Antibodies targeting pd-l1 / pd-1 and uses thereof

Abstract

The present application relates to an antibody targeting PD-L1 / PD-1 and its application, the antibody comprising a first polypeptide chain, a second polypeptide chain, the first polypeptide chain comprising a first targeting moiety, the second polypeptide chain comprising a second targeting moiety, the first targeting moiety and the second targeting moiety each independently binding to an immune cell-associated antigen, the first polypeptide chain being linked to the second polypeptide chain by a disulfide bond; the antibody of the present application can also carry a cytokine, or the antibody of the present application can bind to a tumor-associated antigen (TAA); the present application also provides an immunoconjugate comprising the antibody, a method for preparing the antibody and its use.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antibody targeting PD-L1 / PD-1 and its applications. Background Technology

[0002] Programmed death receptor ligand 1 (PD-L1), also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa type I transmembrane protein that is generally expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, and many non-hematopoietic cells.

[0003] PD-L1 can bind to programmed death receptor 1 (PD-1) and B7-1 (CD80). The PD-L1 / PD-1 signaling pathway is a crucial co-inhibitory signaling pathway in the immune response, negatively regulating T cell immune responses, inhibiting T cell activity, and reducing cytokine secretion. Studies have found that PD-L1 is expressed in many tumor tissues, including gastric cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, colon cancer, mast cell tumors, and malignant melanoma, as well as in bone marrow cells infiltrating the tumor microenvironment, protecting tumor cells from immune attack.

[0004] Currently, anti-PD-L1 and anti-PD-1 antibodies benefit many cancer patients, but some patients still do not respond to monotherapy. Multispecific antibodies that can bind to both PD-1 and PD-L1 proteins hold promise for enhancing anti-tumor activity. Therefore, there is an urgent need to develop PD1 / PD-L1 bispecific antibodies capable of blocking the PD-L1 / PD-1 signaling pathway. Summary of the Invention

[0005] This application provides an isolated antigen-binding protein having one or more of the following properties: 1) it can bind to both PD-1 and PD-L1 proteins; 2) the first and second polypeptide chains pair naturally without the need for unnatural engineered pairing, overcoming the mismatch problem in the general multispecific antibody production process and reducing costs; 3) compared to general multispecific antibodies, it covers an area of ​​30% larger than that of the antigen; and 4) it can efficiently kill tumor cells.

[0006] In some embodiments, the antigen-binding protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain including a first targeting portion and the second polypeptide chain including a second targeting portion, the first targeting portion and the second targeting portion each independently binding to immune cell-associated antigens, and the first polypeptide chain and the second polypeptide chain being linked by disulfide bonds.

[0007] In some embodiments, the immune cells include T cells, macrophages, dendritic cells, and / or NK cells.

[0008] In some embodiments, the immune cell-associated antigens include PD-1, PD-L1, CD3, CD16a, and / or CD28.

[0009] In some implementations, the first and second targeting portions bind to the same immune cell-associated antigen.

[0010] In some embodiments, both the first and second targeting portions bind to either the PD-1 or PD-L1 protein.

[0011] In some implementations, the first targeting portion specifically binds to the PD-1 protein, and the second targeting portion specifically binds to the PD-L1 protein.

[0012] In some implementations, the first targeting portion specifically binds to the PD-L1 protein, and the second targeting portion specifically binds to the PD-1 protein.

[0013] In some implementations, the first targeting portion specifically binds to the PD-1 protein, and the second targeting portion specifically binds to the CD28 or CD16a protein.

[0014] In some implementations, the first targeting portion specifically binds to the CD28 or CD16a protein, and the second targeting portion specifically binds to the PD-1 protein.

[0015] In some embodiments, the first polypeptide chain further includes a third targeting portion.

[0016] In some embodiments, the third targeting portion of the first polypeptide chain is directly or indirectly connected to the C-terminus of the first targeting portion.

[0017] In some embodiments, the third targeting portion of the first polypeptide chain is connected to the C-terminus of the first targeting portion via a linker.

[0018] In some implementations, the third targeting portion targets immune cell-associated antigens.

[0019] In some embodiments, the immune cell-associated antigens include PD-1, PD-L1, CD3, CD16a, and / or CD28.

[0020] In some embodiments, the third targeting portion targets the CD3 or CD16a protein.

[0021] In some implementations, the third targeting portion targets tumor-related antigens.

[0022] In some implementations, the third targeting portion targets EGFR and / or cMet.

[0023] In some implementations, the third targeting portion targets tumor-associated antigens.

[0024] In some embodiments, the tumor-associated antigens include PD-L1, HER2, CD47, CD19, and / or CD20.

[0025] In some embodiments, the second polypeptide chain further includes a fourth targeting portion.

[0026] In some embodiments, the fourth targeting portion of the second polypeptide chain is directly or indirectly connected to the C-terminus of the second targeting portion.

[0027] In some embodiments, the fourth targeting portion of the second polypeptide chain is connected to the C-terminus of the second targeting portion via a linker.

[0028] In some implementations, the fourth targeting portion targets immune cell-associated antigens.

[0029] In some embodiments, the immune cell-associated antigens include PD-1, PD-L1, CD3, CD16a, and / or CD28.

[0030] In some implementations, the fourth targeting portion targets tumor-related antigens.

[0031] In some implementations, the fourth targeting portion targets EGFR and / or cMet.

[0032] In some implementations, the fourth targeting portion targets tumor-associated antigens.

[0033] In some embodiments, the tumor-associated antigens include PD-L1, HER2, CD47, CD19, and / or CD20.

[0034] In some embodiments, the first polypeptide chain contains cytokines.

[0035] In some embodiments, the cytokines include IL-2, IFNα, IFNβ, and / or IL-15.

[0036] In some embodiments, the second polypeptide chain contains cytokines.

[0037] In some embodiments, the cytokines include IL-2, IFNα, IFNβ, and / or IL-15.

[0038] In some implementations, the first targeting portion targets PD-L1, the second targeting portion targets PD-L1, and the fourth targeting portion targets PD-1, CD3, or CD16a.

[0039] In some implementations, the first targeting portion targets PD-1, the second targeting portion targets PD-1, and the fourth targeting portion targets PD-L1, CD3, or CD16a.

[0040] In some implementations, the first targeting portion targets PD-L1, the second targeting portion targets PD-1, and the third targeting portion targets CD3 or CD16a.

[0041] In some implementations, the first targeting portion targets PD-1, the second targeting portion targets PD-L1, and the third targeting portion targets CD3 or CD16a.

[0042] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets CD28 or CD16a, the second targeting portion targets PD-1, and the fourth targeting portion targets PD-L1, HER2, CD47, CD19, and / or CD20.

[0043] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-1, the second targeting portion targets CD28 or CD16a, and the fourth targeting portion targets PD-L1, HER2, CD47, CD19, and / or CD20.

[0044] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets CD28 or CD16a, the second targeting portion targets PD-1, the third targeting portion targets EGFR or cMET, and the fourth targeting portion targets PD-L1, HER2, CD47, CD19, and / or CD20.

[0045] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-1, the second targeting portion targets CD28 or CD16a, the third targeting portion targets PD-L1, HER2, CD47, CD19 and / or CD20, and the fourth targeting portion targets EGFR or cMET.

[0046] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-1, the second targeting portion targets PD-1, and the second polypeptide chain contains IL-2 or IL-15.

[0047] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-L1, the second targeting portion targets PD-L1, and the second polypeptide chain contains IL-2 or IL-15.

[0048] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-1, the second targeting portion targets PD-L1, the first polypeptide chain contains IL-2 or IL-15, and the fourth targeting portion contains CD3 or CD16a.

[0049] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-L1, the second targeting portion targets PD-1, the first polypeptide chain contains IL-2 or IL-15, and the fourth targeting portion contains CD3 or CD16a.

[0050] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-L1, the second targeting portion targets PD-1, and the first polypeptide chain contains IL-2 or IL-15.

[0051] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-1, the second targeting portion targets PD-L1, and the first polypeptide chain contains IL-2 or IL-15.

[0052] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-1, the second targeting portion targets PD-1, the first polypeptide chain contains IL-2 or IL-15, and the fourth targeting portion targets PD-L1.

[0053] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-L1, the second targeting portion targets PD-L1, the first polypeptide chain contains IL-2 or IL-15, and the fourth targeting portion targets PD-1.

[0054] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-1, the second targeting portion targets PD-L1, the third targeting portion targets CD3 or CD16a, and the second polypeptide chain contains IL-2 or IL-15.

[0055] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets PD-L1, the second targeting portion targets PD-1, the third targeting portion targets CD3 or CD16a, and the second polypeptide chain contains IL-2 or IL-15.

[0056] In some embodiments, the first targeting portion comprises HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:51.

[0057] In some embodiments, the first targeting portion comprises HCDR3, which comprises the amino acid sequence shown in either SEQ ID NO:9 or SEQ ID NO:10.

[0058] In some embodiments, the first targeting portion comprises HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:6.

[0059] In some embodiments, the first targeting portion comprises HCDR1, which contains the amino acid sequence shown in SEQ ID NO:3.

[0060] In some embodiments, the first targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO:52.

[0061] In some embodiments, the first targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0062] In some embodiments, the first targeting portion includes a heavy chain variable region VH, the VH comprising HCDR1, HCDR2, and HCDR3, the HCDR3 comprising the amino acid sequence shown in SEQ ID NO:51; the HCDR2 comprising the amino acid sequence shown in SEQ ID NO:6; and the HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3.

[0063] In some embodiments, the first targeting portion includes a heavy chain variable region VH, the VH comprising HCDR1, HCDR2, and HCDR3, the HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10; the HCDR2 comprising the amino acid sequence shown in SEQ ID NO:6; and the HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3.

[0064] In some embodiments, HCDR1, HCDR2, and HCDR3 in the first targeting portion comprise amino acid sequences selected from any of the following groups:

[0065] a) HCDR1: SEQ ID NO:3, HCDR2: SEQ ID NO:6 and HCDR3: SEQ ID NO:9; and

[0066] b) HCDR1: SEQ ID NO:3, HCDR2: SEQ ID NO:6 and HCDR3: SEQ ID NO:10.

[0067] In some embodiments, the first targeting portion comprises H-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:47.

[0068] In some embodiments, the H-FR1 comprises the amino acid sequence shown in either SEQ ID NO:1 or SEQ ID NO:2.

[0069] In some embodiments, the first targeting portion includes H-FR2, which is located between HCDR1 and HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO:48.

[0070] In some embodiments, the H-FR2 comprises the amino acid sequence shown in either SEQ ID NO:4 or SEQ ID NO:5.

[0071] In some embodiments, the first targeting portion includes H-FR3 located between HCDR2 and HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO:49.

[0072] In some embodiments, the H-FR3 in the first targeting portion comprises the amino acid sequence shown in either SEQ ID NO:7 or SEQ ID NO:8.

[0073] In some embodiments, the first targeting portion comprises H-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

[0074] In some embodiments, the H-FR4 in the first targeting portion comprises the amino acid sequence shown in either SEQ ID NO:11 or SEQ ID NO:12.

[0075] In some embodiments, the first targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:47; H-FR2 comprises the amino acid sequence shown in SEQ ID NO:48; H-FR3 comprises the amino acid sequence shown in SEQ ID NO:49; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

[0076] In some embodiments, the first targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2; H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5; H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:8; and H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:12.

[0077] In some embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 in the first targeting portion comprise an amino acid sequence selected from any one of the following groups:

[0078] a) H-FR1: SEQ ID NO: 1, H-FR2: SEQ ID NO: 4, H-FR3: SEQ ID NO: 7 and H-FR4: SEQ ID NO: 11; and

[0079] b) H-FR1: SEQ ID NO:2, H-FR2: SEQ ID NO:5, H-FR3: SEQ ID NO:8 and H-FR4: SEQ ID NO:12.

[0080] In some embodiments, the first targeting portion includes a heavy chain variable region VH, the VH containing the amino acid sequence shown in SEQ ID NO:52.

[0081] In some embodiments, the first targeting portion includes a heavy chain variable region VH, the VH comprising an amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0082] In some embodiments, the first targeting portion comprises LCDR3, which contains the amino acid sequence shown in SEQ ID NO:57.

[0083] In some embodiments, the first targeting portion comprises LCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26.

[0084] In some embodiments, the first targeting portion comprises LCDR2, which contains the amino acid sequence shown in SEQ ID NO:18.

[0085] In some embodiments, the first targeting portion comprises LCDR1, which contains the amino acid sequence shown in SEQ ID NO:15.

[0086] In some embodiments, the first targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in SEQ ID NO:58.

[0087] In some embodiments, the first targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0088] In some embodiments, the first targeting portion includes a light chain variable region VL, the VL comprising LCDR1, LCDR2, and LCDR3, the LCDR3 comprising the amino acid sequence shown in SEQ ID NO:57; the LCDR2 comprising the amino acid sequence shown in SEQ ID NO:18; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:15.

[0089] In some embodiments, the first targeting portion includes a light chain variable region VL, the VL comprising LCDR1, LCDR2, and LCDR3, the LCDR3 comprising an amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26; the LCDR2 comprising an amino acid sequence shown in SEQ ID NO:18; and the LCDR1 comprising an amino acid sequence shown in SEQ ID NO:15.

[0090] In some embodiments, LCDR1, LCDR2, and LCDR3 in the first targeting portion comprise amino acid sequences selected from any of the following groups:

[0091] a) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 21;

[0092] b) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 22;

[0093] c) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 23;

[0094] d) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 24;

[0095] e) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:25; and

[0096] f) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:26.

[0097] In some embodiments, the first targeting portion includes L-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO:53.

[0098] In some embodiments, the L-FR1 in the first targeting portion comprises the amino acid sequence shown in either SEQ ID NO:13 or SEQ ID NO:14.

[0099] In some embodiments, the first targeting portion includes L-FR2 located between LCDR1 and LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO:54.

[0100] In some embodiments, the L-FR2 in the first targeting portion comprises the amino acid sequence shown in either SEQ ID NO:16 or SEQ ID NO:17.

[0101] In some embodiments, the first targeting portion includes L-FR3 located between LCDR2 and LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO:55.

[0102] In some embodiments, the L-FR3 in the first targeting portion comprises the amino acid sequence shown in either SEQ ID NO:19 or SEQ ID NO:20.

[0103] In some embodiments, the first targeting portion comprises L-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0104] In some embodiments, the L-FR4 in the first targeting portion comprises the amino acid sequence shown in either SEQ ID NO:27 or SEQ ID NO:28.

[0105] In some embodiments, the first targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:53; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:54; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:55; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0106] In some embodiments, the first targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:13 and SEQ ID NO:14; L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:16 and SEQ ID NO:17; L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:19 and SEQ ID NO:20; and L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

[0107] In some embodiments, the L-FR1, L-FR2, L-FR3, and L-FR4 in the first targeting portion comprise an amino acid sequence selected from any one of the following groups:

[0108] a) L-FR1: SEQ ID NO: 13, L-FR2: SEQ ID NO: 16, L-FR3: SEQ ID NO: 19 and L-FR4: SEQ ID NO: 27; and

[0109] b) L-FR1: SEQ ID NO: 14, L-FR2: SEQ ID NO: 17, L-FR3: SEQ ID NO: 20 and L-FR4: SEQ ID NO: 28.

[0110] In some embodiments, the first targeting portion includes a VL containing an amino acid sequence shown in any of SEQ ID NO:58.

[0111] In some embodiments, the VL in the first targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0112] In some embodiments, the first targeting portion comprises HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:76.

[0113] In some embodiments, the first targeting portion comprises HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:70.

[0114] In some embodiments, the first targeting portion comprises HCDR1, which contains the amino acid sequence shown in SEQ ID NO:61.

[0115] In some embodiments, the first targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO:122.

[0116] In some embodiments, the first targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

[0117] In some embodiments, the first targeting portion includes a heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence shown in SEQ ID NO:76; the HCDR2 comprising the amino acid sequence shown in SEQ ID NO:70; and the HCDR1 comprising the amino acid sequence shown in SEQ ID NO:61.

[0118] In some embodiments, the first targeting portion comprises H-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:115.

[0119] In some embodiments, the H-FR1 in the first targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:59 to SEQ ID NO:60.

[0120] In some embodiments, the first targeting portion includes H-FR2, which is located between HCDR1 and HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO:116.

[0121] In some embodiments, the H-FR2 in the first targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69.

[0122] In some embodiments, the first targeting portion includes H-FR3 located between HCDR2 and HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO:117.

[0123] In some embodiments, the H-FR3 in the first targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:71 to SEQ ID NO:75.

[0124] In some embodiments, the first targeting portion comprises H-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:118.

[0125] In some embodiments, the H-FR4 in the first targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

[0126] In some embodiments, the first targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:115; H-FR2 comprises the amino acid sequence shown in SEQ ID NO:116; H-FR3 comprises the amino acid sequence shown in SEQ ID NO:117; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:118.

[0127] In some embodiments, the first targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:59 to SEQ ID NO:60; H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69; H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:71 to SEQ ID NO:75; and H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

[0128] In some embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 in the first targeting portion comprise an amino acid sequence selected from any one of the following groups:

[0129] a) H-FR1: SEQ ID NO: 59, H-FR2: SEQ ID NO: 62, H-FR3: SEQ ID NO: 71 and H-FR4: SEQ ID NO: 77;

[0130] b) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 63, H-FR3: SEQ ID NO: 72 and H-FR4: SEQ ID NO: 78;

[0131] c) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 64, H-FR3: SEQ ID NO: 73 and H-FR4: SEQ ID NO: 78;

[0132] d) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 65, H-FR3: SEQ ID NO: 74 and H-FR4: SEQ ID NO: 78;

[0133] e) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 75 and H-FR4: SEQ ID NO: 78;

[0134] f) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:67, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

[0135] g) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:65, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

[0136] h) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 64, H-FR3: SEQ ID NO: 74 and H-FR4: SEQ ID NO: 78;

[0137] i) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:68, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78; and

[0138] j) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:69, H-FR3: SEQ ID NO:73 and H-FR4: SEQ ID NO:78.

[0139] In some embodiments, the first targeting portion includes a heavy chain variable region VH, the VH containing the amino acid sequence shown in SEQ ID NO:122.

[0140] In some embodiments, the VH in the first targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

[0141] In some embodiments, the first targeting portion comprises LCDR3, which contains the amino acid sequence shown in SEQ ID NO:94.

[0142] In some embodiments, the first targeting portion comprises LCDR2, which contains the amino acid sequence shown in SEQ ID NO:87.

[0143] In some embodiments, the first targeting portion comprises LCDR1, which contains the amino acid sequence shown in SEQ ID NO:82.

[0144] In some embodiments, the first targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in any of SEQ ID NO:123.

[0145] In some embodiments, the first targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0146] In some embodiments, the first targeting portion includes a light chain variable region VL, the VL including LCDR1, LCDR2 and LCDR3, the LCDR3 including the amino acid sequence shown in SEQ ID NO:94; the LCDR2 including the amino acid sequence shown in SEQ ID NO:87; and the LCDR1 including the amino acid sequence shown in SEQ ID NO:82.

[0147] In some embodiments, the first targeting portion includes L-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO:119.

[0148] In some embodiments, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:79 to SEQ ID NO:81.

[0149] In some embodiments, the first targeting portion includes L-FR2 located between LCDR1 and LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO:120.

[0150] In some embodiments, the L-FR2 in the first targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:83 to SEQ ID NO:86.

[0151] In some embodiments, the first targeting portion includes L-FR3 located between LCDR2 and LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO:121.

[0152] In some embodiments, the L-FR3 in the first targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:88 to SEQ ID NO:93.

[0153] In some embodiments, the first targeting portion comprises L-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0154] In some embodiments, the L-FR4 in the first targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

[0155] In some embodiments, the first targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:119; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:120; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:121; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0156] In some embodiments, the first targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:79 to SEQ ID NO:81; L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:83 to SEQ ID NO:86; L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:88 to SEQ ID NO:93; and L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

[0157] In some embodiments, the L-FR1, L-FR2, L-FR3, and L-FR4 in the first targeting portion comprise an amino acid sequence selected from any one of the following groups:

[0158] a) L-FR1: SEQ ID NO:79, L-FR2: SEQ ID NO:83, L-FR3: SEQ ID NO:88 and L-FR4: SEQ ID NO:27;

[0159] b) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:84, L-FR3: SEQ ID NO:89 and L-FR4: SEQ ID NO:28;

[0160] c) L-FR1: SEQ ID NO: 81, L-FR2: SEQ ID NO: 85, L-FR3: SEQ ID NO: 90 and L-FR4: SEQ ID NO: 28;

[0161] d) L-FR1: SEQ ID NO:81, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:91 and L-FR4: SEQ ID NO:28;

[0162] e) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:90 and L-FR4: SEQ ID NO:28;

[0163] f) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:92 and L-FR4: SEQ ID NO:28; and

[0164] g) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:86, L-FR3: SEQ ID NO:93 and L-FR4: SEQ ID NO:28.

[0165] In some embodiments, the first targeting portion includes a VL containing the amino acid sequence shown in SEQ ID NO:123.

[0166] In some embodiments, the VL in the first targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0167] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets CD28, and the first targeting portion includes VH, which contains the amino acid sequence shown in SEQ ID NO: 176.

[0168] In some embodiments, the first targeting portion of the isolated antigen-binding protein targets CD16a, and the first targeting portion includes VH, which contains the amino acid sequence shown in SEQ ID NO: 168.

[0169] In some embodiments, the second targeting portion comprises HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:51.

[0170] In some embodiments, the second targeting portion comprises HCDR3, which comprises the amino acid sequence shown in either SEQ ID NO:9 or SEQ ID NO:10.

[0171] In some embodiments, the second targeting portion comprises HCDR2, which contains the amino acid sequence shown in SEQ ID NO:6.

[0172] In some embodiments, the second targeting portion comprises HCDR1, which contains the amino acid sequence shown in SEQ ID NO:3.

[0173] In some embodiments, the second targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO:52.

[0174] In some embodiments, the second targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0175] In some embodiments, the second targeting portion includes a heavy chain variable region VH, the VH comprising HCDR1, HCDR2, and HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6; and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

[0176] In some embodiments, the second targeting portion includes a heavy chain variable region VH, the VH comprising HCDR1, HCDR2, and HCDR3, wherein HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6; and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

[0177] In some embodiments, the HCDR1, HCDR2, and HCDR3 in the second targeting portion comprise amino acid sequences selected from any of the following groups:

[0178] a) HCDR1: SEQ ID NO:3, HCDR2: SEQ ID NO:6 and HCDR3: SEQ ID NO:9; and

[0179] b) HCDR1: SEQ ID NO:3, HCDR2: SEQ ID NO:6 and HCDR3: SEQ ID NO:10.

[0180] In some embodiments, the second targeting portion comprises H-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:47.

[0181] In some embodiments, the H-FR1 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:1 or SEQ ID NO:2.

[0182] In some embodiments, the second targeting portion includes H-FR2 located between HCDR1 and HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO:48.

[0183] In some embodiments, the H-FR2 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:4 or SEQ ID NO:5.

[0184] In some embodiments, the second targeting portion includes H-FR3 located between HCDR2 and HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO:49.

[0185] In some embodiments, the H-FR3 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:7 or SEQ ID NO:8.

[0186] In some embodiments, the second targeting portion comprises H-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

[0187] In some embodiments, the H-FR4 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:11 or SEQ ID NO:12.

[0188] In some embodiments, the second targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:47; H-FR2 comprises the amino acid sequence shown in SEQ ID NO:48; H-FR3 comprises the amino acid sequence shown in SEQ ID NO:49; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

[0189] In some embodiments, the second targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2; H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5; H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:8; and H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:12.

[0190] In some embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 in the second targeting portion comprise an amino acid sequence selected from any of the following groups:

[0191] a) H-FR1: SEQ ID NO: 1, H-FR2: SEQ ID NO: 4, H-FR3: SEQ ID NO: 7 and H-FR4: SEQ ID NO: 11; and

[0192] b) H-FR1: SEQ ID NO:2, H-FR2: SEQ ID NO:5, H-FR3: SEQ ID NO:8 and H-FR4: SEQ ID NO:12.

[0193] In some embodiments, the second targeting portion includes a heavy chain variable region VH, the VH containing the amino acid sequence shown in SEQ ID NO:52.

[0194] In some embodiments, the second targeting portion includes a heavy chain variable region VH, the VH comprising an amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0195] In some embodiments, the second targeting portion comprises LCDR3, which contains the amino acid sequence shown in SEQ ID NO:57.

[0196] In some embodiments, the second targeting portion comprises LCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26.

[0197] In some embodiments, the second targeting portion comprises LCDR2, which contains the amino acid sequence shown in SEQ ID NO:18.

[0198] In some embodiments, the second targeting portion comprises LCDR1, which contains the amino acid sequence shown in SEQ ID NO:15.

[0199] In some embodiments, the second targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in SEQ ID NO:58.

[0200] In some embodiments, the second targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0201] In some embodiments, the second targeting portion includes a light chain variable region VL, the VL comprising LCDR1, LCDR2, and LCDR3, the LCDR3 comprising the amino acid sequence shown in SEQ ID NO:57; the LCDR2 comprising the amino acid sequence shown in SEQ ID NO:18; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:15.

[0202] In some embodiments, the second targeting portion includes a light chain variable region VL, the VL comprising LCDR1, LCDR2, and LCDR3, the LCDR3 comprising an amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26; the LCDR2 comprising an amino acid sequence shown in SEQ ID NO:18; and the LCDR1 comprising an amino acid sequence shown in SEQ ID NO:15.

[0203] In some embodiments, the LCDR1, LCDR2, and LCDR3 in the second targeting portion comprise an amino acid sequence selected from any of the following groups:

[0204] a) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 21;

[0205] b) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 22;

[0206] c) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 23;

[0207] d) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 24;

[0208] e) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:25; and

[0209] f) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:26.

[0210] In some embodiments, the second targeting portion includes L-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO:53.

[0211] In some embodiments, the L-FR1 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:13 or SEQ ID NO:14.

[0212] In some embodiments, the second targeting portion includes L-FR2 located between LCDR1 and LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO:54.

[0213] In some embodiments, the L-FR2 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:16 or SEQ ID NO:17.

[0214] In some embodiments, the second targeting portion includes L-FR3 located between LCDR2 and LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO:55.

[0215] In some embodiments, the L-FR3 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:19 or SEQ ID NO:20.

[0216] In some embodiments, the second targeting portion comprises L-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0217] In some embodiments, the L-FR4 in the second targeting portion comprises the amino acid sequence shown in either SEQ ID NO:27 or SEQ ID NO:28.

[0218] In some embodiments, the second targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:53; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:54; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:55; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0219] In some embodiments, the second targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:13 and SEQ ID NO:14; L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:16 and SEQ ID NO:17; L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:19 and SEQ ID NO:20; and L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

[0220] In some embodiments, the L-FR1, L-FR2, L-FR3, and L-FR4 in the second targeting portion comprise an amino acid sequence selected from any one of the following groups:

[0221] a) L-FR1: SEQ ID NO: 13, L-FR2: SEQ ID NO: 16, L-FR3: SEQ ID NO: 19 and L-FR4: SEQ ID NO: 27; and

[0222] b) L-FR1: SEQ ID NO: 14, L-FR2: SEQ ID NO: 17, L-FR3: SEQ ID NO: 20 and L-FR4: SEQ ID NO: 28.

[0223] In some embodiments, the second targeting portion includes a VL containing an amino acid sequence shown in any of SEQ ID NO:58.

[0224] In some embodiments, the VL in the second targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0225] In some embodiments, the second targeting portion comprises HCDR3, which contains the amino acid sequence shown in SEQ ID NO:76.

[0226] In some embodiments, the second targeting portion comprises HCDR2, which contains the amino acid sequence shown in SEQ ID NO:70.

[0227] In some embodiments, the second targeting portion comprises HCDR1, which contains the amino acid sequence shown in SEQ ID NO:61.

[0228] In some embodiments, the second targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO:122.

[0229] In some embodiments, the second targeting portion includes HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

[0230] In some embodiments, the second targeting portion includes a heavy chain variable region VH, the VH comprising HCDR1, HCDR2, and HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:76; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70; and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:61.

[0231] In some embodiments, the second targeting portion comprises H-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:115.

[0232] In some embodiments, the H-FR1 in the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:59 to SEQ ID NO:60.

[0233] In some embodiments, the second targeting portion includes H-FR2 located between HCDR1 and HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO:116.

[0234] In some embodiments, the H-FR2 in the second targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69.

[0235] In some embodiments, the second targeting portion includes H-FR3 located between HCDR2 and HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO:117.

[0236] In some embodiments, the H-FR3 in the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:71 to SEQ ID NO:75.

[0237] In some embodiments, the second targeting portion comprises H-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:118.

[0238] In some embodiments, the H-FR4 in the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

[0239] In some embodiments, the second targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:115; H-FR2 comprises the amino acid sequence shown in SEQ ID NO:116; H-FR3 comprises the amino acid sequence shown in SEQ ID NO:117; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:118.

[0240] In some embodiments, the second targeting portion comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:59 to SEQ ID NO:60; H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69; H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:71 to SEQ ID NO:75; and H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

[0241] In some embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 in the second targeting portion comprise an amino acid sequence selected from any of the following groups:

[0242] a) H-FR1: SEQ ID NO:59, H-FR2: SEQ ID NO:62, H-FR3: SEQ ID NO:71 and H-FR4: SEQ ID NO:77;

[0243] b) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:63, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

[0244] c) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:64, H-FR3: SEQ ID NO:73 and H-FR4: SEQ ID NO:78;

[0245] d) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:65, H-FR3: SEQ ID NO:74 and H-FR4: SEQ ID NO:78;

[0246] e) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:66, H-FR3: SEQ ID NO:75 and H-FR4: SEQ ID NO:78;

[0247] f) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:67, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

[0248] g) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:65, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

[0249] h) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:64, H-FR3: SEQ ID NO:74 and H-FR4: SEQ ID NO:78;

[0250] i) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:68, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78; and

[0251] j) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:69, H-FR3: SEQ ID NO:73 and H-FR4: SEQ ID NO:78.

[0252] In some embodiments, the second targeting portion includes a heavy chain variable region VH, the VH containing the amino acid sequence shown in SEQ ID NO:122.

[0253] In some embodiments, the VH comprises the amino acid sequence shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

[0254] In some embodiments, the second targeting portion comprises LCDR3, which contains the amino acid sequence shown in SEQ ID NO:94.

[0255] In some embodiments, the second targeting portion comprises LCDR2, which contains the amino acid sequence shown in SEQ ID NO:87.

[0256] In some embodiments, the second targeting portion comprises LCDR1, which contains the amino acid sequence shown in SEQ ID NO:82.

[0257] In some embodiments, the second targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in any of SEQ ID NO:123.

[0258] In some embodiments, the second targeting portion includes LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0259] In some embodiments, the second targeting portion includes a light chain variable region VL, the VL comprising LCDR1, LCDR2, and LCDR3, the LCDR3 comprising the amino acid sequence shown in SEQ ID NO:94; the LCDR2 comprising the amino acid sequence shown in SEQ ID NO:87; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:82.

[0260] In some embodiments, the second targeting portion includes L-FR1, the C-terminus of which is directly or indirectly connected to the N-terminus of LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO:119.

[0261] In some embodiments, the L-FR1 in the second targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:79 to SEQ ID NO:81.

[0262] In some embodiments, the second targeting portion includes L-FR2 located between LCDR1 and LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO:120.

[0263] In some embodiments, the L-FR2 in the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:83 to SEQ ID NO:86.

[0264] In some embodiments, the second targeting portion includes L-FR3 located between LCDR2 and LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO:121.

[0265] In some embodiments, the L-FR3 in the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:88 to SEQ ID NO:93.

[0266] In some embodiments, the second targeting portion comprises L-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0267] In some embodiments, the L-FR4 in the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

[0268] In some embodiments, the second targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:119; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:120; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:121; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

[0269] In some embodiments, the second targeting portion comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:79 to SEQ ID NO:81; L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:83 to SEQ ID NO:86; L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:88 to SEQ ID NO:93; and L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

[0270] In some embodiments, the L-FR1, L-FR2, L-FR3, and L-FR4 in the second targeting portion comprise amino acid sequences selected from any of the following groups:

[0271] a) L-FR1: SEQ ID NO:79, L-FR2: SEQ ID NO:83, L-FR3: SEQ ID NO:88 and L-FR4: SEQ ID NO:27;

[0272] b) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:84, L-FR3: SEQ ID NO:89 and L-FR4: SEQ ID NO:28;

[0273] c) L-FR1: SEQ ID NO: 81, L-FR2: SEQ ID NO: 85, L-FR3: SEQ ID NO: 90 and L-FR4: SEQ ID NO: 28;

[0274] d) L-FR1: SEQ ID NO:81, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:91 and L-FR4: SEQ ID NO:28;

[0275] e) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:90 and L-FR4: SEQ ID NO:28;

[0276] f) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:92 and L-FR4: SEQ ID NO:28; and

[0277] g) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:86, L-FR3: SEQ ID NO:93 and L-FR4: SEQ ID NO:28.

[0278] In some embodiments, the second targeting portion includes a VL containing the amino acid sequence shown in SEQ ID NO:123.

[0279] In some embodiments, the VL of the second targeting portion comprises an amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0280] In some embodiments, the second targeting portion of the isolated antigen-binding protein targets CD28, and the second targeting portion includes VH, which contains the amino acid sequence shown in SEQ ID NO: 176.

[0281] In some embodiments, the second targeting portion of the isolated antigen-binding protein targets CD16a, and the second targeting portion includes VH, which contains the amino acid sequence shown in SEQ ID NO: 168.

[0282] In some embodiments, the isolated antigen-binding protein includes a first targeting portion and a second targeting portion, wherein the first targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38, SEQ ID NO:95 to SEQ ID NO:104, and SEQ ID NO:105 to SEQ ID NO:111; and the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38, SEQ ID NO:95 to SEQ ID NO:104, and SEQ ID NO:105 to SEQ ID NO:111.

[0283] In some embodiments, the first targeting portion and the second targeting portion comprise an amino acid sequence selected from any of the following groups:

[0284] a) First target portion: SEQ ID NO:97 and second target portion: SEQ ID NO:31;

[0285] b) First target portion: SEQ ID NO:98 and second target portion: SEQ ID NO:31;

[0286] c) First target portion: SEQ ID NO:100 and second target portion: SEQ ID NO:31;

[0287] d) First target portion: SEQ ID NO:102 and second target portion: SEQ ID NO:31;

[0288] e) First target portion: SEQ ID NO:103 and second target portion: SEQ ID NO:31;

[0289] f) First target portion: SEQ ID NO:31 and second target portion: SEQ ID NO:97;

[0290] g) First target portion: SEQ ID NO:31 and second target portion: SEQ ID NO:98;

[0291] h) First target portion: SEQ ID NO:31 and second target portion: SEQ ID NO:100;

[0292] i) First target portion: SEQ ID NO:31 and second target portion: SEQ ID NO:102;

[0293] j) First target portion: SEQ ID NO:31 and second target portion: SEQ ID NO:103;

[0294] k) First target portion: SEQ ID NO:97 and second target portion: SEQ ID NO:36;

[0295] l) First target portion: SEQ ID NO:98 and second target portion: SEQ ID NO:36;

[0296] m) First target portion: SEQ ID NO:100 and second target portion: SEQ ID NO:36;

[0297] n) First target portion: SEQ ID NO:102 and second target portion: SEQ ID NO:36;

[0298] o) First target portion: SEQ ID NO:103 and second target portion: SEQ ID NO:36;

[0299] p) First target portion: SEQ ID NO:36 and second target portion: SEQ ID NO:97;

[0300] q) First target portion: SEQ ID NO:36 and second target portion: SEQ ID NO:98;

[0301] r) First target portion: SEQ ID NO:36 and second target portion: SEQ ID NO:100;

[0302] s) First target portion: SEQ ID NO:36 and second target portion: SEQ ID NO:102;

[0303] t) First target portion: SEQ ID NO:36 and second target portion: SEQ ID NO:103;

[0304] u) First target portion: SEQ ID NO:31 and second target portion: SEQ ID NO:176;

[0305] v) First target portion: SEQ ID NO:31 and second target portion: SEQ ID NO:168;

[0306] w) First target portion: SEQ ID NO:176 and second target portion: SEQ ID NO:31;

[0307] x) First target portion: SEQ ID NO:168 and second target portion: SEQ ID NO:31.

[0308] In some embodiments, the second polypeptide chain includes a heavy chain constant region, and the heavy chain constant region includes a constant region derived from IgG or a constant region derived from IgY.

[0309] In some embodiments, the heavy chain constant region in the second polypeptide chain includes a constant region derived from IgG.

[0310] In some embodiments, the heavy chain constant region in the second polypeptide chain includes a constant region derived from IgG1, IgG2, IgG3, or IgG4.

[0311] In some embodiments, the heavy chain constant region in the second polypeptide chain includes an Fc region derived from IgG1.

[0312] In some embodiments, the Fc region in the heavy chain constant region of the second polypeptide chain contains an N297A mutation, and the residues are numbered according to the Kabat system.

[0313] In some embodiments, the Fc region in the heavy chain constant region contains M252Y, S254T, and T256E mutations, with the residues numbered according to the Kabat system.

[0314] In some embodiments, the heavy chain constant region in the second polypeptide chain comprises the amino acid sequences shown in SEQ ID NO:112 to SEQ ID NO:113.

[0315] In some embodiments, the first polypeptide chain includes a light chain constant region, and the light chain constant region includes a constant region derived from Igκ or a constant region derived from Igλ.

[0316] In some implementations, the light chain constant region includes a constant region derived from human Igκ.

[0317] In some embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:114.

[0318] In some embodiments, the third targeting portion of the isolated antigen-binding protein comprises a VH, the VH comprising the amino acid sequence shown in any one of SEQ ID NO: 168, 170, 172, 174, 176, and 178.

[0319] In some embodiments, the third targeting portion of the isolated antigen-binding protein comprises a VL containing an amino acid sequence represented by any one of SEQ ID NO: 169, 171, 173, 175, 177, and 179.

[0320] In some embodiments, the third targeting portion of the isolated antigen-binding protein includes an antibody or an antigen-binding fragment.

[0321] In some embodiments, the antigen-binding fragment in the isolated antigen-binding protein is selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and dAb.

[0322] In some embodiments, the third targeting portion of the isolated antigen-binding protein is scFv.

[0323] In some embodiments, the C-terminus of VH in the third targeting portion of the isolated antigen-binding protein is directly or indirectly connected to the N-terminus of VL.

[0324] In some embodiments, the C-terminus of VL in the third targeting portion of the isolated antigen-binding protein is directly or indirectly connected to the N-terminus of VH.

[0325] In some embodiments, the fourth targeting portion of the isolated antigen-binding protein comprises a VH, the VH comprising the amino acid sequence shown in any one of SEQ ID NO: 29-31, 95-104, 168, 170, 172, 174, 176, and 178.

[0326] In some embodiments, the fourth targeting portion of the isolated antigen-binding protein comprises a VL containing an amino acid sequence represented by any one of SEQ ID NO: 32-38, 105-111, 169, 171, 173, 175, 177, and 179.

[0327] In some embodiments, the fourth targeting portion of the isolated antigen-binding protein includes an antibody or an antigen-binding fragment.

[0328] In some embodiments, the antigen-binding fragment in the isolated antigen-binding protein is selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and dAb.

[0329] In some embodiments, the fourth targeting portion of the isolated antigen-binding protein is scFv.

[0330] In some embodiments, the C-terminus of VH in the fourth targeting portion of the isolated antigen-binding protein is directly or indirectly connected to the N-terminus of VL.

[0331] In some embodiments, the C-terminus of VL in the fourth targeting portion of the isolated antigen-binding protein is directly or indirectly connected to the N-terminus of VH.

[0332] In some embodiments, the antigen-binding protein comprises two first polypeptide chains and two second polypeptide chains, the two first polypeptide chains being linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains forming an IgG-like antibody.

[0333] In some embodiments, the antigen-binding protein includes monoclonal antibodies, single-chain antibodies, chimeric antibodies, humanized antibodies, multispecific antibodies, bispecific antibodies, and / or fully human antibodies.

[0334] On the other hand, this application provides one or more polypeptides comprising the isolated antigen-binding protein.

[0335] On the other hand, this application provides one or more immune conjugates comprising the isolated antigen-binding protein or the polypeptide.

[0336] On the other hand, this application provides one or more isolated nucleic acid molecules that encode the isolated antigen-binding protein or the polypeptide.

[0337] On the other hand, this application provides one or more vectors containing the isolated nucleic acid molecules.

[0338] On the other hand, this application provides one or more cells that contain the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, and / or the carrier.

[0339] On the other hand, this application provides a method for preparing the isolated antigen-binding protein or the polypeptide, the method comprising culturing the cells under conditions in which the isolated antigen-binding protein or the polypeptide is expressed.

[0340] On the other hand, this application provides one or more pharmaceutical compositions comprising the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or a pharmaceutically acceptable adjuvant and / or excipient.

[0341] On the other hand, this application provides a method for inhibiting the interaction between PD-1 and PD-L1, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, and / or the cell.

[0342] On the other hand, this application provides the use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of a disease or condition.

[0343] In some implementations, the disease or condition includes a tumor.

[0344] In some implementations, the tumor includes a solid tumor.

[0345] In some implementations, the tumor includes non-solid tumors.

[0346] In some embodiments, the tumor includes breast cancer, lung cancer, stomach cancer, colorectal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and / or liver cancer.

[0347] On the other hand, this application provides the isolated antigen-binding protein, the polypeptide, the immune conjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition, for the prevention, relief, and / or treatment of diseases or conditions.

[0348] In some implementations, the disease or condition includes a tumor.

[0349] In some implementations, the tumor includes a solid tumor.

[0350] In some implementations, the tumor includes non-solid tumors.

[0351] In some embodiments, the tumor includes breast cancer, lung cancer, stomach cancer, colorectal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and / or liver cancer.

[0352] On the other hand, this application provides a method for preventing and / or treating a disease or condition, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the immune conjugate, the isolated nucleic acid molecule, the carrier, and / or the cell.

[0353] In some implementations, the disease or condition includes a tumor.

[0354] In some implementations, the tumor includes a solid tumor.

[0355] In some implementations, the tumor includes non-solid tumors.

[0356] In some embodiments, the tumor includes breast cancer, lung cancer, stomach cancer, colorectal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and / or liver cancer.

[0357] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0358] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0359] Figure 1A-1B The diagram shown is a schematic representation of the structure of an exemplary antigen-binding protein described in this application.

[0360] Figure 2 The image shows the binding results of the exemplary bispecific antibody described in this application to HEK293 cells with high expression of PD-1, high expression of PDL1, or wild type.

[0361] Figure 3 The results shown are from an ELISA experiment using an exemplary bispecific antibody described in this application. Detailed Implementation

[0362] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0363] Terminology Definition

[0364] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a substance or component is found in nature as an "isolated" substance, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0365] In this application, the term "antigen-binding protein" generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen. For example, in this application, the term "antigen-binding protein" may include "antibody" or "antigen-binding fragment." For example, said antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and may include any molecule containing its antigen-binding portion. The term "antibody" may include monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to murine antibodies, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, single-chain antibodies (e.g., scFv), and antigen-binding antibody fragments (e.g., Fab, Fab', VHH, and (Fab)2 fragments). The term "antibody" may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and their derivatives described herein. Each heavy chain may consist of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may consist of a variable region (VL) and a constant region. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens (e.g., human PD-L1). The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The exact boundaries of the CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a well-defined residue numbering system applicable to any variable region of antigen-binding fragments, but also precise residue boundaries defining CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) found that despite great diversity at the amino acid sequence level, certain sub-regions within Kabat CDRs adopt almost identical peptide backbone conformations.These subregions are named L1, L2, and L3 or H1, H2, and H3, where “L” and “H” refer to the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262 (5):732-45 (1996)). In addition, other CDR boundary definitions may not strictly follow one of the above systems but will still overlap with Kabat CDRs, although they may be shortened or lengthened according to the prediction or experimental findings of antigen binding, even if the specific residue or residue group or even the entire CDR does not significantly affect the specific residue or residue group or even the entire CDR. In this application, the CDRs may be defined by using the Kabat numbering system.

[0366] In this application, the term "antigen-binding fragment" generally refers to one or more fragments in an antibody that perform the function of specifically binding antigens. The antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment including Fv, ScFv, dsFv, Fab, Fab', or F(ab')2, or a light chain comprising a fragment including Fv, scFv, dsFv, Fab, Fab', or F(ab')2. (1) Fab fragment, typically a monovalent fragment consisting of VL, VH, CL and CH domains; (2) F(ab')2 fragment, a divalent fragment consisting of two Fab fragments linked by disulfide bonds at the hinge region; (3) Fd fragment consisting of VH and CH domains; (4) Fv fragment consisting of VL and VH domains of an antibody single arm; (5) dAb fragment consisting of VH domain (Ward et al., (1989) Nature 341: 544-546); (6) separate complementarity-determining regions (CDRs) and (7) combinations of two or more separate CDRs optionally linked by a linker. For example, it may also include a monovalent single-chain molecule Fv (scFv) formed by pairing VL and VH (see Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883). For example, it may also include a class of antibodies VHH that lacks the antibody light chain and only has the variable region of the heavy chain (see, for example, Kang Xiaozhen et al., Chinese Journal of Biotechnology, 2018, 34(12):1974-1984). The “antigen-binding portion” may also include an immunoglobulin fusion protein containing a binding domain selected from the following: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.

[0367] In this application, the term "monoclonal antibody" generally refers to a group of substantially homologous antibodies, meaning that the individual antibodies comprising the group are identical except for the possibility of naturally occurring mutations present in trace amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site directly. For example, said monoclonal antibodies can be prepared by hybridoma technology or produced in bacterial, eukaryotic, or plant cells using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques described, for example, those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0368] In this application, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from an antibody of a specific species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, the variable regions of both the light and heavy chains may originate from the variable region of an antibody from an animal species (e.g., mouse, rat, etc.), while the constant region is homologous to an antibody sequence from another species (e.g., human). For instance, to obtain a chimeric antibody, the variable region can be generated using non-human B cells or hybridoma cells, with the constant region combined with it derived from human cells. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region it is combined with. Furthermore, because the constant region of a chimeric antibody can be derived from humans, the likelihood of the chimeric antibody eliciting an immune response upon injection is lower than with antibodies using a non-human-derived constant region.

[0369] In this application, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity of the xenobiotic antibody when introduced into humans, while maintaining the antibody's complete antigen-binding affinity and specificity. For example, CDR transplantation (Jones et al., Nature 321:522(1986)) and its variants can be used; including “reshaping” (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 19919:266-271), and “hyperchimerization” (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol). Techniques such as veneering (Mark, et al., “Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.” In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), and surface reconstruction (US Patent US5639641) are used to humanize non-human-derived binding domains. If other regions, such as hinge regions and constant region structural domains, are also derived from non-human sources, these regions can also be humanized.

[0370] In this application, the term "mouse antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of this application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random mutations or point mutations or through in vivo somatic mutations.

[0371] In this application, the term "bispecific antibody" generally refers to an antibody or antigen-binding fragment containing two specific antigen-binding sites. In this application, the bispecific antibody can be a homodimer or a heterodimer. For example, in this application, the bispecific antibody can bind to both PD-1 and PD-L1 proteins, effectively inhibiting PD-L1 and PD-1 activation, thereby treating PD1 / PD-L1-related diseases (e.g., tumors).

[0372] In this application, the term "PD-L1" generally refers to programmed death-ligand 1 protein, its functional variants, and / or its functionally active fragments. PD-L1 is also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1) and is a protein encoded by the CD274 gene (in humans). PD-L1 binds to its receptor, such as programmed death-1 (PD-1), which is expressed in activated T cells, B cells, and macrophages (Ishida et al., 1992 EMBO J, 11:3887-3395; Okazaki et al., Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001; 291:319-22). The chelation of PD-L1 and PD-1 exerts immunosuppressive effects by inhibiting T cell proliferation and the production of cytokines IL-2 and IFN-γ (Freeman et al., Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J. Exp. Med. 2000, 192:1027-1034; Carter et al., PD-1: PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur. J. Immunol. 2002, 32:634–643). The term “PD-L1” covers any naturally occurring PD-L1 of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers “full-length”, unprocessed PD-L1, and any form of PD-L1 produced by cellular processing. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also covers variants of naturally occurring PD-L1, such as splice variants or allelic variants. An exemplary full-length human PD-L1 protein amino acid sequence can be found under UniProt accession number Q9NZQ7. For example, the “functionally active fragment” may include a fragment that retains the endogenous function of at least one naturally occurring protein (e.g., binding to the antigen-binding protein described in this application). For example, the “functionally active fragment” may include a domain that binds to the antigen-binding protein of this application.

[0373] In this application, the terms "PD-1 protein," "PD-1," or "PD-1 antigen" are used interchangeably and include any functionally active fragment, variant, and homolog of PD-1, which is naturally expressed in cells or expressed in cells transfected with the PD-1 gene. In this application, PD-1 can be human PD-1, whose accession number in UniProt / Swiss-Prot is Q15116. For example, PD-1 can be a functionally active fragment of human PD-1. For example, the "functionally active fragment" may include a fragment that retains the endogenous function of at least one naturally occurring protein (e.g., binding to the antigen-binding protein described in this application). For example, the "functionally active fragment" may include a domain that binds to the antigen-binding protein of this application.

[0374] In addition to the specific proteins and nucleotides mentioned herein, this application may also include their functionally active fragments, derivatives, analogs, homologs, and fragments thereof.

[0375] The term "functionally active fragment" refers to a polypeptide that has a substantially identical amino acid sequence or is encoded by a substantially identical nucleotide sequence to the naturally occurring sequence and is capable of possessing one or more activities of the naturally occurring sequence. In the context of this application, a functionally active fragment of any given sequence refers to a sequence in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. Sequences encoding functionally active fragments can be obtained by adding, deleting, substituting, modifying, replacing, and / or mutating at least one amino acid residue and / or nucleotide residue present in naturally occurring proteins and / or polynucleotides, as long as the original functional activity is maintained.

[0376] In this application, the term "derivative" generally refers to any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues (or nucleotides) of the polypeptide or polynucleotide of this application, provided that the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

[0377] In this application, the term "analyte" generally refers to a polypeptide or polynucleotide, including any mimic of a polypeptide or polynucleotide, i.e., a chemical compound having at least one endogenous function of the polypeptide or polynucleotide that the mimic mimic has.

[0378] Typically, amino acid substitutions can be made, such as substitutions of at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20), as long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0379] In this application, the term "homologous" generally refers to an amino acid or nucleotide sequence that has a certain degree of homology with a naturally occurring sequence. The term "homology" can be equivalent to sequence "identity." Homologous sequences can include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. Typically, homologous sequences will contain the same active sites, etc., as the subject amino acid sequence. Homology can be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions) or can be expressed in terms of sequence identity. In this application, a sequence having a percentage identity with any of the referenced amino acid or nucleotide sequences in SEQ ID NO means a sequence having said percentage identity across the entire length of the referenced SEQ ID NO. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0380] The proteins or peptides used in this application may also have deletions, insertions, or substitutions of amino acid residues, which produce silent changes and result in functionally equivalent proteins. Intentional amino acid substitutions can be made based on the similarity of the residues' polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric properties, as long as the endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing non-polarized head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0381] In this application, the term "tumor" generally refers to a growth formed by the proliferation of local tissue cells under the influence of various oncogenic factors. For example, the tumor may include a solid tumor. For example, the tumor may include a non-solid tumor. For example, the tumor may include a tumor associated with PD-L1 expression. The term "tumor associated with PD-L1 expression" generally refers to a tumor formed by altered PD-L1 expression leading to disease progression or evasion of immune surveillance. For example, the "tumor associated with PD-L1 expression" may be a tumor formed by upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with PD-L1 protein expression may be a PD-L1 positive tumor. In PD-L1 positive tumors, the protein expression level of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher compared to normal cells.

[0382] In this application, the term "solid tumor" generally refers to a tangible mass that can be detected clinically (e.g., by X-ray, CT scan, ultrasound, or palpation). For example, said solid tumor may include breast cancer, lung cancer, gastric cancer, colorectal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and / or liver cancer.

[0383] In this application, the term "non-solid tumor" generally refers to a tumor that cannot be seen or palpated by X-ray, CT scan, ultrasound, or palpation. For example, a non-solid tumor may include leukemia. For example, a non-solid tumor may include lymphoma. For example, a non-solid tumor may include multiple myeloma.

[0384] In this application, the term "tumor-associated antigen" generally refers to an antigen molecule present on tumor cells or normal cells. In this application, the tumor-associated antigen may be present in trace amounts on normal cells, while being highly expressed in tumor cells. For example, the tumor-associated antigen may include, but is not limited to, embryonic proteins, glycoprotein antigens, and / or squamous cell antigens. For example, the tumor-associated antigen may include CD19, CD20, CD47, HER2, EGFR, CD22, and / or PD-L1.

[0385] In this application, the term "immune cell-associated antigen" generally refers to an antigen molecule present on immune cells. For example, the immune cell-associated antigen may include antigens associated with immunosuppression (e.g., PD-1 antigen). For example, the immune cell-associated antigen may include T cell or NK cell antigens (e.g., CD3, CD16a, etc.).

[0386] In this application, the term "cytokine" generally refers to a class of small molecule proteins with broad biological activity synthesized and secreted by immune cells and certain non-immune cells (e.g., endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. For example, the cytokines can regulate cell growth, differentiation, and effector functions by binding to corresponding receptors, thereby modulating immune responses. For example, the cytokines may include, but are not limited to, interleukins (e.g., IL-2, IL-7, IL-15, etc.), interferons (e.g., IFNα, IFNβ), tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc. For example, the cytokines may include variants of interleukins (e.g., IL-2 variants R38A, I80F, R81D, L85V, I86V, I92F, which can bind to IL-2βγ receptors but have a weaker binding affinity to IL-2α receptors). For example, the cytokines may include interferon variants (e.g., the IFNα variant R144A mutant, which binds significantly less to the IFNAR2 receptor, thus reducing the toxic side effects of INFα).

[0387] In this application, the term "immunoconjugate" generally refers to a conjugate formed by the conjugation (e.g., covalently linked by a linker molecule) of the other therapeutic agent to the isolated antigen-binding protein, which can deliver the other therapeutic agent to the target cell (e.g., tumor cell) through the specific binding of the isolated antigen-binding protein to the antigen on the target cell. Furthermore, the antigen may also be secreted by the target cell and located in an extracellular space.

[0388] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0389] In this application, the term "nucleic acid molecule" generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue, isolated from its natural environment or synthesized artificially.

[0390] In this application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. The vector can transfer an inserted nucleic acid molecule into cells and / or between cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into suitable cells. Typically, by culturing suitable cells containing the vector, the vector can produce a desired expression product. In this application, the vector may comprise a lentiviral vector.

[0391] In this application, the term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector including the nucleic acid molecules described in this application, or that is capable of expressing the polypeptides or antigen-binding proteins described in this application. The cell may include progeny cells of a single cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they must be capable of expressing the polypeptides or antigen-binding proteins described in this application. The cell can be obtained by in vitro transfection of cells using the vector described in this application. The cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some embodiments, the cell may be an immune cell. For example, the immune cells may be selected from the group consisting of: T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0392] In this application, the term “treatment” generally means: (i) preventing a patient who may be susceptible to a disease, condition and / or symptom but has not yet been diagnosed with the disease, from developing such a disease, condition or symptom; (ii) suppressing such a disease, condition or symptom, i.e., curbing its development; and (iii) alleviating such a disease, condition or symptom, i.e., achieving the relief of such a disease, condition and / or symptom and / or symptoms associated with such a disease, condition and / or symptom.

[0393] In this application, the terms “polypeptide,” “peptide,” “protein,” and “protein protein” are used interchangeably and generally refer to a polymer having amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers containing modified amino acids. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeled component). The term “amino acid” includes natural and / or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, as well as amino acid analogs and peptide mimics.

[0394] In this application, the terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. The sequence of a nucleotide may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0395] In this application, the term "K" D (Similarly, "K") D "or "K D The term "affinity constant" or "equilibrium dissociation constant" is typically used in titration measurements at equilibrium, or as a value obtained by dividing the dissociation rate constant (kd) by the binding rate constant (ka). The binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K) are used in various titration measurements. D The term K represents the binding affinity of a binding protein (e.g., the isolated antigen-binding protein described in this application) to an antigen (e.g., PD-L1 protein, PD-1 protein). Methods for determining binding and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K value can be determined by Biacore (Biomolecular Interaction Analysis) (e.g., instruments available from BIAcore International AB, aGE Healthcare Company, Uppsala, Sweden). D The value can also be detected using other experimental methods and instruments, such as Octet. Alternatively, the K value can be determined using KinExA (Kinetic Exclusion Assay), which is available from Sapidyne Instruments (Boise, Idaho). D The value, or the K value, can be determined using a surface plasmon resonance (SPR) spectrometer.D Value. For example, the K value can also be determined using an amine coupling kit. D value.

[0396] In this application, the term "and / or" should be understood to mean any one of the options or both of the options.

[0397] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements. In some cases, "comprising" also covers the situation where only the specified components are included. For example, "comprising" also means "consisting of".

[0398] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0399] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of". Invention Details

[0401] The antigen-binding protein isolated in this application

[0402] On the one hand, this application provides an isolated antigen-binding protein, which may include a first polypeptide chain and a second polypeptide chain. The first polypeptide chain includes a first targeting portion, and the second polypeptide chain includes a second targeting portion. The first targeting portion and the second targeting portion each independently bind to PD-1 protein or PD-L1 protein. The proteins bound by the first targeting portion and the second targeting portion are different. The first polypeptide chain and the second polypeptide chain are connected by disulfide bonds.

[0403] For example, the antigen-binding protein may include, Figure 1A-1B The exemplary structure shown.

[0404] Bispecific antibodies

[0405] In this application, the antibody's CDR, also known as the complementarity-determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or an antigenic epitope. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the CDR encompasses the CDR sequence partitioned according to any CDR partitioning method; it also encompasses its variants, including variations in the amino acid sequence of the CDR where one or more amino acids have been substituted, deleted, and / or added. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the isolated antigen-binding protein described in this application is defined by the Kabat coding system.

[0406] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody can specifically bind to the PD-L1 protein, and the second targeting portion of the second polypeptide chain of the bispecific antibody can specifically bind to the PD-1 protein.

[0407] In this application, the HCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0408] In this application, the HCDR2 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0409] In this application, the HCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0410] For example, the HCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:70; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:76. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0411] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:122. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:122, has an amino acid substitution (e.g., a conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X5, X7, X... 10 X 11 X 12 X 16 X 20 X 37 X 38 X 40 X 46 X 48 X 67 X 68 X 69 X 75 X 81 X 86 X 90 X 115 and X 116 .

[0412] QVQLX5QX7GAX 10 X 11 X 12 KPGX 16 SVKX 20 SCKASGYTFTSNWMHWX 37X 38 QX 40 PGQGLX 46 WX 48 GMIHPNSAIKYNEKFKSRX 67 X 68 X 69 TADKSX 75 STAYMX 81 LSSLX 86 SEDX 90 AVYYCARSYYGSSPYFFDYWGQGTX 115 X 116 TVSS (SEQ ID NO: 122), where X5 can be Q or V, X7 can be P or S, X 10 It can be D or E, X 11 It can be L or V, X 12 It can be K or V, X 16 It can be A or S, X 20 It can be L or V, X 37 It can be D, M, or V, X 38 It can be K or R, X 40 It can be A or R, X 46 It can be D or E, X 48 It can be I or M, X 67 It can be A or V, X 68 It can be S or T, X 69 It can be I or L, X 75 It can be S or T, X 81 It can be E or Q, X 86 It can be R or T, X 90 It can be S or T, X 115 It can be M or T, X 116 It can be L or V.

[0413] In this application, the heavy chain variable region of the first targeting portion of the first polypeptide chain of the bispecific antibody may contain any of the amino acid sequences shown in SEQ ID NO:95 to SEQ ID NO:104.

[0414] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include at least one CDR in the light chain variable region (VL), and the VL may contain the amino acid sequence shown in SEQ ID NO:123.

[0415] For example, the VL in the first targeting portion of the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111. In this application, the LCDR of the first targeting portion of the first polypeptide chain of the bispecific antibody can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111, and the LCDR obtained by dividing in any form falls within the protection scope of this application.

[0416] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0417] In this application, the LCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0418] In this application, the LCDR2 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:87. For example, the LCDR2 of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0419] In this application, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:82. For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0420] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:87; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:94. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0421] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO:123, the VL of the first targeting portion of the first polypeptide chain of the bispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X7, X8, X... 15 X 22 X 41 X 42 X 43 X 44 X 46 X 60 X 71 X 72 X 77 X 79 X 80 X 83 X 87 and X 104 .

[0422] DIQMTQX7X8SSLSASX 15 GDRVTIX 22 CRASQDISKYLNWYQQKPX 41 X 42 X 43 X 44 KX 46 LIY YTSRLHSGVPX 60 RFSGSGSGTDX 71 X 72 LTISX 77 LX 79 X 80 EDX 83 ATYX 87 CQQGDTLPWTFGGGT KX 104 EIK (SEQ ID NO: 123), where X7 can be S or T, X8 can be P or T, X 15 It can be L or V, X 22 It can be S or T, X 41 It can be D or G, X 42 It can be G or K, X 43 It can be A or T, X 44 It can be P or V, X 46 It can be F or L, X 60 It can be A or S, X 71 It can be F or Y, X 72 It can be F or T, X77 It can be N or S, X 79 It can be E or Q, X 80 It can be P or Q, X 83 It can be F or V, X 87 It can be F or Y, X 104 It can be L or V.

[0423] In this application, the light chain variable region of the first targeting portion in the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0424] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a light chain constant region, which may include a constant region derived from Igκ or a constant region derived from Igλ.

[0425] For example, the light chain constant region may include a constant region derived from Igκ.

[0426] For example, the light chain constant region of the first targeting portion in the first polypeptide chain of the bispecific antibody contains the amino acid sequence shown in any one of SEQ ID NO: 114.

[0427] For example, the first polypeptide chain may contain the amino acid sequence shown in any one of SEQ ID NO:134 to 140 and SEQ ID NO:144 to 153.

[0428] For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0429] For example, the second targeting portion of the second polypeptide chain of the bispecific antibody has an amino acid substitution (e.g., a conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 51, compared to the sequence shown in SEQ ID NO: 51: X1.

[0430] X1HYGTSPFVY(SEQ ID NO: 51), where X1 can be D or E.

[0431] In this application, the HCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in either SEQ ID NO:9 or SEQ ID NO:10. For example, the HCDR3 sequence of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0432] In this application, the HCDR2 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0433] In this application, the HCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0434] For example, the HCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:9. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0435] For example, the HCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:10. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4-25-3C11, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0436] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:52. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:52, has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X1, X5, X 13 X 16 X 17 X20 X 37 X 48 X 67 X 68 X 73 X 76 X 79 X 82 X 83 X 85 X 86 X 87 X 88 X 92 X 98 and X 118 .

[0437] X1VQLX5ESGPGLVX 13 PSX 16 X 17 LSX 20 TCTVSGFSLTSYAISWX 37 RQPPGKGLEWX 48 GVI WTGGGTNYNSALKSRX 67 X 68 ISKDX 73 SKX 76 QVX 79 LKX 82 X 83 SX 85 X 86 X 87 X 88 DTAX 92 YYCARX 98 HYGTSPFVYWGQGTLVTVSX 118 (SEQ ID NO: 52), where X1 can be E or Q, X5 can be K or Q, X 13 It can be A or K, X 16 It can be E or Q, X 17 It can be S or T, X 20 It can be I or L, X 37 It can be I or V, X 48 It can be I or L, X 67 It can be L or V, X 68 It can be S or T, X 73 It can be N or T, X 76 It can be N or S, X 79 It can be F or S, X 82 It can be L or M, X 83 It can be N or S, X 85 It can be L or V, X 86It can be Q or T, X 87 It can be A or T, X 88 It can be A or E, X 92 It can be S or V, X 98 It can be D or E, X 118 It can be A or S.

[0438] In this application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0439] For example, the second polypeptide chain contains the amino acid sequence shown in either SEQ ID NO:39 or SEQ ID NO:40.

[0440] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include at least one CDR in the variable region (VL) of the antibody light chain, and the VL may contain the amino acid sequence shown in SEQ ID NO:58.

[0441] For example, the VL may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In this application, the LCDR of the second targeting portion in the isolated bispecific antibody second polypeptide chain can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38, and the LCDR obtained by dividing in any form falls within the protection scope of this application.

[0442] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0443] In this application, the LCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0444] For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X1, X4, X5, X7, and X9, compared to the sequence shown in SEQ ID NO:57.

[0445] X1QSX4X5VX7WX9 (SEQ ID NO: 57), wherein X1 can be Q or S, X4 can be K, L or S, X5 can be E, H, K or R, X7 can be N or P, and X9 can be S or T.

[0446] In this application, the LCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0447] In this application, the LCDR2 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:18. For example, the LCDR2 of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0448] In this application, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:15. For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0449] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:21. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0450] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:22. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0451] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:23. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0452] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:24. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4-25-1C2, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0453] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:25. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0454] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:26. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0455] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO:58, the VL of the second targeting portion of the second polypeptide chain of the bispecific antibody has amino acid substitutions (e.g., conserved amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: X7, X9, X 12 X 14 X 15 X 18 X 20 X 41 X 49 X 64 X 76 X 78 X 80 X 81 X 82 X 84 X 85 X 87 X 88 X 89 X 93 X 96 X 97 X 99 X 101 and X 108 .

[0456] DIVLTQX7PX9SLX 12 VX 14 X 15 GQX 18 AX 20 ISCRASESVDNYGISFMNWFX 41 QKPGQPPX 49 LLIYAASNQGSGVPX 64 RFSGSGSGTDFX 76 LX 78 IX 80 X 81 X 82 EX 84 X 85 DX 87 X 88 X 89 YFCX 93 QSX 96 X 97 VX 99 WX 101 FGGGTKX 108EIK (SEQ ID NO: 58), where X7 can be S or T, X9 can be A or L, X 12 It can be A or S, X 14 It can be S or T, X 15 It can be L or P, X 18 It can be P or R, X 20 It can be S or T, X 41 It can be L or Q, X 49 It can be K or Q, X 64 It can be A or D, X 76 It can be S or T, X 78 It can be K or N, X 80 It can be H or S, X 81 It can be P or R, X 82 It can be M or V, X 84 It can be A or E, X 85 It can be D or E, X 87 It can be T or V, X 88 It can be A or G, X 89 It can be M or V, X 93 It can be Q or S, X 96 It can be K, L, or S, X 97 It can be E, H, K or R, X 99 It can be N or P, X 101 It can be S or T, X 108 It can be L or V.

[0457] In this application, the light chain variable region of the second targeting portion in the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0458] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a heavy chain constant region, which may include a constant region derived from IgG or a constant region derived from IgY.

[0459] For example, the heavy chain constant region of the second targeting portion in the second polypeptide chain of the bispecific antibody may contain the amino acid sequences shown in SEQ ID NO:112 to SEQ ID NO:113.

[0460] For example, the second polypeptide chain may contain the amino acid sequence shown in any one of SEQ ID NO:39 to SEQ ID NO:40, SEQ ID NO:154 to SEQ ID NO:160.

[0461] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise any of the amino acid sequences shown in SEQ ID NO:95 to SEQ ID NO:104, SEQ ID NO:105 to SEQ ID NO:111, and the second targeting portion in the second polypeptide chain may comprise any of the amino acid sequences shown in SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38.

[0462] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0463] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0464] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0465] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0466] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0467] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0468] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0469] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0470] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0471] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0472] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:146, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

[0473] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:147, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

[0474] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:149, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

[0475] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:151, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

[0476] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:152, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

[0477] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:146, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

[0478] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:147, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

[0479] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:149, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

[0480] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:151, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

[0481] For example, the bispecific antibody may comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:152, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

[0482] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody can specifically bind to the PD-1 protein, and the second targeting portion of the second polypeptide chain of the bispecific antibody can specifically bind to the PD-L1 protein.

[0483] For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0484] For example, the HCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody has an amino acid substitution (e.g., a conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 51, compared to the sequence shown: X1.

[0485] X1HYGTSPFVY(SEQ ID NO: 51), where X1 can be D or E.

[0486] In this application, the HCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in either SEQ ID NO:9 or SEQ ID NO:10. For example, the HCDR3 sequence of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0487] In this application, the HCDR2 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0488] In this application, the HCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0489] For example, the HCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:9. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0490] For example, the HCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:10. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4-25-3C11, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0491] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:52. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:52, has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X1, X5, X 13 X 16 X 17 X 20 X 37 X 48 X 67 X 68 X 73 X 76 X 79 X 82 X83 X 85 X 86 X 87 X 88 X 92 X 98 and X 118 .

[0492] X1VQLX5ESGPGLVX 13 PSX 16 X 17 LSX 20 TCTVSGFSLTSYAISWX 37 RQPPGKGLEWX 48 GVI WTGGGTNYNSALKSRX 67 X 68 ISKDX 73 SKX 76 QVX 79 LKX 82 X 83 SX 85 X 86 X 87 X 88 DTAX 92 YYCARX 98 HYGTSPFVYWGQGTLVTVSX 118 (SEQ ID NO: 52), where X1 can be E or Q, X5 can be K or Q, X 13 It can be A or K, X 16 It can be E or Q, X 17 It can be S or T, X 20 It can be I or L, X 37 It can be I or V, X 48 It can be I or L, X 67 It can be L or V, X 68 It can be S or T, X 73 It can be N or T, X 76 It can be N or S, X 79 It can be F or S, X 82 It can be L or M, X 83 It can be N or S, X 85 It can be L or V, X 86 It can be Q or T, X 87 It can be A or T, X 88 It can be A or E, X 92 It can be S or V, X 98 It can be D or E, X 118 It can be A or S.

[0493] In this application, the heavy chain variable region of the first targeting portion of the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0494] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include at least one CDR in the variable region (VL) of the antibody light chain, and the VL may contain the amino acid sequence shown in SEQ ID NO:58.

[0495] For example, the VL of the first targeting portion in the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In this application, the LCDR of the first targeting portion in the first polypeptide chain of the bispecific antibody can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38, and the LCDR obtained by dividing in any form falls within the protection scope of this application.

[0496] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0497] In this application, the LCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0498] For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X1, X4, X5, X7, and X9, compared to the sequence shown in SEQ ID NO:57.

[0499] X1QSX4X5VX7WX9 (SEQ ID NO: 57), wherein X1 can be Q or S, X4 can be K, L or S, X5 can be E, H, K or R, X7 can be N or P, and X9 can be S or T.

[0500] In this application, the LCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0501] In this application, the LCDR2 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:18. For example, the LCDR2 of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0502] In this application, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:15. For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0503] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:21. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0504] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:22. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0505] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:23. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0506] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:24. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4-25-1C2, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0507] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:25. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0508] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:26. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may include antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0509] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO:58, the VL of the first targeting portion of the first polypeptide chain of the bispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X7, X9, X 12 X 14 X 15 X 18 X 20 X 41 X 49 X 64 X 76 X 78 X 80 X 81 X 82 X 84 X 85 X 87 X 88 X 89 X 93 X 96 X 97 X 99 X 101 and X 108 .

[0510] DIVLTQX7PX9SLX 12 VX 14 X 15 GQX 18 AX 20 ISCRASESVDNYGISFMNWFX 41 QKPGQPPX 49 LLIYAASNQGSGVPX 64 RFSGSGSGTDFX 76 LX 78 IX 80 X 81 X 82 EX 84 X 85 DX 87 X 88 X 89 YFCX 93 QSX 96 X 97 VX 99 WX 101 FGGGTKX 108EIK (SEQ ID NO: 58), where X7 can be S or T, X9 can be A or L, X 12 It can be A or S, X 14 It can be S or T, X 15 It can be L or P, X 18 It can be P or R, X 20 It can be S or T, X 41 It can be L or Q, X 49 It can be K or Q, X 64 It can be A or D, X 76 It can be S or T, X 78 It can be K or N, X 80 It can be H or S, X 81 It can be P or R, X 82 It can be M or V, X 84 It can be A or E, X 85 It can be D or E, X 87 It can be T or V, X 88 It can be A or G, X 89 It can be M or V, X 93 It can be Q or S, X 96 It can be K, L, or S, X 97 It can be E, H, K or R, X 99 It can be N or P, X 101 It can be S or T, X 108 It can be L or V.

[0511] In this application, the light chain variable region of the first targeting portion in the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0512] In this application, the first targeting portion of the first polypeptide chain of the bispecific antibody may include a light chain constant region, which may include a constant region derived from Igκ or a constant region derived from Igλ.

[0513] For example, the light chain constant region may include a constant region derived from Igκ.

[0514] For example, the light chain constant region of the first targeting portion in the first polypeptide chain of the bispecific antibody contains the amino acid sequence shown in SEQ ID NO: 114.

[0515] For example, the first polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO:141 to 143 and SEQ ID NO:41 to SEQ ID NO:46. In this application, the HCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0516] In this application, the HCDR2 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0517] In this application, the HCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat coding system.

[0518] For example, the HCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:70; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:76. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0519] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:122. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:122, has an amino acid substitution (e.g., a conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X5, X7, X... 10 X 11 X 12 X 16 X 20 X 37 X38 X 40 X 46 X 48 X 67 X 68 X 69 X 75 X 81 X 86 X 90 X 115 and X 116 .

[0520] QVQLX5QX7GAX 10 X 11 X 12 KPGX 16 SVKX 20 SCKASGYTFTSNWMHWX 37 X 38 QX 40 PGQGLX 46 WX 48 GMIHPNSAIKYNEKFKSRX 67 X 68 X 69 TADKSX 75 STAYMX 81 LSSLX 86 SEDX 90 AVYYCARSYYGSSPYFFDYWGQGTX 115 X 116 TVSS (SEQ ID NO: 122), where X5 can be Q or V, X7 can be P or S, X 10 It can be D or E, X 11 It can be L or V, X 12 It can be K or V, X 16 It can be A or S, X 20 It can be L or V, X 37 It can be D, M, or V, X 38 It can be K or R, X 40 It can be A or R, X 46 It can be D or E, X 48 It can be I or M, X 67 It can be A or V, X 68 It can be S or T, X 69 It can be I or L, X 75 It can be S or T, X 81 It can be E or Q, X 86 It can be R or T, X 90 It can be S or T, X 115It can be M or T, X 116 It can be L or V.

[0521] In this application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

[0522] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include at least one CDR in the variable region (VL) of the antibody light chain, and the VL may contain the amino acid sequence shown in SEQ ID NO:123.

[0523] For example, the VL in the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111. In this application, the LCDR of the second targeting portion of the second polypeptide chain of the bispecific antibody can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111, and the LCDR obtained by dividing in any form falls within the protection scope of this application.

[0524] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0525] In this application, the LCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0526] In this application, the LCDR2 of the second targeting portion of the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:87. For example, the LCDR2 of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0527] In this application, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO:82. For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody may be defined according to the Kabat numbering system.

[0528] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the bispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:87; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:94. For example, the second targeting portion of the second polypeptide chain of the bispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0529] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO:123, the VL of the second targeting portion of the second polypeptide chain of the bispecific antibody has amino acid substitutions (e.g., conserved amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: X7, X8, X... 15 X 22 X 41 X 42 X 43 X 44 X 46 X 60 X 71 X 72 X 77 X 79 X 80 X 83 X 87 and X 104 .

[0530] DIQMTQX7X8SSLSASX 15 GDRVTIX 22 CRASQDISKYLNWYQQKPX 41 X 42 X 43 X 44 KX 46 LIY YTSRLHSGVPX 60 RFSGSGSGTDX 71 X 72 LTISX 77 LX 79 X 80 EDX 83 ATYX 87CQQGDTLPWTFGGGT KX 104 EIK (SEQ ID NO: 123), where X7 can be S or T, X8 can be P or T, X 15 It can be L or V, X 22 It can be S or T, X 41 It can be D or G, X 42 It can be G or K, X 43 It can be A or T, X 44 It can be P or V, X 46 It can be F or L, X 60 It can be A or S, X 71 It can be F or Y, X 72 It can be F or T, X 77 It can be N or S, X 79 It can be E or Q, X 80 It can be P or Q, X 83 It can be F or V, X 87 It can be F or Y, X 104 It can be L or V.

[0531] In this application, the light chain variable region of the second targeting portion in the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0532] In this application, the second targeting portion of the second polypeptide chain of the bispecific antibody may include a heavy chain constant region, which may include a constant region derived from IgG or a constant region derived from IgY.

[0533] For example, the heavy chain constant region of the second targeting portion in the second polypeptide chain of the bispecific antibody may contain the amino acid sequences shown in SEQ ID NO:112 to SEQ ID NO:113.

[0534] For example, the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:124 to 133, SEQ ID NO:161 to SEQ ID NO:167.

[0535] In this application, the bispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. For example, the first targeting portion of the first polypeptide chain may comprise any of the amino acid sequences shown in SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38, and the second targeting portion of the second polypeptide chain may comprise any of the amino acid sequences shown in SEQ ID NO:95 to SEQ ID NO:104, SEQ ID NO:105 to SEQ ID NO:111.

[0536] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97.

[0537] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98.

[0538] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100.

[0539] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102.

[0540] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103.

[0541] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97.

[0542] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98.

[0543] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100.

[0544] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102.

[0545] For example, the bispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103.

[0546] In this application, the bispecific antibody may comprise two of the aforementioned first polypeptide chains and two of the aforementioned second polypeptide chains. For example, the two aforementioned first polypeptide chains may be linked by disulfide bonds, and the two aforementioned first polypeptide chains and the two aforementioned second polypeptide chains may form an IgG-like antibody. For example, the bispecific antibody may be a homodimer.

[0547] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:126.

[0548] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:127.

[0549] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:129.

[0550] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:131.

[0551] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:132.

[0552] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:126.

[0553] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:127.

[0554] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:129.

[0555] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:131.

[0556] For example, the bispecific antibody may comprise two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:132.

[0557] For example, the first targeting portion of the first polypeptide chain of the bispecific antibody can specifically bind to the PD-1 protein, and the second targeting portion of the second polypeptide chain of the bispecific antibody can specifically bind to the CD28 or CD16a protein. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 31, and the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 176 or SEQ ID NO: 168. For example, the bispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

[0558] For example, the first targeting portion of the first polypeptide chain of the bispecific antibody can specifically bind to CD28 or CD16a protein, and the second targeting portion of the second polypeptide chain of the bispecific antibody can specifically bind to PD-1 protein. For example, the first targeting portion of the first polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in either SEQ ID NO: 176 or SEQ ID NO: 168, and the second targeting portion of the second polypeptide chain of the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 31. For example, the bispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

[0559] Multispecific antibodies

[0560] On the other hand, this application provides one or more multispecific antibodies comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first targeting portion and the second polypeptide chain comprises a second targeting portion, wherein the first targeting portion and the second targeting portion each independently bind to immune cell-associated antigens, and the first polypeptide chain and the second polypeptide chain are linked by disulfide bonds.

[0561] For example, the immune cells may include T cells, macrophages, dendritic cells, and / or NK cells. For example, the immune cell-associated antigens may include PD-1, PD-L1, CD3, CD16a, and / or CD28.

[0562] In this application, the first targeting portion of the multispecific antibody may be the same as the first targeting portion of the aforementioned bispecific antibody. In this application, the second targeting portion of the multispecific antibody may be the same as the second targeting portion of the aforementioned bispecific antibody.

[0563] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to the PD-L1 protein, and the second targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to the PD-1 protein.

[0564] In this application, the HCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0565] In this application, the HCDR2 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0566] In this application, the HCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0567] For example, the HCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:70; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:76. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0568] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:122. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:122, has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X5, X7, X... 10 X 11 X 12 X 16 X 20 X 37 X 38 X 40 X 46 X 48 X 67 X 68 X 69 X 75 X 81 X 86 X 90 X 115 and X 116 .

[0569] QVQLX5QX7GAX 10 X 11 X 12 KPGX 16 SVKX 20 SCKASGYTFTSNWMHWX 37 X 38 QX 40 PGQGLX 46 WX 48 GMIHPNSAIKYNEKFKSRX 67 X 68 X 69TADKSX 75 STAYMX 81 LSSLX 86 SEDX 90 AVYYCARSYYGSSPYFFDYWGQGTX 115 X 116 TVSS (SEQ ID NO: 122), where X5 can be Q or V, X7 can be P or S, X 10 It can be D or E, X 11 It can be L or V, X 12 It can be K or V, X 16 It can be A or S, X 20 It can be L or V, X 37 It can be D, M, or V, X 38 It can be K or R, X 40 It can be A or R, X 46 It can be D or E, X 48 It can be I or M, X 67 It can be A or V, X 68 It can be S or T, X 69 It can be I or L, X 75 It can be S or T, X 81 It can be E or Q, X 86 It can be R or T, X 90 It can be S or T, X 115 It can be M or T, X 116 It can be L or V.

[0570] In this application, the heavy chain variable region of the first targeting portion of the first polypeptide chain of the multispecific antibody may contain an amino acid sequence shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

[0571] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include at least one CDR in the light chain variable region (VL), and the VL may contain the amino acid sequence shown in SEQ ID NO:123.

[0572] For example, the VL in the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111. In this application, the LCDR of the first targeting portion of the first polypeptide chain of the multispecific antibody can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111, and the LCDR obtained by dividing in any form can fall within the protection scope of this application.

[0573] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0574] In this application, the LCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0575] In this application, the LCDR2 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:87. For example, the LCDR2 of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0576] In this application, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:82. For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0577] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:87; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:94. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0578] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO:123, the VL of the first targeting portion of the first polypeptide chain of the multispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X7, X8, X... 15 X 22 X 41 X 42 X43 X 44 X 46 X 60 X 71 X 72 X 77 X 79 X 80 X 83 X 87 and X 104 .

[0579] DIQMTQX7X8SSLSASX 15 GDRVTIX 22 CRASQDISKYLNWYQQKPX 41 X 42 X 43 X 44 KX 46 LIY YTSRLHSGVPX 60 RFSGSGSGTDX 71 X 72 LTISX 77 LX 79 X 80 EDX 83 ATYX 87 CQQGDTLPWTFGGGT KX 104 EIK (SEQ ID NO: 123), where X7 can be S or T, X8 can be P or T, X 15 It can be L or V, X 22 It can be S or T, X 41 It can be D or G, X 42 It can be G or K, X 43 It can be A or T, X 44 It can be P or V, X 46 It can be F or L, X 60 It can be A or S, X 71 It can be F or Y, X 72 It can be F or T, X 77 It can be N or S, X 79 It can be E or Q, X 80 It can be P or Q, X 83 It can be F or V, X 87 It can be F or Y, X 104 It can be L or V.

[0580] In this application, the light chain variable region of the first targeting portion in the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0581] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a light chain constant region, which may include a constant region derived from Igκ or a constant region derived from Igλ.

[0582] For example, the light chain constant region may include a constant region derived from Igκ.

[0583] For example, the light chain constant region of the first targeting portion in the first polypeptide chain of the multispecific antibody contains the amino acid sequence shown in any one of SEQ ID NO: 114.

[0584] For example, the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:134 to 140 and SEQ ID NO:144 to 153.

[0585] For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0586] For example, the second targeting portion of the second polypeptide chain of the multispecific antibody has an amino acid substitution (e.g., a conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 51, compared to the sequence shown in SEQ ID NO: 51: X1.

[0587] X1HYGTSPFVY(SEQ ID NO: 51), where X1 can be D or E.

[0588] In this application, the HCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in either SEQ ID NO:9 or SEQ ID NO:10. For example, the HCDR3 sequence of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0589] In this application, the HCDR2 of the second targeting portion of the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0590] In this application, the HCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0591] For example, the HCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:9. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0592] For example, the HCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:10. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4-25-3C11, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0593] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:52. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:52, has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X1, X5, X 13 X 16 X 17 X 20 X 37 X 48 X 67 X 68 X 73 X 76 X 79 X 82 X83 X 85 X 86 X 87 X 88 X 92 X 98 and X 118 .

[0594] X1VQLX5ESGPGLVX 13 PSX 16 X 17 LSX 20 TCTVSGFSLTSYAISWX 37 RQPPGKGLEWX 48 GVI WTGGGTNYNSALKSRX 67 X 68 ISKDX 73 SKX 76 QVX 79 LKX 82 X 83 SX 85 X 86 X 87 X 88 DTAX 92 YYCARX 98 HYGTSPFVYWGQGTLVTVSX 118 (SEQ ID NO: 52), where X1 can be E or Q, X5 can be K or Q, X 13 It can be A or K, X 16 It can be E or Q, X 17 It can be S or T, X 20 It can be I or L, X 37 It can be I or V, X 48 It can be I or L, X 67 It can be L or V, X 68 It can be S or T, X 73 It can be N or T, X 76 It can be N or S, X 79 It can be F or S, X 82 It can be L or M, X 83 It can be N or S, X 85 It can be L or V, X 86 It can be Q or T, X 87 It can be A or T, X 88 It can be A or E, X 92 It can be S or V, X 98 It can be D or E, X 118 It can be A or S.

[0595] In this application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0596] For example, the second polypeptide chain of the multispecific antibody contains the amino acid sequence shown in either SEQ ID NO:39 or SEQ ID NO:40.

[0597] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include at least one CDR in the variable region (VL) of the antibody light chain, and the VL may contain the amino acid sequence shown in SEQ ID NO:58.

[0598] For example, the VL may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In this application, the LCDR of the second targeting portion in the isolated multispecific antibody second polypeptide chain can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38, and the LCDR obtained by dividing in any form can fall within the protection scope of this application.

[0599] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0600] In this application, the LCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0601] For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X1, X4, X5, X7, and X9, compared to the sequence shown in SEQ ID NO:57.

[0602] X1QSX4X5VX7WX9 (SEQ ID NO: 57), wherein X1 can be Q or S, X4 can be K, L or S, X5 can be E, H, K or R, X7 can be N or P, and X9 can be S or T.

[0603] In this application, the LCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0604] In this application, the LCDR2 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:18. For example, the LCDR2 of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0605] In this application, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:15. For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0606] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:21. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0607] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:22. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0608] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:23. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0609] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:24. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4-25-1C2, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0610] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:25. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0611] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:26. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0612] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO:58, the VL of the second targeting portion of the second polypeptide chain of the multispecific antibody has amino acid substitutions (e.g., conserved amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: X7, X9, X 12 X 14 X 15 X 18 X 20 X 41 X 49 X 64 X 76 X 78 X 80 X 81 X 82 X 84 X 85 X 87 X 88 X 89 X 93 X 96 X 97 X 99 X 101 and X 108 .

[0613] DIVLTQX7PX9SLX 12 VX 14 X 15 GQX 18 AX 20 ISCRASESVDNYGISFMNWFX 41 QKPGQPPX 49 LLIYAASNQGSGVPX 64 RFSGSGSGTDFX 76 LX 78 IX 80 X 81 X 82 EX 84 X 85 DX 87 X 88 X 89 YFCX 93 QSX 96 X 97 VX 99 WX 101 FGGGTKX 108EIK (SEQ ID NO: 58), where X7 can be S or T, X9 can be A or L, X 12 It can be A or S, X 14 It can be S or T, X 15 It can be L or P, X 18 It can be P or R, X 20 It can be S or T, X 41 It can be L or Q, X 49 It can be K or Q, X 64 It can be A or D, X 76 It can be S or T, X 78 It can be K or N, X 80 It can be H or S, X 81 It can be P or R, X 82 It can be M or V, X 84 It can be A or E, X 85 It can be D or E, X 87 It can be T or V, X 88 It can be A or G, X 89 It can be M or V, X 93 It can be Q or S, X 96 It can be K, L, or S, X 97 It can be E, H, K or R, X 99 It can be N or P, X 101 It can be S or T, X 108 It can be L or V.

[0614] In this application, the light chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0615] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a heavy chain constant region, which may include a constant region derived from IgG or a constant region derived from IgY.

[0616] For example, the heavy chain constant region of the second targeting portion in the second polypeptide chain of the multispecific antibody may contain the amino acid sequences shown in SEQ ID NO:112 to SEQ ID NO:113.

[0617] For example, the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:39 to SEQ ID NO:40, SEQ ID NO:154 to SEQ ID NO:160.

[0618] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain of the multispecific antibody and a second targeting portion in a second polypeptide chain of the multispecific antibody. The first targeting portion in the first polypeptide chain of the multispecific antibody may comprise any of the amino acid sequences shown in SEQ ID NO:95 to SEQ ID NO:104, SEQ ID NO:105 to SEQ ID NO:111, and the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise any of the amino acid sequences shown in SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38.

[0619] For example, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. The first targeting portion of the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97, and the second targeting portion of the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0620] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0621] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0622] For example, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. The first targeting portion of the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102, and the second targeting portion of the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0623] For example, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. The first targeting portion of the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103, and the second targeting portion of the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

[0624] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0625] For example, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. The first targeting portion of the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98, and the second targeting portion of the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0626] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0627] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0628] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36.

[0629] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to the PD-1 protein, and the second targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to the PD-L1 protein.

[0630] For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0631] For example, the HCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody has an amino acid substitution (e.g., a conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 51, compared to the sequence shown: X1.

[0632] X1HYGTSPFVY(SEQ ID NO: 51), where X1 can be D or E.

[0633] In this application, the HCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in either SEQ ID NO:9 or SEQ ID NO:10. For example, the HCDR3 sequence of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0634] In this application, the HCDR2 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0635] In this application, the HCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0636] For example, the HCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:9. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0637] For example, the HCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:10. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4-25-3C11, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0638] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:52. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:52, has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X1, X5, X 13 X 16 X 17 X 20 X 37 X 48 X 67 X 68 X 73 X 76 X 79 X 82 X 83 X 85 X 86 X 87 X 88 X 92 X 98 and X 118 .

[0639] X1VQLX5ESGPGLVX 13 PSX 16 X 17 LSX 20 TCTVSGFSLTSYAISWX 37 RQPPGKGLEWX 48 GVI WTGGGTNYNSALKSRX 67 X 68 ISKDX 73 SKX 76 QVX 79 LKX 82 X 83 SX 85 X 86 X 87 X 88 DTAX 92 YYCARX 98 HYGTSPFVYWGQGTLVTVSX 118 (SEQ ID NO: 52), where X1 can be E or Q, X5 can be K or Q, X 13 It can be A or K, X 16 It can be E or Q, X 17 It can be S or T, X 20 It can be I or L, X 37 It can be I or V, X 48 It can be I or L, X 67 It can be L or V, X 68 It can be S or T, X 73 It can be N or T, X 76 It can be N or S, X 79 It can be F or S, X 82 It can be L or M, X 83 It can be N or S, X 85 It can be L or V, X 86 It can be Q or T, X 87 It can be A or T, X 88 It can be A or E, X 92 It can be S or V, X 98 It can be D or E, X 118 It can be A or S.

[0640] In this application, the heavy chain variable region of the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

[0641] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include at least one CDR in the variable region (VL) of the antibody light chain, and the VL may contain the amino acid sequence shown in SEQ ID NO:58.

[0642] For example, the VL of the first targeting portion in the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In this application, the LCDR of the first targeting portion in the first polypeptide chain of the multispecific antibody can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38, and the LCDR obtained by dividing in any form falls within the protection scope of this application.

[0643] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0644] In this application, the LCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0645] For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X1, X4, X5, X7, and X9, compared to the sequence shown in SEQ ID NO:57.

[0646] X1QSX4X5VX7WX9 (SEQ ID NO: 57), wherein X1 can be Q or S, X4 can be K, L or S, X5 can be E, H, K or R, X7 can be N or P, and X9 can be S or T.

[0647] In this application, the LCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0648] In this application, the LCDR2 of the first targeting portion of the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:18. For example, the LCDR2 of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0649] In this application, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:15. For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0650] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:21. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4F1, hu19D4-25, 19D4-25-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0651] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:22. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0652] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:23. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0653] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:24. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4-25-1C2, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0654] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:25. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0655] For example, the LCDR1 of the first targeting portion of the first polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:26. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may include antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0656] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO:58, the VL of the first targeting portion of the first polypeptide chain of the multispecific antibody has an amino acid substitution (e.g., conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X7, X9, X 12 X 14 X 15 X 18 X 20 X 41 X 49 X 64 X 76 X 78 X80 X 81 X 82 X 84 X 85 X 87 X 88 X 89 X 93 X 96 X 97 X 99 X 101 and X 108 .

[0657] DIVLTQX7PX9SLX 12 VX 14 X 15 GQX 18 AX 20 ISCRASESVDNYGISFMNWFX 41 QKPGQPPX 49 LLIYAASNQGSGVPX 64 RFSGSGSGTDFX 76 LX 78 IX 80 X 81 X 82 EX 84 X 85 DX 87 X 88 X 89 YFCX 93 QSX 96 X 97 VX 99 WX 101 FGGGTKX 108 EIK (SEQ ID NO: 58), where X7 can be S or T, X9 can be A or L, X 12 It can be A or S, X 14 It can be S or T, X 15 It can be L or P, X 18 It can be P or R, X 20 It can be S or T, X 41 It can be L or Q, X 49 It can be K or Q, X 64 It can be A or D, X 76 It can be S or T, X 78 It can be K or N, X 80 It can be H or S, X 81 It can be P or R, X 82 It can be M or V, X 84 It can be A or E, X 85It can be D or E, X 87 It can be T or V, X 88 It can be A or G, X 89 It can be M or V, X 93 It can be Q or S, X 96 It can be K, L, or S, X 97 It can be E, H, K or R, X 99 It can be N or P, X 101 It can be S or T, X 108 It can be L or V.

[0658] In this application, the light chain variable region of the first targeting portion in the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

[0659] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may include a light chain constant region, which may include a constant region derived from Igκ or a constant region derived from Igλ.

[0660] For example, the light chain constant region may include a constant region derived from Igκ.

[0661] For example, the light chain constant region of the first targeting portion in the first polypeptide chain of the multispecific antibody contains the amino acid sequence shown in SEQ ID NO: 114.

[0662] In this application, the HCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0663] In this application, the HCDR2 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the second targeting portion in the second polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0664] In this application, the HCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat coding system.

[0665] For example, the HCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:70; and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO:76. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3).

[0666] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a heavy chain variable region, which may contain the amino acid sequence shown in SEQ ID NO:122. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody includes VH, which, compared to the sequence shown in SEQ ID NO:122, has an amino acid substitution (e.g., a conserved amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X5, X7, X... 10 X 11 X 12 X 16 X 20 X 37 X 38 X 40 X 46 X 48 X 67 X 68 X 69 X 75 X 81 X 86 X 90 X 115 and X 116 .

[0667] QVQLX5QX7GAX 10 X 11 X 12 KPGX 16 SVKX 20 SCKASGYTFTSNWMHWX 37 X 38 QX 40 PGQGLX 46 WX 48 GMIHPNSAIKYNEKFKSRX 67 X 68 X 69TADKSX 75 STAYMX 81 LSSLX 86 SEDX 90 AVYYCARSYYGSSPYFFDYWGQGTX 115 X 116 TVSS (SEQ ID NO: 122), where X5 can be Q or V, X7 can be P or S, X 10 It can be D or E, X 11 It can be L or V, X 12 It can be K or V, X 16 It can be A or S, X 20 It can be L or V, X 37 It can be D, M, or V, X 38 It can be K or R, X 40 It can be A or R, X 46 It can be D or E, X 48 It can be I or M, X 67 It can be A or V, X 68 It can be S or T, X 69 It can be I or L, X 75 It can be S or T, X 81 It can be E or Q, X 86 It can be R or T, X 90 It can be S or T, X 115 It can be M or T, X 116 It can be L or V.

[0668] In this application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

[0669] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include at least one CDR in the variable region (VL) of the antibody light chain, and the VL may contain the amino acid sequence shown in SEQ ID NO:123.

[0670] For example, the VL in the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111. In this application, the LCDR of the second targeting portion of the second polypeptide chain of the multispecific antibody can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111, and the LCDR obtained by dividing in any form falls within the protection scope of this application.

[0671] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.

[0672] In this application, the LCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0673] In this application, the LCDR2 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:87. For example, the LCDR2 of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0674] In this application, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO:82. For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody may be defined according to the Kabat numbering system.

[0675] For example, the LCDR1 of the second targeting portion of the second polypeptide chain of the multispecific antibody described in this application may contain the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may contain the amino acid sequence shown in SEQ ID NO:87; and the LCDR3 may contain the amino acid sequence shown in SEQ ID NO:94. For example, the second targeting portion of the second polypeptide chain of the multispecific antibody may include antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or an antigen-binding fragment having the same LCDR3 (e.g., having the same LCDR1-3).

[0676] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a light chain variable region (VL), which may contain the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO:123, the VL of the second targeting portion of the second polypeptide chain of the multispecific antibody has amino acid substitutions (e.g., conserved amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of X7, X8, X... 15 X 22 X 41 X 42 X43 X 44 X 46 X 60 X 71 X 72 X 77 X 79 X 80 X 83 X 87 and X 104 .

[0677] DIQMTQX7X8SSLSASX 15 GDRVTIX 22 CRASQDISKYLNWYQQKPX 41 X 42 X 43 X 44 KX 46 LIY YTSRLHSGVPX 60 RFSGSGSGTDX 71 X 72 LTISX 77 LX 79 X 80 EDX 83 ATYX 87 CQQGDTLPWTFGGGT KX 104 EIK (SEQ ID NO: 123), where X7 can be S or T, X8 can be P or T, X 15 It can be L or V, X 22 It can be S or T, X 41 It can be D or G, X 42 It can be G or K, X 43 It can be A or T, X 44 It can be P or V, X 46 It can be F or L, X 60 It can be A or S, X 71 It can be F or Y, X 72 It can be F or T, X 77 It can be N or S, X 79 It can be E or Q, X 80 It can be P or Q, X 83 It can be F or V, X 87 It can be F or Y, X 104 It can be L or V.

[0678] In this application, the light chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO:105 to SEQ ID NO:111.

[0679] In this application, the second targeting portion of the second polypeptide chain of the multispecific antibody may include a heavy chain constant region, which may include a constant region derived from IgG or a constant region derived from IgY.

[0680] For example, the heavy chain constant region of the second targeting portion in the second polypeptide chain of the multispecific antibody may contain the amino acid sequences shown in SEQ ID NO:112 to SEQ ID NO:113.

[0681] In this application, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. For example, the first targeting portion of the first polypeptide chain may comprise any of the amino acid sequences shown in SEQ ID NO:29 to SEQ ID NO:31 and SEQ ID NO:32 to SEQ ID NO:38, and the second targeting portion of the second polypeptide chain may comprise any of the amino acid sequences shown in SEQ ID NO:95 to SEQ ID NO:104 and SEQ ID NO:105 to SEQ ID NO:111.

[0682] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97.

[0683] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98.

[0684] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100.

[0685] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102.

[0686] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103.

[0687] For example, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. The first targeting portion of the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion of the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:97.

[0688] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:98.

[0689] For example, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. The first targeting portion of the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion of the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:100.

[0690] For example, the multispecific antibody may comprise a first targeting portion in a first polypeptide chain and a second targeting portion in a second polypeptide chain. The first targeting portion in the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:102.

[0691] For example, the multispecific antibody may comprise a first targeting portion of a first polypeptide chain and a second targeting portion of a second polypeptide chain. The first targeting portion of the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:36, and the second targeting portion of the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:103.

[0692] For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to the PD-1 protein, and the second targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to the CD28 or CD16a protein. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 31, and the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 176 or SEQ ID NO: 168.

[0693] For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to CD28 or CD16a protein, and the second targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to PD-1 protein. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in either SEQ ID NO: 176 or SEQ ID NO: 168, and the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 31.

[0694] In this application, the first polypeptide chain of the multispecific antibody may further include a third targeting portion. For example, the third targeting portion in the first polypeptide chain of the multispecific antibody may be directly or indirectly linked to the C-terminus of the first targeting portion in the first polypeptide chain of the multispecific antibody. For example, the third targeting portion in the first polypeptide chain of the multispecific antibody may be linked to the C-terminus of the first targeting portion in the first polypeptide chain of the multispecific antibody via a linker. For example, the linker may be a linker commonly used in the art. For example, the linker may be a flexible linker. For example, the linker may be a G4S linker.

[0695] In this application, the third targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to immune cell-associated antigens. For example, the immune cell-associated antigens may include PD-1, PD-L1, CD3, CD16a, and / or CD28. For example, the third targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to CD3 or CD16a protein.

[0696] In this application, the third targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to tumor-related antigens. For example, the third targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to EGFR and / or cMet.

[0697] In this application, the third targeting portion of the first polypeptide chain of the multispecific antibody can specifically bind to tumor-associated antigens. For example, the tumor-associated antigens may include PD-L1, HER2, CD47, CD19, and / or CD20.

[0698] In this application, the second polypeptide chain of the multispecific antibody may further include a fourth targeting portion. For example, the fourth targeting portion of the second polypeptide chain of the multispecific antibody may be directly or indirectly linked to the C-terminus of the second targeting portion of the second polypeptide chain of the multispecific antibody. For example, the fourth targeting portion of the second polypeptide chain of the multispecific antibody may be linked to the C-terminus of the second targeting portion of the second polypeptide chain of the multispecific antibody via a linker. For example, the linker may be a linker commonly used in the art. For example, the linker may be a flexible linker. For example, the linker may be a G4S linker.

[0699] In this application, the fourth targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to immune cell-associated antigens. For example, the immune cell-associated antigens may include PD-1, PD-L1, CD3, CD16a, and / or CD28. For example, the fourth targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to CD3 or CD16a protein.

[0700] In this application, the fourth targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to tumor-related antigens. For example, the fourth targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to EGFR and / or cMet.

[0701] In this application, the fourth targeting portion of the second polypeptide chain of the multispecific antibody can specifically bind to tumor-associated antigens. For example, the tumor-associated antigens may include PD-L1, HER2, CD47, CD19, and / or CD20.

[0702] In this application, the multispecific antibody may comprise two of the aforementioned first polypeptide chains and two of the aforementioned second polypeptide chains. For example, the two aforementioned first polypeptide chains may be linked by disulfide bonds, and the two aforementioned first polypeptide chains and the two aforementioned second polypeptide chains may form an IgG-like antibody. For example, the multispecific antibody may be a homodimer.

[0703] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1, CD3, or CD16a. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 105-111; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; and the fourth targeting portion of the second polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 29-31, 168, and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO: 32-38, 169, and 173. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains can form an IgG-like antibody.

[0704] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1, CD3, or CD16a. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 32-38; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; and the fourth targeting portion of the second polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 95-104, 168, and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO: 105-111, 169, and 173. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains can form an IgG-like antibody.

[0705] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1, and the third targeting portion of the first polypeptide chain of the multispecific antibody can target CD3 or CD16a. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 95-104 and SEQ ID NO: 105-111; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31 and SEQ ID NO: 32-38; and the third targeting portion of the first polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 168 and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO: 169 and 173. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains can form an IgG-like antibody.

[0706] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody targets PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody targets PD-L1, and the third targeting portion of the first polypeptide chain of the multispecific antibody targets CD3 or CD16a. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequences shown in any one of SEQ ID NO:29-31 and SEQ ID NO:32-38; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequences shown in any one of SEQ ID NO:95-104 and SEQ ID NO:105-111; and the third targeting portion of the first polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO:168 and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO:169 and 173. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains may form an IgG-like antibody.

[0707] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may target CD28 or CD16a, the second targeting portion of the second polypeptide chain of the multispecific antibody may target PD-1, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody may target PD-L1, HER2, CD47, CD19, and / or CD20. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; and the fourth targeting portion of the second polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 95-104, 170, 174, and 178, and the amino acid sequence of VL shown in any one of SEQ ID NO: 105-111, 171, 175, and 179. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains can form IgG-like antibodies.

[0708] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody targets PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody targets CD28 or CD16a, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody targets PD-L1, HER2, CD47, CD19, and / or CD20. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; and the fourth targeting portion of the second polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 95-104, 170, 174, and 178 and the amino acid sequence of VL shown in any one of SEQ ID NO: 105-111, 171, 175, and 179. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains can form IgG-like antibodies.

[0709] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody may target CD28 or CD16a, the second targeting portion of the second polypeptide chain of the multispecific antibody may target PD-1, the third targeting portion of the first polypeptide chain of the multispecific antibody may target EGFR or cMET, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody may target PD-L1, HER2, CD47, CD19, and / or CD20. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; and the fourth targeting portion of the second polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 95-104, 170, 174, and 178 and the amino acid sequence of VL shown in any one of SEQ ID NO: 105-111, 171, 175, and 179. For example, the multispecific antibody may comprise two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains may form an IgG-like antibody.

[0710] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target CD28 or CD16a, the third targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, HER2, CD47, CD19 and / or CD20, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody can target EGFR or cMET. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; and the third targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 95-104, 170, 174 and 178 and the amino acid sequence of VL shown in any one of SEQ ID NO: 105-111, 171, 175 and 179. For example, the multispecific antibody may comprise two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains may form an IgG-like antibody.

[0711] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1, and the C-terminus of the second polypeptide chain of the multispecific antibody can contain an IL-2 variant, IFNα-2b, or IL-15. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 32-38; the second targeting portion of the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; and the cytokine in the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181. For example, the multispecific antibody can contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains can form an IgG-like antibody.

[0712] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1, and the C-terminus of the second polypeptide chain of the multispecific antibody can contain an IL-2 variant, IFNα-2b, or IL-15. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 105-111; the second targeting portion of the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; and the cytokine in the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181. For example, the multispecific antibody can contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains can form an IgG-like antibody.

[0713] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1, the C-terminus of the first polypeptide chain of the multispecific antibody can contain IL-2, IFNα-2b, or IL-15, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody can contain CD3 or CD16a. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second targeting portion of the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; the cytokine in the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181; and the fourth targeting portion of the second polypeptide chain can contain the amino acid sequence of VH shown in any one of SEQ ID NO: 168 and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO: 169 and 173. For example, the multispecific antibody may comprise two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains may form an IgG-like antibody.

[0714] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1, the C-terminus of the first polypeptide chain of the multispecific antibody can contain IL-2, IFNα-2b, or IL-15, and the fourth targeting portion can contain CD3 or CD16a. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; the second targeting portion of the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the cytokine in the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181; and the fourth targeting portion of the second polypeptide chain can contain the amino acid sequence of VH shown in any one of SEQ ID NO: 168 and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO: 169 and 173. For example, the multispecific antibody can contain two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains can form IgG-like antibodies.

[0715] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1. The C-terminus of the first polypeptide chain of the multispecific antibody may contain IL-2, IFNα-2b, or IL-15. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; and the cytokine in the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains may form an IgG-like antibody.

[0716] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1. The C-terminus of the first polypeptide chain of the multispecific antibody may contain IL-2, IFNα-2b, or IL-15. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; the cytokine in the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181. For example, the multispecific antibody may contain two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and the two second polypeptide chains may form an IgG-like antibody.

[0717] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1, the C-terminus of the first polypeptide chain of the multispecific antibody can contain IL-2, IFNα-2b, or IL-15, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 32-38; the second targeting portion of the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the cytokine in the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181; and the fourth targeting portion of the second polypeptide chain can contain the amino acid sequence of VH shown in any one of SEQ ID NO: 95-104 and the amino acid sequence of VL shown in any one of SEQ ID NO: 105-111. For example, the multispecific antibody can contain two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains can form IgG-like antibodies.

[0718] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1, the C-terminus of the first polypeptide chain of the multispecific antibody can contain IL-2, IFNα-2b, or IL-15, and the fourth targeting portion of the second polypeptide chain of the multispecific antibody targets PD-1. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 105-111; the second targeting portion of the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; the cytokine in the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181; and the fourth targeting portion of the second polypeptide chain can contain the amino acid sequence of VH shown in any one of SEQ ID NO: 29-31 and the amino acid sequence of VL shown in any one of SEQ ID NO: 32-38. For example, the multispecific antibody can contain two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains can form IgG-like antibodies.

[0719] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-L1, the third targeting portion of the first polypeptide chain of the multispecific antibody can target CD3 or CD16a, and the C-terminus of the second polypeptide chain of the multispecific antibody can contain IL-2, IFNα-2b, or IL-15. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second targeting portion of the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; the third targeting portion of the first polypeptide chain can contain the amino acid sequence of VH shown in any one of SEQ ID NO: 168 and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO: 169 and 173; and the cytokine in the second polypeptide chain of the multispecific antibody can contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181. For example, the multispecific antibody may comprise two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains may form an IgG-like antibody.

[0720] In this application, the first targeting portion of the first polypeptide chain of the multispecific antibody can target PD-L1, the second targeting portion of the second polypeptide chain of the multispecific antibody can target PD-1, the third targeting portion of the first polypeptide chain of the multispecific antibody targets CD3 or CD16a, and the C-terminus of the second polypeptide chain of the multispecific antibody contains IL-2, IFNα-2b, or IL-15. For example, the first targeting portion of the first polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 95-104; the second targeting portion of the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in any one of SEQ ID NO: 29-31; the third targeting portion of the first polypeptide chain may contain the amino acid sequence of VH shown in any one of SEQ ID NO: 168 and 172 and the amino acid sequence of VL shown in any one of SEQ ID NO: 169 and 173; and the cytokine in the second polypeptide chain of the multispecific antibody may contain the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181. For example, the multispecific antibody may comprise two first polypeptide chains and two second polypeptide chains. For example, the two first polypeptide chains may be linked by disulfide bonds, and the two first polypeptide chains and two second polypeptide chains may form an IgG-like antibody.

[0721] Peptides and Immunoconjugates

[0722] On the other hand, this application provides one or more polypeptides that may comprise the isolated antigen-binding protein of this application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptide may comprise a bispecific antibody.

[0723] On the other hand, this application provides one or more immunoconjugates that may contain the isolated antigen-binding protein of this application. In some embodiments, the immunoconjugate may also contain pharmaceutically acceptable therapeutic agents, markers, and / or detection agents.

[0724] Nucleic acid, vector, cell and drug composition

[0725] On the other hand, this application also provides one or more isolated nucleic acid molecules that can encode the isolated antigen-binding protein described in this application. For example, each of the one or more nucleic acid molecules can encode the complete antigen-binding protein or a portion thereof (e.g., one or more of HCDR1-3 and the heavy chain variable region).

[0726] For example, when nucleic acid molecules encode a portion of the antigen-binding protein, the products encoded by the nucleic acid molecules can be combined to form a functional (e.g., capable of binding PD-L1 and PD-1) isolated antigen-binding protein of this application.

[0727] The nucleic acid molecules described in this application can be isolated. For example, they can be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR), (ii) clonal recombination, (iii) purification, such as separation by enzyme digestion and gel electrophoresis, or (iv) synthesis, such as by chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology.

[0728] In this application, nucleic acids encoding the isolated antigen-binding protein can be prepared by a variety of methods known in the art, including but not limited to reverse transcription PCR and PCR to obtain the nucleic acid molecules of the isolated antigen-binding protein described in this application.

[0729] On the other hand, this application provides one or more vectors containing one or more nucleic acid molecules described in this application. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. In some embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the species or cell type and function, but typically includes 5' non-transcriptional sequences and 5' and 3' non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcriptional expression control sequence may contain a promoter region, which may contain a promoter sequence for transcriptionally controlling functionally linked nucleic acids. The expression control sequence may also include enhancer sequences or upstream activator sequences. In this application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6RNA promoters, CMV promoters, and their artificial hybrid promoters (such as CMV), wherein a portion of the promoter may be fused to a portion of a promoter of another cellular protein (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, and may or may not contain additional introns. One or more nucleic acid molecules described in this application may be operatively linked to the expression control element.

[0730] The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the vector may be a viral vector. Viral vectors can be administered directly to a patient (in vivo) or indirectly, for example, by treating cells with a virus in vitro and then administering the treated cells to a patient (ex vivo). Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Conventional virus-based systems may include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transfer. In some cases, gene transfer can be integrated into the host genome using retroviral, lentiviral, and adeno-associated virus methods, resulting in long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. Lentiviral vectors may contain long terminal repeat sequences (5'LTRs) and truncated 3'LTRs, RREs, rev response elements (cPPTs), central termination sequences (CTSs), and / or post-translational regulatory elements (WPREs). The vectors described in this application can be introduced into cells.

[0731] On the other hand, this application provides a cell. The cell may contain the isolated antigen-binding protein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules, and / or one or more vectors as described in this application. For example, each or every cell may contain one or more nucleic acid molecules or vectors as described in this application. For example, each or every cell may contain multiple (e.g., two or more) or more (e.g., two or more) nucleic acid molecules or vectors as described in this application. For example, the vectors as described in this application can be introduced into the host cell, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS / O cells, HEK293T cells, 293F cells, or HEK293A cells, or other eukaryotic cells, such as cells from plants, fungi, or yeast cells. The vectors as described in this application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the cell may include yeast cells. For example, the cell may include Escherichia coli cells. For example, the cell may include mammalian cells. For example, the cell may include immune cells.

[0732] The cells may include immune cells. In some cases, the cells may include immune cells. For example, the cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells.

[0733] On the other hand, this application provides a pharmaceutical composition. The pharmaceutical composition may comprise the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or pharmaceutically acceptable adjuvants and / or excipients described in this application. In this application, the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants. Unless incompatible with the cell described in this application, any conventional media or reagent may be considered for use in the pharmaceutical composition of this application. In this application, the pharmaceutically acceptable excipient may include additives other than the active pharmaceutical ingredient in a pharmaceutical formulation, also referred to as excipients. For example, the excipient may include binders, fillers, disintegrants, and lubricants in tablets. For example, the excipient may include wine, vinegar, or medicinal juice in traditional Chinese medicine pills. For example, the excipient may include the base portion in semi-solid preparations such as ointments or creams. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid formulations.

[0734] Uses and methods

[0735] On the other hand, this application provides a method for modulating an immune response, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the immune conjugate, the isolated nucleic acid molecule, the carrier, the cell and / or the pharmaceutical composition, and / or a pharmaceutically acceptable therapeutic agent.

[0736] In this application, the method for modulating the immune response may include in vitro methods, ex vivo methods, and methods for non-diagnostic or non-therapeutic purposes.

[0737] On the other hand, this application provides a method for modulating an immune response, comprising administering an effective amount of the said combination of drugs and / or a pharmaceutically acceptable therapeutic agent to a subject in need.

[0738] In this application, the method for modulating the immune response may include in vitro methods, ex vivo methods, and methods for non-diagnostic or non-therapeutic purposes.

[0739] On the other hand, this application provides isolated antigen-binding proteins, the aforementioned polypeptides, the aforementioned immunoconjugates, the aforementioned isolated nucleic acid molecules, the aforementioned carriers, and the aforementioned pharmaceutical compositions for the prevention, relief, and / or treatment of diseases or conditions.

[0740] In this application, the disease or condition may include a tumor. For example, the tumor may include a non-solid tumor. For example, the tumor may include a tumor associated with PD-L1 expression. The term "tumor associated with PD-L1 expression" generally refers to a tumor formed due to altered PD-L1 expression leading to disease progression or evasion of immune surveillance. For example, the "tumor associated with PD-L1 expression" may be a tumor formed due to upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with PD-L1 protein expression may be a PD-L1 positive tumor. In PD-L1 positive tumors, the protein expression level of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher compared to normal cells.

[0741] On the other hand, this application provides the use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament for the prevention, relief, and / or treatment of a disease or condition.

[0742] In this application, the disease or condition may include a tumor. For example, the tumor may include a solid tumor. For example, the tumor may include a non-solid tumor. For example, the tumor may include a tumor associated with PD-L1 expression. The term "tumor associated with PD-L1 expression" generally refers to a tumor formed due to altered PD-L1 expression leading to disease progression or evasion of immune surveillance. For example, the "tumor associated with PD-L1 expression" may be a tumor formed due to upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with PD-L1 protein expression may be a PD-L1 positive tumor. In PD-L1 positive tumors, the protein expression level of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher compared to normal cells.

[0743] On the other hand, this application provides a method for preventing and / or treating diseases or conditions, comprising administering to a subject in need the isolated antigen-binding protein, the isolated nucleic acid molecule, the carrier, the cell, and the pharmaceutical composition.

[0744] In this application, the disease or condition may include a tumor. For example, the tumor may include a non-solid tumor. For example, the tumor may include a tumor associated with PD-L1 expression. The term "tumor associated with PD-L1 expression" generally refers to a tumor formed due to altered PD-L1 expression leading to disease progression or evasion of immune surveillance. For example, the "tumor associated with PD-L1 expression" may be a tumor formed due to upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with PD-L1 protein expression may be a PD-L1-positive tumor. In PD-L1-positive tumors, the protein expression level of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher compared to normal cells. The pharmaceutical compositions and methods described in this application can be used in combination with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, etc. The pharmaceutical compositions and methods described in this application can be used for other disease conditions that depend on immune responses, such as inflammation, immune diseases, and infectious diseases.

[0745] In this application, the subject may include humans or non-human animals. For example, the non-human animal may be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice, and rats. Furthermore, non-human animals may also include any animal species other than humans, such as livestock, rodents, primates, domesticated animals, or poultry. The human may be Caucasian, African, Asian, Semitic, or of other races, or a hybrid of various races. For example, the human may be elderly, adult, adolescent, child, or infant.

[0746] The effective dose in humans can be inferred from the effective dose in laboratory animals. For example, Freireich et al. described the dose-response relationship between animals and humans (based on milligrams per square meter of body surface area) (Freireich et al., Cancer Chemother. Rep. 50, 219 (1966)). Body surface area can be approximated from the patient's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, NY, 537 (1970).

[0747] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0748] Example

[0749] Example 1: Construction of a bispecific antibody vector

[0750] 1.1 Obtaining 5BM and 19D4-25-3C11 monoclonal antibodies

[0751] The antigen-binding proteins 13H6D3 and 19D4F1 were obtained using hybridoma technology. Subsequently, through antibody humanization design and affinity maturation, humanized antibodies against 13H6D3 (hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM) and humanized antibodies against 19D4F1 (hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, 19D4-25-3C11, 19D4-25-1C2-3C11) were obtained. The methods for hybridoma preparation and humanization design can be based on methods published in numerous documents in this field.

[0752] Construction of 1.2pCP-5BM-Lh vector

[0753] Using the primers Lh5BM-VL-S+Lh5BM-VL-AS corresponding to Table 1 below, a 417bp fragment was amplified from the antibody 5BM heavy chain vector pCP-5BM-H-agYTE (agYTE represents YTE mutation and N297A mutation). PCR enzymes and systems were as per Takara Bio's instructions. Preparation and use of the HS (Premix) kit. Fragment amplification is followed by gel purification for later use.

[0754] A 350 bp light chain Kappa constant region sequence was amplified from a vector containing the Kappa light chain constant region using PCP-CK1-S+PCP-CK1-AS primers. The amplified fragment was then purified using a gel electrophoresis.

[0755] The light chain expression vector pCP-L was digested with KpnI and NotI (KpnI and NotI were from Takara Bio), and the approximately 5.5 kb vector fragment was recovered by gel excision.

[0756] Homologous recombination of the three fragments was performed using the ClonExpress Ultra One Step Cloning Kit (Vazyme). Ten positive clones were picked from LB plates, sequenced, and selected from the correct plasmids to proceed to the subsequent Lh vector amplification preparation process.

[0757] Table 1 Primer sequences

[0758]

[0759] Construction of 1.3pCP-3C11-H-agYTE vector

[0760] Using the primers 3C11-VH-S+3C11-VH-AS corresponding to Table 2 below, a 411bp fragment was amplified from the antibody 19D4-25-3C11 VH template (agYTE represents the YTE mutation and N297A mutation). PCR enzymes and systems were as per Takara Bio's instructions. Preparation and use of the HS (Premix) kit. Fragment amplification is followed by gel purification for later use.

[0761] The light chain expression vector pCP-H-agYTE was double-digested with KpnI and NheI (KpnI and NheI were from TakaraBio), and the approximately 6.5 kb vector fragment was recovered by gel digestion.

[0762] Homologous recombination of the three fragments was performed using the ClonExpress Ultra One Step Cloning Kit (Vazyme). Ten positive clones were picked from LB plates and sequenced for identification. One of the correctly constructed plasmids was selected to proceed to the subsequent pCP-3C11-H-agYTE amplification preparation process.

[0763] Table 2 Primer sequences

[0764]

[0765] Example 2: Bispecific Antibody Characterization Experiment

[0766] Using antibody expression plasmids, the sequence of the 5BMVH region of the anti-PDL1 antibody (SEQ ID NO: 97) was ligated to the N-terminus of the constant region (CL) of the human IgG1 kappa chain, and the sequence of the 19D4-25-3C11VH region of the anti-PD1 antibody (SEQ ID NO: 31) was ligated to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. HEK293 cells were transfected with the plasmids expressing the bispecific antibodies and cultured. The culture medium was then used to separate and purify the antibodies. The expression level of the 2xVH bispecific antibody (5BMVH-3C11VH, also called Lh5BM-3C11) was no different from that of a general antibody. To characterize this molecule, HEK293 cells highly expressing human PD1, PDL1, or wild-type cells were co-incubated with Lh5BM-3C11 for one hour. After washing, fluorescently labeled anti-human IgG1 Fc was added and the cells were developed. The results showed that (…). Figure 2 Lh5BM-3C11 can stain cells expressing PD1 or PDL1 with high intensity but has no staining on wild-type HEK293, indicating that this bispecific antibody can bind to PD1 and PDL1.

[0767] In vitro PD1 antagonism assays were performed to further characterize Lh5BM-3C11. ELISA plates were coated with recombinant PDL-1-Fc as the capture agent, a PD1-biotin tag, and horseradish peroxidase HRP-streptavidin as the detection agent. Anti-PD1 monoclonal antibody (1C2-3C11-AgYTE, referring to the antigen-binding protein obtained by fusing 19D4-25-1C2-3C11 with YTE-mutated and N297-mutated Fc) and anti-PD1 / PDL1 bispecific antibody (Lh5BM-3C11) were both used as blocking agents. Results ( Figure 3 The results showed that anti-PD1 antibodies could dose-dependently reduce the binding of PD1 to coated PDL-1. Bispecific antibodies dose-dependently increased HRP signaling, which can be interpreted as bispecific anti-PDL-1 binding to coated PDL1 and anti-PD1 binding to PD1-biotin bound to HRP-avidin; therefore, bispecific antibodies dose-dependently increased HRP signaling.

[0768] Example 3 Characterization experiment of trispecific antibody (anti-PD1 / PDL1 / CD16a trispecific antibody)

[0769] Using an antibody expression plasmid, the sequence of the 5BMVH region of the anti-PDL1 antibody (SEQ ID NO: 97) was ligated to the N-terminus of the constant region (CL) of the human IgG1 kappa chain, and the sequence of the 19D4-25-3C11VH region of the anti-PD1 antibody (SEQ ID NO: 31) was ligated to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. The VH (SEQ ID NO: 168) and VL (SEQ ID NO: 169) of the anti-CD16a antibody were linked together using a linker (GGGGS)3, and the N-terminus of VH was ligated to the C-terminus of Fc. This constituted a trispecific antibody against PD1 / PDL1 / CD16a. Staining was performed on cells expressing PD1, PDL1, CD16a, or wild-type HEK293 cells. The results showed that this trispecific antibody specifically stained cells with high expression of PD1, PDL1, or CD16a, but did not stain wild-type HEK293 cells.

[0770] To characterize that this trispecific antibody can simultaneously bind to PD1, PDL-1, and CD16a proteins, Biacore experiments showed that after coating the chip with the trispecific antibody and adding PD1, PDL-1, or CD16a in sequence, the peak value increased sequentially, indicating that this molecule can bind to PD1, PDL-1, and CD16a simultaneously.

[0771] Example 4: Characterization experiment of bispecific antibody against PD1 / PDL1 / IL-2

[0772] Using antibody expression plasmids, the sequence of the 5BMVH region of the anti-PDL-1 antibody (SEQ ID NO: 97) was ligated to the N-terminus of the constant region (CL) of the human IgG1 kappa chain, and the sequence of the 19D4-25-3C11VH region of the anti-PD1 antibody (SEQ ID NO: 31) was ligated to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. The IL-2 (SEQ ID NO: 180) expression gene was ligated to the C-terminus of Fc using a linker (GGGGS)3. This constitutes a bispecific antibody immunocytokine against PD1 / PDL1 / IL-2. Biacore experiments showed that when a chip was coated with the bispecific antibody against PD1 / PDL1 / IL-2 and PD1, PDL-1, or IL-2R were added sequentially, the peak values ​​increased sequentially, indicating that this molecule can bind to PD1, PDL-1, and IL-2R simultaneously.

[0773] When bispecific antibodies against PD1 / PDL1 / IL-2 were incubated with human peripheral blood PBMCs, the proliferation of T cells and the production of cytokines were significantly higher than those stimulated with PD1-PDL1 bispecific antibodies, indicating the immune-enhancing effect of IL-2.

[0774] Example 5: Characterization experiments of the bispecific antibody against PD1 / PDL1 / IFN-α

[0775] Using antibody expression plasmids, the sequence of the 5BM VH region of the anti-PDL-1 antibody (SEQ ID NO: 97) was ligated to the N-terminus of the constant region (CL) of the human IgG1 kappa chain, and the sequence of the 19D4-25-3C11 VH region of the anti-PD1 antibody (SEQ ID NO: 31) was ligated to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. The IFN-α (SEQ ID NO: 181) expression gene was ligated to the C-terminus of Fc using a linker (GGGGS)3. This constitutes a bispecific antibody immunocytokine against PD1 / PDL1 / IFN-α. Biacore experiments showed that when a chip was coated with the bispecific antibody against PD1 / PDL1 / IFN-α and PD1, PDL-1, or IFNR were added sequentially, the peak values ​​increased sequentially, indicating that this molecule can bind to PD1, PDL-1, and IFNR simultaneously.

[0776] When bispecific antibodies against PD1 / PDL1 / IFN-α were incubated with human peripheral blood PBMCs, the proliferation of T cells and the production of cytokines were significantly higher than those stimulated with PD1-PDL1 bispecific antibodies, indicating the immune-enhancing effect of IFN-α.

Claims

1. An isolated antigen-binding protein comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a first targeting portion and the second polypeptide chain comprising a second targeting portion, wherein the first targeting portion and the second targeting portion each independently bind to an immune cell-associated antigen, and the first polypeptide chain and the second polypeptide chain are linked by a disulfide bond; The first targeting portion is an antibody targeting the immune cell-associated antigen PD-1, the antibody comprising a heavy chain variable region VH and a light chain variable region VL; the VH comprises HCDR1, HCDR2, and HCDR3; the VL comprises LCDR1, LCDR2, and LCDR3; the HCDR1 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:10; the LCDR1 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:

21. The second targeting portion is an antibody targeting the immune cell-associated antigen PD-L1, the antibody comprising a heavy chain variable region VH and a light chain variable region VL; the VH comprises HCDR1, HCDR2, and HCDR3; the VL comprises LCDR1, LCDR2, and LCDR3; the HCDR1 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:61; the HCDR2 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:70; the HCDR3 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:76; the LCDR1 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:82; the LCDR2 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:87; and the LCDR3 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:

94.

2. An isolated antigen-binding protein comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a first targeting portion and the second polypeptide chain comprising a second targeting portion, wherein the first targeting portion and the second targeting portion each independently bind to an immune cell-associated antigen, and the first polypeptide chain and the second polypeptide chain are linked by a disulfide bond; The first targeting portion is an antibody targeting the immune cell-associated antigen PD-L1, the antibody comprising a heavy chain variable region VH and a light chain variable region VL; the VH comprises HCDR1, HCDR2, and HCDR3; the VL comprises LCDR1, LCDR2, and LCDR3; the HCDR1 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:61; the HCDR2 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:70; the HCDR3 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:76; the LCDR1 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:82; the LCDR2 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:87; and the LCDR3 of the first targeting portion has the amino acid sequence shown in SEQ ID NO:

94. The second targeting portion is an antibody targeting the immune cell-associated antigen PD-1, the antibody comprising a heavy chain variable region VH and a light chain variable region VL; the VH comprises HCDR1, HCDR2, and HCDR3; the VL comprises LCDR1, LCDR2, and LCDR3; the HCDR1 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:10; the LCDR1 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:18; and the LCDR3 of the second targeting portion has the amino acid sequence shown in SEQ ID NO:

21.

3. The isolated antigen-binding protein according to any one of claims 1-2, wherein the immune cells include T cells, macrophages, dendritic cells and / or NK cells.

4. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion specifically binds to PD-1 protein, and the second targeting portion specifically binds to PD-L1 protein.

5. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion specifically binds to PD-L1 protein, and the second targeting portion specifically binds to PD-1 protein.

6. The isolated antigen-binding protein according to any one of claims 1-2, wherein the first polypeptide chain further comprises a third targeting portion; the third targeting portion of the first polypeptide chain is linked to the C-terminus of the first targeting portion via a linker; the third targeting portion targets an antibody against the immune cell-associated antigen CD16a; the antibody comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO:168; and VL comprises the amino acid sequence shown in SEQ ID NO:

169.

7. The isolated antigen-binding protein according to claim 6, wherein the VH and VL of the anti-CD16a antibody are linked by a linker and the N-terminus of VH is linked to the C-terminus of Fc by a linker.

8. The isolated antigen-binding protein according to any one of claims 1-2, wherein the first polypeptide chain comprises cytokines, the cytokines comprising IL-2 and IFNα, wherein the IL-2 comprises the amino acid sequence shown in SEQ ID NO:180, and the IFNα comprises the amino acid sequence shown in SEQ ID NO:

181.

9. The isolated antigen-binding protein according to any one of claims 1-2, wherein the second polypeptide chain comprises cytokines, the cytokines comprising IL-2 and IFNα, wherein the IL-2 comprises the amino acid sequence shown in SEQ ID NO:180, and the IFNα comprises the amino acid sequence shown in SEQ ID NO:

181.

10. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises H-FR1, the C-terminus of H-FR1 being directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprising the amino acid sequence shown in SEQ ID NO:

2.

11. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises H-FR2, the H-FR2 being located between HCDR1 and HCDR2, and the H-FR2 comprising the amino acid sequence shown in SEQ ID NO:

5.

12. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises H-FR3, the H-FR3 being located between HCDR2 and HCDR3, and the H-FR3 comprising the amino acid sequence shown in SEQ ID NO:

8.

13. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises H-FR4, the N-terminus of H-FR4 being directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprising the amino acid sequence shown in SEQ ID NO:

12.

14. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 2; H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5; H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 8; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

12.

15. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises a heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO:

31.

16. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises L-FR1, the C-terminus of L-FR1 being directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprising the amino acid sequence shown in SEQ ID NO:

14.

17. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises L-FR2, the L-FR2 being located between LCDR1 and LCDR2, and the L-FR2 comprising the amino acid sequence shown in SEQ ID NO:

17.

18. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises L-FR3, the L-FR3 being located between LCDR2 and LCDR3, and the L-FR3 comprising the amino acid sequence shown in SEQ ID NO:

20.

19. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises L-FR4, the N-terminus of L-FR4 being directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprising the amino acid sequence shown in SEQ ID NO:

28.

20. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:14; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:17; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:20; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

28.

21. The isolated antigen-binding protein according to claim 1, wherein the first targeting portion comprises VL, the VL comprising the amino acid sequence shown in SEQ ID NO:

33.

22. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises H-FR1, the C-terminus of H-FR1 being directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprising the amino acid sequence shown in SEQ ID NO:

60.

23. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises H-FR2, the H-FR2 being located between HCDR1 and HCDR2, and the H-FR2 comprising the amino acid sequence shown in SEQ ID NO:

64.

24. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises H-FR3, the H-FR3 being located between HCDR2 and HCDR3, and the H-FR3 comprising the amino acid sequence shown in SEQ ID NO:

73.

25. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises H-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

78.

26. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:60; H-FR2 comprises the amino acid sequence shown in SEQ ID NO:64; H-FR3 comprises the amino acid sequence shown in SEQ ID NO:73; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

78.

27. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises a heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO:

97.

28. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises L-FR1, the C-terminus of L-FR1 being directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprising the amino acid sequence shown in SEQ ID NO:

81.

29. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises L-FR2, the L-FR2 being located between LCDR1 and LCDR2, and the L-FR2 comprising the amino acid sequence shown in SEQ ID NO:

85.

30. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises L-FR3, the L-FR3 being located between LCDR2 and LCDR3, and the L-FR3 comprising the amino acid sequence shown in SEQ ID NO:

90.

31. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises L-FR4, the N-terminus of L-FR4 being directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprising the amino acid sequence shown in SEQ ID NO:

28.

32. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:81; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:85; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:90; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

28.

33. The isolated antigen-binding protein according to claim 2, wherein the first targeting portion comprises VL, the VL comprising the amino acid sequence shown in SEQ ID NO:

107.

34. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises H-FR1, the C-terminus of which is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:

2.

35. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises H-FR2, the H-FR2 being located between HCDR1 and HCDR2, and the H-FR2 comprising the amino acid sequence shown in SEQ ID NO:

5.

36. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises H-FR3, the H-FR3 being located between HCDR2 and HCDR3, and the H-FR3 comprising the amino acid sequence shown in SEQ ID NO:

8.

37. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises H-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

12.

38. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 2; H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5; H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 8; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

12.

39. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises a heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO:

31.

40. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises L-FR1, the C-terminus of L-FR1 being directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprising the amino acid sequence shown in SEQ ID NO:

14.

41. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises L-FR2, the L-FR2 being located between LCDR1 and LCDR2, and the L-FR2 comprising the amino acid sequence shown in SEQ ID NO:

17.

42. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises L-FR3, the L-FR3 being located between LCDR2 and LCDR3, and the L-FR3 comprising the amino acid sequence shown in SEQ ID NO:

20.

43. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises L-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

28.

44. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:14; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:17; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:20; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

28.

45. The isolated antigen-binding protein according to claim 2, wherein the second targeting portion comprises VL, the VL comprising the amino acid sequence shown in SEQ ID NO:

33.

46. ​​The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises H-FR1, the C-terminus of which is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:

60.

47. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises H-FR2, the H-FR2 being located between HCDR1 and HCDR2, and the H-FR2 comprising the amino acid sequence shown in SEQ ID NO:

64.

48. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises H-FR3, the H-FR3 being located between HCDR2 and HCDR3, and the H-FR3 comprising the amino acid sequence shown in SEQ ID NO:

73.

49. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises H-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

78.

50. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein HFR1 comprises the amino acid sequence shown in SEQ ID NO: 60; H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 64; H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 73; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

78.

51. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises a heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO:

97.

52. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises L-FR1, the C-terminus of L-FR1 being directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprising the amino acid sequence shown in SEQ ID NO:

81.

53. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises L-FR2, the L-FR2 being located between LCDR1 and LCDR2, and the L-FR2 comprising the amino acid sequence shown in SEQ ID NO:

85.

54. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises L-FR3, the L-FR3 being located between LCDR2 and LCDR3, and the L-FR3 comprising the amino acid sequence shown in SEQ ID NO:

90.

55. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises L-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

28.

56. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:81; L-FR2 comprises the amino acid sequence shown in SEQ ID NO:85; L-FR3 comprises the amino acid sequence shown in SEQ ID NO:90; and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

28.

57. The isolated antigen-binding protein according to claim 1, wherein the second targeting portion comprises VL, the VL comprising the amino acid sequence shown in SEQ ID NO:

107.

58. The isolated antigen-binding protein according to claim 1, comprising a first targeting portion and a second targeting portion, wherein the first targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:31 and SEQ ID NO:33; and the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:97 and SEQ ID NO:

107.

59. The isolated antigen-binding protein according to claim 2, comprising a first targeting portion and a second targeting portion, wherein the first targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:97 and SEQ ID NO:107; and the second targeting portion comprises the amino acid sequence shown in any one of SEQ ID NO:31 and SEQ ID NO:

33.

60. The isolated antigen-binding protein according to any one of claims 1-2, wherein the second polypeptide chain comprises a heavy chain constant region, and the heavy chain constant region comprises a constant region derived from IgG or a constant region derived from IgY; the heavy chain constant region comprises the amino acid sequences shown in SEQ ID NO: 112 to SEQ ID NO:

113.

61. The isolated antigen-binding protein of claim 60, wherein the heavy chain constant region comprises a constant region derived from IgG.

62. The isolated antigen-binding protein of claim 60, wherein the heavy chain constant region comprises a constant region derived from IgG1, IgG2, IgG3 or IgG4.

63. The isolated antigen-binding protein of claim 60, wherein the heavy chain constant region comprises an Fc region derived from IgG1.

64. The isolated antigen-binding protein according to claim 63, wherein the Fc region in the heavy chain constant region contains the N297A mutation, and the residues are numbered according to the Kabat system.

65. The isolated antigen-binding protein according to claim 63, wherein the Fc region in the constant region of the heavy chain contains M252Y, S254T and T256E mutations, the residues being numbered according to the Kabat system.

66. The isolated antigen-binding protein according to any one of claims 1-2, wherein the first polypeptide chain comprises a light chain constant region, and the light chain constant region comprises a constant region derived from Igκ or a constant region derived from Igλ; the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

114.

67. The isolated antigen-binding protein of claim 66, wherein the light chain constant region comprises a constant region derived from human Igκ.

68. The isolated antigen-binding protein of claim 7, wherein the third targeting portion comprises an antibody.

69. The isolated antigen-binding protein according to claim 68, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and dAb.

70. The isolated antigen-binding protein according to claim 69, wherein the third targeting portion is scFv.

71. The isolated antigen-binding protein according to any one of claims 1-2, comprising two first polypeptide chains and two second polypeptide chains, the two first polypeptide chains being linked by disulfide bonds, the two first polypeptide chains and the two second polypeptide chains forming an IgG-like antibody.

72. The isolated antigen-binding protein according to any one of claims 1-2, comprising multispecific antibodies and bispecific antibodies.

73. A polypeptide comprising the isolated antigen-binding protein of any one of claims 1-2.

74. An isolated nucleic acid molecule encoding the isolated antigen-binding protein of any one of claims 1-2, or the polypeptide of claim 73.

75. A vector comprising the isolated nucleic acid molecule of claim 74.

76. A cell comprising the isolated antigen-binding protein of any one of claims 1-2, the polypeptide of claim 73, the isolated nucleic acid molecule of claim 74, and / or the vector of claim 75.

77. A method for preparing the isolated antigen-binding protein of any one of claims 1-2 or the polypeptide of claim 73, the method comprising culturing the cells of claim 76 under conditions in which the isolated antigen-binding protein of any one of claims 1-2 or the polypeptide of claim 73 is expressed.

78. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-2, the polypeptide of claim 73, the isolated nucleic acid molecule of claim 74, the carrier of claim 75, the cell of claim 76, and / or a pharmaceutically acceptable adjuvant and / or excipient.

79. The isolated antigen-binding protein of any one of claims 1-2, the polypeptide of claim 73, the isolated nucleic acid molecule of claim 74, the carrier of claim 75, the cell of claim 76, and / or the pharmaceutical composition of claim 78, for use in the preparation of a medicament for treating solid tumor diseases or conditions.

80. The use according to claim 79, wherein the solid tumor includes breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and / or liver cancer.

81. The use according to claim 80, wherein the lung cancer is non-small cell lung cancer.

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