region-CH 2-CH3; and

A third polypeptide comprising a first light chain;

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

A second polypeptide comprising a first scFv-hinge region-CH 2-CH3; and

A third polypeptide comprising a second light chain;

(ggg) a first polypeptide comprising a first scFv-hinge region-CH 2-CH3; and

A second polypeptide comprising a second scFv-hinge region-CH 2-CH3;

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.

35. The bispecific antibody of claim 34, wherein the hinge region-CH 2-CH3 is derived from human IgG1 into which the L234A, L a and T366W mutations have been introduced; and/or the number of the groups of groups,

The hinge region-CH 2-CH3 is derived from human IgG1 having introduced mutations L234A, L A and T366W;

and/or the number of the groups of groups,

The hinge region-CH 2-CH3 was derived from human IgG1, into which the L234A, L A and T366W mutations had been introduced.

36. The bispecific antibody of claim 34, wherein the second peptide linker is a GS linker.

37. The bispecific antibody of claim 36, wherein the amino acid sequence of the second peptide linker is: GGGGSGGGGSGGGGSGGGGS.

38. The bispecific antibody of claim 34, comprising a combination of polypeptides selected from any one of (jjj) - (ppp):

(jjj) and SEQ ID NO:152, a first polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no; and

And SEQ ID NO:75, a second polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no;

(kkk) and SEQ ID NO:153, a first polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no;

And SEQ ID NO:75, a second polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no; and

And SEQ ID NO:154, a third polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no;

(lll) and SEQ ID NO:155, a first polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no;

And SEQ ID NO:145, a second polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no; and

And SEQ ID NO:156, a third polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no;

(mmm) and SEQ ID NO:157, a first polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no; and

(nnn) and SEQ ID NO:158, a first polypeptide having at least 80% identity to the amino acid sequence depicted in seq id no; and

(ooo) and SEQ ID NO:159, a first polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no; and

And

(Ppp) and SEQ ID NO:160, a first polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no; and

And SEQ ID NO:75, and a second polypeptide having at least 80% identity to the amino acid sequence set forth in seq id no.

39. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 38 and at least one pharmaceutically acceptable carrier, diluent, buffer or excipient; or alternatively

The pharmaceutical composition comprises a first antibody comprising a first antigen binding domain as defined in any one of claims 1 to 38 and a second antibody comprising a second antigen binding domain as defined in any one of claims 1 to 38.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition further comprises a chemotherapeutic agent, a radiopharmaceutical, and/or other agents for cancer immunotherapy.

41. A nucleic acid composition comprising at least one nucleic acid molecule encoding the bispecific antibody of any one of claims 1-38.

42. An expression vector composition comprising at least one expression vector comprising at least one nucleic acid molecule as defined in claim 41.

43. A host cell comprising the expression vector composition of claim 42, or transformed or transfected with the expression vector composition of claim 42.

44. The host cell of claim 43, wherein the host cell is selected from the group consisting of a bacterium, a yeast, an insect cell, and a mammalian cell.

45. A host cell according to claim 44, wherein the bacterium is E.coli, the yeast is Pichia pastoris, and the mammalian cell is selected from CHO cells, NSO cells, HEK293 cells or COS cells.

46. A method of making a bispecific antibody according to any one of claims 1 to 38, comprising:

Culturing a host cell as defined in any one of claims 43-45 under conditions such that the antigen binding molecule is expressed; and

Recovering the bispecific antibody.

47. The method of claim 46, wherein the method comprises a step of purifying the bispecific antibody.

48. Use of the bispecific antibody of any one of claims 1 to 38, the pharmaceutical composition of claim 39 or 40, the nucleic acid composition of claim 41 or the expression vector composition of claim 42 in the manufacture of a medicament for the treatment and/or prevention of a tumor or cancer;

Wherein the tumor or cancer is selected from: head and neck squamous cell carcinoma, head and neck carcinoma, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatocellular carcinoma, hepatobiliary carcinoma, pancreatic carcinoma, gastric carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma.

49. The use of claim 48, wherein the lymphoma is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinum large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma.

50. The use of claim 48, wherein the lung cancer is selected from the group consisting of: non-small cell lung cancer and small cell lung cancer.

51. The use of claim 48, wherein the tumor or cancer is selected from the group consisting of: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, kidney cancer, bladder cancer and bowel cancer and colon cancer.

52. The use of claim 48, wherein the tumor or cancer is associated with PD-1 and/or PD-L1.