Bispecific antibody and application thereof

Abstract

The invention provides a bispecific antibody. The bispecific antibody comprises (a) a first antibody specifically binding to a first antigen or an antigen binding fragment thereof; and (b) a second antibody or antigen-binding fragment thereof that specifically binds to a second antigen; wherein the first antigen is a tumor associated antigen (TAA), and the second antigen is a TNFR2 (Tumor Necrosis Factor Receptor 2).

Description

A bispecific antibody and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a bispecific antibody that binds to a tumor-associated antigen (TAA) and TNFR2 and its application. Background Art

[0002] Monoclonal antibodies (mAbs) have been widely used to treat a variety of human diseases, including cancer, autoimmune diseases, infectious diseases, and cardiovascular diseases. Currently, there are more than 80 monoclonal antibodies, including mouse, fully humanized, and chimeric antibodies, which have been approved by the FDA for therapeutic use. Most of these antibodies are monospecific antibodies that recognize a single epitope and can be selected to activate or inhibit the activity of the target molecule through this single epitope. For example, trastuzumab is one of the best-selling anti-cancer protein therapeutics. It blocks the attachment of human epidermal growth factor to Her2 by attaching itself to Her2, thereby blocking the growth of cancer cells. Trastuzumab can also stimulate the body's own immune cells to destroy cancer cells. However, many physiological reactions require the cross-linking or co-engagement of two or more different proteins or protein subunits to be triggered. Taking the activation of heteromeric cell-surface receptor complexes as an example, for these receptor complexes, activation is usually achieved through the interaction of ligands with multiple domains on different proteins, thereby causing the close-related activation of one or two receptor components.

[0003] Bispecific antibodies (BsAbs) are antibody molecules that can simultaneously bind to two (or more) different antigenic epitopes. Compared with traditional monoclonal antibodies, bispecific antibodies have a unique mechanism of action: (1) Bispecific antibodies can simultaneously bind to two or more different antigenic molecules or different epitopes of the same molecule, while combination therapy often does not have this effect. (2) They mediate cell-to-cell interactions. Bispecific antibodies can bind to two antigens on effector cells and target cells respectively, building a bridge between effector cells and target cells, promoting cell-to-cell interactions, such as mediating immune cell killing of tumor cells. Therefore, bispecific antibodies have unique advantages that traditional monoclonal antibodies do not have.

[0004] Tumor necrosis factor receptor 2 (TNFR2), also known as tumor necrosis factor receptor superfamily 1B (TNFRSF1B) and CD120b, is a 75-kDa type I transmembrane protein and a member of the tumor necrosis factor receptor superfamily. It contains an extracellular domain (ECD, residues 1-257) and an intracellular domain (ICD, residues 288-461) with the TRAF2 binding domain. In normal T cells, TNFA-TNFR2 interaction triggers cell survival signals through the NFKB signaling pathway. However, in autoimmune T cells, TNFA-TNFR2 interaction triggers apoptosis signals through the caspase pathway.

[0005] Currently, TNFR2 has been shown to enhance the activation of effector T cells (Teff) and reduce the inhibitory effects mediated by regulatory T cells (Treg). TNFR2 activation induces signaling through the mitogen-activated protein kinase (MAPK) signaling pathway, which coordinates the transcription of genes that promote evasion of apoptosis and cell proliferation mediated by TRAF2 / 3 signaling and NFκB. TNFR2 is expressed not only on cancer cells and tumor-infiltrating Tregs, but also on effector Teff cells. Studies have shown that agonistic antibodies against TNFR2 can stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFNγ and IL2; antagonistic antibodies against TNFR2 can block TNF-TNFR2 and inhibit Treg proliferation.

[0006] The present invention aims to develop a bispecific antibody that can not only specifically recognize tumor-associated antigens, but also use TNFR2 agonistic antibodies to stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFNγ and IL2; or use TNFR2 antagonistic antibodies to block TNF-TNFR2 and inhibit Treg proliferation.

[0007] Summary of the Invention

[0008] In the present application, the inventors have developed a bispecific antibody with good performance that can bind to tumor-associated antigens (TAA) and TNFR2. The bispecific antibody of the present invention can target tumor-associated antigens (TAA) and at the same time can use TNFR2 agonistic antibodies to stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFNγ and IL2; or use TNFR2 antagonistic antibodies to block TNF-TNFR2 and inhibit Treg proliferation.

[0009] The present invention provides a bispecific antibody, comprising: (a) a first antibody or an antigen-binding fragment thereof that specifically binds to a first antigen; and (b) a second antibody or an antigen-binding fragment thereof that specifically binds to a second antigen; wherein the first antigen is a tumor-associated antigen (TAA) and the second antigen is TNFR2.

[0010] In some embodiments, the first antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; and the second antibody or antigen-binding fragment thereof comprises a scFv or a VHH.

[0011] In some embodiments, the heavy chain variable region of one heavy chain of the first antibody forms an antigen binding site with the light chain variable region of one light chain, and the heavy chain variable region of the other heavy chain forms an antigen binding site with the light chain variable region of the other light chain.

[0012] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more scFvs.

[0013] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and a scFv, wherein the scFv is linked to the N-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof.

[0014] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and a scFv, wherein the scFv is linked to the C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof.

[0015] In some embodiments, the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and two of the scFvs.

[0016] In some embodiments, the two scFvs are respectively linked to the N-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.

[0017] In some embodiments, the two scFvs are respectively linked to the C-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.

[0018] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to a tumor-associated antigen (TAA) and two scFvs that specifically bind to TNFR2, wherein the two scFvs that specifically bind to TNFR2 are respectively linked to the N-termini of the two heavy chains of the first antibody.

[0019] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to a tumor-associated antigen (TAA) and two scFvs that specifically bind to TNFR2, wherein the two scFvs that specifically bind to TNFR2 are respectively linked to the C-termini of the two heavy chains of the first antibody.

[0020] In some embodiments, the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, for each of the polypeptide chains: (a) the first polypeptide chains each independently comprise a light chain of the first antibody or antigen-binding fragment thereof; and (b) the second polypeptide chains each independently comprise a heavy chain of the first antibody or antigen-binding fragment thereof and the scFv.

[0021] In some embodiments, the two first polypeptide chains of the bispecific antibody are identical or different, and / or the two second polypeptide chains are identical or different.

[0022] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more of the VHHs.

[0023] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and a VHH, wherein the VHH is linked to the N-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof.

[0024] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and a VHH, wherein the VHH is linked to the C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof.

[0025] In some embodiments, the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and two of the VHHs.

[0026] In some embodiments, the two VHHs are respectively linked to the N-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.

[0027] In some embodiments, the two VHHs are respectively linked to the C-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.

[0028] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to a tumor-associated antigen (TAA) and two VHHs that specifically bind to TNFR2, wherein the two VHHs that specifically bind to TNFR2 are respectively linked to the N-termini of the two heavy chains of the first antibody.

[0029] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to a tumor-associated antigen (TAA) and two VHHs that specifically bind to TNFR2, wherein the two VHHs that specifically bind to TNFR2 are respectively linked to the C-termini of the two heavy chains of the first antibody.

[0030] In some embodiments, the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, for each of the polypeptide chains: (a) the first polypeptide chains each independently comprise the light chain of the first antibody or antigen-binding fragment thereof; and (b) the second polypeptide chains each independently comprise the heavy chain of the first antibody or antigen-binding fragment thereof and the VHH.

[0031] In some embodiments, the two first polypeptide chains of the bispecific antibody are identical or different, and / or the two second polypeptide chains are identical or different.

[0032] In some embodiments, the tumor-associated antigen (TAA) is selected from GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2, AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-ab1, BR CA1, BORIS, CA9, CA125, carbonic anhydrase IX, caspase-8, CALR, CCR5, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, cyclin-B1, CYP1B1, EGF R, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IL13Rα2, LMP2, κ-Light, LeY, MAGE-1, MAGE-2, MAGE- 3, MAGE-4, MAGE-6, MAGE-12, MART-1, mesothelin, ML-IAP, MOv-γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16, MUM1, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Tang-Nordstein antigen, TRP-1, TRP-2, tyrosinase and urolysin-3, 5T4. Preferably, the tumor-associated antigen (TAA) is selected from B7H3, CD24 or Claudin18.2.

[0033] In some embodiments, the heavy chain variable region and the light chain variable region of the scFv are connected by a linker L1.

[0034] In some embodiments, the scFv is linked to the N-terminus or C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof via a linker L2.

[0035] In some embodiments, the VHH is connected to the N-terminus or C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof via a linker L2.

[0036] In some embodiments, the linker L1 and linker L2 are the same or different. In some embodiments, the linker L1 and / or linker L2 have a sequence such as (G4S) x The amino acid sequence shown, x is an integer selected from 1-6; preferably, the linker L1 and / or linker L2 is (G4S)2, (G4S)3 or (G4S)4.

[0037] In some embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region; preferably, the first antibody or antigen-binding fragment thereof is a full-length antibody.

[0038] In some embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof comprises a first Fc region and a second Fc region. In some embodiments, the first Fc region and the second Fc region are the same or different. In some embodiments, the first Fc region or the second Fc region is selected from IgG, IgA, IgD, IgE, IgM or a variant thereof. In some embodiments, the first Fc region or the second Fc region is selected from IgG1, IgG2, IgG3, IgG4 or a variant thereof. In some embodiments, the first Fc region or the second Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

[0039] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to B7H3, wherein HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:2, or a sequence at least 80% identical to SEQ ID NO:2; HCDR2 is as shown in SEQ ID NO:3, or a sequence at least 80% identical to SEQ ID NO:3; HCDR3 is as shown in SEQ ID NO:4, or a sequence at least 80% identical to SEQ ID NO:4; LCDR1 is as shown in SEQ ID NO:6, or a sequence at least 80% identical to SEQ ID NO:6; LCDR2 is as shown in SEQ ID NO:7, or a sequence at least 80% identical to SEQ ID NO:7; and LCDR3 is as shown in SEQ ID NO:8, or a sequence at least 80% identical to SEQ ID NO:8.

[0040] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to CD24, wherein HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:10, or a sequence at least 80% identical to SEQ ID NO:10; HCDR2 is as shown in SEQ ID NO:11, or a sequence at least 80% identical to SEQ ID NO:11; HCDR3 is as shown in SEQ ID NO:12, or a sequence at least 80% identical to SEQ ID NO:12; LCDR1 is as shown in SEQ ID NO:14, or a sequence at least 80% identical to SEQ ID NO:14; LCDR2 is as shown in SEQ ID NO:15, or a sequence at least 80% identical to SEQ ID NO:15; and LCDR3 is as shown in SEQ ID NO:16, or a sequence at least 80% identical to SEQ ID NO:16.

[0041] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:18, or a sequence having at least 80% identity with SEQ ID NO:18; HCDR2 is as shown in SEQ ID NO:19, or a sequence having at least 80% identity with SEQ ID NO:19; HCDR3 is as shown in SEQ ID NO:20, or a sequence having at least 80% identity with SEQ ID NO:20; LCDR1 is as shown in SEQ ID NO:22, or a sequence having at least 80% identity with SEQ ID NO:22; LCDR2 is as shown in SEQ ID NO:23, or a sequence having at least 80% identity with SEQ ID NO:23; LCDR3 is as shown in SEQ ID NO:24, or a sequence having at least 80% identity with SEQ ID NO:24.

[0042] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:26, or a sequence having at least 80% identity with SEQ ID NO:26; HCDR2 is as shown in SEQ ID NO:27, or a sequence having at least 80% identity with SEQ ID NO:27; HCDR3 is as shown in SEQ ID NO:28, or a sequence having at least 80% identity with SEQ ID NO:28; LCDR1 is as shown in SEQ ID NO:30, or a sequence having at least 80% identity with SEQ ID NO:30; LCDR2 is as shown in SEQ ID NO:31, or a sequence having at least 80% identity with SEQ ID NO:31; LCDR3 is as shown in SEQ ID NO:32, or a sequence having at least 80% identity with SEQ ID NO:32.

[0043] In some embodiments, the scFv specifically binds TNFR2, and the scFv has a HCDR1 as set forth in SEQ ID NO:34, or a sequence at least 80% identical to SEQ ID NO:34; a HCDR2 as set forth in SEQ ID NO:35, or a sequence at least 80% identical to SEQ ID NO:35; a HCDR3 as set forth in SEQ ID NO:36, or a sequence at least 80% identical to SEQ ID NO:36; and a LCDR1 as set forth in SEQ ID NO:38, or a sequence at least 80% identical to SEQ ID NO:38; a LCDR2 as set forth in SEQ ID NO:39, or a sequence at least 80% identical to SEQ ID NO:39; and a LCDR3 as set forth in SEQ ID NO:40, or a sequence at least 80% identical to SEQ ID NO:40.

[0044] In some embodiments, the VHH specifically binds to TNFR2, and the HCDR1 of the VHH is set forth in SEQ ID NO:42, or a sequence at least 80% identical to SEQ ID NO:42; the HCDR2 is set forth in SEQ ID NO:43, or a sequence at least 80% identical to SEQ ID NO:43; and the HCDR3 is set forth in SEQ ID NO:44, or a sequence at least 80% identical to SEQ ID NO:44.

[0045] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to B7H3, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 1, or a sequence having at least 80% identity with SEQ ID NO: 1; the light chain variable region VL is as shown in SEQ ID NO: 5, or a sequence having at least 80% identity with SEQ ID NO: 5.

[0046] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to CD24, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:9, or a sequence having at least 80% identity with SEQ ID NO:9; and the light chain variable region VL is as shown in SEQ ID NO:13, or a sequence having at least 80% identity with SEQ ID NO:13.

[0047] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 17, or a sequence having at least 80% identity with SEQ ID NO: 17; the light chain variable region VL is as shown in SEQ ID NO: 21, or a sequence having at least 80% identity with SEQ ID NO: 21.

[0048] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:25, or a sequence having at least 80% identity with SEQ ID NO:25; the light chain variable region VL is as shown in SEQ ID NO:29, or a sequence having at least 80% identity with SEQ ID NO:29.

[0049] In some embodiments, the scFv specifically binds to TNFR2, and the heavy chain variable region VH of the scFv is shown in SEQ ID NO: 33, or a sequence with at least 80% identity to SEQ ID NO: 33; the light chain variable region VL is shown in SEQ ID NO: 37, or a sequence with at least 80% identity to SEQ ID NO: 37.

[0050] In some embodiments, the VHH specifically binds to TNFR2, and the VHH heavy chain variable region VH is as shown in SEQ ID NO:41, or a sequence that is at least 80% identical to SEQ ID NO:41.

[0051] In some embodiments, the first polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 46-50, 59-63, 71-76, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 46-50, 59-63, 71-76; the second polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 51-58, 64-70, 77-82, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 51-58, 64-70, 77-82.

[0052] In some embodiments, the bispecific antibody comprises:

[0053] (1) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:51; or

[0054] (2) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:56; or

[0055] (3) the first polypeptide chain shown in SEQ ID NO:46 and the second polypeptide chain shown in SEQ ID NO:53; or

[0056] (4) the first polypeptide chain shown in SEQ ID NO:46 and the second polypeptide chain shown in SEQ ID NO:54; or

[0057] (5) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:57; or

[0058] (6) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:55; or

[0059] (7) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:58; or

[0060] (8) the first polypeptide chain shown in SEQ ID NO:47, the second polypeptide chain shown in SEQ ID NO:52; or

[0061] (9) the first polypeptide chain shown in SEQ ID NO: 50, the second polypeptide chain shown in SEQ ID NO: 52; or

[0062] (10) the first polypeptide chain shown in SEQ ID NO: 48, the second polypeptide chain shown in SEQ ID NO: 52; or

[0063] (11) the first polypeptide chain shown in SEQ ID NO: 49 and the second polypeptide chain shown in SEQ ID NO: 52; or

[0064] (12) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 64; or

[0065] (13) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 69; or

[0066] (14) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 66; or

[0067] (15) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 67; or

[0068] (16) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 68; or

[0069] (17) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 70; or

[0070] (18) the first polypeptide chain as shown in SEQ ID NO: 60, the second polypeptide chain as shown in SEQ ID NO: 65; or

[0071] (19) the first polypeptide chain as shown in SEQ ID NO: 61, the second polypeptide chain as shown in SEQ ID NO: 65; or

[0072] (20) the first polypeptide chain as shown in SEQ ID NO: 62, the second polypeptide chain as shown in SEQ ID NO: 65; or

[0073] (21) the first polypeptide chain as shown in SEQ ID NO: 63, the second polypeptide chain as shown in SEQ ID NO: 65; or

[0074] (22) the first polypeptide chain as shown in SEQ ID NO: 71, the second polypeptide chain as shown in SEQ ID NO: 77; or

[0075] (23) the first polypeptide chain as shown in SEQ ID NO: 72, the second polypeptide chain as shown in SEQ ID NO: 78; or

[0076] (24) the first polypeptide chain as shown in SEQ ID NO: 71, the second polypeptide chain as shown in SEQ ID NO: 81; or

[0077] (25) the first polypeptide chain as shown in SEQ ID NO: 72, the second polypeptide chain as shown in SEQ ID NO: 82; or

[0078] (26) the first polypeptide chain as shown in SEQ ID NO: 73, the second polypeptide chain as shown in SEQ ID NO: 79; or

[0079] (27) the first polypeptide chain as shown in SEQ ID NO: 74, the second polypeptide chain as shown in SEQ ID NO: 80; or

[0080] (28) a first polypeptide chain as shown in SEQ ID NO: 75, a second polypeptide chain as shown in SEQ ID NO: 79; or

[0081] (29) The first polypeptide chain shown in SEQ ID NO:76, and the second polypeptide chain shown in SEQ ID NO:80.

[0082] The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above-described bispecific antibodies. Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of any of the above-described bispecific antibodies. Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the second polypeptide chain of any of the above-described bispecific antibodies.

[0083] The present invention provides a multifunctional fusion protein comprising any of the above-mentioned bispecific antibodies.

[0084] In some embodiments, the multifunctional fusion protein further comprises one or more third antibodies or antigen binding portions thereof that specifically bind to other antigens. In some embodiments, the antigen that binds to the third antibody or its antigen binding portion is selected from tumor-associated antigens (TAA) or immune checkpoints. In some embodiments, the antigen that binds to the third antibody or its antigen binding portion is selected from GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2, AFP, ALK, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin (β-catenin), brc-ab1, BRCA1, BORIS, CA9, CA12 5. Carbonic anhydrase IX, caspase-8, CALR, CCR5, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV 6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTE RT, IL13Rα2, LMP2, κ-Light, LeY, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-12, MART-1, mesothelin, ML-IAP, MOv-γ, Muc1, Muc 2, Muc3, Muc4, Muc5, Muc16, MUM1, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Tang-Nordstein antigen, TRP-1, TRP-2, tyrosinase and urokinin-3, 5T4, PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, ILT4, TREM2, LAG3, CD27, or B7H4.

[0085] In some embodiments, the multifunctional fusion protein further comprises a cytokine. In some embodiments, the cytokine is selected from IL-1, IL-2, IL-2Rα, IL-2Rβ, IL-3, IL-3Rα, IL-4, IL-4Rα, IL-5, IL-5Rα, IL-6, IL-6Rα, IL-7, IL-7Rα, IL-8, IL-9, IL-9Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11Rα, IL-12, IL-12Rα, IL-12Rβ2 , IL-12Rβ1, IL-13, IL-13Rα, IL-13Rα2, IL-14, IL-15, IL-15Rαsushi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21Rα, IL-22, IL-23, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFNγ, IFNα, or GM-CSF.

[0086] The present invention also provides the use of the bispecific antibody described in any one of the above or the multifunctional fusion protein described in any one of the above in the preparation of a drug for treating cancer. In some embodiments, the cancer is selected from human brain astroglioblastoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor, Kaposi sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer.

[0087] The present invention also provides use of any of the above-described bispecific antibodies or multifunctional fusion proteins in the preparation of a medicament for treating an autoimmune disease. In some embodiments, the autoimmune disease is selected from graft-versus-host disease, rheumatoid arthritis, Crohn's disease, multiple sclerosis, colitis, psoriasis, autoimmune uveitis, pemphigus, epidermolysis bullosa, or type I diabetes.

[0088] In some embodiments, the use is achieved by one or more of tumor immunotherapy, cell therapy, or gene therapy.

[0089] The present invention also provides a pharmaceutical composition comprising any of the above-described bispecific antibodies and a pharmaceutically acceptable carrier, diluent or excipient.

[0090] The present invention also provides a pharmaceutical composition comprising any of the multifunctional fusion proteins described above and a pharmaceutically acceptable carrier, diluent or excipient.

[0091] The present invention also provides an antibody-drug conjugate comprising any of the above-mentioned bispecific antibodies.

[0092] In some embodiments, the conjugated drug is selected from a cytotoxin, a small molecule chemical drug, or an immunotoxin.

[0093] Abbreviations and Definitions

[0094] The following abbreviations are used herein: VH: antibody heavy chain variable region; VL: antibody light chain variable region; CDR: complementarity determining region in an immunoglobulin variable region; FR: antibody framework region, i.e., amino acid residues in an antibody variable region other than CDR residues; IgG: immunoglobulin G.

[0095] The term "antibody" refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be reconstructed and separated from natural resources such as plasma or serum in which the antibody is naturally present, or from the culture supernatant of hybridoma cells that produce the antibody, in animal immune serum, or by phage library screening. Alternatively, they can be synthesized partially or completely using techniques such as genetic recombination. Preferred antibodies include, for example, antibodies of the isotype of an immunoglobulin or subclasses of these isotypes. Known human immunoglobulins include 9 categories (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Among these isotypes, the antibodies of the present invention can include IgG1, IgG2, IgG3, and / or IgG4.

[0096] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell culture system.

[0097] As used herein, some antibodies are immunoglobulin molecules consisting of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating 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 (C1q) of the classical complement system. The VH and VL regions can also be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site.

[0098] The term "bispecific antibody" refers to a protein molecule that can specifically bind to two target antigens or target antigen epitopes. In the present invention, "bispecific antigen-binding protein" comprising an antibody or antigen-binding fragment (e.g., Fab, scFv, etc.) is used interchangeably with "bispecific antibody" and "bispecific antibody".

[0099] The term "antigen-binding fragment" of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical fragmentation of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (e.g., technology from Ablynx), domain antibodies (e.g., technology from Domantis), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0100] The term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or antigen-binding fragment thereof) linked to one or more conjugated drugs (which may optionally be therapeutic agents or cytotoxic agents). Its structure generally consists of three parts: an antibody or antibody-like ligand, a drug moiety, and a linker that couples the antibody or antibody-like ligand and the drug. ADCs typically have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs conjugated to the antibody.

[0101] The term "polypeptide" refers to an amino acid chain of any length, regardless of modifications such as phosphorylation or glycosylation. The term polypeptide includes proteins and fragments thereof. Polypeptides can be "exogenous," meaning they are "heterologous," i.e., foreign to the host cell being utilized, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written from left to right in the direction of amino terminus to carboxyl terminus. Amino acid residue sequences are designated by three-letter or one-letter codes according to standard nomenclature.

[0102] The term "scFv" refers to a molecule comprising an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated GGGGS amino acid sequences or variants thereof, for example, using 1-6 repeated GGGGS amino acid sequences or variants thereof.

[0103] The term "VHH" refers to a single antigen-binding polypeptide comprising only one heavy chain variable region (VHH), which is derived from the variable domain of a heavy chain molecule that is naturally free of light chains, as distinguished from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in species of the Camelidae family, such as camels, llamas, llamas, dromedaries, alpacas, and guanacos. Other species outside the Camelidae family can produce heavy chain molecules that naturally lack light chains, and such VHHs are within the scope of this application. The term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby expresses a selected gene of interest. The term includes the offspring of a parent cell, regardless of whether the offspring is identical to the original parent cell in morphology or genetic composition, as long as the offspring has the selected gene of interest. Commonly used host cells include bacteria, yeast, mammalian cells, etc.

[0104] The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked and are referred to herein as "expression vectors."

[0105] The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, and emulsions, as well as various types of wetting agents and the like.

[0106] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Comparisons for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST software or the FASTA program package.

[0107] The term "at least 80% identity" means that the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence is greater than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0108] The term "specific" means that one of the molecules involved in specific binding does not show any significant binding to molecules other than one or more of the binding partner molecules. In addition, this term is also used when the domain containing the antibody variable region has specificity for a particular epitope among multiple epitopes in an antigen. When the epitope bound by the domain containing the antibody variable region is contained in several different antigens, the antigen-binding molecule containing the domain containing the antibody variable region can bind to various antigens containing the epitope.

[0109] The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed in whole or as a fragment on the surface of cancerous cells and which can be used to preferentially target pharmacological agents to cancerous cells.

[0110] The term "epitope" refers to an antigenic determinant in an antigen and refers to the antigenic site bound by the domains of the antigen-binding molecules comprising the antibody variable regions disclosed herein. Therefore, an epitope can be defined based on its structure. Alternatively, an epitope can be defined based on the antigen-binding activity of an antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that form the epitope; when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.

[0111] The term "positive control" refers to a natural or engineered cell or antibody that can bind to or express a target protein. The positive control referred to herein refers to a single-target positive control.

[0112] The term "negative control" refers to the use of the same species, subtype, dose, immunoglobulin and subtype of immunoglobulin, and the same marker as the experimental sample in the same experiment to eliminate the experimental background effect of non-specific binding samples on the experimental values, and serve as a control to better illustrate the experimental effect.

[0113] The terms "Treg", "Treg cells" or "regulatory T cells" are sometimes referred to as suppressor T cells, characterized by the expression of the biomarkers CD4, FOXP3 and CD25, representing a T cell subset that regulates the immune system, maintains tolerance to self-antigens and prevents autoimmune diseases. Tregs are immunosuppressive and typically inhibit or downregulate the induction and proliferation of effector T (Teff) cells. Tregs can develop in the thymus (so-called CD4+Foxp3+ "natural" Tregs) or differentiate from naive CD4+ T cells in the periphery, for example, after exposure to TGFβ or retinoic acid.

[0114] The terms "Teff," "Teff cells," or "effector T cells" refer to cells formed by proliferation and differentiation after T cells are stimulated by antigens. Effector T cells release lymphokines, and during this process, a small percentage of T cells become memory T cells. When effector T cells come into contact with target cells, they trigger granule exocytosis. The released perforins polymerize to form pores on the target cell surface, mediating the killing effect. The target cell death process is similar to apoptosis. Effector T cells also release immunoreactive substances—lymphokines, such as interleukins and interferons.

[0115] There are a variety of methods / systems in the art to define and describe CDRs, and these systems and / or definitions have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on sequence variability based on the position of the structural loop region; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on the AbM antibody modeling software of Oxford Molecular Company and is a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in many aspects. Unless otherwise specified, this article uses Kabat to define CDRs. Special note, some CDRs in this application are divided using the definition standard, such as the HCDR1 shown in SEQ ID NO: 2, 10, 18, 34, and 42, which starts at the fourth position after the first cysteine ​​in the heavy chain variable region. The length of HCDR1 is generally 10-12, ending at the amino acid before tryptophan. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] FIG1 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the C-termini of the two heavy chains of the full-length antibody via a linker.

[0117] FIG2 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the N-termini of the two heavy chains of the full-length antibody via a linker.

[0118] FIG3 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the C-termini of the two heavy chains of the full-length antibody via a linker.

[0119] FIG4 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the N-termini of the two heavy chains of the full-length antibody via a linker.

[0120] FIG5 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the C-termini of two light chains of the full-length antibody via a linker.

[0121] FIG6 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the N-termini of two light chains of the full-length antibody via a linker.

[0122] FIG7 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the C-termini of the two light chains of the full-length antibody via a linker.

[0123] FIG8 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the N-termini of the two light chains of the full-length antibody via a linker.

[0124] FIG9 shows the binding activity of antibodies TB-A1, TB-E, TB-B, and TB-F to TNFR2 protein.

[0125] FIG10 shows the binding activity of antibodies TB-G, TB-D1, and TB-H to TNFR2 protein.

[0126] FIG11 shows the binding activity of antibody TB-A2 to TNFR2 protein.

[0127] FIG12 shows the binding activity of antibodies TB-C1, TB-C2, and TB-D2 to TNFR2 protein.

[0128] FIG13 shows the binding activity of antibodies TB-A1, TB-E, TB-B, and TB-F to B7H3 protein.

[0129] FIG14 shows the binding activity of antibodies TB-G, TB-D1, and TB-H to B7H3 protein.

[0130] FIG15 shows the binding activity of antibodies TB-A2 and TB-C1 to B7H3 protein.

[0131] FIG16 shows the binding activity of antibodies TB-C2 and TB-D2 to B7H3 protein.

[0132] FIG17 shows the dual-terminal binding activities of antibodies TB-A1, TB-E, TB-B, and TB-F.

[0133] FIG18 shows the dual-terminal binding activities of antibodies TB-G, TB-D1, and TB-H.

[0134] FIG19 shows the dual-terminal binding activity of antibody TB-C1.

[0135] FIG20 shows the dual-terminal binding activities of antibodies TB-A1, TB-D1, and TB-C1.

[0136] FIG21 shows the anti-tumor growth curves of antibodies TB-C1 and TB-C2.

[0137] FIG22 shows the binding activity of antibodies TD-A1, TD-E, TD-B, and TD-F to TNFR2 protein.

[0138] FIG23 shows the binding activity of antibodies TD-C, TD-G, TD-D1, and TD-H to TNFR2 protein.

[0139] FIG24 shows the binding activity of antibodies TD-A1 and TD-A2 to TNFR2 protein.

[0140] FIG25 shows the binding activity of antibodies TD-D1 and TD-D2 to TNFR2 protein.

[0141] FIG26 shows the ADCC activities of antibodies TD-A1 and TD-D1. DETAILED DESCRIPTION

[0142] The present invention will be further described below with reference to the accompanying drawings and specific examples, and the protection content of the present invention is not limited to the following examples. It should also be understood that the terms used in the examples of the present invention are for the purpose of describing specific embodiments, rather than for the purpose of limiting the scope of protection of the present invention. Without departing from the spirit and scope of the inventive concept, variations and advantages that can be imagined by those skilled in the art are all included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0143] Example 1 Preparation of bispecific antibodies with different structures

[0144] Targeting TNFR2 and B7H3, bispecific antibodies were constructed based on the eight structures in Figures 1-8, named TB-A to TB-H, respectively. Targeting TNFR2 and CD24, bispecific antibodies were constructed based on the eight structures in Figures 1-8, named TD-A to TD-H, respectively. Targeting TNFR2 and Claudin18.2, bispecific antibodies were constructed based on the four structures in Figures 1, 2, 5, and 6, named TC-A, TC-B, TC-E, and TC-F, respectively. Some of the bispecific antibodies in this patent have both IgG1 and IgG4 subtypes. Antibodies with a "1" after their names are IgG1 subtypes, and antibodies with a "2" after their names are IgG4 subtypes. For example, antibody TB-A1 is an IgG1 subtype antibody targeting TNFR2 and B7H3, constructed based on the structure in Figure 1. Antibody TB-A2 is an IgG4 subtype antibody targeting TNFR2 and B7H3, constructed based on the structure in Figure 1.

[0145] The sequences of the anti-B7H3 antibody, anti-CD24 antibody, and anti-Claudin18.2 antibody as full-length antibodies, and the sequences of the TNFR2 antibody as scFv and VHH are shown in Table 1.

[0146] Table 1 Sequence Listing

[0147] The sequences of the TNFR2 antibody and the B7H3 antibody, CD24 antibody, and Claudin18.2 antibody are connected in a certain order through a linker to form the peptide chains shown in Tables 2-4. In this embodiment, the linker used in the bispecific antibody has two GGGGS repeats (i.e., GGGGSGGGGS, hereinafter abbreviated as (G4S)2) or three GGGGS repeats (i.e., GGGGSGGGGSGGGGS, hereinafter abbreviated as (G4S)3). The CL used in this embodiment is of the kappa (κ) type, and its sequence is shown in SEQ ID NO:45.

[0148] Table 2 Bispecific antibody (TNFR2+B7H3) peptide chain sequence list

[0149] Table 3 Bispecific antibody (TNFR2 + CD24) peptide chain sequence list

[0150] Table 4 Bispecific antibody (TNFR2 + Claudin18.2) peptide chain sequence list

[0151] Based on the peptide chain combinations in Tables 2-4, the amino acid sequences of the bispecific antibodies shown in Table 5 were designed.

[0152] Table 5 Bispecific antibody sequences

[0153] Each chain of the bispecific antibody with different structures designed above was genetically synthesized. Using molecular cloning techniques, the antibody fragments were inserted into the PCDNA3.1 vector to construct a mammalian cell expression plasmid. This was then transfected into the host cell line, CHO cells, using liposome transfection. Fermentation supernatant was obtained using a fed-batch assay. The supernatant was then purified through a series of steps, including affinity chromatography and ion exchange chromatography, to ultimately obtain the constructed antibody. The purified antibody was tested for expression level, purity, and SDS-PAGE analysis to confirm the bispecificity.

[0154] Example 2 ELISA detection of antibody binding activity to TNFR2 protein (TNFR2+B7H3)

[0155] Dilute human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) to 0.3 μg / mL in coating buffer (1× PBS, pH 7.4) and coat in a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating buffer and wash the plate with 1× PBST (300 μL / well), wash four times using a plate washer, and pat dry on paper. Block with 3% skim milk powder (300 μL / well), incubate at 37°C for 1 hour, discard the blocking buffer, wash four times using a plate washer, and pat dry on paper. Negative and positive controls were set up. The negative control was commercially available trastuzumab for injection (Herceptin). The variable region of the antibody sequence in positive control 1 consisted of SEQ ID NO:83 and SEQ ID NO:84, with the human IgG1 constant region added (see SEQ ID NO:85 and SEQ ID NO:86); the variable region of the antibody sequence in positive control 2 consisted of SEQ ID NO:41. The positive control and antibody were diluted to 10 μg / mL with 3% nonfat dry milk. From this initial concentration, three-fold dilutions were performed, totaling 11 dilutions. A blank well was set up, with only the diluent added. 100 μL / well was added, and the cells were incubated at 37°C for 1 hour. The liquid in the wells was discarded, and the plates were washed four times with a plate washer and patted dry on paper. A goat anti-human IgG Fc was diluted 1:20,000 with 3% nonfat dry milk, 100 μL / well was added, and the cells were incubated at 37°C for 1 hour. The plates were washed six times with a plate washer and patted dry on paper. Add 100 μL / well of TMB colorimetric solution, wrap with aluminum foil, and incubate at 37°C in the dark for 8 min. Terminate the colorimetric reaction by adding 100 μL / well of 1M HCl stop solution. Read the plate at 450 nm on a microplate reader. The ELISA results for the antibodies are shown in Figures 9-12, respectively. These results demonstrate that antibodies TB-A1 to TB-H bind to human TNFR2 protein.

[0156] Example 3 ELISA detection of antibody binding activity to B7H3 protein (TNFR2+B7H3)

[0157] Human-B7H3His (manufacturer: Acro, CAT: TB73-H52E2, LOT: 2052-21AXF1-YS) was diluted to 0.3 μg / mL with coating solution (1×PBS, pH 7.4) and coated into a 96-well ELISA plate at 100 μL / well at 4°C overnight. Discard the coating solution, wash the plate with 1×PBST (300 μL per well), wash four times with a plate washer, and pat dry on a paper plate. Block with 3% skim milk powder (300 μL / well), incubate at 37°C for 1 hour, discard the blocking solution, wash four times with a plate washer, and pat dry on a paper plate. Negative and positive controls were set up. The negative control was commercially available trastuzumab for injection (Herceptin). The variable region of the positive control antibody sequence consisted of SEQ ID NO: 1 and SEQ ID NO: 5, with the constant region of human IgG1 added (see SEQ ID NO: 85 and SEQ ID NO: 86). Dilute the positive control and test sample to 10 μg / mL with 3% skim milk powder. Perform three-fold dilutions from this starting concentration for a total of 11 dilutions. Set up a blank well with only the diluent. Add 100 μL / well (100 μL) and incubate at 37°C for 1 hour. Discard any liquid from the wells, wash the plate four times with a plate washer, and pat dry on paper. Dilute goat anti-human IgG Fc 1:20,000 with 3% skim milk powder, add 100 μL / well (100 μL) and incubate at 37°C for 1 hour. Wash the plate six times with a plate washer, and pat dry on paper. Add 100 μL / well (100 μL) of TMB colorimetric solution, wrap the plate with aluminum foil, and develop at 37°C in the dark for 8 minutes. Stop the color reaction with 100 μL / well of 1M HCl. Read on a microplate reader at 450 nm. The ELISA results of the antibody molecules are shown in Figures 13-16, respectively. The results show that antibodies TB-A1 to TB-H can bind to human B7H3 protein.

[0158] Example 4 Construction of Antibody Dual-Target Binding Activity (TNFR2+B7H3)

[0159] Human-B7H3Fc (Manufacturer: Acro, CAT: B73-H5253, LOT: 2053b-201XF1-UD) was diluted to 0.5 μg / mL in coating solution (1× PBS, pH 7.4) and coated into a 96-well microtiter plate at 100 μL / well, incubating overnight at 4°C. Discard the coating solution and wash the plate with 1× PBST (300 μL / well) for 1 hour at 37°C. Discard the blocking solution, wash the plate four times with a plate washer, and pat dry on a tissue paper. Block with 3% skim milk powder (300 μL / well) and incubate at 37°C for 1 hour. Discard the blocking solution, wash the plate four times with a plate washer, and pat dry on a tissue paper. A negative control was established using commercially available trastuzumab for injection (Herceptin). Starting at 300 nM, a three-fold dilution series was performed, with a total of eight dilutions. A blank well was also prepared, with only the diluent added. Incubate at 37°C for 1 hour at 100 μL / well. Discard any liquid from the wells, wash the plate four times with a plate washer, and pat dry on paper. Dilute human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) to 0.5 μg / mL and add 100 μL per well. Incubate at room temperature for 1 hour. Wash the plate three times with PBST. Then, dilute HRP-conjugated his antibody 1:10,000 in sample diluent and add 100 μL per well. Incubate at room temperature for 1 hour. Wash the plate six times with a plate washer and pat dry on paper. Add 100 μL / well of TMB colorimetric solution, wrap with aluminum foil, and develop at 37°C in the dark for 8 minutes. Terminate the color reaction by adding 100 μL / well of 1M HCl stop solution. Read at 450 nm on a microplate reader. The ELISA results of the antibody molecules are shown in Figures 17 to 19, respectively. The results show that antibodies TB-A1 to TB-H all have binding activity at both ends.

[0160] Example 5 Construction of Antibody Dual-Target Binding Activity (TNFR2+B7H3)

[0161] Dilute human-TNFR2mFc (Manufacturer: Kaixia, CAT: TNF-HM3R2, LOT: 031202) to 0.5 μg / mL in coating buffer (1× PBS, pH 7.4) and coat into a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating buffer and wash the plate with 300 μL / well of 1× PBST. Wash four times with a plate washer and pat dry on paper. Block with 3% skim milk powder at 300 μL / well and incubate at 37°C for 1 hour. Discard the blocking buffer, wash four times with a plate washer, and pat dry on paper. Starting at 100 nM, perform a three-fold dilution series for a total of 11 dilutions. Set up a blank well with only the diluent. Incubate at 37°C for 1 hour at 100 μL / well. Discard the liquid in the wells, wash four times with a plate washer, and pat dry on paper. Human-B7H3His (Manufacturer: Acro, CAT: TB73-H52E2, LOT: 2052-21AXF1-YS) protein was diluted to 0.5 μg / mL and 100 μL was added to each well. Incubate at room temperature for 1 hour, then wash the plate three times with PBST. HRP-labeled His antibody was then diluted 1:10,000 in sample diluent and 100 μL was added to each well. Incubate at room temperature for 1 hour. Wash the plate six times in a plate washer and pat dry on paper. TMB colorimetric solution was added at 100 μL / well, wrapped with aluminum foil, and allowed to develop at 37°C in the dark for 8 minutes. The color reaction was terminated by adding 100 μL / well of 1M HCl stop solution. The plate was read at 450 nm on a microplate reader. The ELISA results for the antibody molecules are shown in Figure 20. The results show that antibodies TB-A1, TB-D1, and TB-C1 all have binding activity at both ends.

[0162] Example 6 CD8+ T cell activation experiment (TNFR2+B7H3)

[0163] The antibody was diluted to 70nM with PBS (final experimental concentration 35nM, 50μL / well); OKT3 was diluted to 10μg / mL with PBS (final experimental concentration 5μg / mL, 50μL / well). 50μLOKT3 diluent and 50μL antibody diluent were set up with irrelevant antibodies, which were human IgG1kappa Isotype control (HG1K, Sino Biological). A negative control group was set up and coated into a 96-well flat-bottom plate with a total volume of 100μL / well and incubated overnight at 4°C. The next day, CD8+T cells were negatively isolated from frozen PBMCs (ORiCELLS, ID: PCH20210300033, with a separation efficiency of 30%), and the sorted CD8+T cells were tested for purity; CFSE was used to label CD8+T cells at a concentration of 5μM, incubated at 37°C for 10min, and complete culture medium was added to terminate staining. The plate used for coating was washed once with PBS and 2×10 5CFSE-labeled or unlabeled T cells were added at 100 μL / well. Anti-CD28 was added at a final concentration of 1 μg / mL at 100 μL / well. The total experimental volume was 200 μL / well. The cells were incubated at 37°C for 96 hours. After centrifugation, the supernatant was collected for detection of IFNγ and IL-2 cytokines. The cells were washed with PBS and analyzed by flow cytometry. The results are shown in Table 6. The agonistic antibodies TB-A1, TB-B, TB-E, and TB-F all activated CD8+ T cells.

[0164] Table 6 Results of CD8+ T cell activation experiment

[0165] Example 7 BLI method to detect the affinity of antibodies to TNFR2 protein (TNFR2+B7H3)

[0166] Before use, the AHC sensor was soaked in 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) for 600 seconds to remove any sucrose coating on the sensor surface. The AHC sensor was equilibrated in 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) for 60 seconds, followed by 300 seconds of immobilization of the antibody in the sample plate, and then a second equilibration buffer for 180 seconds. 100 nM human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) protein was allowed to bind to the antibody for 300 seconds, followed by 600 seconds of dissociation. Following dissociation, the sensor was regenerated in 10 mM glycine (pH 2.0) for 30 seconds. The sensor was then regenerated with 10 mM glycine (pH 2.0). The affinity results of the antibody molecules are shown in Table 7. The results show that the affinities of the TNFR2 end of antibodies TB-A1, TB-C1 and TB-D1 are all at the nanomolar or sub-nanomolar level.

[0167] Table 7 Affinity of antibodies to TNFR2 protein

[0168] Example 8 BLI method to detect the affinity of antibodies to B7H3 protein (TNFR2+B7H3)

[0169] Before use, the AHC sensor was soaked in 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) for 600 seconds to remove any sucrose coating on the sensor surface. The AHC sensor was equilibrated in 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) for 60 seconds, followed by 300 seconds of immobilization of the antibody in the sample plate, and then a second equilibration buffer for 180 seconds. 100 nM human-B7H3His (Manufacturer: Acro, CAT: TB73-H52E2, LOT: 2052-21AXF1-YS) protein was allowed to bind to the antibody for 300 seconds, followed by 600 seconds of dissociation. After dissociation, the sensor was regenerated in 10 mM glycine (pH 2.0) for 30 seconds. The sensor was then regenerated with 10 mM glycine (pH 2.0). The affinity results of the antibody molecules are shown in Table 8, which show that antibodies TB-A1, TB-C1 and TB-D1 all have affinity for the B7H3 protein.

[0170] Table 8 Affinity of antibodies to B7H3 protein

[0171] Example 9 Pharmacodynamic Study of Candidate Test Substances in a Tumor Model of MC38-hB7H3 Subcutaneously Transplanted Mouse Colon Cancer Cell Line in hTNFR2 Mice (TNFR2+B7H3)

[0172] Female C57BL / 6-hTNFR2 mice aged 6-7 weeks were subcutaneously inoculated with MC38-hB7H3 tumors. 3 The mice were randomly divided into four groups. Each group consisted of 5 mice, including: (1) G1: PBS group; (2) G2: control antibody group, the variable region of which consisted of SEQ ID NO:83 and SEQ ID NO:84, with the constant region of human IgG1 added (see SEQ ID NO:85 and SEQ ID NO:86); (3) G3: antibody TB-C1 group; (4) G4: antibody TB-C2 group. The negative control group was intratumorally administered with PBS, and the samples of the other groups were intratumorally administered at 10 mg / kg. The frequency of administration was twice a week for 4 consecutive weeks, for a total of 8 times; the tumor volume and body weight of the mice were measured every other day, and the tumor volume was calculated according to a·b 2 / 2 (a is the long diameter, b is the short diameter). The experimental design is shown in Table 9.

[0173] Table 9 Experimental design

[0174] The results are shown in FIG21 . From the tumor growth curves, it can be seen that antibodies TB-C1 and TB-C2 have significant anti-tumor effects.

[0175] Example 10 ELISA detection of antibody binding activity to TNFR2 protein (TNFR2+CD24)

[0176] Dilute human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) to 0.3 μg / mL in coating buffer (1× PBS, pH 7.4) and coat in a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating buffer and wash the plate with 1× PBST (300 μL / well), wash four times using a plate washer, and pat dry on paper. Block with 3% skim milk powder (300 μL / well), incubate at 37°C for 1 hour, discard the blocking buffer, wash four times using a plate washer, and pat dry on paper. Negative and positive controls were set up. The negative control was commercially available trastuzumab for injection (Herceptin). The variable region of the antibody sequence in positive control 1 consisted of SEQ ID NO:83 and SEQ ID NO:84, with the human IgG1 constant region added (see SEQ ID NO:85 and SEQ ID NO:86); the variable region of the antibody sequence in positive control 2 consisted of SEQ ID NO:41. The positive control and antibody were diluted to 10 μg / mL with 3% nonfat dry milk. From this initial concentration, three-fold dilutions were performed, totaling 11 dilutions. A blank well was set up, with only the diluent added. 100 μL / well was added, and the cells were incubated at 37°C for 1 hour. The liquid in the wells was discarded, and the plates were washed four times with a plate washer and patted dry on paper. A goat anti-human IgG Fc was diluted 1:20,000 with 3% nonfat dry milk, 100 μL / well was added, and the cells were incubated at 37°C for 1 hour. The plates were washed six times with a plate washer and patted dry on paper. Add 100 μL / well of TMB colorimetric solution, wrap with aluminum foil, and incubate at 37°C in the dark for 8 min. Terminate the colorimetric reaction by adding 100 μL / well of 1M HCl stop solution. Read the plate at 450 nm on a microplate reader. The ELISA results for the antibodies are shown in Figures 22-25, respectively. These results demonstrate that antibodies TD-A1 to TD-H bind to human TNFR2 protein.

[0177] Example 11 Flow cytometric detection of antibody binding activity to CD24 protein (TNFR2+CD24)

[0178] A positive control was set up. The variable region of the antibody sequence of the positive control consisted of SEQ ID NO: 9 and SEQ ID NO: 13. The constant region of human IgG1 (see SEQ ID NO: 85 and SEQ ID NO: 86) was added and diluted. The actual final concentration of the positive control and the test antibody was set to 150nM, and 100μL was added to each well. The tumor cells MCF-7 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number: TCHu74) with high expression of human CD24 were washed twice and 1×10 5100 μL / tube was added to each well of a 96-well plate. The positive control and test antibody were added to the 96-well plate at 50 μL / well. An equal volume of diluent was added to the blank and negative control groups. After mixing, the 96-well plate was incubated at 4°C for 1 hour. Wash once with pre-chilled diluent, centrifuge (3000 rpm, 3 min), discard the supernatant, and harvest the cells. Fluorescent secondary antibody goat anti-human IgG Fc (Biolegend, Catalog No. 398004, Lot No. B340957) was diluted to a working concentration of 1:500. 100 μL of diluent was added to the blank control group, and 100 μL of fluorescent secondary antibody diluent was added to each of the remaining sample groups. After incubation at room temperature at 4°C for 0.5 hour in the dark, the cells were washed with PBS and centrifuged twice (3000 rpm, 3 min). The supernatant was discarded and the cells were resuspended in 80 μL of diluent. The cells in each group were sequentially subjected to flow cytometry detection. The results of the antibody molecules are shown in Table 10. The results showed that antibodies TD-A1 to TD-H could bind to MCF-7 tumor cells that highly expressed human CD24.

[0179] Table 10 Binding activity of antibodies TD-A1 to TD-H to CD24 protein

[0180] Example 12 Construction of Antibody Binding Activity at Both Ends (TNFR2+CD24)

[0181] Negative control groups were set up, negative control 1 (PBS group), negative control 2, the variable region of the antibody sequence consisted of SEQ ID NO: 83 and SEQ ID NO: 84, and the constant region of human IgG1 was added (see SEQ ID NO: 85 and SEQ ID NO: 86); the variable region of the antibody sequence of negative control 3 consisted of SEQ ID NO: 9 and SEQ ID NO: 13, and the constant region of human IgG1 was added (SEQ ID NO: 85 and SEQ ID NO: 86). Diluted, the actual final concentration of the negative control and the test antibody was set to 150nM, 100μL per well. The tumor cells MCF-7 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number: TCHu74) that highly express human CD24 were washed twice, and 1×10 5100 μL / well of the dilution solution was added to a 96-well plate. Negative controls and test antibodies were added sequentially at 50 μL / well. An equal volume of diluent was added to the blank and negative control groups. Human TNFR2 protein (SINO, CAT: 10417-H08H) was added sequentially at 50 μL / well to the 96-well plate. After mixing, the 96-well plate was incubated at 4°C for 1 hour. Wash cells once with pre-chilled diluent, centrifuge at 3000 rpm for 3 minutes, discard the supernatant, and harvest the cells. Fluorescent secondary antibody Anti-6X-his tag antibody (FITC) (manufacturer: abcam, catalog number: ab1206, batch number: GR3420599-3) was diluted to a working concentration of 1:500. 100 μL of diluent was added to the blank control group, and 100 μL of fluorescent secondary antibody diluent was added to each of the other sample groups. After incubation at room temperature in the dark at 4°C for 0.5 h, the cells were washed with PBS and centrifuged twice (3000 rpm, 3 min). The supernatant was discarded and the cells were resuspended in 80 μL of diluent. The cells in each group were sequentially analyzed by flow cytometry. The results for the antibody molecules are shown in Table 11. The results show that antibodies TD-A1 to TD-H can simultaneously bind to the TNFR2 protein and MCF-7 tumor cells that overexpress human CD24.

[0182] Table 11 Antibody Binding Activity at Both Ends

[0183] Example 13 BLI method to detect the affinity of antibodies to TNFR2 protein (TNFR2+CD24)

[0184] Positive control groups were set up. The variable regions of the antibody sequence in positive control 1 consisted of SEQ ID NO:83 and SEQ ID NO:84, with the addition of the human IgG1 constant region (see SEQ ID NO:85 and SEQ ID NO:86); the variable regions of the antibody sequence in positive control 2 consisted of SEQ ID NO:41. The AHC sensor was soaked in 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) for 600 seconds to remove the sucrose coating on the sensor surface. The AHC sensor was equilibrated in 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) for 60 seconds, followed by 300 seconds of solidification of the antibody in the sample plate, and a secondary equilibration in buffer for 180 seconds. 100 nM human-TNFR2-His (manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) protein was allowed to bind to the antibody for 300 seconds, followed by 600 seconds of dissociation. After dissociation, the sensor was regenerated for 30 seconds using 10 mM glycine (pH 2.0) as a regeneration buffer. The affinity results for the antibody molecules are shown in Table 12. The results show that antibodies TD-A1, TD-C, TD-D1, TD-G, and TD-H all have nanomolar or subnanomolar affinities for the TNFR2 end.

[0185] Table 12 Affinity of antibodies to TNFR2 protein

[0186] Example 14 CD8+ T cell activation experiment (TNFR2+CD24)

[0187] Dilute the antibody to 70 nM in PBS (final experimental concentration 35 nM, 50 μL / well); dilute OKT3 to 10 μg / mL in PBS (final experimental concentration 5 μg / mL, 50 μL / well). An unrelated antibody, human IgG1 kappa isotype control (HG1K, Sino Biological), was used as a negative control and a positive control. The variable regions of the positive control antibody sequence consisted of SEQ ID NO:83 and SEQ ID NO:84, and the constant region of human IgG1 (see SEQ ID NO:85 and SEQ ID NO:86) was added. 50 μL of OKT3 dilution and 50 μL of antibody dilution were coated in a 96-well flat-bottom plate in a total volume of 100 μL / well and incubated overnight at 4°C. On the next day, CD8+ T cells were negatively isolated from frozen PBMC (ORiCELLS, ID: PCH20210300033, isolation efficiency 30%), and the sorted CD8+ T cells were tested for purity; CD8+ T cells were labeled with CFSE at a concentration of 5 μM, incubated at 37°C for 10 minutes, and complete medium was added to terminate the staining. The plate used for coating was washed once with PBS and 2×10 5 CFSE-labeled or unlabeled T cells were added to each well at 100 μL / well. Anti-CD28 was added at a final concentration of 1 μg / mL at 100 μL / well. The total experimental volume was 200 μL / well. The cells were incubated at 37°C for 96 hours. After centrifugation, the supernatant was collected for detection of IFNγ and IL-2 cytokines. The cells were washed with PBS and analyzed by flow cytometry. The results are shown in Table 13, indicating that the antibody TD-A1 activated CD8+ T cells.

[0188] Table 13 CD8+ T cell activation experimental results

[0189] Example 15 ADCC activity of antibodies against MCF-7 cells (TNFR2+CD24)

[0190] A negative control (IgG1 isotype control) and a positive control (the variable region of the antibody sequence consists of SEQ ID NO: 9 and SEQ ID NO: 13, and the constant region of human IgG1 is added, see SEQ ID NO: 85 and SEQ ID NO: 86) were set up. MCF-7 cells overexpressing human CD24 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number: TCHu74) were used as target cells. After centrifugation at 1000 rpm for 4 minutes at room temperature, the cells were resuspended in RPMI1640 basal medium (containing 5% FBS) and diluted to 1×10 4 / well, 50μL / well were plated in a 96-well plate; the antibody was diluted in RPMI1640 basal medium (containing 5% FBS) with a starting concentration of 120nM, followed by a 5-fold serial dilution, for a total of 8 concentration gradients, 100μL / well; NK158V cells were resuspended and added to the corresponding wells at 50μL / well, with an effector-target ratio of 5:1. At the same time, the target cell maximum lysis well (M), target cell spontaneous release well (ST), effector cell spontaneous release well (SE), total volume correction blank well (BV), and medium blank control well (BM) were set. After standing for 10 minutes, the cells were centrifuged at 1000rpm at room temperature for 4 minutes and incubated in a 5% CO2, 37°C carbon dioxide cell culture incubator for 4 hours. 45 minutes in advance, 20μL of lysis buffer was added to the M and BV wells, mixed, and centrifuged at 1000rpm at room temperature for 4 minutes after the incubation. Pipette 50 μL of supernatant into the LDH assay plate and add 50 μL / well of substrate dissolved in assay buffer. Incubate at room temperature in the dark for 30 minutes. Add 50 μL / well of stop solution and let stand for 10 minutes. Read at 490 nm to calculate cell mortality.

[0191] The logarithm of the antibody concentration was used as the abscissa, and the Sigmoidal dose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, San Diego, California) was used for nonlinear regression to obtain the ADCC activity curve of the target antibody against target cells.

[0192] As shown in FIG26 , the negative control showed no killing effect on MCF-7 cells, while the positive control, antibody TD-A1 and antibody TD-D1 showed obvious lytic and lethal effects on MCF-7 cells in a concentration-dependent manner.

[0193] Example 16 Antibody Binding Activity at Both Ends (TNFR2+Claudin18.2)

[0194] Negative control groups were set up, including negative control 1 (PBS group), negative control 2 (antibody sequence consisting of SEQ ID NO: 87 and SEQ ID NO: 88), negative control 3 (antibody sequence consisting of SEQ ID NO: 79 and SEQ ID NO: 71), and irrelevant antibody (human IgG1kappa Isotype control, manufacturer: Sino Biological, product number: HG1K, batch number: MA140C2803). The negative control, irrelevant antibody and the test antibody were diluted to a final concentration of 150 nM, with 100 μL per well. The tumor cells CHOK1-18.2 (P6-4-1) that highly express human Claudin18.2 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number: TCHu74) were washed twice and 1×10 5 100 μL / well was added to a 96-well plate. Negative control 2, negative control 3, an unrelated antibody, and the test antibody were sequentially added to the 96-well plate at 50 μL / well. An equal volume of diluent was added to the blank and negative control groups. Human TNFR2 protein (SINO, CAT: 10417-H08H) was sequentially added to the 96-well plate at 100 μL / well. After mixing, the 96-well plate was incubated at 4°C for 1 hour. Wash once with pre-chilled diluent, centrifuge (3000 rpm, 3 minutes), and discard the supernatant. Fluorescent secondary antibody Anti-6X-his tag antibody (FITC) (Manufacturer: abcam, Catalog No. ab1206, Lot No. GR3420599-3) was diluted to a working concentration of 1:500. 100 μL of diluent was added to the blank control group, and 100 μL of fluorescent secondary antibody diluent was added to each of the remaining sample groups. After incubation at room temperature in the dark at 4°C for 0.5 h, cells were washed with PBS and centrifuged twice (3000 rpm, 3 min). The supernatant was discarded and the cells were resuspended in 80 μL of diluent. The cells in each group were sequentially analyzed by flow cytometry. The results for the antibody molecules are shown in Table 14. The results show that all antibodies can simultaneously bind to the TNFR2 protein and CHOK1-18.2 tumor cells that overexpress human claudin18.2.

[0195] Table 14 Antibody Binding Activity at Both Ends

[0196] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A bispecific antibody, characterized in that: The bispecific antibody comprises: (a) a first antibody or an antigen-binding fragment thereof that specifically binds to a first antigen; and (b) a second antibody or an antigen-binding fragment thereof that specifically binds to a second antigen; the first antigen is a tumor-associated antigen (TAA), and the second antigen is TNFR2.

2. The bispecific antibody according to claim 1, characterized in that The first antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; and the second antibody or antigen-binding fragment thereof comprises a scFv or a VHH.

3. The bispecific antibody according to claim 1 or 2, characterized in that The heavy chain variable region of one heavy chain of the first antibody forms an antigen binding site with the light chain variable region of one light chain, and the heavy chain variable region of the other heavy chain forms an antigen binding site with the light chain variable region of the other light chain.

4. The bispecific antibody according to claim 2 or 3, characterized in that It comprises a first antibody or an antigen-binding fragment thereof and one or more of the scFv or VHH.

5. The bispecific antibody according to claim 4, characterized in that It comprises a first antibody or an antigen-binding fragment thereof and a scFv or a VHH, wherein the scFv or VHH is connected to the N-terminus or C-terminus of the heavy chain of the first antibody or the antigen-binding fragment thereof.

6. The bispecific antibody according to claim 4, characterized in that It comprises a first antibody or an antigen-binding fragment thereof and two of the scFvs or two of the VHHs.

7. The bispecific antibody according to claim 6, characterized in that The two scFvs or the two VHHs are respectively connected to the N-termini or C-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.

8. The bispecific antibody according to claim 7, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, characterized in that: For each of the polypeptide chains: (a) the first polypeptide chains each independently comprise the light chain of the first antibody or antigen-binding fragment thereof; and (b) the second polypeptide chains each independently comprise the heavy chain of the first antibody or antigen-binding fragment thereof and the scFv or VHH.

9. The bispecific antibody according to claim 8, characterized in that The two first polypeptide chains are identical or different, and / or the two second polypeptide chains are identical or different.

10. The bispecific antibody according to any one of claims 1 to 9, characterized in that The tumor-associated antigen (TAA) is selected from GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2, AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-ab1, BRCA1, BO RIS, CA9, CA125, carbonic anhydrase IX, caspase-8, CALR, CCR5, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, cyclin-B1, CYP1B1, EGFR, EG FRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, GM3, gp1 00, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IL13Rα2, LMP2, κ-Light, LeY, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-12, MART-1, mesothelin, ML-IAP, MOv-γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16, MUM1, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Tang-Nordstein antigen, TRP-1, TRP-2, tyrosinase, and urokinin-3, 5T4.

11. The bispecific antibody according to claim 10, characterized in that The tumor-associated antigen (TAA) is selected from B7H3, CD24 or Claudin18.

2.

12. The bispecific antibody according to any one of claims 2 to 11, characterized in that The heavy chain variable region and the light chain variable region of the scFv are connected by a linker L1.

13. The bispecific antibody according to any one of claims 2 to 12, characterized in that The scFv or VHH is connected to the N-terminus or C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof through a linker L2.

14. The bispecific antibody according to claim 13, characterized in that The linker L1 and linker L2 are the same or different.

15. The bispecific antibody according to any one of claims 12 to 14, characterized in that The linker L1 and / or linker L2 have (G4S) x The amino acid sequence shown, x is an integer selected from 1-6; preferably, the linker L1 and / or linker L2 is (G4S)2, (G4S)3 or (G4S)4.

16. The bispecific antibody according to any one of claims 1 to 15, characterized in that The heavy chain of the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region; preferably, the first antibody or antigen-binding fragment thereof is a full-length antibody.

17. The bispecific antibody according to claim 16, characterized in that The heavy chain of the first antibody or antigen-binding fragment thereof comprises a first Fc region and a second Fc region.

18. The bispecific antibody according to claim 17, characterized in that The first Fc region and the second Fc region are the same or different.

19. The bispecific antibody according to claim 17 or 18, characterized in that The first Fc region or the second Fc region is selected from IgG, IgA, IgD, IgE, IgM or variants thereof.

20. The bispecific antibody according to claim 19, characterized in that The first Fc region or the second Fc region is selected from IgG1, IgG2, IgG3, IgG4 or variants thereof.

21. The bispecific antibody according to claim 19 or 20, characterized in that The first Fc region or the second Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

22. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to B7H3, wherein HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:2, or a sequence having at least 80% identity with SEQ ID NO:2; HCDR2 is as shown in SEQ ID NO:3, or a sequence having at least 80% identity with SEQ ID NO:3; HCDR3 is as shown in SEQ ID NO:4, or a sequence having at least 80% identity with SEQ ID NO:4; LCDR1 is as shown in SEQ ID NO:6, or a sequence having at least 80% identity with SEQ ID NO:6; LCDR2 is as shown in SEQ ID NO:7, or a sequence having at least 80% identity with SEQ ID NO:7; and LCDR3 is as shown in SEQ ID NO:8, or a sequence having at least 80% identity with SEQ ID NO:

8.

23. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to CD24, wherein HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:10, or a sequence at least 80% identical to SEQ ID NO:10; HCDR2 is as shown in SEQ ID NO:11, or a sequence at least 80% identical to SEQ ID NO:11; HCDR3 is as shown in SEQ ID NO:12, or a sequence at least 80% identical to SEQ ID NO:12; LCDR1 is as shown in SEQ ID NO:14, or a sequence at least 80% identical to SEQ ID NO:14; LCDR2 is as shown in SEQ ID NO:15, or a sequence at least 80% identical to SEQ ID NO:15; and LCDR3 is as shown in SEQ ID NO:16, or a sequence at least 80% identical to SEQ ID NO:

16.

24. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:18, or a sequence having at least 80% identity with SEQ ID NO:18; HCDR2 is shown in SEQ ID NO:19, or a sequence having at least 80% identity with SEQ ID NO:19; HCDR3 is shown in SEQ ID NO:20, or a sequence having at least 80% identity with SEQ ID NO:20; LCDR1 is shown in SEQ ID NO:22, or a sequence having at least 80% identity with SEQ ID NO:22; LCDR2 is shown in SEQ ID NO:23, or a sequence having at least 80% identity with SEQ ID NO:23; and LCDR3 is shown in SEQ ID NO:24, or a sequence having at least 80% identity with SEQ ID NO:

24.

25. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:26, or a sequence having at least 80% identity with SEQ ID NO:26; HCDR2 is shown in SEQ ID NO:27, or a sequence having at least 80% identity with SEQ ID NO:27; HCDR3 is shown in SEQ ID NO:28, or a sequence having at least 80% identity with SEQ ID NO:28; LCDR1 is shown in SEQ ID NO:30, or a sequence having at least 80% identity with SEQ ID NO:30; LCDR2 is shown in SEQ ID NO:31, or a sequence having at least 80% identity with SEQ ID NO:31; and LCDR3 is shown in SEQ ID NO:32, or a sequence having at least 80% identity with SEQ ID NO:

32.

26. The bispecific antibody according to any one of claims 22 to 25, characterized in that The scFv specifically binds to TNFR2, and the scFv has HCDR1 as set forth in SEQ ID NO:34, or a sequence at least 80% identical to SEQ ID NO:34; HCDR2 as set forth in SEQ ID NO:35, or a sequence at least 80% identical to SEQ ID NO:35; HCDR3 as set forth in SEQ ID NO:36, or a sequence at least 80% identical to SEQ ID NO:36; and LCDR1 as set forth in SEQ ID NO:38, or a sequence at least 80% identical to SEQ ID NO:38; LCDR2 as set forth in SEQ ID NO:39, or a sequence at least 80% identical to SEQ ID NO:39; and LCDR3 as set forth in SEQ ID NO:40, or a sequence at least 80% identical to SEQ ID NO:

40.

27. The bispecific antibody according to any one of claims 22 to 25, characterized in that The VHH specifically binds to TNFR2, and the HCDR1 of the VHH is shown in SEQ ID NO:42, or a sequence with at least 80% identity to SEQ ID NO:42; HCDR2 is shown in SEQ ID NO:43, or a sequence with at least 80% identity to SEQ ID NO:43; and HCDR3 is shown in SEQ ID NO:44, or a sequence with at least 80% identity to SEQ ID NO:

44.

28. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to B7H3, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 1, or a sequence having at least 80% identity with SEQ ID NO: 1; the light chain variable region VL is shown in SEQ ID NO: 5, or a sequence having at least 80% identity with SEQ ID NO:

5.

29. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to CD24, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 9, or a sequence having at least 80% identity with SEQ ID NO: 9; and the light chain variable region VL is shown in SEQ ID NO: 13, or a sequence having at least 80% identity with SEQ ID NO:

13.

30. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 17, or a sequence having at least 80% identity with SEQ ID NO: 17; the light chain variable region VL is shown in SEQ ID NO: 21, or a sequence having at least 80% identity with SEQ ID NO:

21.

31. The bispecific antibody according to any one of claims 1 to 21, characterized in that The first antibody or its antigen-binding fragment specifically binds to Claudin18.2, wherein the heavy chain variable region VH of the first antibody or its antigen-binding fragment is shown in SEQ ID NO:25, or a sequence having at least 80% identity with SEQ ID NO:25; the light chain variable region VL is shown in SEQ ID NO:29, or a sequence having at least 80% identity with SEQ ID NO:

29.

32. The bispecific antibody according to any one of claims 28 to 31, characterized in that The scFv specifically binds to TNFR2, and the heavy chain variable region VH of the scFv is shown in SEQ ID NO: 33, or a sequence having at least 80% identity with SEQ ID NO: 33; the light chain variable region VL is shown in SEQ ID NO: 37, or a sequence having at least 80% identity with SEQ ID NO:

37.

33. The bispecific antibody according to any one of claims 28 to 31, characterized in that The VHH specifically binds to TNFR2, and the VHH heavy chain variable region VH is shown in SEQ ID NO: 41, or a sequence having at least 80% identity with SEQ ID NO:

41.

34. The bispecific antibody according to any one of claims 8 to 33, characterized in that The first polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 46-50, 59-63, and 71-76, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 46-50, 59-63, and 71-76; the second polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 51-58, 64-70, and 77-82, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 51-58, 64-70, and 77-82.

35. The bispecific antibody according to claim 34, characterized in that The bispecific antibody comprises: (1) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:51; or (2) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:56; or (3) the first polypeptide chain shown in SEQ ID NO:46 and the second polypeptide chain shown in SEQ ID NO:53; or (4) the first polypeptide chain shown in SEQ ID NO:46 and the second polypeptide chain shown in SEQ ID NO:54; or (5) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:57; or (6) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:55; or (7) the first polypeptide chain shown in SEQ ID NO:46, the second polypeptide chain shown in SEQ ID NO:58; or (8) the first polypeptide chain shown in SEQ ID NO:47, the second polypeptide chain shown in SEQ ID NO:52; or (9) the first polypeptide chain shown in SEQ ID NO: 50, the second polypeptide chain shown in SEQ ID NO: 52; or (10) the first polypeptide chain shown in SEQ ID NO: 48, the second polypeptide chain shown in SEQ ID NO: 52; or (11) the first polypeptide chain shown in SEQ ID NO: 49 and the second polypeptide chain shown in SEQ ID NO: 52; or (12) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 64; or (13) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 69; or (14) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 66; or (15) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 67; or (16) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 68; or (17) the first polypeptide chain as shown in SEQ ID NO: 59, the second polypeptide chain as shown in SEQ ID NO: 70; or (18) the first polypeptide chain as shown in SEQ ID NO: 60, the second polypeptide chain as shown in SEQ ID NO: 65; or (19) the first polypeptide chain as shown in SEQ ID NO: 61, the second polypeptide chain as shown in SEQ ID NO: 65; or (20) the first polypeptide chain as shown in SEQ ID NO: 62, the second polypeptide chain as shown in SEQ ID NO: 65; or (21) the first polypeptide chain as shown in SEQ ID NO: 63, the second polypeptide chain as shown in SEQ ID NO: 65; or (22) the first polypeptide chain as shown in SEQ ID NO: 71, the second polypeptide chain as shown in SEQ ID NO: 77; or (23) the first polypeptide chain as shown in SEQ ID NO: 72, the second polypeptide chain as shown in SEQ ID NO: 78; or (24) the first polypeptide chain as shown in SEQ ID NO: 71, the second polypeptide chain as shown in SEQ ID NO: 81; or (25) the first polypeptide chain as shown in SEQ ID NO: 72, the second polypeptide chain as shown in SEQ ID NO: 82; or (26) the first polypeptide chain as shown in SEQ ID NO: 73, the second polypeptide chain as shown in SEQ ID NO: 79; or (27) the first polypeptide chain as shown in SEQ ID NO: 74, the second polypeptide chain as shown in SEQ ID NO: 80; or (28) a first polypeptide chain as shown in SEQ ID NO: 75, a second polypeptide chain as shown in SEQ ID NO: 79; or (29) The first polypeptide chain shown in SEQ ID NO:76, and the second polypeptide chain shown in SEQ ID NO:

80.

36. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1 to 35; preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of the bispecific antibody of any one of claims 8 to 35; preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the second polypeptide chain of the bispecific antibody of any one of claims 8 to 35. A multifunctional fusion protein comprising the bispecific antibody according to any one of claims 1 to 35.

38. The multifunctional fusion protein according to claim 37, characterized in that One or more third antibodies or antigen-binding portions thereof that specifically bind to other antigens are also included.

39. The multifunctional fusion protein according to claim 38, characterized in that The antigen that binds to the third antibody or its antigen-binding portion is selected from a tumor-associated antigen (TAA) or an immune checkpoint.

40. The multifunctional fusion protein according to claim 39, characterized in that The antigen that binds to the third antibody or its antigen-binding portion is selected from GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2, AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-ab1, BRCA1, BORIS, CA9, CA125, carbonic anhydrase IX, caspase-8, CALR, CCR5, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p 53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, GM3, gp10 0. Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IL13Rα2, LMP2, κ-Light, LeY, MAGE-1, MAGE-2, MAG E-3, MAGE-4, MAGE-6, MAGE-12, MART-1, mesothelin, ML-IAP, MOv-γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16, MUM1, Ra s, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Tang-Novo antigen, TRP-1, TRP-2, tyrosinase and urokinin-3, 5T4, PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, ILT4, TREM2, LAG3, CD27, or B7H4.

41. The multifunctional fusion protein according to any one of claims 37 to 40, characterized in that Also contains cytokines.

42. The multifunctional fusion protein according to claim 41, characterized in that The cytokine is selected from the group consisting of IL-1, IL-2, IL-2 Rα, IL-2 Rβ, IL-3, IL-3 Rα, IL-4, IL-4 Rα, IL-5, IL-5 Rα, IL-6, IL-6 Rα, IL-7, IL-7 Rα, IL-8, IL-9, IL-9 Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11 Rα, IL-12, IL-12 Rα, IL-12 Rβ2, IL-12 Rβ1, IL-13, IL-13 Rα, IL-13 Rα2, IL-14, IL-15, IL-15Rαsushi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21 Rα, IL-22, IL-23, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFNγ, IFNα, or GM-CSF.

43. Use of the bispecific antibody according to any one of claims 1 to 35 or the multifunctional fusion protein according to any one of claims 37 to 42 in the preparation of a medicament for treating cancer.

44. The use according to claim 43, characterized in that The cancer is selected from the group consisting of glioblastoma, astrocytoma, cystic fibrosis, fibromyalgia, fibrosarcoma, fibromyalgia ... tumor, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer.

45. Use of the bispecific antibody according to any one of claims 1 to 35 or the multifunctional fusion protein according to any one of claims 37 to 42 in the preparation of a medicament for treating an autoimmune disease.

46. ​​The use according to claim 45, characterized in that The autoimmune disease is selected from graft-versus-host disease, rheumatoid arthritis, Crohn's disease, multiple sclerosis, colitis, psoriasis, autoimmune uveitis, pemphigus, epidermolysis bullosa, or type I diabetes.

47. The use according to any one of claims 43 to 46, characterized in that The use is achieved through one or more of tumor immunotherapy, cell therapy or gene therapy.

48. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-35 and a pharmaceutically acceptable carrier, diluent or excipient.

49. A pharmaceutical composition comprising the multifunctional fusion protein according to any one of claims 37 to 42 and a pharmaceutically acceptable carrier, diluent or excipient.

50. An antibody-drug conjugate comprising the bispecific antibody of any one of claims 1-35.

51. The antibody drug conjugate according to claim 50, characterized in that The conjugated drug is selected from cytotoxins, small molecule chemical drugs or immunotoxins.

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