Bispecific antibody combining TNFR2 and 4-1BB

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; (b) a second antibody or antigen-binding fragment thereof that specifically binds to a second antigen; wherein the first antigen is TNFR2 (Tumor Necrosis Factor Receptor 2), and the second antigen is 4-1BB; or, the first antigen is 4-1BB, and the second antigen is TNFR2 (Tumor Necrosis Factor Receptor 2).

Description

A bispecific antibody that binds to TNFR2 and 4-1BB Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a bispecific antibody that binds TNFR2 and 4-1BB and its application. Background Art

[0002] 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.

[0003] Currently, TNFR2 has been shown to enhance the activation of effector T cells (Teff) and reduce the inhibitory effects mediated by regulatory T cells (Treg). TNFR2 activation induces signaling through the mitogen-activated protein kinase (MAPK) signaling pathway, which coordinates the transcription of genes that promote evasion of apoptosis and cell proliferation mediated by TRAF2 / 3 signaling and NFκB. TNFR2 is expressed not only on cancer cells and tumor-infiltrating Tregs, but also on effector Teff cells. Studies have shown that TNFR2 agonist monoclonal antibodies can stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFNγ and IL2; TNFR2 antagonist monoclonal antibodies can block TNF-TNFR2 and inhibit Treg proliferation.

[0004] The body's immune response process requires the participation of multiple immune cells and immune molecules, among which the activation of T cells is the core of the immune response. Generally, two different signals are required for T cells to be fully activated: the first signal is generated by the combination of TCR expressed by T cells and antigens presented by the major histocompatibility complex (MHC). The second is various stimulation signals that activate or turn off the switch of T cell function. PD-L1 is a ligand that turns off T cell function. Among the ligands that activate T cell function, 4-1BBL (the ligand of 4-1BB) is an important member.

[0005] 4-1BB is a T cell co-stimulatory molecule and a key member of the tumor necrosis factor receptor (TNF) superfamily. It is primarily expressed on activated CD4+ and CD8+ T cells, as well as natural killer (NK) cells. 4-1BB is primarily expressed on activated antigen-presenting cells (APCs), such as monocytes, phagocytes, dendritic cells (DCs), and B cells. The interaction between 4-1BB and its ligand provides a secondary signal for T cell activation, independent of the CD28 signal. When the two interact, the co-stimulatory signal promotes T cell proliferation and activation and inhibits activation-induced apoptosis (AICD), a major type of programmed cell death (T cell) death, thereby enhancing the immune-killing function of T cells. Furthermore, it induces the activation of monocytes and dendritic cells, promoting cytokine secretion and enabling these cells to play a role in immune regulation. Therefore, signaling pathways involving 4-1BB are involved in immune regulation in autoimmune diseases, tumors, viral infections, transplant rejection, and inflammation. 4-1BB is also considered a promising target for enhancing anti-tumor immunity.

[0006] Summary of the Invention

[0007] In this application, the inventors have developed a bispecific antibody with good performance that can bind to TNFR2 and 4-1BB. The bispecific antibody of the present invention is a T cell regulator of the tumor microenvironment. It can use agonistic anti-TNFR2 monoclonal antibodies to stimulate the proliferation of CD8+T cells, activate CD8+T cells, and release anti-tumor factors such as IFNγ and IL2; TNFR2 antagonists block TNF-TNFR2 and inhibit Treg proliferation. The bispecific antibody of the present invention simultaneously targets 4-1BB, activates the 4-1BB signaling pathway of T cells and NK cells, enhances the killing ability of cytotoxic T cells and NK cells, and produces anti-tumor activity.

[0008] The present invention provides a bispecific antibody that can specifically bind to TNFR2 and 4-1BB, wherein 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 TNFR2 and the second antigen is 4-1BB; or, the first antigen is 4-1BB and the second antigen is TNFR2.

[0009] 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.

[0010] 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.

[0011] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more scFvs.

[0012] 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 or light chain of the first antibody or antigen-binding fragment thereof.

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

[0014] In some embodiments, the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and two of the scFvs.

[0015] 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.

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

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

[0018] In some embodiments, the two scFvs are respectively linked to the C-termini of the two light chains of the first antibody or antigen-binding fragment thereof.

[0019] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to TNFR2 and two scFvs that specifically bind to 4-1BB, wherein the two scFvs that specifically bind to 4-1BB are respectively linked to the N-termini of the two heavy chains of the first antibody.

[0020] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to TNFR2 and two scFvs that specifically bind to 4-1BB, wherein the two scFvs that specifically bind to 4-1BB are respectively linked to the C-termini of the two heavy chains of the first antibody.

[0021] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to TNFR2 and two scFvs that specifically bind to 4-1BB, wherein the two scFvs that specifically bind to 4-1BB are respectively linked to the N-termini of the two light chains of the first antibody.

[0022] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to TNFR2 and two scFvs that specifically bind to 4-1BB, wherein the two scFvs that specifically bind to 4-1BB are respectively linked to the C-termini of the two light chains of the first antibody.

[0023] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB 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.

[0024] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB 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.

[0025] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB 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 light chains of the first antibody.

[0026] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB 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 light chains of the first antibody.

[0027] 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.

[0028] In some embodiments, wherein 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 the scFv; and (b) the second polypeptide chains each independently comprise the heavy chain of the first antibody or antigen-binding fragment thereof.

[0029] 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.

[0030] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more of the VHHs.

[0031] 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 or light chain of the first antibody or antigen-binding fragment thereof.

[0032] 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 or light chain of the first antibody or antigen-binding fragment thereof.

[0033] In some embodiments, the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and two of the VHHs.

[0034] 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.

[0035] In some embodiments, the two VHHs are respectively linked to the N-termini of the two light chains of the first antibody or antigen-binding fragment thereof.

[0036] 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.

[0037] In some embodiments, the two VHHs are respectively linked to the C-termini of the two light chains of the first antibody or antigen-binding fragment thereof.

[0038] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB and two VHHs that specifically bind to TNFR2, wherein the two VHHs that specifically bind to TNFR2 are respectively connected to the N-termini of the two heavy chains of the first antibody.

[0039] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB and two VHHs that specifically bind to TNFR2, wherein the two VHHs that specifically bind to TNFR2 are respectively connected to the C-termini of the two heavy chains of the first antibody.

[0040] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB 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 light chains of the first antibody.

[0041] In some embodiments, the bispecific antibody comprises a first antibody that specifically binds to 4-1BB and two VHHs that specifically bind to TNFR2, wherein the two VHHs that specifically bind to TNFR2 are respectively connected to the C-termini of the two light chains of the first antibody.

[0042] 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.

[0043] 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 the VHH; and (b) the second polypeptide chains each independently comprise the heavy chain of the first antibody or antigen-binding fragment thereof.

[0044] 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.

[0045] In some embodiments, the heavy chain variable region and the light chain variable region of the scFv are connected by a linker L1.

[0046] In some embodiments, the scFv is linked to the N-terminus or C-terminus of the heavy chain or light chain of the first antibody or antigen-binding fragment thereof via a linker L2.

[0047] In some embodiments, the linker L1 and linker L2 are the same or different.

[0048] In some embodiments, 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.

[0049] 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.

[0050] 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 Fc region is selected from IgG, IgA, IgD, IgE, IgM and variants thereof. In some embodiments, the Fc region is selected from IgG1, IgG2, IgG3, IgG4 and variants thereof. In some embodiments, the Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

[0051] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds 4-1BB, wherein HCDR1 of the first antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 16, or a sequence at least 80% identical to SEQ ID NO: 16; HCDR2 is as set forth in SEQ ID NO: 17, or a sequence at least 80% identical to SEQ ID NO: 17; HCDR3 is as set forth in SEQ ID NO: 18, or a sequence at least 80% identical to SEQ ID NO: 18; LCDR1 is as set forth in SEQ ID NO: 20, or a sequence at least 80% identical to SEQ ID NO: 20; LCDR2 is as set forth in SEQ ID NO: 21, or a sequence at least 80% identical to SEQ ID NO: 21; and LCDR3 is as set forth in SEQ ID NO: 22, or a sequence at least 80% identical to SEQ ID NO: 22.

[0052] In some embodiments, the scFv specifically binds TNFR2, and the scFv has HCDR1 as set forth in SEQ ID NO:2, or a sequence at least 80% identical to SEQ ID NO:2; HCDR2 as set forth in SEQ ID NO:3, or a sequence at least 80% identical to SEQ ID NO:3; HCDR3 as set forth in SEQ ID NO:4, or a sequence at least 80% identical to SEQ ID NO:4; and LCDR1 as set forth in SEQ ID NO:6, or a sequence at least 80% identical to SEQ ID NO:6; LCDR2 as set forth in SEQ ID NO:7, or a sequence at least 80% identical to SEQ ID NO:7; and LCDR3 as set forth in SEQ ID NO:8, or a sequence at least 80% identical to SEQ ID NO:8.

[0053] In some embodiments, the VHH specifically binds to TNFR2, and the HCDR1 of the VHH is set forth in SEQ ID NO: 12, or a sequence at least 80% identical to SEQ ID NO: 12; the HCDR2 is set forth in SEQ ID NO: 13, or a sequence at least 80% identical to SEQ ID NO: 13; and the HCDR3 is set forth in SEQ ID NO: 14, or a sequence at least 80% identical to SEQ ID NO: 14.

[0054] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to 4-1BB, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 16; the HCDR2 is set forth in SEQ ID NO: 17; the HCDR3 is set forth in SEQ ID NO: 18; the LCDR1 is set forth in SEQ ID NO: 20; the LCDR2 is set forth in SEQ ID NO: 21; and the LCDR3 is set forth in SEQ ID NO: 22. The scFv specifically binds to TNFR2, wherein the HCDR1 of the scFv is set forth in SEQ ID NO: 2; the HCDR2 is set forth in SEQ ID NO: 3; the HCDR3 is set forth in SEQ ID NO: 4; the LCDR1 is set forth in SEQ ID NO: 6; the LCDR2 is set forth in SEQ ID NO: 7; and the LCDR3 is set forth in SEQ ID NO: 8.

[0055] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to 4-1BB, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 16; the HCDR2 is set forth in SEQ ID NO: 17; the HCDR3 is set forth in SEQ ID NO: 18; the LCDR1 is set forth in SEQ ID NO: 20; the LCDR2 is set forth in SEQ ID NO: 21; and the LCDR3 is set forth in SEQ ID NO: 22. The VHH specifically binds to TNFR2, wherein the HCDR1 of the VHH is set forth in SEQ ID NO: 12; the HCDR2 is set forth in SEQ ID NO: 13; and the HCDR3 is set forth in SEQ ID NO: 14.

[0056] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to TNFR2, wherein HCDR1 of the first antibody or antigen-binding fragment thereof is set forth in SEQ ID NO:2, or a sequence at least 80% identical to SEQ ID NO:2; HCDR2 is set forth in SEQ ID NO:3, or a sequence at least 80% identical to SEQ ID NO:3; HCDR3 is set forth in SEQ ID NO:4, or a sequence at least 80% identical to SEQ ID NO:4; LCDR1 is set forth in SEQ ID NO:6, or a sequence at least 80% identical to SEQ ID NO:6; LCDR2 is set forth in SEQ ID NO:7, or a sequence at least 80% identical to SEQ ID NO:7; and LCDR3 is set forth in SEQ ID NO:8, or a sequence at least 80% identical to SEQ ID NO:8.

[0057] In some embodiments, the scFv specifically binds 4-1BB, and the HCDR1 of the scFv is set forth in SEQ ID NO: 16, or a sequence at least 80% identical to SEQ ID NO: 16; HCDR2 is set forth in SEQ ID NO: 17, or a sequence at least 80% identical to SEQ ID NO: 17; HCDR3 is set forth in SEQ ID NO: 18, or a sequence at least 80% identical to SEQ ID NO: 18; LCDR1 is set forth in SEQ ID NO: 20, or a sequence at least 80% identical to SEQ ID NO: 20; LCDR2 is set forth in SEQ ID NO: 21, or a sequence at least 80% identical to SEQ ID NO: 21; and LCDR3 is set forth in SEQ ID NO: 22, or a sequence at least 80% identical to SEQ ID NO: 22.

[0058] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to TNFR2, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 2; the HCDR2 is set forth in SEQ ID NO: 3; the HCDR3 is set forth in SEQ ID NO: 4; the LCDR1 is set forth in SEQ ID NO: 6; the LCDR2 is set forth in SEQ ID NO: 7; and the LCDR3 is set forth in SEQ ID NO: 8. The scFv specifically binds to 4-1BB, wherein the HCDR1 of the scFv is set forth in SEQ ID NO: 16; the HCDR2 is set forth in SEQ ID NO: 17; the HCDR3 is set forth in SEQ ID NO: 18; the LCDR1 is set forth in SEQ ID NO: 20; the LCDR2 is set forth in SEQ ID NO: 21; and the LCDR3 is set forth in SEQ ID NO: 22.

[0059] 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: 15, or a sequence having at least 80% identity with SEQ ID NO: 15; the light chain variable region VL is as shown in SEQ ID NO: 19, or a sequence having at least 80% identity with SEQ ID NO: 19.

[0060] In some embodiments, the scFv specifically binds to TNFR2, and the heavy chain variable region VH of the scFv is set forth in SEQ ID NO: 9, or a sequence at least 80% identical to SEQ ID NO: 9; the light chain variable region VL is set forth in SEQ ID NO: 10, or a sequence at least 80% identical to SEQ ID NO: 10.

[0061] In some embodiments, the VHH specifically binds to TNFR2, and the VHH heavy chain variable region VH is as shown in SEQ ID NO: 11, or a sequence that is at least 80% identical to SEQ ID NO: 11.

[0062] In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds to TNFR2, 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 to SEQ ID NO: 1; and the light chain variable region VL is as shown in SEQ ID NO: 5, or a sequence having at least 80% identity to SEQ ID NO: 5.

[0063] In some embodiments, the scFv specifically binds to 4-1BB, and the scFv heavy chain variable region VH is shown in SEQ ID NO: 23, or a sequence having at least 80% identity with SEQ ID NO: 23; the light chain variable region VL is shown in SEQ ID NO: 24, or a sequence having at least 80% identity with SEQ ID NO: 24.

[0064] In some embodiments, the first polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 26-33, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 26-33; the second polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 35-45, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 35-45.

[0065] In some embodiments, the bispecific antibody comprises:

[0066] (1) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 35; or

[0067] (2) the first polypeptide chain as shown in SEQ ID NO: 27, the second polypeptide chain as shown in SEQ ID NO: 36; or

[0068] (3) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 37; or

[0069] (4) the first polypeptide chain as shown in SEQ ID NO: 28, the second polypeptide chain as shown in SEQ ID NO: 36; or

[0070] (5) the first polypeptide chain as shown in SEQ ID NO: 29, the second polypeptide chain as shown in SEQ ID NO: 38; or

[0071] (6) the first polypeptide chain as shown in SEQ ID NO: 29, the second polypeptide chain as shown in SEQ ID NO: 43; or

[0072] (7) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 39; or

[0073] (8) the first polypeptide chain as shown in SEQ ID NO: 29, the second polypeptide chain as shown in SEQ ID NO: 40; or

[0074] (9) the first polypeptide chain as shown in SEQ ID NO: 31, the second polypeptide chain as shown in SEQ ID NO: 39; or

[0075] (10) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 41; or

[0076] (11) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 44; or

[0077] (12) the first polypeptide chain as shown in SEQ ID NO: 32, the second polypeptide chain as shown in SEQ ID NO: 36; or

[0078] (13) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 42; or

[0079] (14) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 45; or

[0080] (15) The first polypeptide chain shown in SEQ ID NO: 33, and the second polypeptide chain shown in SEQ ID NO: 36.

[0081] The present invention also provides a bispecific antibody that can specifically bind to TNFR2 and 4-1BB, wherein 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 TNFR2 and the second antigen is 4-1BB.

[0082] 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.

[0083] In some embodiments, the Fab region of the first antibody or antigen-binding fragment thereof is replaced with a VHH that is capable of specifically binding to TNFR2.

[0084] In some embodiments, it comprises a first antibody or antigen-binding fragment thereof and one or more of said scFvs.

[0085] In some embodiments, it comprises a first antibody or an 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 the antigen-binding fragment thereof.

[0086] In some embodiments, it comprises a first antibody or antigen-binding fragment thereof and two of the scFvs.

[0087] 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.

[0088] In some embodiments, the bispecific antibody comprises two polypeptide chains, characterized in that each of the polypeptide chains independently comprises the first antibody or antigen-binding fragment thereof and the scFv.

[0089] In some embodiments, the two polypeptide chains are the same or different.

[0090] In some embodiments, the scFv is linked to the C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof via linker L3.

[0091] In some embodiments, the linker L3 has a structure 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.

[0092] 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 Fc region is selected from IgG, IgA, IgD, IgE and / or IgM. In some embodiments, the Fc region is selected from IgG1, IgG2, IgG3 and / or IgG4. In some embodiments, the Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

[0093] In some embodiments, the VHH that specifically binds to TNFR2 has a HCDR1 as shown in SEQ ID NO: 12, or a sequence at least 80% identical to SEQ ID NO: 12; a HCDR2 as shown in SEQ ID NO: 13, or a sequence at least 80% identical to SEQ ID NO: 13; and a HCDR3 as shown in SEQ ID NO: 14, or a sequence at least 80% identical to SEQ ID NO: 14.

[0094] In some embodiments, the scFv specifically binds 4-1BB, and the HCDR1 of the scFv is set forth in SEQ ID NO: 16, or a sequence at least 80% identical to SEQ ID NO: 16; HCDR2 is set forth in SEQ ID NO: 17, or a sequence at least 80% identical to SEQ ID NO: 17; HCDR3 is set forth in SEQ ID NO: 18, or a sequence at least 80% identical to SEQ ID NO: 18; LCDR1 is set forth in SEQ ID NO: 20, or a sequence at least 80% identical to SEQ ID NO: 20; LCDR2 is set forth in SEQ ID NO: 21, or a sequence at least 80% identical to SEQ ID NO: 21; and LCDR3 is set forth in SEQ ID NO: 22, or a sequence at least 80% identical to SEQ ID NO: 22.

[0095] In some embodiments, the VHH that specifically binds to TNFR2 has a heavy chain variable region VH as shown in SEQ ID NO: 11, or a sequence that has at least 80% identity to SEQ ID NO: 11.

[0096] In some embodiments, the scFv specifically binds to 4-1BB, and the scFv heavy chain variable region VH is shown in SEQ ID NO: 23, or a sequence having at least 80% identity with SEQ ID NO: 23; the light chain variable region VL is shown in SEQ ID NO: 24, or a sequence having at least 80% identity with SEQ ID NO: 24.

[0097] In some embodiments, the polypeptide chain of the bispecific antibody is selected from any one of the amino acid sequences of SEQ ID NOs: 34, 48-55, or a sequence that is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 34, 48-55.

[0098] 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 a polypeptide chain of any of the above-described bispecific antibodies.

[0099] The present invention provides a multifunctional fusion protein comprising any of the above-mentioned bispecific antibodies.

[0100] 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.

[0101] In some embodiments, the antigen that binds to the third antibody or antigen-binding portion thereof is selected from a tumor-associated antigen (TAA) or an immune checkpoint.

[0102] In some embodiments, the antigen that binds to the third antibody or its antigen-binding portion is selected from GPC3, CD19, CD20 (MS4A1), CD22, 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, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her 2. 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, kappa-Light, LeY, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-12, MART-1, mesothelin, ML-IAP, MOv-γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16, MUM1, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Tang-Nordstein antigen, TRP-1, TRP-2, tyrosinase and urokinin-3, 5T4, PD-L1, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, ILT4, TREM2, LAG3, CD27, CD24, B7H3, or B7H4.

[0103] 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.

[0104] The present invention also provides use of any of the above-mentioned bispecific antibodies or any of the above-mentioned multifunctional fusion proteins in the preparation of a drug for treating cancer.

[0105] In some embodiments, the cancer is selected from the group consisting of 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.

[0106] Use of any of the above-mentioned bispecific antibodies or any of the above-mentioned multifunctional fusion proteins in the preparation of a medicament for treating an autoimmune disease.

[0107] 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.

[0108] In some embodiments, the use is achieved by one or more of tumor immunotherapy, cell therapy, or gene therapy.

[0109] The present invention also provides a pharmaceutical composition comprising any of the above-described bispecific antibodies and a pharmaceutically acceptable carrier, diluent or excipient.

[0110] 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.

[0111] The present invention also provides an antibody-drug conjugate comprising any of the above-mentioned bispecific antibodies.

[0112] In some embodiments, the conjugated drug is selected from a cytotoxin, a small molecule chemical drug, or an immunotoxin.

[0113] Abbreviations and Definitions

[0114] The following abbreviations are used herein: VH: antibody heavy chain variable region; VL: antibody light chain variable region; CDR: complementarity determining region in an immunoglobulin variable region; FR: antibody framework region, i.e., amino acid residues in an antibody variable region other than CDR residues; IgG: immunoglobulin G.

[0115] 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.

[0116] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell culture system.

[0117] 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.

[0118] 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".

[0119] 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.

[0120] 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.

[0121] The term "polypeptide" refers to an amino acid chain of any length, regardless of modifications (e.g., 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.

[0122] 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.

[0123] 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.

[0124] The term "Fab" is composed of an intact L chain and the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent for antigen binding, i.e., it has a single antigen binding site. For example, Fab fragments can be produced recombinantly or by papain digestion of a full-length antibody.

[0125] 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.

[0126] 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."

[0127] 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.

[0128] 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.

[0129] 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%.

[0130] There are a variety of methods / systems in the art to define and describe CDRs, and these systems and / or definitions have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on sequence variability based on the position of the structural loop region; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on the AbM antibody modeling software from Oxford Molecular, and is a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in many aspects. 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, 12, and 16, which starts at the fourth position after the first cysteine ​​in the heavy chain variable region. The length of HCDR1 is generally 10-12, ending at the amino acid before tryptophan.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] FIG1 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the C-termini of the two heavy chains of the full-length antibody via a linker.

[0138] FIG2 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the C-termini of the two light chains of the full-length antibody via a linker.

[0139] FIG3 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB 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.

[0140] FIG4 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB and a scFv capable of specifically recognizing TNFR2, wherein the scFv is connected to the N-termini of the two light chains of the full-length antibody via a linker.

[0141] FIG5 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing TNFR2 and a scFv capable of specifically recognizing 4-1BB, wherein the scFv is connected to the C-termini of the two heavy chains of the full-length antibody via a linker.

[0142] FIG6 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing TNFR2 and a scFv capable of specifically recognizing 4-1BB, wherein the scFv is connected to the C-termini of the two light chains of the full-length antibody via a linker.

[0143] FIG7 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing TNFR2 and a scFv capable of specifically recognizing 4-1BB, wherein the scFv is connected to the N-termini of the two heavy chains of the full-length antibody via a linker.

[0144] FIG8 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing TNFR2 and a scFv capable of specifically recognizing 4-1BB, wherein the scFv is connected to the N-termini of the two light chains of the full-length antibody via a linker.

[0145] FIG9 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB 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.

[0146] FIG10 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the C-termini of the two light chains of the full-length antibody via a linker.

[0147] FIG11 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB 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.

[0148] FIG12 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing 4-1BB and a VHH capable of specifically recognizing TNFR2, wherein the VHH is connected to the N-termini of the two light chains of the full-length antibody via a linker.

[0149] FIG13 depicts an exemplary bispecific antibody, the Fab region of which is replaced by a VHH capable of specifically recognizing TNFR2, and further comprising a scFv capable of specifically recognizing 4-1BB, the scFv being connected to the C-termini of the two heavy chains of the antibody via a linker.

[0150] FIG14 shows the binding activity of antibodies A, B, and C to TNFR2 protein.

[0151] FIG15 shows the binding activity of antibodies D, E1, and F to TNFR2 protein.

[0152] FIG16 shows the binding activity of antibodies G and H to TNFR2 protein.

[0153] FIG17 shows the binding activity of antibodies J1 and K to TNFR2 protein.

[0154] FIG18 shows the binding activity of antibodies M1 and N to TNFR2 protein.

[0155] FIG19 shows the binding activity of antibodies E1 and E2 to TNFR2 protein.

[0156] FIG20 shows the binding activity of antibodies J2 and M2 to TNFR2 protein.

[0157] FIG21 shows the binding activity of antibody O to TNFR2 protein.

[0158] FIG22 shows the binding activity of antibodies A, B, and C to 4-1BB protein.

[0159] FIG23 shows the binding activity of antibodies D, E1, and F to 4-1BB protein.

[0160] FIG24 shows the binding activity of antibodies G, H, and J1 to 4-1BB protein.

[0161] FIG25 shows the binding activity of antibodies K, M1, and N to 4-1BB protein.

[0162] FIG26 shows the binding activity of antibodies E1, E2, and J2 to 4-1BB protein.

[0163] FIG27 shows the binding activity of antibody O to 4-1BB protein.

[0164] FIG28 shows the dual-terminal binding activities of antibodies A, B, C, and D.

[0165] FIG29 shows the dual-terminal binding activities of antibodies E1, F, G, and H.

[0166] FIG30 shows the dual-terminal binding activities of antibodies J1, K, M1, and N.

[0167] FIG31 shows the dual-terminal binding activities of antibodies A, B, C, and D.

[0168] FIG32 shows the dual-terminal binding activities of antibodies E1, F, G, and H.

[0169] FIG33 shows the dual-terminal binding activities of antibodies J1, K, M1, and N.

[0170] FIG34 shows the activation effects of antibodies E1, H, J1, and M1 on HEK-293 / NFκB-Luci / 4-1BB cells.

[0171] FIG35 shows the activation effect of antibodies O and J1 on HEK-293 / NFκB-Luci / 4-1BB cells.

[0172] FIG36 shows the anti-tumor growth curves of antibodies E1, E2, J2, M2, and O.

[0173] FIG37 shows the binding activities of antibodies O1, O2, O3, and O4 to TNFR2 protein.

[0174] FIG38 shows the binding activity of antibodies O1, O5, and O6 to TNFR2 protein.

[0175] FIG39 shows the binding activity of antibodies O1, O7, and O8 to TNFR2 protein.

[0176] FIG40 shows the binding activity of antibodies O1, O2, O3, and O4 to 4-1BB protein.

[0177] FIG41 shows the binding activity of antibodies O1, O5, and O6 to 4-1BB protein.

[0178] FIG42 shows the binding activity of antibodies O1, O7, and O8 to 4-1BB protein. DETAILED DESCRIPTION

[0179] 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.

[0180] Example 1 Preparation of bispecific antibodies with different structures

[0181] The sequences of the TNFR2 antibody as a full-length antibody, scFv, and VHH, and the sequences of the 4-1BB antibody as a full-length antibody and scFv are shown in Table 1.

[0182] Table 1 Sequence list of TNFR2 antibody and 4-1BB antibody

[0183] The peptide chains shown in Table 2 can be formed by connecting the sequences of the TNFR2 antibody and the 4-1BB antibody through a linker in a certain order. 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: 25.

[0184] Table 2 Peptide chain sequence list

[0185] Based on the peptide chain combinations in Table 2, the bispecific antibody sequences shown in Table 3 were designed, wherein antibody O contains only two peptide chains (i.e., one first peptide chain and one second peptide chain), and all antibodies except antibody O contain four peptide chains (i.e., two identical first peptide chains and two identical second peptide chains).

[0186] Table 3 Bispecific antibody sequences

[0187] Each chain of the bispecific antibody with different structures designed above was gene-synthesized. Molecular cloning techniques were then used to insert the antibody fragments into the pcDNA3.1 vector to construct a mammalian cell expression plasmid. This was then transfected into the host cell line, CHO cells, using liposome transfection. Fermentation supernatant was obtained using a fed-batch assay. The supernatant was then purified through a series of steps, including affinity chromatography and ion exchange chromatography, to ultimately obtain the constructed antibody. The purified antibody was tested for expression level, purity, and SDS-PAGE analysis to confirm the bispecific antibody.

[0188] Example 2 ELISA detection of antibody binding activity to TNFR2 protein

[0189] 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 in a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating buffer and wash the plate with 300 μL per well of 1× PBST, wash four times using a plate washer, and pat dry on paper. Block with 3% skim milk powder, 300 μL / well, incubate at 37°C for 1 hour, discard the blocking buffer, wash four times using a plate washer, and pat dry on paper. Negative and positive controls were set up. The negative control was commercially available trastuzumab for injection (Herceptin). The variable region of the antibody sequence in positive control 1 consisted of SEQ ID NO:5 and SEQ ID NO:1, with the human IgG1 constant region (see SEQ ID NO:46 and SEQ ID NO:47) added. The variable region of the antibody sequence in positive control 2 consisted of SEQ ID NO:11. 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 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% skim milk powder was added, 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 14-21, respectively, demonstrating that all antibodies bind to human TNFR2 protein.

[0190] Example 3 ELISA detection of antibody binding activity to 4-1BB protein

[0191] Human 4-1BB His (Manufacturer: Acro, CAT: 41B-H5258, LOT: 198-2146F1-W6) was diluted to 0.2 μg / mL in 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 tissue paper. 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 tissue paper. Negative and positive controls were set up. The negative control was commercially available trastuzumab for injection (Herceptin). The positive control had an antibody sequence with variable regions consisting of SEQ ID NO: 15 and SEQ ID NO: 19, with the human IgG1 constant region (see SEQ ID NO: 46 and SEQ ID NO: 47) added. 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 by adding 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 22-27, respectively. All antibodies bind to the human 4-1BB protein.

[0192] Example 4 Construction of antibody binding activity at both ends

[0193] 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 into 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) 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 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). Starting at 50 nM, three-fold dilutions were performed, with a total of 10 dilutions. A blank well was also prepared, with only the diluent added. Incubate at 37°C for 1 hour using 100 μL / well of the diluent. 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 to each 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 to each 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 the plate at 450 nm on a microplate reader. ELISA results for the antibody molecules are shown in Figures 28-30, respectively, demonstrating that all antibodies possess binding activity at both ends.

[0194] Example 5 Construction of antibody binding activity at both ends

[0195] 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). Starting at 50 nM, three-fold dilutions were performed, with a total of 10 dilutions. A blank well was also established, with only the diluent added. Incubate at 37°C for 1 hour using 100 μL / well of the diluent. Discard any liquid from the wells, wash the plate 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 and add 100 μL to each 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 to each 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 the plate at 450 nm on a microplate reader. ELISA results for the antibody molecules are shown in Figures 31-33, respectively, demonstrating that all antibodies possess binding activity at both ends.

[0196] Example 6: Detection of Antibody Affinity for TNFR2 Protein Using BLI Method

[0197] The variable region of the antibody sequence for positive control 1 consisted of SEQ ID NO:5 and SEQ ID NO:1, with the constant region of human IgG1 (see SEQ ID NO:46 and SEQ ID NO:47) added; the variable region of the antibody sequence for positive control 2 consisted of SEQ ID NO:11. The AHC sensor was soaked in 0.02% PBST (0.02% Tween 20, pH 7.4, 1× PBS) for 600 s 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 s, followed by 300 s of solidification of the antibody in the sample plate, and a secondary equilibration buffer for 180 s. 100 nM human-TNFR2-His (manufacturer: SINO, CAT: 10417-H08H, LOT: LC15NO0412) protein was allowed to bind to the antibody for 300 s, followed by 600 s of dissociation. After dissociation, the sensor was regenerated with 10 mM glycine (pH 2.0) as a regeneration buffer for 30 seconds. The affinity results of the antibody molecules are shown in Table 4. The results show that all antibodies have affinity for the TNFR2 protein.

[0198] Table 4 Affinity of antibodies to TNFR2 protein

[0199] Example 7 BLI method to detect the affinity of antibodies to 4-1BB protein

[0200] The variable regions of the positive control antibody sequence consisted of SEQ ID NO:15 and SEQ ID NO:19, with the constant region of human IgG1 added (see SEQ ID NO:46 and SEQ ID NO:47). The AHC sensor was soaked in 0.02% PBST (0.02% Tween 20, pH 7.4, 1× PBS) for 600 s 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 s, followed by immobilization of the antibody in the sample plate for 300 s and a secondary equilibration buffer for 180 s. 100 nM human 4-1BB His (Manufacturer: Acro, CAT: 41B-H5258, LOT: 198-2146F1-W6) protein was bound to the antibody for 300 s, followed by dissociation for 600 s. After dissociation, the sensor was regenerated in 10 mM glycine (pH 2.0) for 30 s. The sensor was regenerated with 10 mM glycine (pH 2.0). The affinity results of the antibody molecules are shown in Table 5, which show that all antibodies have affinity for the 4-1BB protein.

[0201] Table 5 Affinity of antibodies to TNFR2 protein

[0202] Example 8 HEK-293 / NFκB-Luci / 4-1BB cell activation experiment

[0203] The variable region of the positive control antibody sequence consists of SEQ ID NO: 15 and SEQ ID NO: 19, and the constant region of human IgG1 (see SEQ ID NO: 46 and SEQ ID NO: 47) was added. The actual initial concentration of the antibody was 60 nM, and 100 μL was used per well, with 4-fold dilutions and 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, and 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 34, indicating that antibodies E1, H, J1, and M1 all activated HEK-293 / NFκB-Luci / 4-1BB effector cells.

[0204] Example 9 HEK-293 / NFκB-Luci / 4-1BB cell activation experiment

[0205] The actual starting and ending concentration of the antibody was 60 nM, 100 μL per well, 5-fold dilution, 7 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 / 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 35, demonstrating that both Antibody O and Antibody J1 activated HEK-293 / NFκB-Luci / 4-1BB effector cells.

[0206] Example 10 Pharmacodynamic Study of Each Test Substance in a C57BL / 6-h4-1BB / hTNFR2 Mice Subcutaneously Transplanted Mouse Colon Cancer Cell Line MC38 Tumor Model

[0207] Female C57BL / 6-h4-1BB / hTNFR2 mice aged 6-7 weeks were subcutaneously inoculated with MC38 tumor cells (from the cell bank of Shenghe (China) Biopharmaceutical Co., Ltd.) and the tumor volume was approximately 100±50mm. 3 The mice were randomly divided into seven groups. The groups included: (1) G1: PBS group; (2) G2: control antibody group, the variable region of which consisted of SEQ ID NO: 15 and SEQ ID NO: 19, and the constant region of human IgG1 (see SEQ ID NO: 46 and SEQ ID NO: 47) was added; (3) G3: antibody E1 group; (4) G4: antibody E2 group; (5) G5: antibody J2 group; (6) G6: antibody M2 group; (7) G7: antibody O 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 for 4 consecutive weeks, for a total of 8 times; the tumor volume and body weight of the mice were measured every other day, and the tumor volume was calculated according to a·b 2 / 2 (a is the long diameter, b is the short diameter). The experimental design is shown in Table 6.

[0208] Table 6 Experimental design

[0209] The results are shown in Figure 36 , showing that antibody M2 and antibody O had significant anti-tumor effects.

[0210] Example 11 Sequence Optimization for Antibody O

[0211] The peptide chains in Table 7 can be formed by connecting the sequences of the TNFR2 antibody and the 4-1BB antibody through a linker in a certain order. In this embodiment, the linker used in the bispecific antibody is 3 GGGGS repeats (i.e., GGGGSGGGGSGGGGS, hereinafter abbreviated as (G4S)3) or 4 GGGGS repeats (i.e., GGGGSGGGGSGGGGSGGGGS, hereinafter abbreviated as (G4S)4), or SPNSASHSGSAPQTSSAPGSQ or GGGSAAA. Cysteine ​​introduction refers to the introduction of one cysteine ​​in each of VH (4-1BB) and VL (4-1BB). The CL used in this embodiment is a kappa (κ) type, and the sequence is shown in SEQ ID NO: 25.

[0212] Table 7 Peptide chain sequence list

[0213] According to the peptide chain combinations in Table 7, the bispecific antibody sequences shown in Table 8 were designed, and each antibody contained only two peptide chains (i.e., one first peptide chain and one second peptide chain).

[0214] Table 8 Bispecific antibody sequences

[0215] Each chain of the bispecific antibody with different structures designed above was gene-synthesized. Molecular cloning techniques were then used to insert the antibody fragments into the pcDNA3.1 vector to construct a mammalian cell expression plasmid. This was then transfected into the host cell line, CHO cells, using liposome transfection. Fermentation supernatant was obtained using a fed-batch assay. The supernatant was then purified through a series of steps, including affinity chromatography and ion exchange chromatography, to ultimately obtain the constructed antibody. The purified antibody was tested for expression level, purity, and SDS-PAGE analysis to confirm the bispecific antibody.

[0216] Example 12 ELISA detection of antibody binding activity to TNFR2 protein

[0217] Dilute human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC15SE2206) to 0.1 μg / mL in coating buffer (1× PBS, pH 7.4) and coat in a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating buffer and wash the plate with 1× PBST (300 μL / well), wash four times using a plate washer, and pat dry on paper. Block with 3% skim milk powder (300 μL / well), incubate at 37°C for 1 hour, discard the blocking buffer, wash four times using a plate washer, and pat dry on paper. Negative and positive controls were set up. The negative control was commercially available trastuzumab for injection (Herceptin), and the positive control was antibody O1. The negative control, positive control, and test antibody were diluted to 10 μg / mL with 3% skim milk powder. Three-fold dilutions were performed from this starting concentration, resulting in 11 dilution steps. A blank well was set up, containing only the diluent. 100 μL / well was added, and the plate was incubated at 37°C for 1 hour. The wells were discarded, the plates were washed four times with a plate washer, and the plates were patted dry on paper. Goat anti-human IgG Fc was diluted 1:20,000 with 3% skim milk powder, 100 μL / well was added, and the plates were incubated at 37°C for 1 hour. The plates were washed six times with a plate washer, and the plates were patted dry on paper. TMB colorimetric solution (100 μL / well) was added, the plates were wrapped with aluminum foil, and the plates were incubated at 37°C in the dark for 8 minutes. The color reaction was terminated with 100 μL / well of 1M HCl. The plate was read at 450 nm on a microplate reader. The ELISA results of the antibody molecules are shown in Figures 37-39, respectively, and the results show that the antibodies can bind to the human TNFR2 protein.

[0218] Example 13 ELISA detection of antibody binding activity to 4-1BB protein

[0219] Human 4-1BB His (manufacturer: Acro, CAT: 41B-H5258, LOT: 2407c-214FF1-YC) 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 paper plate. 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 paper plate. Set up negative and positive controls. The negative control is commercially available trastuzumab for injection (Herceptin), and the positive control is antibody O1. The negative control, positive control, and test antibody were diluted to 10 μg / mL with 3% skim milk powder. Three-fold dilutions were performed from this initial concentration, resulting in 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 by adding 100 μL / well of 1M HCl as the stop solution. The assay was read at 450 nm on a microplate reader. The ELISA results for the antibodies are shown in Figures 40-42, respectively. All antibodies bind to the human 4-1BB protein.

[0220] 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 that specifically binds to TNFR2 and 4-1BB, 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 antigen-binding fragment thereof that specifically binds to a second antigen; in: The first antigen is TNFR2, and the second antigen is 4-1BB; or, the first antigen is 4-1BB, and the second antigen is TNFR2.

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

3. The bispecific antibody according to claim 2, 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.

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 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 or light 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 of the two heavy chains or the two light chains of the first antibody or antigen-binding fragment thereof.

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

10. The bispecific antibody according to claim 8 or 9, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, characterized in that: For each polypeptide chain described: (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 the heavy chain of the first antibody or antigen-binding fragment thereof and the scFv or VHH.

11. The bispecific antibody according to claim 8 or 9, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, characterized in that: For each polypeptide chain described: (a) the first polypeptide chains each independently comprise the light chain of the first antibody or antigen-binding fragment thereof and the scFv or VHH; and (b) the second polypeptide chains each independently comprise a heavy chain of the first antibody or antigen-binding fragment thereof.

12. The bispecific antibody according to claim 10 or 11, characterized in that The two first polypeptide chains are identical or different, and / or the two second polypeptide chains are identical or different.

13. The bispecific antibody according to any one of claims 3 to 9, characterized in that The Fab region of the first antibody or antigen-binding fragment thereof is replaced by a VHH capable of specifically binding to TNFR2.

14. The bispecific antibody according to claim 13, wherein the bispecific antibody comprises two polypeptide chains, characterized in that: Each of the polypeptide chains independently comprises the first antibody or antigen-binding fragment thereof and the scFv.

15. The bispecific antibody according to claim 14, characterized in that The two polypeptide chains may be identical or different.

16. The bispecific antibody according to any one of claims 2 to 15, characterized in that The heavy chain variable region and the light chain variable region of the scFv are connected by a linker L1.

17. The bispecific antibody according to any one of claims 2 to 15, characterized in that The scFv is connected to the N-terminus or C-terminus of the heavy chain or light chain of the first antibody or antigen-binding fragment thereof through a linker L2.

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

19. The bispecific antibody according to claim 17 or 18, characterized in that The linker L1 and linker L2 are the same or different.

20. The bispecific antibody according to any one of claims 16 to 19, 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.

21. The bispecific antibody according to any one of claims 2 to 20, 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.

22. The bispecific antibody according to claim 21, characterized in that The first Fc region and the second Fc region are the same or different.

23. The bispecific antibody according to claim 21 or 22, characterized in that The Fc region is selected from the group consisting of IgG, IgA, IgD, IgE, IgM and variants thereof.

24. The bispecific antibody according to claim 23, characterized in that The Fc region is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 and variants thereof.

25. The bispecific antibody according to claim 23 or 24, characterized in that The Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

26. The bispecific antibody according to any one of claims 4 to 12, 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: 16, or a sequence having at least 80% identity with SEQ ID NO: 16; HCDR2 is as shown in SEQ ID NO: 17, or a sequence having at least 80% identity with SEQ ID NO: 17; HCDR3 is as shown in SEQ ID NO: 18, or a sequence having at least 80% identity with SEQ ID NO: 18; LCDR1 is as shown in SEQ ID NO: 20, or a sequence having at least 80% identity with SEQ ID NO: 20; LCDR2 is as shown in SEQ ID NO: 21, or a sequence having at least 80% identity with SEQ ID NO: 21; and LCDR3 is as shown in SEQ ID NO: 22, or a sequence having at least 80% identity with SEQ ID NO:

22.

27. The bispecific antibody according to claim 26, characterized in that The scFv specifically binds to TNFR2, wherein HCDR1 of the scFv is set forth in SEQ ID NO:2, or a sequence at least 80% identical to SEQ ID NO:2; HCDR2 is set forth in SEQ ID NO:3, or a sequence at least 80% identical to SEQ ID NO:3; HCDR3 is set forth in SEQ ID NO:4, or a sequence at least 80% identical to SEQ ID NO:4; and LCDR1 is set forth in SEQ ID NO:6, or a sequence at least 80% identical to SEQ ID NO:6; LCDR2 is set forth in SEQ ID NO:7, or a sequence at least 80% identical to SEQ ID NO:7; and LCDR3 is set forth in SEQ ID NO:8, or a sequence at least 80% identical to SEQ ID NO:

8.

28. The bispecific antibody according to claim 26, characterized in that The VHH specifically binds to TNFR2, and the HCDR1 of the VHH is set forth in SEQ ID NO: 12, or a sequence having at least 80% identity to SEQ ID NO: 12; the HCDR2 is set forth in SEQ ID NO: 13, or a sequence having at least 80% identity to SEQ ID NO: 13; and the HCDR3 is set forth in SEQ ID NO: 14, or a sequence having at least 80% identity to SEQ ID NO:

14.

29. The bispecific antibody according to any one of claims 4 to 12, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to TNFR2, wherein HCDR1 of the first antibody or antigen-binding fragment thereof is set forth in SEQ ID NO:2, or a sequence at least 80% identical to SEQ ID NO:2; HCDR2 is set forth in SEQ ID NO:3, or a sequence at least 80% identical to SEQ ID NO:3; HCDR3 is set forth in SEQ ID NO:4, or a sequence at least 80% identical to SEQ ID NO:4; LCDR1 is set forth in SEQ ID NO:6, or a sequence at least 80% identical to SEQ ID NO:6; LCDR2 is set forth in SEQ ID NO:7, or a sequence at least 80% identical to SEQ ID NO:7; and LCDR3 is set forth in SEQ ID NO:8, or a sequence at least 80% identical to SEQ ID NO:

8.

30. The bispecific antibody according to claim 29, characterized in that The scFv specifically binds to 4-1BB, and the HCDR1 of the scFv is set forth in SEQ ID NO: 16, or a sequence at least 80% identical to SEQ ID NO: 16; HCDR2 is set forth in SEQ ID NO: 17, or a sequence at least 80% identical to SEQ ID NO: 17; HCDR3 is set forth in SEQ ID NO: 18, or a sequence at least 80% identical to SEQ ID NO: 18; LCDR1 is set forth in SEQ ID NO: 20, or a sequence at least 80% identical to SEQ ID NO: 20; LCDR2 is set forth in SEQ ID NO: 21, or a sequence at least 80% identical to SEQ ID NO: 21; and LCDR3 is set forth in SEQ ID NO: 22, or a sequence at least 80% identical to SEQ ID NO:

22.

31. The bispecific antibody according to any one of claims 4 to 12, 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 shown in SEQ ID NO: 15, or a sequence having at least 80% identity with SEQ ID NO: 15; the light chain variable region VL is shown in SEQ ID NO: 19, or a sequence having at least 80% identity with SEQ ID NO:

19.

32. The bispecific antibody according to claim 31, characterized in that The scFv specifically binds to TNFR2, and the heavy chain variable region VH of the scFv is shown in SEQ ID NO: 9, or a sequence having at least 80% identity with SEQ ID NO: 9; the light chain variable region VL is shown in SEQ ID NO: 10, or a sequence having at least 80% identity with SEQ ID NO:

10.

33. The bispecific antibody according to claim 31, characterized in that The VHH specifically binds to TNFR2, and the VHH heavy chain variable region VH is shown in SEQ ID NO: 11, or a sequence having at least 80% identity with SEQ ID NO:

11.

34. The bispecific antibody according to any one of claims 4 to 12, characterized in that The first antibody or antigen-binding fragment thereof specifically binds to TNFR2, 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; and 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.

35. The bispecific antibody according to claim 34, characterized in that The scFv specifically binds to 4-1BB, and the scFv heavy chain variable region VH is shown in SEQ ID NO: 23, or a sequence having at least 80% identity with SEQ ID NO: 23; the light chain variable region VL is shown in SEQ ID NO: 24, or a sequence having at least 80% identity with SEQ ID NO:

24.

36. The bispecific antibody according to any one of claims 26 to 35, characterized in that The first polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 26-33, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 26-33; the second polypeptide chain of the bispecific antibody is selected from any one of SEQ ID NOs: 35-45, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 35-45.

37. The bispecific antibody according to claim 36, characterized in that The bispecific antibody comprises: (1) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 35; or (2) the first polypeptide chain as shown in SEQ ID NO: 27, the second polypeptide chain as shown in SEQ ID NO: 36; or (3) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 37; or (4) a first polypeptide chain as shown in SEQ ID NO: 28, a second polypeptide chain as shown in SEQ ID NO: 36 peptide chain; or (5) the first polypeptide chain as shown in SEQ ID NO: 29, the second polypeptide chain as shown in SEQ ID NO: 38; or (6) the first polypeptide chain as shown in SEQ ID NO: 29, the second polypeptide chain as shown in SEQ ID NO: 43; or (7) the first polypeptide chain as shown in SEQ ID NO: 30, the second polypeptide chain as shown in SEQ ID NO: 39; or (8) the first polypeptide chain as shown in SEQ ID NO: 29, the second polypeptide chain as shown in SEQ ID NO: 40; or (9) the first polypeptide chain as shown in SEQ ID NO: 31, the second polypeptide chain as shown in SEQ ID NO: 39; or (10) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 41; or (11) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 44; or (12) the first polypeptide chain as shown in SEQ ID NO: 32, the second polypeptide chain as shown in SEQ ID NO: 36; or (13) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 42; or (14) the first polypeptide chain as shown in SEQ ID NO: 26, the second polypeptide chain as shown in SEQ ID NO: 45; or (15) The first polypeptide chain shown in SEQ ID NO: 33, and the second polypeptide chain shown in SEQ ID NO:

36.

38. The bispecific antibody according to any one of claims 13 to 15, characterized in that The VHH that specifically binds to TNFR2, its HCDR1 is shown as SEQ ID NO: 12, or a sequence having at least 80% identity with SEQ ID NO: 12; its HCDR2 is shown as SEQ ID NO: 13, or a sequence having at least 80% identity with SEQ ID NO: 13; and its HCDR3 is shown as SEQ ID NO: 14, or a sequence having at least 80% identity with SEQ ID NO:

14.

39. The bispecific antibody according to claim 38, characterized in that The scFv specifically binds to 4-1BB, and the HCDR1 of the scFv is set forth in SEQ ID NO: 16, or a sequence at least 80% identical to SEQ ID NO: 16; HCDR2 is set forth in SEQ ID NO: 17, or a sequence at least 80% identical to SEQ ID NO: 17; HCDR3 is set forth in SEQ ID NO: 18, or a sequence at least 80% identical to SEQ ID NO: 18; LCDR1 is set forth in SEQ ID NO: 20, or a sequence at least 80% identical to SEQ ID NO: 20; LCDR2 is set forth in SEQ ID NO: 21, or a sequence at least 80% identical to SEQ ID NO: 21; and LCDR3 is set forth in SEQ ID NO: 22, or a sequence at least 80% identical to SEQ ID NO:

22.

40. The bispecific antibody according to any one of claims 13 to 15, characterized in that The VHH that specifically binds to TNFR2 has a heavy chain variable region VH as shown in SEQ ID NO: 11, or a sequence that has at least 80% identity with SEQ ID NO:

11.

41. The bispecific antibody according to claim 40, characterized in that The scFv specifically binds to 4-1BB, and the scFv heavy chain variable region VH is shown in SEQ ID NO: 23, or a sequence having at least 80% identity with SEQ ID NO: 23; the light chain variable region VL is shown in SEQ ID NO: 24, or a sequence having at least 80% identity with SEQ ID NO:

24.

42. The bispecific antibody according to any one of claims 38 to 41, characterized in that The polypeptide chain of the bispecific antibody is selected from any one of the amino acid sequences of SEQ ID NOs: 34, 48-55, or a sequence having at least 80% identity with any one of the amino acid sequences of SEQ ID NOs: 34, 48-55.

43. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1-42; Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide chain of the bispecific antibody according to any one of claims 10-42. A multifunctional fusion protein comprising the bispecific antibody according to any one of claims 1 to 42.

45. The multifunctional fusion protein according to claim 44, characterized in that One or more third antibodies or antigen-binding portions thereof that specifically bind to other antigens are also included.

46. ​​The multifunctional fusion protein according to claim 45, 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.

47. The multifunctional fusion protein according to claim 46, characterized in that The antigen that binds to the third antibody or its antigen-binding portion is selected from GPC3, CD19, CD20 (MS4A1), CD22, 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, p53, PAP, P AX3, 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, MAG E-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-Novo antigen, TRP-1, TRP-2, tyrosinase and urokinin-3, 5T4, PD-L1, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, ILT4, TREM2, LAG3, CD27, CD24, B7H3, or B7H4.

48. The multifunctional fusion protein according to any one of claims 44 to 47, characterized in that Also contains cytokines.

49. The multifunctional fusion protein according to claim 48, 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.

50. Use of the bispecific antibody according to any one of claims 1 to 42 or the multifunctional fusion protein according to any one of claims 44 to 49 in the preparation of a medicament for treating cancer.

51. The use according to claim 50, 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.

52. Use of the bispecific antibody according to any one of claims 1 to 42 or the multifunctional fusion protein according to any one of claims 44 to 49 in the preparation of a medicament for treating an autoimmune disease.

53. The use according to claim 52, 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.

54. The use according to any one of claims 50 to 53, characterized in that The use is achieved through one or more of tumor immunotherapy, cell therapy or gene therapy.

55. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-42 and a pharmaceutically acceptable carrier, diluent or excipient.

56. A pharmaceutical composition comprising the multifunctional fusion protein according to any one of claims 44 to 49 and a pharmaceutically acceptable carrier, diluent or excipient.

57. An antibody-drug conjugate comprising the bispecific antibody of any one of claims 1-42.

58. The antibody drug conjugate according to claim 57, characterized in that The conjugated drug is selected from cytotoxins, small molecule chemical drugs or immunotoxins.

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