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 T cell immunomodulator, and the second antigen is 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 combining a T cell immunomodulator 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 signal transduction 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 TNFR2 agonistic antibodies can stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFNγ and IL2; TNFR2 antagonistic antibodies can block TNF-TNFR2 and inhibit Treg proliferation.

[0006] T cell co-stimulatory immunomodulators are a class of auxiliary molecules involved in immune responses. In the process of cell recognition of antigens, the specific binding of T cell co-stimulatory immunomodulators can effectively enhance the adhesion of T cells to other cells, transmit antigen stimulation information, participate in the immune activation process of cells, and play an important role in cell antigen recognition and immune response.

[0007] T cell co-inhibitory immunomodulators negatively regulate the immune response. Multiple studies have shown that co-inhibitory molecules are an important strategy for tumors to evade immune surveillance. They inhibit immune cell activity through co-inhibitory signals, thereby promoting cancer development, cancer cell proliferation, metastasis, and further tumor progression. In some cases, co-inhibitory receptors (such as CTLA-4Ig) or monoclonal antibodies against co-inhibitory molecules can relieve immunosuppressive signals and restore immune cell function. By combining with T cell modulators, they can further enhance the activity and number of effector cells, eliminate Tregs in the tumor microenvironment, and increase effector T cells that recognize and kill tumors, thereby achieving the goal of tumor treatment.

[0008] Similarly, some studies have shown that the loss and mutation of co-inhibitory molecules lead to autoimmune diseases in mice and humans, indicating that the downregulation of co-inhibitory molecules plays an important role in preventing and treating autoimmunity. In some cases, monoclonal antibodies against co-inhibitory receptors or agonistic anti-co-inhibitory modulators can inhibit the function of autoreactive T cells. Negative regulation of immune responses by T cell co-inhibitory immunomodulators is an effective way to induce tolerance and thus treat autoimmunity.

[0009] Research has shown that immunotherapies targeting the tumor microenvironment can be successfully applied to cancer. The tumor microenvironment is heterogeneous, influenced by age, environment, and tumor type, requiring the coordinated action of multiple mechanisms. T cells are a crucial component of anti-tumor therapy, and the synergistic use of multiple mechanisms can benefit cancer patients.

[0010] Summary of the Invention

[0011] In the present invention, the inventors have developed a bispecific antibody with good performance. The bispecific antibody of the present invention 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. At the same time, it cooperates with T cell immunomodulators at the target site or the whole body system to further relieve immunosuppression, increase the number of anti-tumor cells, and solve the limitations of TME heterogeneity. The present invention combines TNFR2 agonistic or antagonistic antibodies with other T cell co-stimulatory immunomodulators or T cell co-inhibitory immunomodulators to form a bispecific antibody, thereby regulating the ratio of Teff and Treg, reversing the ratio of immune cells in the tumor microenvironment, further relieving immunosuppression, increasing the number of anti-tumor immune cells, and promoting tumor cell apoptosis in multiple ways.

[0012] 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 T cell immunomodulator and the second antigen is TNFR2.

[0013] In some embodiments, the T cell immunomodulator is a T cell co-stimulatory immunomodulator or a T cell co-inhibitory immunomodulator.

[0014] 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.

[0015] 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.

[0016] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more scFvs.

[0017] 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.

[0018] 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.

[0019] In some embodiments, the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and two of the scFvs.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more of the VHHs.

[0027] 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.

[0028] 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.

[0029] In some embodiments, the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and two of the VHHs.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In some embodiments, the T cell co-stimulatory immunomodulator is selected from 4-1BB, OX40, B7H2, GITR, CD48, HVEM, Nectin-2, CD40, CD30, CD28, CD27, B7-1, B7-2, LIGHT, B7H6, 2B4, CD28H or NKp30.

[0037] In some embodiments, the T cell co-inhibitory immunomodulator is selected from CCR8, PD-1, PD-L1, PD-L2, B7H3, B7H4, B7H5, BLTA, CD96, CD47, CD155, CTLA-4, LAG-3, TIGIT, TIM-3, CD111, DNAM-1, Galectin-9, Nectin-3, PVRIG, SIRPα, SIRPβ, SIRPαV2 or CD160.

[0038] In some embodiments, the heavy chain variable region and the light chain variable region of the scFv are connected by a linker L1.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to 4-1BB, 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.

[0045] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to CCR8, 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.

[0046] In some embodiments, the scFv specifically binds TNFR2, and the scFv's HCDR1 is set forth in SEQ ID NO:18, or a sequence at least 80% identical to SEQ ID NO:18; HCDR2 is set forth in SEQ ID NO:19, or a sequence at least 80% identical to SEQ ID NO:19; HCDR3 is set forth in SEQ ID NO:20, or a sequence at least 80% identical to SEQ ID NO:20; LCDR1 is set forth in SEQ ID NO:22, or a sequence at least 80% identical to SEQ ID NO:22; LCDR2 is set forth in SEQ ID NO:23, or a sequence at least 80% identical to SEQ ID NO:23; and LCDR3 is set forth in SEQ ID NO:24, or a sequence at least 80% identical to SEQ ID NO:24.

[0047] In some embodiments, the VHH specifically binds to TNFR2, and the HCDR1 of the VHH is set forth in SEQ ID NO:26, or a sequence at least 80% identical to SEQ ID NO:26; the HCDR2 is set forth in SEQ ID NO:27, or a sequence at least 80% identical to SEQ ID NO:27; and the HCDR3 is set forth in SEQ ID NO:28, or a sequence at least 80% identical to SEQ ID NO:28.

[0048] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to 4-1BB, 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.

[0049] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to CCR8, 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; 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.

[0050] 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: 17, or a sequence with at least 80% identity to SEQ ID NO: 17; the light chain variable region VL is shown in SEQ ID NO: 21, or a sequence with at least 80% identity to SEQ ID NO: 21.

[0051] In some embodiments, the VHH specifically binds to TNFR2, and the VHH heavy chain variable region VH is as shown in SEQ ID NO: 25, or a sequence that is at least 80% identical to SEQ ID NO: 25.

[0052] In some embodiments, the first polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 30-34, 42-46, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 30-34, 42-46; the second polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 35-41, 47-51, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 35-41, 47-51.

[0053] In some embodiments, the bispecific antibody comprises:

[0054] (1) the first polypeptide chain shown in SEQ ID NO: 30, and the second polypeptide chain shown in SEQ ID NO: 35; or

[0055] (2) the first polypeptide chain as shown in SEQ ID NO: 31, the second polypeptide chain as shown in SEQ ID NO: 36; or

[0056] (3) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 37; or

[0057] (4) the first polypeptide chain shown in SEQ ID NO: 32, the second polypeptide chain shown in SEQ ID NO: 36; or

[0058] (5) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 38; or

[0059] (6) the first polypeptide chain shown in SEQ ID NO: 30, the second polypeptide chain shown in SEQ ID NO: 40; or

[0060] (7) the first polypeptide chain as shown in SEQ ID NO: 33, the second polypeptide chain as shown in SEQ ID NO: 36; or

[0061] (8) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 39; or

[0062] (9) the first polypeptide chain shown in SEQ ID NO: 30, the second polypeptide chain shown in SEQ ID NO: 41; or

[0063] (10) the first polypeptide chain as shown in SEQ ID NO: 34, the second polypeptide chain as shown in SEQ ID NO: 36; or

[0064] (11) the first polypeptide chain as shown in SEQ ID NO: 42, the second polypeptide chain as shown in SEQ ID NO: 48; or

[0065] (12) a first polypeptide chain as shown in SEQ ID NO: 43, a second polypeptide chain as shown in SEQ ID NO: 47; or

[0066] (13) the first polypeptide chain as shown in SEQ ID NO:42, the second polypeptide chain as shown in SEQ ID NO:49; or

[0067] (14) the first polypeptide chain as shown in SEQ ID NO: 44, the second polypeptide chain as shown in SEQ ID NO: 47; or

[0068] (15) the first polypeptide chain shown in SEQ ID NO:42, the second polypeptide chain shown in SEQ ID NO:50; or

[0069] (16) the first polypeptide chain as shown in SEQ ID NO: 45, the second polypeptide chain as shown in SEQ ID NO: 47; or

[0070] (17) the first polypeptide chain as shown in SEQ ID NO:42, the second polypeptide chain as shown in SEQ ID NO:51; or

[0071] (18) The first polypeptide chain shown in SEQ ID NO:46, and the second polypeptide chain shown in SEQ ID NO:47.

[0072] 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.

[0073] The present invention provides a multifunctional fusion protein comprising any of the bispecific antibodies described above. 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 bound to the third antibody or antigen-binding portion thereof is selected from a tumor-associated antigen (TAA) or an immune checkpoint molecule.In some embodiments, the antigen that binds to the third antibody, or antigen-binding portion thereof, is selected from GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2, AFP, ALK, B7H2, B7H3, B7H5, 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, p53, 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, 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, Thomson-Knowledge 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, B7H4, CD48, HVEM, nectin-2, CD28, B7-1, B7-2, B7H6, 2B4, CD28H, NKp30, BLTA, CD96, CD155, CTLA-4, LAG-3, TIM-3, CD111, DNAM-1, Galectin-9, nectin-3, PVRIG, SIRPα, SIRPβ, SIRPαV2, or CD160.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] In some embodiments, the use is achieved by one or more of tumor immunotherapy, cell therapy, or gene therapy.

[0078] The present invention also provides a pharmaceutical composition comprising any of the above-described bispecific antibodies and a pharmaceutically acceptable carrier, diluent or excipient.

[0079] 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.

[0080] The present invention also provides an antibody-drug conjugate comprising any of the above-mentioned bispecific antibodies.

[0081] In some embodiments, the conjugated drug is selected from a cytotoxin, a small molecule chemical drug, or an immunotoxin.

[0082] Abbreviations and Definitions

[0083] 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; IgG: immunoglobulin G.

[0084] 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.

[0085] 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.

[0086] 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".

[0087] 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.

[0088] The term "T cell immunomodulator" refers to T cell regulatory molecules that modulate the T cell response of the human immune system to monitor and kill tumor cells, foreign pathogens, or prevent overactivation of the immune system on healthy cells. T cell immunomodulators include T cell co-stimulatory immunomodulators and T cell co-inhibitory immunomodulators. Removing immune system co-inhibition and enhancing co-stimulation can enhance anti-tumor immune responses.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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 naturally does not contain a light chain, to distinguish it 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.

[0093] The term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence to express a selected gene of interest. The term includes the progeny of a parent cell, regardless of whether the progeny is identical in morphology or genetic makeup to the original parent cell, as long as the gene of interest is present in the progeny. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0094] 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."

[0095] The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate-buffered saline solutions, water, and emulsions, as well as various types of wetting agents.

[0096] 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.

[0097] 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%.

[0098] 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.

[0099] 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. Non-limiting examples of "tumor-associated antigens" include, for example, 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, 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, FOL R1, 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-Novogene 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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 exocytosis of granules. The released perforins polymerize to form pores on the target cell surface, mediating their killing effect. The target cell death process is similar to apoptosis. Effector T cells also release immunoreactive substances—lymphokines, such as interleukins and interferons.

[0105] 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 defined based on Oxford Molecular's AbM antibody modeling software, which 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. The CDRs of this application are mainly divided using Kabat, and some CDRs are divided using definition standards, such as the HCDR1 shown in SEQ ID NO: 2, 10, 18, and 26. The division starts at the fourth position after the first cysteine ​​in the heavy chain variable region, and the HCDR1 length is generally 10-12, ending at the amino acid before tryptophan. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG1 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the C-termini of two heavy chains of the full-length antibody via a linker.

[0107] FIG2 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator 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.

[0108] FIG3 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator 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.

[0109] FIG4 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator 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.

[0110] FIG5 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator 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.

[0111] FIG6 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator 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.

[0112] FIG7 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the C-termini of two light chains of the full-length antibody via a linker.

[0113] FIG8 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a T cell immunomodulator and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the N-termini of two light chains of the full-length antibody via a linker.

[0114] FIG9 shows the binding activity of antibodies TB-A, TB-E, TB-B, and TB-F to TNFR2 protein.

[0115] FIG10 shows the binding activity of antibodies TB-C1 and TB-G to TNFR2 protein.

[0116] FIG11 shows the binding activity of antibodies TB-D1 and TB-H to TNFR2 protein.

[0117] FIG12 shows the binding activity of antibodies TB-C2 and TB-D2 to TNFR2 protein.

[0118] FIG13 shows the binding activity of antibodies TB-A, TB-E, TB-B, and TB-F to 4-1BB protein.

[0119] FIG14 shows the binding activity of antibodies TB-C1, TB-G, TB-D1, and TB-H to 4-1BB protein.

[0120] FIG15 shows the binding activity of antibody TB-C2 to 4-1BB protein.

[0121] FIG16 shows the dual-terminal binding activities of antibodies TB-A, TB-E, TB-B, and TB-F.

[0122] FIG17 shows the dual-terminal binding activities of antibodies TB-C1, TB-G, TB-D1, and TB-H.

[0123] FIG18 shows the dual-terminal binding activities of antibodies TB-A, TB-E, TB-B, and TB-F.

[0124] FIG19 shows the dual-terminal binding activities of antibodies TB-C1, TB-G, TB-D1, and TB-H.

[0125] FIG20 shows the activation effect of antibodies TB-C1 and TB-D1 on HEK-293 / NFκB-Luci / 4-1BB cells.

[0126] FIG21 shows the anti-tumor growth curves of antibodies TB-C2 and TB-D2.

[0127] FIG22 shows the ADCC activities of antibodies TB-C1 and TB-D1.

[0128] FIG23 shows the binding activity of antibodies TR-A to TR-H to CCR8-CHO-K1 cells.

[0129] FIG24 shows the binding activity of antibodies TR-A to TR-H to TNFR2-CHO-K1 cells.

[0130] FIG25 shows the ADCC activities of antibodies TR-A, TR-E, TR-B, and TR-F.

[0131] FIG26 shows the ADCC activities of antibodies TR-C, TR-G, TR-D, and TR-H.

[0132] FIG27 shows the blocking activity of antibodies TR-C, TR-G, and TR-D on TNFR2-TNF binding.

[0133] FIG28 shows the blocking activity of antibody TR-H on TNFR2-TNF binding.

[0134] FIG29 shows the results of an activation experiment in which antibodies TR-A, TR-E, TR-B, and TR-F activate CD8+ T cells to release IFN-γ. DETAILED DESCRIPTION

[0135] 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.

[0136] Example 1 Preparation of bispecific antibodies with different structures

[0137] Targeting TNFR2 and 4-1BB, bispecific antibodies were constructed based on the eight structures in Figures 1-8, and were named TB-A to TB-H, respectively. Targeting TNFR2 and CCR8, bispecific antibodies were constructed based on the eight structures in Figures 1-8, and were named TR-A to TR-H, respectively. Some of the bispecific antibodies in this patent simultaneously constructed 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-C1 is an IgG1 subtype antibody targeting TNFR2 and 4-1BB targets, constructed based on the structure in Figure 3. Antibody TB-C2 is an IgG4 subtype antibody targeting TNFR2 and 4-1BB targets, constructed based on the structure in Figure 3.

[0138] The sequences of the anti-4-1BB antibody and anti-CCR8 antibody as full-length antibodies, and the sequences of the TNFR2 antibody as scFv and VHH are shown in Table 1.

[0139] Table 1 Sequence Listing

[0140] The TNFR2 antibody, the anti-4-1BB antibody, and the anti-CCR8 antibody are connected in a certain order through a linker to form the peptide chains shown in Tables 2 and 3. 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), and the CL used in this embodiment is of kappa (κ) type, and the sequence is shown in SEQ ID NO:29.

[0141] Table 2 Amino acid sequence of bispecific antibody (TNFR2+4-1BB) peptide chain

[0142] Table 3 Amino acid sequence of bispecific antibody (TNFR2+CCR8) peptide chain

[0143] Based on the peptide chain combinations in Tables 2 and 3, the amino acid sequences of the bispecific antibodies shown in Table 4 were designed.

[0144] Table 4 Amino acid sequences of bispecific antibodies

[0145] Each chain of the bispecific antibody with different structures designed above was gene-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 and characterize the bispecific antibody.

[0146] Example 2 ELISA detection of antibody binding activity to TNFR2 protein (TNFR2+4-1BB)

[0147] Dilute human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC15NO0412) to 0.1 μg / mL in coating buffer (1× PBS, pH 7.4) and coat the plate 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 microplate washer and pat dry on paper. Block the plate with 3% skim milk powder (300 μL / well) and incubate at 37°C for 1 hour. Discard the blocking buffer, wash four times using a microplate 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:52 and SEQ ID NO:53, with the constant region of human IgG1 (see SEQ ID NO:54 and SEQ ID NO:55) added. The variable region of the antibody sequence in positive control 2 consisted of SEQ ID NO:25. 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 plate was incubated at 37°C for 1 hour. The liquid in the well was discarded, the plate was washed four times with a plate washer, and the plate was patted dry on paper. A goat anti-human IgG Fc was diluted 1:10,000 with 3% nonfat dry milk, 100 μL / well was added, and the plate was incubated at 37°C for 1 hour. The plate was washed six times with a plate washer, and the plate was 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-A through TB-H bind to human TNFR2 protein.

[0148] Example 3 ELISA detection of antibody binding activity to 4-1BB protein (TNFR2+4-1BB)

[0149] Human 4-1BB His (manufacturer: Acro, CAT: 41B-H5258, LOT: 198-2146F1-W6) was diluted to 0.2 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 at 300 μL per well, wash 4 times with a plate washer, and pat dry on a flat sheet. Block with 3% skim milk powder at 300 μL / well, incubate at 37°C for 1 hour, discard the blocking solution, wash 4 times with a plate washer, and pat dry on a flat sheet. Set up negative and positive controls. The negative control is commercially available trastuzumab for injection (Herceptin). The variable region of the antibody sequence of the positive control consists of SEQ ID NO: 1 and SEQ ID NO: 5, with the constant region of human IgG1 added (see SEQ ID NO: 54 and SEQ ID NO: 55). The positive control and antibody were diluted to 10 μg / mL with 3% skim milk powder. From this initial concentration, three-fold dilutions were performed, totaling 11 dilution steps. A blank well was also 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, the plates were washed four times on a plate washer, and the plates were patted dry on paper. A goat anti-human IgG was diluted 1:20,000 with 3% skim milk powder was added, 100 μL / well was added, and the cells were incubated at 37°C for 1 hour. The plates were washed six times on a plate washer, and the plates were patted dry on paper. TMB colorimetric solution was added (100 μL / well), the plates were wrapped with aluminum foil, and the cells were allowed to develop at 37°C in the dark for 8 minutes. The colorimetric reaction was terminated with 100 μL / well of 1M HCl stop solution. The assay was read at 450 nm on a microplate reader. The ELISA results for the antibodies are shown in Figures 13-15, respectively. Antibodies TB-A through TB-H all bind to the human 4-1BB protein.

[0150] Example 4 Construction of Antibody Binding Activity at Both Ends (TNFR2+4-1BB)

[0151] Human 4-1BB Fc (Manufacturer: Acro, CAT: 41B-H5258, LOT: 198-2146F1-W6) was diluted to 0.3 μg / mL using coating solution (1× PBS, pH 7.4) and coated onto a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating solution and wash the plate with 1× PBST (300 μL / well) at 300 μL per well. Wash four times using a plate washer and pat dry on a tissue paper. Block with 3% skim milk powder at 300 μL / well and incubate at 37°C for 1 hour. Discard the blocking solution, wash four times using a plate washer, and pat dry on a tissue paper. A negative control was established using commercially available trastuzumab for injection (Herceptin). A three-fold dilution was performed starting at 50 nM, resulting in a total of 10 dilutions. A blank well was also prepared, containing only the diluent. Add 100 μL / well of the diluent 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 human-TNFR2-His (Manufacturer: Sino, CAT: 10417-H08H, LOT: LC15NO0412) to 0.3 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 1:5000 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. 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 16 and 17, respectively. The results show that antibodies TB-A to TB-H all have binding activity at both ends.

[0152] Example 5 Construction of Antibody Binding Activity at Both Ends (TNFR2+4-1BB)

[0153] Human TNFR2mFc (Manufacturer: Kaixia, CAT: TN-HM3R2, LOT: 031202) was diluted to 0.3 μg / mL using 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 using a plate washer. Discard the blocking solution, wash the plate four times using a plate washer, and pat dry on a tissue paper. Block the plate 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 using a plate washer, and pat dry on a tissue paper. A negative control was established using commercially available trastuzumab for injection (Herceptin). Three-fold dilutions were performed starting at 50 nM, resulting in a total of 10 dilutions. A blank well was also prepared, containing only the diluent. Incubate at 37°C for 1 hour using 100 μL / well of the diluent. Discard the liquid from the wells, wash the plates four times with a plate washer, and pat dry on paper. Dilute human 4-1BB His (Manufacturer: Acro, CAT: 41B-H5258, LOT: 198-2146F1-W6) to 0.3 μg / mL, add 100 μL to each well, incubate at room temperature for 1 hour, then wash the plates three times with PBST. Then, dilute HRP-labeled His antibody 1:5000 in sample diluent, add 100 μL to each well, and incubate at room temperature for 1 hour. Wash the plates six times with a plate washer, and pat dry on paper. Add TMB colorimetric solution (100 μL / well), wrap with aluminum foil, and develop at 37°C in the dark for 8 minutes. Add 100 μL / well of 1M HCl stop solution to terminate the color reaction. Read at 450 nm on a microplate reader. The ELISA results of the antibody molecules are shown in Figures 18 and 19, respectively, and the results show that antibodies TB-A to TB-H all have binding activity at both ends.

[0154] Example 6 BLI method to detect the affinity of antibodies to TNFR2 protein (TNFR2+4-1BB)

[0155] The variable region of the positive control antibody sequence consists of SEQ ID NO: 25. 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 immobilization of the antibody in the sample plate, and a second equilibration buffer for 180 seconds. 100 nM human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC15NO0412) 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 5. The results show that both antibody TB-C1 and antibody TB-D1 have affinity for TNFR2 protein.

[0156] Table 5 Affinity of antibodies to TNFR2 protein

[0157] Example 7 BLI method to detect the affinity of antibodies to 4-1BB protein (TNFR2+4-1BB)

[0158] The amino acid sequences of the antibody variable regions in the positive control consist of SEQ ID NO:1 and SEQ ID NO:5, with the addition of the human IgG1 constant region (see SEQ ID NO:54 and SEQ ID NO:55). 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 immobilization of the antibody in the sample plate, and a secondary equilibration buffer for 180 seconds. 100 nM human 4-1BB His (Manufacturer: Acro, CAT: 41B-H5258, LOT: 198-2146F1-W6) protein was bound 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 regenerated with 10 mM glycine (pH 2.0). The affinity results of the antibody molecules are shown in Table 6. The results show that both antibody TB-C1 and antibody TB-D1 have affinity for the 4-1BB protein.

[0159] Table 6 Affinity of antibodies to TNFR2 protein

[0160] Example 8 HEK-293 / NFκB-Luci / 4-1BB cell activation experiment (TNFR2+4-1BB)

[0161] The variable region of the positive control antibody sequence consists of SEQ ID NO: 1 and SEQ ID NO: 5, with the constant region of human IgG1 added (see SEQ ID NO: 54 and SEQ ID NO: 55). The actual initial concentration of the antibody was 60 nM, 100 μL per well, 4-fold dilution, 8 gradients. HEK-293 / NFκB-Luci / 4-1BB effector cells (from the cell bank of Shenghe (China) Biopharmaceutical Co., Ltd.) were added to the plate, 3×10 4 / well, 40 μL per well; CHOK1-TNFR2 target cells were added to the plate at a cell density of 1×10 4 40 μL per well; 20 μL of diluted antibody was added to the plate. After loading, the 96-well black plate was placed in a 37°C incubator and incubated for 20 hours. Lumiescence fluorescent agent was added at 100 μL per well. The results are shown in Figure 20, indicating that both antibodies TB-C1 and TB-D1 activated HEK-293 / NFκB-Luci / 4-1BB effector cells.

[0162] Example 9 Pharmacodynamic Study of Each Test Substance in a Tumor Model of MC38 Mouse Colon Cancer Cell Line Subcutaneously Transplanted in C57BL / 6-h4-1BB / hTNFR2 Mice (TNFR2+4-1BB)

[0163] Female C57BL / 6-h4-1BB / hTNFR2 mice aged 6-7 weeks (from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with MC38 tumor cells (from the cell bank of Shenghe (China) Biopharmaceutical Co., Ltd.). 3 The mice were randomly divided into four groups. The groups included: (1) G1: PBS group; (2) G2: control antibody group, the sequence variable region consisted of SEQ ID NO: 1 and SEQ ID NO: 5, and the constant region of human IgG1 was added (see SEQ ID NO: 54 and SEQ ID NO: 55); (3) G3: antibody TB-C2 group; (4) G4: antibody TB-D2 group. The negative control group was intratumorally administered with PBS, and the samples of the other groups were intratumorally administered with 10 mg / kg. The frequency of administration was twice a week, and the administration was continued for 4 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 7.

[0164] Table 7 Experimental design

[0165] The results are shown in Figure 21, which show that antibody TB-D2 has a significant anti-tumor effect.

[0166] Example 10 Antibody ADCC Activity on CHO-4-1BB Cells (TNFR2+4-1BB)

[0167] A negative control (IgG1 isotype control) and a reference control (the variable region of the antibody sequence consists of SEQ ID NO: 1 and SEQ ID NO: 5, and the constant region of human IgG4 is added, see SEQ ID NO: 55 and SEQ ID NO: 56) were set up. CHO-4-1BB cells overexpressing human 4-1BB (from Hefei Hanke Maibo) 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 the suspension was 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 60nM, followed by a 5-fold gradient dilution, for a total of 7 concentration gradients, 100μL / well; the NK cells were resuspended and added to the corresponding wells at 50μL / well, with an effector-target ratio of 3:1. 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, centrifuged at 1000rpm at room temperature for 4 minutes and incubated in a 5% CO2, 37℃ 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.

[0168] 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.

[0169] As shown in FIG22 , both antibody TB-C1 and antibody TB-D1 showed significant lytic and cell death effects on CHO-4-1BB cells in a concentration-dependent manner.

[0170] Example 11 Flow cytometry detection of antibody binding activity to CCR8-CHO-K1 cells (TNFR2+CCR8)

[0171] CCR8-CHO-K1 cells overexpressing human CCR8 were constructed by lentiviral transduction of CHO cells. The CCR8 antigen sequence is shown in SEQ ID NO: 57. Flow cytometry was used to detect the binding activity of the antibody to CCR8-CHO-K1 cells.

[0172] Take CCR8-CHO-K1 cells with normal morphology in logarithmic growth phase, transfer them to centrifuge tube and centrifuge at 1000 rpm for 5 min, resuspend the cells in diluent, and take 1×10 5 / well was added to a 96-well cell culture plate. The purified antibody was diluted to 120nM with FACS buffer. This was used as the starting concentration and a 5-fold serial dilution was performed for a total of 6 steps. An irrelevant antibody negative control and a positive control (the variable region of the antibody sequence consisted of SEQ ID NO: 9 and SEQ ID NO: 13, and the constant region of human IgG1 was added, see SEQ ID NO: 54 and SEQ ID NO: 55) were set up, and 100μL of antibody diluent was added. The cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100μL FACS buffer and the fluorescent secondary antibody against human IgG FC - APC (Biolegend, Cat: 109306) was added to the sample, incubated for 30 minutes and washed twice with excess FACS buffer. The cells were resuspended in flow cytometry buffer and then detected and analyzed by flow cytometry. The FACS method was used to detect the binding activity of the antibody to CCR8-CHO-K1 cells.

[0173] The FACs test results of the antibody binding activity to CCR8-CHO-K1 cells are shown in Figure 23. Antibodies TR-A to TR-H can specifically bind to CCR8-CHO-K1 cells in multiple concentration ranges, indicating that antibodies TR-A to TR-H have CCR8 binding activity.

[0174] Example 12 Flow cytometry detection of antibody binding activity to TNFR2-CHO-K1 cells (TNFR2+CCR8)

[0175] TNFR2-CHO-K1 cells overexpressing human TNFR2 were constructed by lentiviral transduction of CHO cells. The TNFR2 antigen sequence is shown in SEQ ID NO: 58. Flow cytometry was used to detect the binding activity of the antibody to TNFR2-CHO-K1 cells.

[0176] Take the TNFR2-CHO-K1 cells with normal morphology in logarithmic growth phase, transfer them to a centrifuge tube and centrifuge at 1000 rpm for 5 min. After resuspending the cells in diluent, take 1×10 5 The purified antibody was diluted to 120 nM in FACS buffer. This was used as the starting concentration for a 6-fold serial dilution, resulting in seven steps. A negative control and a positive control (an unrelated antibody sequence consisting of SEQ ID NO: 52 and SEQ ID NO: 53, with the human IgG1 constant region added, see SEQ ID NO: 54 and SEQ ID NO: 55) were also prepared. 100 μL of antibody dilution was then added. The cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL of FACS buffer, and a fluorescent secondary antibody against human IgG FC, APC (Biolegend, Cat: 109306), was added to the sample, incubated for 30 minutes, and washed twice with excess FACS buffer. The cells were resuspended in flow cytometry buffer and analyzed by flow cytometry. Antibody binding activity to TNFR2-CHO-K1 cells was determined by FACS.

[0177] The FACs test results of the antibody binding activity to TNFR2-CHO-K1 cells are shown in Figure 24. Antibodies TR-A to TR-H can specifically bind to TNFR2-CHO-K1 cells in multiple concentration ranges, indicating that antibodies TR-A to TR-H have TNFR2 binding activity.

[0178] Example 13 Antibody Dual-Target Binding Activity (TNFR2+CCR8)

[0179] Take CCR8-CHO-K1 cells (from the same source) in logarithmic phase and normal in morphology, transfer them to a centrifuge tube and centrifuge at 1000 rpm for 5 min. Resuspend the cells in diluent and take 1×10 550 μL of purified antibody was diluted to 300 nM in FACS buffer, and 50 μL of human TNFR2-His protein (Sino Biological, CAT: 10417-H08H) diluted to 900 nM was added to the cells. A negative control group 1 (PBS group), a negative control 2 (the variable region of the antibody sequence consisted of SEQ ID NO: 9 and SEQ ID NO: 13, with the constant region of human IgG1 added, see SEQ ID NO: 54 and SEQ ID NO: 55), and a negative control 3 (the antibody sequence consisted of SEQ ID NO: 52 and SEQ ID NO: 53, with the constant region of human IgG1 added, see SEQ ID NO: 54 and SEQ ID NO: 55) were set up. After the addition of the sample, the cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL FACS buffer and a FITC-anti-His tag (Abcam, Cat: ab1206) fluorescent secondary antibody was added to the sample. The cells were incubated for 30 minutes and washed twice with excess FACS buffer. The cells were resuspended in flow cytometry buffer and analyzed by flow cytometry.

[0180] The results of FACS detection of the binding of antibodies TR-A to TR-H to both ends of CCR8 and TNFR2 are shown in Table 8. Compared with the negative control, antibodies TR-A to TR-H have binding activity at both ends, indicating that antibodies TR-A to TR-H can bind to CCR8 and TNFR2 simultaneously, among which antibodies TR-E and TR-C have the best binding activity at both ends.

[0181] Table 8 Average fluorescence values ​​of antibody binding at both ends

[0182] Example 14 ADCC activity of antibodies against CCR8-CHO-K1 cells (TNFR2+CCR8)

[0183] 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: 54 and SEQ ID NO: 55) were set up. CCR8-CHO-K1 cells overexpressing human CCR8 (source as above) 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 1×10 450 μL / well were plated in a 96-well plate. Antibody was diluted in RPMI1640 basal medium (containing 5% FBS) to a starting concentration of 60 μg / mL, followed by a 10-fold serial dilution series of seven concentrations at 100 μL / well. NK cells were resuspended and added to the corresponding wells at 50 μL / well, with an effector-target ratio of 3:1. Wells for maximum target cell lysis (M), spontaneous release of target cells (ST), spontaneous release of effector cells (SE), a total volume correction blank well (BV), and a medium blank control well (BM) were also set. After 10 minutes of quiescence, the wells were centrifuged at 1000 rpm for 4 minutes at room temperature and incubated for 4 hours in a 5% CO2, 37°C CO2 cell culture incubator. Lysis buffer was added to wells M and BV 45 minutes in advance, mixed thoroughly, and centrifuged at 1000 rpm for 4 minutes at the end of the incubation period. 50 μL of supernatant was transferred to the LDH assay plate, and 50 μL / well of substrate dissolved in assay buffer was added. The wells were reacted at room temperature in the dark for 30 minutes. Add 50 μL / well stop solution, let it stand for 10 minutes, read at 490 nm, and calculate the cell death rate.

[0184] 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.

[0185] As shown in Figures 25 and 26, the negative control showed no killing of CCR8-CHO-K1 cells, while the positive control and antibodies TR-A to TR-H showed lysis and cell death of CCR8-CHO-K1 cells in a concentration-dependent manner.

[0186] Example 15 ELSIA determination of the blocking activity of antibodies against TNFR2 (TNFR2+CCR8)

[0187] Human-TNFR2-mFC (CAT: TNF-HM3R2) was diluted to 3.5 μg / mL with coating solution (1× PBS, pH 7.4) and coated into a 96-well microtiter plate at 100 μL / well at 4°C overnight. The coating solution was discarded, and the plate was washed with 1× PBST (300 μL / well) for 4 minutes using a plate washer. The plate was then patted dry on a paper plate. The plate was blocked with 3% skim milk powder (300 μL / well) and incubated at 37°C for 1 hour. The blocking solution was discarded, and the plate was washed 4 times using a plate washer. The plate was then patted dry on a paper plate. Negative and positive controls were set up. The negative control was commercially available trastuzumab for injection (Herceptin), and the positive control antibody consisted of the variable region sequence of SEQ ID NO: 25. The positive control and antibody were diluted to 100 nM with 3% skim milk powder. Three-fold dilutions were performed from this initial concentration, for a total of 11 dilutions. A blank well was set up, containing only the diluent. Add 50 μL / well of each of the antibody and human TNF alpha His protein (novoprotein, Cat: C008) diluted to 1 μg / mL to an ELISA plate coated with human-TNFR2-mFC protein and incubate at 37°C for 1 hour. Discard the liquid from the wells, wash four times with a plate washer, and pat dry on paper. Dilute anti-His antibody (Abcam, Cat: ab1187) 1:5000 with 3% nonfat dry milk and add 100 μL / well. Incubate at 37°C for 1 hour. Wash 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. Stop the reaction with 100 μL / well of 1M HCl. Read on a microplate reader at 450 nm.

[0188] The ELISA results of antibody molecules blocking TNFR2-TNF are shown in Figures 27 and 28. The results show that antibodies TR-C, TR-D, TR-G and TR-H can all block the binding of TNFR2 and TNF.

[0189] Example 16 CD8+ T cell activation experiment (TNFR2+CCR8)

[0190] Dilute the antibody to 40 μg / mL in PBS (final experimental concentration 20 μg / mL, 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:52 and SEQ ID NO:53, and the constant region of human IgG1 (see SEQ ID NO:54 and SEQ ID NO:55) was added. 50 μL of OKT3 dilution and 50 μL of antibody dilution were coated into 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: PCH20201200031), and the purity of the negative CD8+ T cells was tested. CD8+ T cells were labeled with CFSE at a concentration of 5 μM, incubated at 37°C for 10 min, and complete medium was added to terminate the staining. The plate used for coating was washed once with PBS and 2×10 5 / well, 100 μL / well added CFSE-labeled or unlabeled T cells, and added anti-CD28 at a final concentration of 1 μg / mL, 100 μL / well. The total experimental system was 200 μL / well. Incubate at 37°C for 96 hours; collect the supernatant after centrifugation for detection of IFN-γ and IL2 cytokines, and wash the cells with PBS before flow cytometry detection. The results are shown in Figure 29. Antibodies TR-A, TR-E, TR-B, and TR-F all have an activating effect on CD8+ T cells. Compared with the negative control, they have the ability to promote CD8+ T cells to release more IFN-γ.

[0191] 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; wherein the first antigen is a T cell immunomodulator and the second antigen is TNFR2.

2. The bispecific antibody according to claim 1, characterized in that The T cell immunomodulator is a T cell co-stimulatory immunomodulator or a T cell co-inhibitory immunomodulator.

3. The bispecific antibody according to claim 1 or 2, 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.

4. The bispecific antibody according to any one of claims 1 to 3, 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.

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

6. The bispecific antibody according to claim 5, 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.

7. The bispecific antibody according to claim 5, characterized in that It comprises a first antibody or an antigen-binding fragment thereof and two of the scFvs or two of the VHHs.

8. The bispecific antibody according to claim 7, 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.

9. The bispecific antibody according to claim 8, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein: 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.

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

11. The bispecific antibody according to any one of claims 2 to 10, characterized in that The T cell co-stimulatory immunomodulator is selected from 4-1BB, OX40, B7H2, GITR, CD48, HVEM, Nectin-2, CD40, CD30, CD28, CD27, B7-1, B7-2, LIGHT, B7H6, 2B4, CD28H or NKp30.

12. The bispecific antibody according to any one of claims 2 to 10, characterized in that The T cell co-inhibitory immunomodulator is selected from CCR8, PD-1, PD-L1, PD-L2, B7H3, B7H4, B7H5, BLTA, CD96, CD47, CD155, CTLA-4, LAG-3, TIGIT, TIM-3, CD111, DNAM-1, Galectin-9, Nectin-3, PVRIG, SIRPα, SIRPβ, SIRPαV2 or CD160.

13. The bispecific antibody according to any one of claims 3 to 12, characterized in that The heavy chain variable region and the light chain variable region of the scFv are connected by a linker L1.

14. The bispecific antibody according to any one of claims 3 to 13, 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.

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

16. The bispecific antibody according to any one of claims 13 to 15, 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.

17. The bispecific antibody according to any one of claims 1 to 16, 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.

18. The bispecific antibody according to claim 17, 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.

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

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

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

22. The bispecific antibody according to claim 20 or 21, 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.

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

8.

24. The bispecific antibody according to any one of claims 1 to 22, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to CCR8, 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.

25. The bispecific antibody according to claim 23 or 24, characterized in that The scFv specifically binds to TNFR2, and the HCDR1 of the scFv is set forth in SEQ ID NO:18, or a sequence at least 80% identical to SEQ ID NO:18; HCDR2 is set forth in SEQ ID NO:19, or a sequence at least 80% identical to SEQ ID NO:19; HCDR3 is set forth in SEQ ID NO:20, or a sequence at least 80% identical to SEQ ID NO:20; LCDR1 is set forth in SEQ ID NO:22, or a sequence at least 80% identical to SEQ ID NO:22; LCDR2 is set forth in SEQ ID NO:23, or a sequence at least 80% identical to SEQ ID NO:23; and LCDR3 is set forth in SEQ ID NO:24, or a sequence at least 80% identical to SEQ ID NO:

24.

26. The bispecific antibody according to claim 23 or 24, characterized in that The VHH specifically binds to TNFR2, and the HCDR1 of the VHH is shown in SEQ ID NO:26, or a sequence with at least 80% identity to SEQ ID NO:26; HCDR2 is shown in SEQ ID NO:27, or a sequence with at least 80% identity to SEQ ID NO:27; and HCDR3 is shown in SEQ ID NO:28, or a sequence with at least 80% identity to SEQ ID NO:

28.

27. The bispecific antibody according to any one of claims 1 to 22, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to 4-1BB, 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.

28. The bispecific antibody according to any one of claims 1 to 22, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to CCR8, 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.

29. The bispecific antibody according to claim 27 or 28, 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: 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.

30. The bispecific antibody according to claim 27 or 28, characterized in that The VHH specifically binds to TNFR2, and the VHH heavy chain variable region VH is shown in SEQ ID NO: 25, or a sequence having at least 80% identity with SEQ ID NO:

25.

31. The bispecific antibody according to any one of claims 9 to 30, characterized in that The first polypeptide chain of the bispecific antibody is selected from any one of the amino acid sequences of SEQ ID NOs: 30-34, 42-46, or an amino acid sequence that is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 30-34, 42-46; the second polypeptide chain of the bispecific antibody is selected from any one of the amino acid sequences of SEQ ID NOs: 35-41, 47-51, or an amino acid sequence that is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 35-41, 47-51.

32. The bispecific antibody according to claim 31, characterized in that The bispecific antibody comprises: (1) the first polypeptide chain shown in SEQ ID NO: 30, and the second polypeptide chain shown in SEQ ID NO: 35; or (2) the first polypeptide chain as shown in SEQ ID NO: 31, the second polypeptide chain as shown in SEQ ID NO: 36; or (3) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 37; or (4) the first polypeptide chain shown in SEQ ID NO: 32, the second polypeptide chain shown in SEQ ID NO: 36; or (5) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 38; or (6) the first polypeptide chain shown in SEQ ID NO: 30, the second polypeptide chain shown in SEQ ID NO: 40; or (7) the first polypeptide chain as shown in SEQ ID NO: 33, the second polypeptide chain as shown in SEQ ID NO: 36; or (8) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 39; or (9) a first polypeptide chain as shown in SEQ ID NO: 30, and a second polypeptide as shown in SEQ ID NO: 41 chain; or (10) the first polypeptide chain as shown in SEQ ID NO: 34, the second polypeptide chain as shown in SEQ ID NO: 36; or (11) the first polypeptide chain as shown in SEQ ID NO: 42, the second polypeptide chain as shown in SEQ ID NO: 48; or (12) a first polypeptide chain as shown in SEQ ID NO: 43, a second polypeptide chain as shown in SEQ ID NO: 47; or (13) the first polypeptide chain as shown in SEQ ID NO:42, the second polypeptide chain as shown in SEQ ID NO:49; or (14) the first polypeptide chain as shown in SEQ ID NO: 44, the second polypeptide chain as shown in SEQ ID NO: 47; or (15) the first polypeptide chain shown in SEQ ID NO:42, the second polypeptide chain shown in SEQ ID NO:50; or (16) the first polypeptide chain as shown in SEQ ID NO: 45, the second polypeptide chain as shown in SEQ ID NO: 47; or (17) the first polypeptide chain as shown in SEQ ID NO:42, the second polypeptide chain as shown in SEQ ID NO:51; or (18) The first polypeptide chain shown in SEQ ID NO:46, and the second polypeptide chain shown in SEQ ID NO:

47.

33. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1 to 32; 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 32; 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 32. A multifunctional fusion protein comprising the bispecific antibody according to any one of claims 1 to 32.

35. The multifunctional fusion protein according to claim 34, characterized in that One or more third antibodies or antigen-binding portions thereof that specifically bind to other antigens are also included.

36. The multifunctional fusion protein according to claim 35, 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 molecule.

37. The multifunctional fusion protein according to claim 36, 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, B7H2, B7H3, B7H5, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-ab1, BRCA1, BORIS, C A9, 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, EGFRvIII, ErbB2 / Her2, ErbB3, E rbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAG E-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-Nossin 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, B7H4, CD48, HVEM, Nectin-2, CD28, B7-1, B7-2, B7H6, 2B4, CD28H, NKp30, BLTA, CD96, CD155, C TLA-4, LAG-3, TIM-3, CD111, DNAM-1, Galectin-9, Nectin-3, PVRIG, SIRPα, SIRPβ, SIRPαV2 or CD160.

38. The multifunctional fusion protein according to any one of claims 34 to 37, characterized in that Also contains cytokines.

39. The multifunctional fusion protein according to claim 38, 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.

40. Use of the bispecific antibody according to any one of claims 1 to 32 or the multifunctional fusion protein according to any one of claims 34 to 39 in the preparation of a medicament for treating cancer.

41. The use according to claim 40, characterized in that The cancer is selected from the group consisting of glioblastoma, astrocytoma, cystic fibrosis, fibromyalgia, fibrosarcoma, fibromyalgia ... , 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.

42. Use of the bispecific antibody according to any one of claims 1 to 32 or the multifunctional fusion protein according to any one of claims 34 to 39 in the preparation of a medicament for treating an autoimmune disease.

43. The use according to claim 42, 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.

44. The use according to any one of claims 40 to 43, characterized in that The use is achieved through one or more of tumor immunotherapy, cell therapy or gene therapy.

45. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-32 and a pharmaceutically acceptable carrier, diluent or excipient.

46. ​​A pharmaceutical composition comprising the multifunctional fusion protein according to any one of claims 34 to 39 and a pharmaceutically acceptable carrier, diluent or excipient.

47. An antibody-drug conjugate comprising the bispecific antibody of any one of claims 1-32.

48. The antibody drug conjugate according to claim 47, characterized in that The conjugated drug is selected from cytotoxins, small molecule chemical drugs or immunotoxins.

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