Bispecific antibody, and cytokine fusion protein and use thereof

Abstract

Provided are a fusion protein and a use thereof. The fusion protein comprises a bispecific antibody and a cytokine.

Description

Bispecific antibody and cytokine fusion protein and its application Technical Field

[0001] The present application relates to the field of molecular immunology in biomedicine, and specifically to a fusion protein and its application. Background Art

[0002] Bifunctional antibodies, also known as bispecific antibodies (BsAbs), are specific drugs that simultaneously target two different antigens and can be produced through immunosorting and purification. BsAbs can be designed to simultaneously target both tumor cells and effector cells, activating effector cells to kill tumor cells. The role of the immune system, particularly T cell-mediated cytotoxicity, in tumor control is well established, with growing evidence demonstrating that T cells play a central role in controlling tumor growth and survival in cancer patients. Over the past few decades, treatments for various tumor types have aimed to reshape and improve T cell function and activity, conferring stronger and longer-lasting anti-tumor properties. Bispecific T-cell engagers (BiTEs) are among these bispecific antibodies composed of a T cell-binding domain (such as CD3) and a tumor-binding domain (primarily a tumor cell surface-associated antigen, or TAA). These bispecific antibodies establish a physical connection between T cells and tumor cells, promoting tumor killing by cytotoxic T cells. In 2014, the U.S. Food and Drug Administration (FDA) approved the first BiTE, Blinatumomab (an anti-CD3 and anti-CD19 bispecific antibody), for the treatment of relapsed / refractory B-cell acute lymphoblastic leukemia (ALL). This demonstrated that killing endogenous T cells in tumors is achievable without the need for in vitro immune cell editing, which is one aspect of BiTE's advantage over cell therapy.

[0003] Although Blinatumomab has been successfully used in B-cell acute lymphoblastic leukemia (ALL), the effect of BiTE in solid tumors has been mediocre, encountering a dilemma similar to that of cell therapy. On the one hand, this may be due to the limited penetration of BiTE into tumor tissues, and on the other hand, there may be many immunosuppressive factors in the tumor microenvironment that limit the restoration of T cell function in the tumor by BiTE. There are currently reports of adding co-stimulatory factors such as anti-CD28 to BiTE to enhance the function of BiTE. Cytokines, as the third signal in T cell activation, play a vital role in improving the tumor microenvironment. However, the function of cytokines on BiTE is unknown. Based on this, we explored the function of cytokines in BiTE therapy. Summary of the Invention

[0004] Based on the above problems and therapeutic potential of BiTE, it is necessary to further develop it in clinical applications to make it more effective and safe.

[0005] Specifically, the present invention provides the following aspects:

[0006] 1. A fusion protein comprising structural unit A and structural unit B, wherein structural unit A is an IgG bispecific antibody, the bispecific antibody comprising structural unit 1 and structural unit 2, wherein structural unit 1 is an anti-CD3 antibody, and structural unit 2 is an anti-tumor cell surface antigen antibody, wherein the antibodies of structural unit 1 and structural unit 2 are in Fab or scFv form, and the structural unit B is one or more (preferably 2) cytokines or cytokine prodrugs, and the structural unit B is connected to the N-terminus and / or C-terminus of the bispecific antibody via a cleavable linker or a non-cleavable linker, wherein the cytokine prodrug is an antibody or receptor of the cytokine connected to the cytokine via a cleavable linker. Preferably, the fusion protein exists in the form of a protein drug, mRNA or Car T.

[0007] 2. The fusion protein according to item 1, wherein when the structural units 1 and 2 are both in the form of scFv, the structural unit B is located at the N-terminus or C-terminus of the structural unit 1;

[0008] When the structural unit 1 and / or structural unit 2 is in the form of scFv and / or Fab, the bispecific antibody further comprises a structural unit 4, which is an antibody Fc fragment or human serum albumin.

[0009] 1) When the structural unit 4 is an Fc fragment of an antibody:

[0010] The structural unit 1 and the structural unit 2 are both located at the N-terminus of the bispecific antibody; the structural unit 4 is connected to the C-terminus of the structural unit 1 and the structural unit 2 respectively;

[0011] The structural unit B is located at the N-terminus of the structural unit 1 and / or structural unit 2 or at the C-terminus of the structural unit 4, and the structural unit B is one or two cytokines or cytokine prodrugs;

[0012] II) When the structural unit 4 is human serum albumin:

[0013] The bispecific antibody is as follows from N-terminus to C-terminus:

[0014] a) Structural unit 2-Structural unit 4-Structural unit 1,

[0015] b) Structural unit 4-structural unit 1-structural unit 2,

[0016] c) Structural unit 2 - Structural unit 1 - Structural unit 4, or

[0017] d) Structural unit 1-structural unit 4-structural unit 2.

[0018] 3. The fusion protein according to item 1, wherein the structural unit A further comprises a structural unit 4, the structural unit 4 is the Fc fragment of an antibody, the structural unit 1 and the structural unit 2 are in the form of scFv, and the structural unit 2 is located at the N-terminus of the structural unit 1, the C-terminus of the structural unit 1 is connected to one chain of the Fc fragment, and the structural unit B is connected to the other chain of the Fc fragment, preferably the structural unit B is 1 or 2 cytokines or cytokine prodrugs.

[0019] 4. The fusion protein according to item 1 or 2, wherein the structural unit A further comprises structural unit 3 and / or structural unit 4,

[0020] The structural unit 3 is a tumor cell targeting peptide, which is connected to the N-terminus of the structural unit 1 through a linker, and the linker is selected from the group consisting of:

[0021] (1) Linker 1, which is a pH-sensitive linker,

[0022] (2) Linker 2, which is a cleavable linker,

[0023] (3) Linker 3, which is a combination of a pH-sensitive linker and a cleavable linker, wherein the cleavable linker and the pH-sensitive linker are sequentially connected (wherein the cleavable linker is located at the N-terminus or the C-terminus), or the Linker 3 sequence is as shown in SEQ ID NO: 13 or 14,

[0024] The structural unit 4 is an antibody Fc fragment or human serum albumin,

[0025] 1) When the structural unit 4 is an Fc fragment of an antibody:

[0026] The structural unit 1, structural unit 2 and structural unit 3 are simultaneously located at the N-terminus or C-terminus of the bispecific antibody;

[0027] The structural unit 4 is connected to the C-terminal of the structural unit 1 and the structural unit 2, respectively, and the N-terminal of the structural unit 1 is connected to the structural unit 3, or the structural unit 4 is connected to the N-terminal of the structural unit 1 and the structural unit 2, respectively, and the C-terminal of the structural unit 1 is connected to the structural unit 3;

[0028] II) When the structural unit 4 is human serum albumin:

[0029] The bispecific antibody is as follows from N-terminus to C-terminus:

[0030] a) Structural unit 2-Structural unit 4-Structural unit 1-Structural unit 3,

[0031] b) structural unit 3-structural unit 4-structural unit 1-structural unit 2,

[0032] c) structural unit 2 - structural unit 1 - structural unit 4 - structural unit 3, or

[0033] d) Structural unit 3 - structural unit 1 - structural unit 4 - structural unit 2.

[0034] 5. The fusion protein according to item 4, wherein (1) the bispecific antibody is composed of structural units 1-4, wherein the structural units 1-3 are located at the N-terminus of the bispecific antibody, the structural unit 4 is located at the C-terminus of the bispecific antibody, the structural unit 3 is connected to the N-terminus of the structural unit 1 via a linker, the structural units 1 and 3 are in the Fab form, the structural unit 2 is in the scFv form, and the structural unit 4 is the Fc fragment of the antibody,

[0035] wherein the cytokine is connected to the C-terminus of the light chain variable region of the structural unit 1 or to the C-terminus of any chain of the structural unit 4 via a cleavable linker or a non-cleavable linker;

[0036] (2) The bispecific antibody is composed of structural unit 1, structural unit 2 and structural unit 4, wherein the structural units 1 and 2 are located at the N-terminus of the bispecific antibody, the structural unit 4 is located at the C-terminus of the bispecific antibody, the structural unit 1 is in the form of Fab, the structural unit 2 is in the form of scFv, and the structural unit 4 is the Fc fragment of the antibody.

[0037] The cytokine is connected to the N-terminus of one chain of the structural unit 1 through a cleavable linker or a non-cleavable linker, and the cytokine receptor is connected to the N-terminus of the other chain of the structural unit 1 through a cleavable linker or a non-cleavable linker.

[0038] 6. The fusion protein according to item 5, wherein when the cytokine is linked to the C-terminus of the light chain variable region of structural unit 1, the other end of the cytokine is further linked to a cytokine receptor via a cleavable or non-cleavable linker; or

[0039] When the cytokine is linked to the C-terminus of any chain of the structural unit 4, the structural unit B further comprises the cytokine receptor, wherein the cytokine receptor is linked to the C-terminus of the other chain of the structural unit 4 via a cleavable or non-cleavable linker, or the cytokine receptor is linked to the other end of the cytokine via a cleavable or non-cleavable linker.

[0040] 7. The fusion protein according to item 4, wherein the structural unit 3 is selected from an anti-tumor cell surface antigen antibody, NGR, CNGRC, RGD, MT1-AF7p, YSA or TNYL. When the structural unit 3 is an anti-tumor cell surface antigen antibody, it is the same as or different from the tumor cell surface antigen targeted by the structural unit 2, and is in the form of Fab or scFv.

[0041] 8. The fusion protein according to item 4 or 7, wherein the structural unit B is located at the C-terminus of the structural unit A, and the structural unit B is one or two cytokines or cytokine prodrugs.

[0042] 9. The fusion protein according to item 1 or 2, wherein the cytokine is selected from the wild type or mutant thereof of IL2 (such as ACCESSION P60568), IL15 (such as ACCESSION P40933), IL-21 (such as ACCESSION Q9HBE4-1 or Q9HBE4-2), IL-10 (such as ACCESSION P22301), or IL-4 (such as ACCESSION P05112).

[0043] 10. The fusion protein according to item 5 or 6, wherein the cytokine is a wild type or mutant of IL2 or IL15, preferably, the sequence of IL2 is as shown in ACCESSION P60568, the sequence of IL15 is as shown in SEQ ID NO: 135 or 137, the sequence of the IL2 mutant is as shown in SEQ ID NO: 110, 171-177, and the sequence of the IL15 mutant is as shown in SEQ ID NO: 147, 148, 178. Preferably, when the structural unit B comprises a cytokine receptor, the sequence of the IL15 receptor is as shown in SEQ ID NO: 136 or 138, the IL15-IL15 receptor complex is selected from the sequence shown in SEQ ID NO: 133 or 146, and the sequence of the IL2 receptor is as shown in SEQ ID NO: 167 or 168.

[0044] 11. The fusion protein according to any one of items 1 to 10, wherein the sequence of the anti-CD3 antibody is selected from the group consisting of:

[0045] (1) OKT3, wherein the CDR1 sequence of its VL is shown in SEQ ID NO:48, the CDR2 sequence of its VL is DTS, the CDR3 sequence of its VL is shown in SEQ ID NO:49, the CDR1 sequence of its VH is shown in SEQ ID NO:50, the CDR2 sequence of its VH is shown in SEQ ID NO:51, and the CDR3 sequence of its VH is shown in SEQ ID NO:52; preferably, the amino acid sequence of its VL is shown in SEQ ID NO:35, and the amino acid sequence of its VH is shown in SEQ ID NO:36;

[0046] (2) L2K, whose VL amino acid sequence is shown in SEQ ID NO: 37, and whose VH amino acid sequence is shown in SEQ ID NO: 38;

[0047] (3) UCHT1, wherein the CDR1 sequence of its VL is shown in SEQ ID NO: 53, the CDR2 sequence of its VL is YTS, the CDR3 sequence of its VL is shown in SEQ ID NO: 54, the CDR1 sequence of its VH is shown in SEQ ID NO: 55, the CDR2 sequence of its VH is shown in SEQ ID NO: 56, and the CDR3 sequence of its VH is shown in SEQ ID NO: 57; preferably, the amino acid sequence of its VL is shown in SEQ ID NO: 39, and the amino acid sequence of its VH is shown in SEQ ID NO: 40;

[0048] (4) SP34, wherein the CDR1 sequence of its VL is shown in SEQ ID NO: 58, the CDR2 sequence of its VL is GTN, the CDR3 sequence of its VL is shown in SEQ ID NO: 59, the CDR1 sequence of its VH is shown in SEQ ID NO: 60, the CDR2 sequence of its VH is shown in SEQ ID NO: 61, and the CDR3 sequence of its VH is shown in SEQ ID NO: 62; preferably, the amino acid sequence of its VL is shown in SEQ ID NO: 41, and the amino acid sequence of its VH is shown in SEQ ID NO: 42;

[0049] (5) 17A2, wherein the CDR1 sequence of its VL is shown in SEQ ID NO:63, the CDR2 sequence of its VL is YAS, the CDR3 sequence of its VL is shown in SEQ ID NO:64, the CDR1 sequence of its VH is shown in SEQ ID NO:65, the CDR2 sequence of its VH is shown in SEQ ID NO:66, and the CDR3 sequence of its VH is shown in SEQ ID NO:67; preferably, the amino acid sequence of its VL is shown in SEQ ID NO:43, and the amino acid sequence of its VH is shown in SEQ ID NO:44; and

[0050] (6) 145-2C11, the CDR1 sequence of its VL is shown in SEQ ID NO:68, the CDR2 sequence of its VL is YTN, the CDR3 sequence of its VL is shown in SEQ ID NO:69, the CDR1 sequence of its VH is shown in SEQ ID NO:70, the CDR2 sequence of its VH is shown in SEQ ID NO:71, and the CDR3 sequence of its VH is shown in SEQ ID NO:72; preferably, the amino acid sequence of its VL is shown in SEQ ID NO:45, and the amino acid sequence of its VH is shown in SEQ ID NO:46.

[0051] 12. The fusion protein according to any one of items 4 to 10, wherein the pH-sensitive linker is selected from HA, or the cleavable linker is selected from the cleavage site of MMP, uPA or CD13,

[0052] Preferably, the MMP is selected from the group consisting of:

[0053] (1) any one of SEQ ID NOs: 6-8 and 83-94, or

[0054] (2) any two combinations of any one sequence selected from SEQ ID NOs: 6-8 and any one sequence selected from SEQ ID NOs: 83-94, or

[0055] (3) Any one sequence selected from SEQ ID NOs: 1-3, 24, 28, 30 and 32 in combination with any one sequence selected from SEQ ID NOs: 6-8 and 83-94.

[0056] 13. The fusion protein according to any one of items 1 to 12, wherein the tumor cell surface antigen is selected from Claudin18.2 (such as ACCESSION: P56856), PDL1 (such as ACCESSION: Q9NZQ7), CD139 (such as ACCESSION: P15144), MSLN (such as ACCESSION: Q12321), GPC3 (such as ACCESSION: Q8CFZ4), Her29r (such as ACCESSION: P04626), E GFR (such as ACCESSION: P00533), GD2 (such as ACCESSION: Q9UI17), Trop2 (such as ACCESSION: P09758), CD19 (such as ACCESSION: P15391), CD20 (such as ACCESSION: P11836), Her3 ACCESSION: P21860, FolR1 (such as ACCESSION: P15328), Nectin4 (such as ACCESSION: Q96NY8), C D276 (such as ACCESSION: Q5ZPR3), CD30 (such as ACCESSION: P28908), CD70 (such as ACCESSION: P32970), BCMA (such as ACCESSION: Q02223), CD22 (such as ACCESSION: P20273), CD33 (such as ACCESSION: P20138), GPRC5D (such as ACCESSION: Q9NZD1), HLADR (such as ACCESSION: Q2974 4), a group consisting of VTCN1 (such as ACCESSION: Q7Z7D3), CD79B (such as ACCESSION: P40295), CDH3 ACCESSION: P22223, DR5 ACCESSION: P21918, MUC1 (such as ACCESSION: P15941), NT5E (such as ACCESSION: P21589), FAP (such as ACCESSION: Q12884) and PSMA (such as ACCESSION: Q04609).

[0057] 14. The fusion protein according to any one of items 4 to 13, wherein the tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by the structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by the structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by the structural unit 2 is PDL1, and the tumor cell surface antigen targeted by the structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by the structural unit 2 is PDL1, and the tumor cell surface antigen targeted by the structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by the structural unit 2 is MSLN, and the tumor cell surface antigen targeted by the structural unit 3 is MSLN; or wherein the tumor cell surface antigen targeted by the structural unit 2 is FAP, and the tumor cell surface antigen targeted by the structural unit 3 is FAP, preferably, the LCDR1 sequence of the structural unit 2 or 3 targeting the tumor cell surface antigen PDL1 is as shown in SEQ ID NO:73, the LCDR2 sequence is SAS, the LCDR3 sequence is shown in SEQ ID NO:74, the HCDR1 sequence is shown in SEQ ID NO:75, the HCDR2 sequence is shown in SEQ ID NO:76, and the HCDR3 sequence is shown in SEQ ID NO:77. The LCDR1 sequence of structural unit 2 or 3 for the tumor cell surface antigen Claudin18.2 is shown in SEQ ID NO:78, the LDR2 sequence is WAS, the LCDR3 sequence is shown in SEQ ID NO:79, the HCDR1 sequence is shown in SEQ ID NO:80, the VCDR2 sequence is shown in SEQ ID NO:81, and the HCDR3 sequence is shown in SEQ ID NO:82; or the LCDR1 sequence of structural unit 2 is shown in SEQ ID NO:78, the LCDR2 sequence is WAS, the LCDR3 sequence is shown in SEQ ID NO:79, the HCDR1 sequence is shown in SEQ ID NO:80, the HCDR2 sequence is shown in SEQ ID NO:81, and the HCDR3 sequence is shown in SEQ ID NO:82. NO:82, the LCDR1 sequence of the structural unit 3 is shown in SEQ ID NO:73, the LCDR2 sequence is SAS, the LCDR3 sequence is shown in SEQ ID NO:74, the HCDR1 sequence is shown in SEQ ID NO:75, the HCDR2 sequence is shown in SEQ ID NO:76, and the HCDR3 sequence is shown in SEQ ID NO:77.

[0058] 15. The fusion protein according to any one of items 4 to 13, wherein

[0059] The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2 shown in ACCESSION: P56856, and the structural unit 3 is CNGRC;

[0060] The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2, and the structural unit 3 is RGD;

[0061] The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2 shown in ACCESSION: P56856, and the structural unit 3 is TNYL;

[0062] The tumor cell surface antigen targeted by structural unit 2 is MSLN, and the structural unit 3 is CNGRC.

[0063] 16. The fusion protein according to any one of items 4 to 15, wherein the Fc fragment is an Fc fragment of IgG1, IgG2, IgG3 or IgG4, preferably the Fc fragment is a humanized Fc fragment.

[0064] 17. The fusion protein according to any one of items 4 to 16, wherein the heavy chain constant region of the Fc fragment adopts the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts the Ig kappa chain C region, ACCESSION: P01834,

[0065] Preferably, according to the EU numbering system, based on Ig gamma-1 chain C region, ACCESSION: P01857, the heavy chain constant region of the Fc fragment has the following mutations at positions 234, 235 and / or 237:

[0066] L234A and L235A,

[0067] L234A and G237A,

[0068] L235A and G237A,

[0069] or

[0070] L234A, L235A, and G237A;

[0071] Preferably, according to the EU numbering system, the heavy chain constant region of the Fc fragment further has one or more mutations selected from the following:

[0072] N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, H268A, E318A, and K320A.

[0073] 18. The fusion protein according to any one of items 4 to 17, wherein the Fc fragment is selected from SEQ ID NO: 19 and SEQ ID NO: 20.

[0074] 19. The fusion protein according to any one of items 4 to 18, wherein when the structural unit 4 is human serum albumin, it is the human serum albumin of ACCESSION: P02768.

[0075] 20. The fusion protein according to any one of items 4 to 19, wherein the structural unit 2 is selected from SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 156 and / or the structural unit 3 is selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 156, SEQ ID NO: 33 or SEQ ID NO: 34,

[0076] Preferably, the bispecific antibody is selected from the group consisting of:

[0077] (1) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 17 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 18,

[0078] (2) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 18 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 17,

[0079] (3) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 17 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 33,

[0080] (4) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 18 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 33,

[0081] (5) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 18 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 34, and

[0082] (6) The structural unit 2 is selected from the sequence shown in SEQ ID NO: 17 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 34.

[0083] 21. The fusion protein according to any one of items 4 to 20, wherein the sequence of the bispecific antibody is selected from the group consisting of:

[0084] (1) A bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23,

[0085] (2) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 25,

[0086] (3) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27,

[0087] (4) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 29,

[0088] (5) A bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 31,

[0089] (6) A bispecific antibody comprising or consisting of SEQ ID NO: 22 and a sequence selected from any one of SEQ ID NOs: 95-109,

[0090] (7) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22, SEQ ID NO: 107 and SEQ ID NO: 108, and

[0091] (8) A bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22, SEQ ID NO: 107, and SEQ ID NO: 109,

[0092] (9) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 162, and SEQ ID NO: 163, wherein sequence A is selected from any one of SEQ ID NOs: 4-14, 83-94, or 149-155;

[0093] (10) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, wherein sequence A is a sequence selected from any one of SEQ ID NOs: 4-14, 83-94, or 149-155;

[0094] (11) A bispecific antibody prodrug comprising a sequence represented by SEQ ID NO: 156, sequence A, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, and SEQ ID NO: 165, or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, and SEQ ID NO: 165, wherein sequence A is a sequence selected from any one of SEQ ID NOs: 4-14, 83-94, or 149-155,

[0095] (12) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 156, sequence A, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, and SEQ ID NO: 165, wherein sequence A is selected from any one of SEQ ID NOs: 4-14 or 83-94 and 149-155,

[0096] Preferably, the fusion protein is selected from the group consisting of:

[0097] (1) A fusion protein comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26 or 156 and a sequence selected from the group consisting of SEQ ID NO: 111;

[0098] (2) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112 and SEQ ID NO: 113;

[0099] (3) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112 and SEQ ID NO: 114;

[0100] (4) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112 and SEQ ID NO: 115;

[0101] (5) a fusion protein comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26 or 156 and a sequence selected from the group consisting of SEQ ID NO: 116;

[0102] (6) a fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 117, and SEQ ID NO: 108, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 117, and SEQ ID NO: 108;

[0103] (7) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112, SEQ ID NO: 118, and SEQ ID NO: 107;

[0104] (8) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112, SEQ ID NO: 119, and SEQ ID NO: 107;

[0105] (9) A fusion protein comprising or consisting of a sequence selected from any one of SEQ ID NOs: 120-125 and a sequence selected from any one of SEQ ID NOs: 25, 29, or 95-109;

[0106] (10) A fusion protein comprising a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 126-128, or consisting of a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 126-128;

[0107] (11) A fusion protein comprising or consisting of SEQ ID NO: 129 and a sequence selected from any one of SEQ ID NOs: 23, 25, 27, 29, 95-109, or 157;

[0108] (12) A fusion protein comprising or consisting of SEQ ID NO: 130, SEQ ID NO: 109, and a sequence selected from SEQ ID NO: 22, 26, or 156;

[0109] (13) A fusion protein comprising or consisting of SEQ ID NO: 131 and a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156;

[0110] (14) A fusion protein comprising or consisting of SEQ ID NO: 132 and a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156;

[0111] (15) A fusion protein comprising a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 134, 139, 140, or 143, or a fusion protein comprising a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 134, 139, 140, or 143;

[0112] (16) A fusion protein comprising or consisting of SEQ ID NO: 141 and a sequence selected from any one of SEQ ID NOs: 25, 29, 95-109, or 157;

[0113] (17) A fusion protein comprising or consisting of SEQ ID NO: 142 and a sequence selected from any one of SEQ ID NOs: 25, 29, 95-109, or 157;

[0114] (18) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 139 and SEQ ID NO: 141;

[0115] (19) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 140 and SEQ ID NO: 142;

[0116] (20) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 108, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 108;

[0117] (21) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 109, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 109;

[0118] (22) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 145, and SEQ ID NO: 108, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 145, and SEQ ID NO: 108;

[0119] (23) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NOs: 22, 26, and 156, SEQ ID NOs: 145, and SEQ ID NOs: 109, or consisting of a sequence selected from the group consisting of SEQ ID NOs: 22, 26, and 156, SEQ ID NOs: 145, and SEQ ID NOs: 109;

[0120] (24) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 185, SEQ ID NO: 186, and SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161;

[0121] (25) A fusion protein comprising an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody is selected from the sequence shown in any one of SEQ ID NOs: 23, 25, 27, 29, 95-109 or 157, the anti-tumor cell surface antigen antibody is selected from the sequence shown in any one of SEQ ID NOs: 22, 26 or 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 146-148, 166 or 169-184, and the linker is selected from the sequence shown in any one of SEQ ID NOs: 1-14, 24, 28, 30, 32, 83-94 or 149-155, preferably, the anti-CD3 antibody is in the form of scFv, and the cytokine is located at the C-terminus of the Fc fragment;

[0122] (26) A fusion protein comprising an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises SEQ ID NO: 158 and SEQ ID NO: 159, the anti-tumor cell surface antigen antibody is selected from the sequence shown in any one of SEQ ID NO: 22, 26, or 156, the cytokine is selected from the sequence shown in any one of SEQ ID NO: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NO: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and the cytokine and the cytokine receptor are located at the N-terminus of the anti-CD3 antibody;

[0123] (27) A fusion protein comprising an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises SEQ ID NO: 160 and SEQ ID NO: 161, the anti-tumor cell surface antigen antibody is selected from the sequence shown in any one of SEQ ID NO: 22, 26, or 156, the cytokine is selected from the sequence shown in any one of SEQ ID NO: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NO: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and the cytokine and the cytokine receptor are located at the N-terminus of the anti-CD3 antibody;

[0124] (28) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 162 and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NO: 110, 133, 135, 137, 146-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody or at the C-terminus of the Fc fragment;

[0125] (29) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162 and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 146-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody;

[0126] (30) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164 and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody;

[0127] (31) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164 and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody;

[0128] (32) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 162, and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: The sequence shown in any one of 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody;

[0129] wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or

[0130] wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody,

[0131] (33) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody,

[0132] wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or

[0133] wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody;

[0134] (34) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody,

[0135] wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or

[0136] wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody;

[0137] (35) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody,

[0138] wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or

[0139] wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody;

[0140] (36) a fusion protein comprising SEQ ID NO: 22, SEQ ID NO: 160, and SEQ ID NO: 161;

[0141] (37) A fusion protein comprising SEQ ID NO: 22, sequence a, and sequence b, wherein sequence a comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence b comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus;

[0142] (38) A fusion protein comprising sequence a, sequence b, and sequence c, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3, and SEQ ID NO: 110 from the N-terminus to the C-terminus, sequence b comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence c comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus;

[0143] (39) A fusion protein comprising sequence a, sequence b, and sequence c, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3, and SEQ ID NO: 176 from the N-terminus to the C-terminus, sequence b comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence c comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus;

[0144] (40) a fusion protein comprising sequence a, SEQ ID NO: 160 and SEQ ID NO: 161, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 176 from the N-terminus to the C-terminus;

[0145] (41) a fusion protein comprising SEQ ID NO: 22, sequence a and sequence b, wherein sequence a comprises SEQ ID NO: 167, SEQ ID NO: 149 and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence b comprises SEQ ID NO: 110, SEQ ID NO: 32 and SEQ ID NO: 161 from the N-terminus to the C-terminus;

[0146] (42) a fusion protein comprising SEQ ID NO: 22, sequence a and sequence b, wherein sequence a comprises SEQ ID NO: 163, SEQ ID NO: 149 and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence b comprises SEQ ID NO: 162, SEQ ID NO: 149 and SEQ ID NO: 161 from the N-terminus to the C-terminus;

[0147] (43) a fusion protein comprising SEQ ID NO: 185, SEQ ID NO: 186 and sequence a, wherein sequence a comprises SEQ ID NO: 162, SEQ ID NO: 149 and SEQ ID NO: 161 from the N-terminus to the C-terminus;

[0148] (44) A fusion protein comprising sequence a, sequence b, and sequence c, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3, and SEQ ID NO: 137 from the N-terminus to the C-terminus, sequence b comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence c comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus;

[0149] (45) a fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 23, SEQ ID NO: 3, and SEQ ID NO: 137 from the N-terminus to the C-terminus;

[0150] (46) a fusion protein comprising SEQ ID NO: 22 and SEQ ID NO: 23;

[0151] (47) a fusion protein comprising SEQ ID NO: 22 and SEQ ID NO: 157;

[0152] (48) a fusion protein comprising SEQ ID NO: 129 and SEQ ID NO: 157;

[0153] (49) a fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 181, SEQ ID NO: 32, SEQ ID NO: 149, and SEQ ID NO: 157 from the N-terminus to the C-terminus;

[0154] (50) a fusion protein comprising SEQ ID NO: 22 and SEQ ID NO: 116;

[0155] (51) A fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 184, SEQ ID NO: 3 and SEQ ID NO: 23 from the N-terminus to the C-terminus, or

[0156] (52) A fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 184, SEQ ID NO: 32, SEQ ID NO: 149 and SEQ ID NO: 23 from N-terminus to C-terminus.

[0157] 22. A biomaterial selected from the group consisting of:

[0158] (1) a polynucleotide encoding the fusion protein according to any one of items 1 to 21,

[0159] (2) A vector comprising the fusion protein according to any one of items 1 to 21, and

[0160] (3) A host cell comprising the fusion protein according to any one of items 1 to 21.

[0161] 23. A conjugate comprising the fusion protein of any one of items 1 to 21 and a conjugated moiety, wherein the conjugated moiety is selected from a purification tag (such as a His tag, a streptavidin tag), a cytotoxic agent, a detectable label, a radioactive isotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

[0162] 24. A pharmaceutical composition comprising the fusion protein of any one of items 1 to 21 and a pharmaceutical carrier, preferably, the pharmaceutical composition is in a dosage form suitable for oral administration to the gastrointestinal tract (GI), preferably, the dosage form is selected from tablets, capsules, pills, powders, granules, emulsions, microemulsions, solutions, suspensions, syrups and elixirs; or the drug is in a dosage form suitable for subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

[0163] 25. A kit comprising the fusion protein of any one of items 1 to 21 and a therapeutic agent for treating tumors, preferably the therapeutic agent for treating tumors is selected from chemotherapeutics, targeted therapeutics, T cells expressing chimeric antigen receptors, or angiogenesis inhibitors.

[0164] 26. Use of the fusion protein according to any one of items 1 to 21 in treating tumors, or in preparing a drug for treating tumors.

[0165] 27. The use according to item 26, wherein the tumor is selected from gastric cancer, pancreatic cancer, lung cancer, colon cancer, melanoma, microglioma.

[0166] definition

[0167] The antibodies described herein can be from any animal origin, including birds and mammals, including primates. Preferably, the antibodies are human, baboon, rhesus monkey, cynomolgus monkey, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies.

[0168] The antibodies involved in the present invention are IgG antibodies, wherein the CDRs involved therein (based on the heavy chain variable region and the light chain variable region, a person of ordinary skill in the art can define the CDRs sequence according to numbering systems known in the art, such as the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, etc.), heavy chain variable region, light chain variable region, light chain constant region, heavy chain constant region, hinge region, and disulfide bond are all terms well known in the art.

[0169] The term "detectable label" as used herein refers to a compound or composition that can be detected directly or indirectly, and the compound or composition is directly or indirectly bound to a composition to be detected (e.g., a polynucleotide or protein, such as an antibody) to obtain a "labeled" composition. The term also includes a sequence that is bound to the polynucleotide, which provides a signal by the expression of an inserted sequence, such as green fluorescent protein (GFP). The label itself can be detected (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze a chemical change in a substrate compound or composition that can be detected. The label can be used for small-scale detection or is more suitable for high-throughput screening. Similarly, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). The label can only be detected or can be quantified. The reaction that is only detected generally includes a reaction that can only confirm its presence, and the reaction that can be quantified generally includes a reaction with a quantifiable value (e.g., that can be reported digitally) such as intensity, polarization, and / or other properties. In luminescent or fluorescent assays, the detectable reaction can be directly using a luminophore or fluorophore associated with a component of the assay that actually involves binding, or indirectly using a luminophore or fluorophore linked to another (eg, a reporter molecule or indicator) component.

[0170] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In certain aspects, these regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and also contain serine or threonine for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, and vice versa. The protein retains the properties of the original immunoglobulin, except that the constant regions have been removed and a linker has been introduced. ScFv molecules are known in the art, such as those described in U.S. Patent No. 5,892,019.

[0171] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like.

[0172] Those skilled in the art understand that the expression "cleavable linker" means that some amino acid sequences can be recognized by some enzymes in vivo or in vitro, and then disconnected in the middle, so that the sequences connecting the two ends can be separated, for example, selected from MMP enzymes, uPA enzymes, serine hydrolases, etc.

[0173] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.

[0174] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D ) binds to the antigen.

[0175] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind with a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binds to an antigen (e.g., PD-1 protein). K can be determined using methods known to those skilled in the art. D , for example, using the Fortebio molecular interaction instrument.

[0176] In the present invention, the structural unit A is a bispecific antibody. In some embodiments, the bispecific antibody exists in the form of a bispecific antibody prodrug, wherein the anti-CD3 antibody is connected to a cytokine, a cytokine prodrug or a tumor cell targeting peptide via a cleavable linker, and the activity of the anti-CD3 antibody is restored by cleaving the cytokine, cytokine prodrug or the tumor cell targeting peptide. Description of the drawings:

[0177] Figure 1. Schematic diagram of several structures of bispecific antibodies that bind to the same tumor surface-specific antigen.

[0178] Figure 2. Schematic diagrams of several structures of bispecific antibodies that bind to different tumor surface-specific antigens.

[0179] Figure 3. Schematic diagrams of several structures of bispecific antibodies, including a tumor-targeting peptide and an antibody that binds to a specific antigen on the tumor surface.

[0180] Figure 4. Schematic diagrams of several bispecific antibody structures, including a tumor-targeting peptide and an antibody that binds to a tumor-specific antigen on the surface in tandem.

[0181] Figure 5. SEC-HPLC analysis of the purity of the bispecific antibody.

[0182] Figure 6. Flow cytometry was used to detect the binding ability of bispecific antibodies to the CLDN18.2 antigen overexpressed on the colorectal cancer cell line MC38. The bispecific antibodies used were PDL1×CD3 (UCHT1) with the structure shown in Figure 2A, PDL1×CLDN-CD3 (UCHT1) with the structure shown in Figure 2D, CLDN×CD3 (UCHT1) with the structure shown in Figure 1A, and CLDN×CLDN-CD3 (UCHT1) with the structure shown in Figure 1A.

[0183] Figure 7. SDS-PAGE electrophoresis analysis of the efficiency and results of fusion protein digestion with MMP14. The bispecific antibody used was PDL1×CLDN-MMP(double)-CD3(UCHT1), as shown in Figure 2D. Lane 1 shows the electrophoresis of PDL1×CLDN-MMP(double)-CD3(UCHT1) alone, lane 2 shows the electrophoresis of PDL1×CLDN-MMP(double)-CD3(UCHT1) digested overnight with MMP14, and lane 3 shows the electrophoresis of PDL1×CD3(UCHT1) alone, as shown in Figure 1A.

[0184] Figure 8. The biological activity recovery of the fusion protein under conditions of enzymatic cleavage and non-enzymatic cleavage was identified using spleen cells from Human CD3KI mice. The bispecific antibodies used were PDL1×CLDN-MMP(double)-CD3(UCHT1)N with the structure shown in Figure 2D and PDL1×CLDN-MMP(double)-CD3(UCHT1)C with the structure shown in Figure 2D, where N indicates no MMP addition and C indicates MMP addition and cleavage, and the structure was PDL1×CD3(UCHT1) with the structure shown in Figure 2A.

[0185] Figure 9. Compared with the bispecific antibody CLDN*CD3, the bispecific antibodies of the present invention have better in vivo anti-tumor effects. The bispecific antibodies used are CLDN×CD3 (UCHT1) with the structure shown in Figure 1A and CLDN×CLDN-CD3 (UCHT1) with the structure shown in Figure 1D.

[0186] Figure 10. The experimental process is shown in Figure 10A. 24 hours after administration, peripheral blood was collected and serum was collected for cytokine analysis. Figure 10B shows the cytokine levels in peripheral serum. The results show that compared to the bispecific antibody PDL1*CD3, the bispecific antibody PDL1×CLDN-MMP(double)-CD3(OKT3) with the structure shown in Figure 2D secreted lower levels of inflammatory cytokines (including IL-6, IFN, TNF, etc.) in serum, indicating a better safety profile. The bispecific antibody used was PDL1×CD3(OKT3) with the structure shown in Figure 1A.

[0187] Figure 11. SDS-PAGE images of bispecific antibodies cleaved or not by MMP14, wherein Figure 11A shows the SDS PAGE image of PDL1×CLDN-MMP(double)-CD3(OKT3) with the structure shown in Figure 2D after cleavage (+) or not (-) by MMP14, wherein lanes 3, 4, 6, and 7 from the left are irrelevant samples; Figure 11B shows the SDS PAGE image of PDL1×targeting peptide-HA-MMP(double)-CD3(OKT3) with the structure shown in Figure 3C after cleavage (+) or not (-) by MMP14, wherein lanes 3 and 4 from the left are irrelevant samples.

[0188] Figure 12. PBMC activation by bispecific antibodies after MMP14 cleavage or not, wherein Figure 12A shows the PBMC activation of PDL1×CLDN-MMP(double)-CD3(OKT3) with the structure shown in Figure 2D with or without MMP14 cleavage; Figure 12B shows the PBMC activation of PDL1×NGR-HA-MMP(double)-CD3(OKT3) with the structure shown in Figure 3C with or without MMP14 cleavage, wherein N indicates no MMP addition and C indicates cleavage with MMP addition.

[0189] Figure 13. Schematic diagram of an exemplary structure of the fusion protein of the present invention, wherein Figure 13A shows that the cytokine is connected to the anti-CD3 antibody via a cleavable linker; Figure 13B shows that the cytokine prodrug is connected to the anti-CD3 antibody via a cleavable linker; Figure 13C shows that the cytokine is connected to the anti-CD3 antibody via a non-cleavable linker; Figure 13D shows that the cytokine prodrug is connected to the anti-CD3 antibody via a non-cleavable linker; Figure 13E shows that the cytokine is connected to the anti-CD3 antibody via a cleavable linker, and the anti-CD3 antibody and the antibody against the tumor cell surface antigen are in the form of scFv and are connected in series.

[0190] Figure 14. Schematic diagrams of exemplary structures of fusion proteins of the present invention, wherein Figure 14A shows an anti-CD3 antibody linked to an anti-TAA (or tumor cell targeting peptide) via a cleavable linker and a cytokine at the C-terminus of the Fc; Figure 14B shows an anti-CD3 antibody linked to an anti-TAA (or tumor cell targeting peptide) via a non-cleavable linker and a cytokine at the C-terminus of the Fc; Figure 14C shows an anti-CD3 antibody linked to an anti-TAA (or tumor cell targeting peptide) via a cleavable linker and a cytokine prodrug at the C-terminus of the Fc; Figure 14D shows an anti-CD3 antibody linked to an anti-TAA (or tumor cell targeting peptide) via a non-cleavable linker and a cytokine prodrug at the C-terminus of the Fc; Figure 14E shows an anti-CD3 antibody linked to an anti-TAA (or tumor cell targeting peptide) via a cleavable linker, without an Fc, and a cytokine tandemly linked to an anti-TAA (or tumor cell targeting peptide)

[0191] Figure 15. Schematic diagram of an exemplary structure of the fusion protein of the present invention, wherein Figure 15A shows that one side of the Fc is connected to an anti-CD3 antibody, and is connected to an anti-TAA (or tumor cell targeting peptide) through a non-cleavable linker, and a cytokine is connected to the other side of the Fc to form a heterodimer; Figure 15B shows that one side of the Fc is connected to an anti-CD3 antibody, and is connected to an anti-TAA (or tumor cell targeting peptide) through a non-cleavable linker, and a cytokine prodrug is connected to the other side of the Fc to form a heterodimer.

[0192] FIG16 Flow cytometry was used to detect the binding ability of the exemplary fusion protein of the present invention to the CLDN18.2 antigen overexpressed on the colorectal cancer cell line MC38.

[0193] FIG17 shows the in vivo anti-tumor effect of the exemplary fusion protein of the present invention. FIG17A shows the changes in tumor volume, and FIG17B shows the changes in body weight.

[0194] Figure 18. Schematic diagram of exemplary structures of fusion proteins of the present invention, wherein Figure 18A shows that the cytokine is fused to the C-terminus of the anti-CD3 heavy chain (it can also be fused to the other side of the C-terminus); Figure 18B shows that the cytokine is fused to the C-terminus of the anti-CD3 light chain; Figure 18C shows that the cytokine and receptor are fused to the C-terminus of the anti-TAA heavy chain (wherein the positions of the cytokine and receptor can be interchanged); Figure 18D shows that the cytokine and cytokine receptor are fused to the hinge region of Fc and the middle of the anti-CD3 / anti-TAA light and heavy chains (wherein the positions of the cytokine and receptor are interchangeable). Figure 18E shows that the cytokine and receptor are fused at the C-terminus of the anti-TAA heavy chain (wherein the positions of the cytokine and receptor are interchangeable); Figure 18F shows that the cytokine and cytokine receptor are fused at the C-terminus of the anti-CD3 light chain (wherein the positions of the cytokine and receptor are interchangeable); Figure 18G shows that the cytokine and receptor are fused at the N-terminus of the anti-CD3 light and heavy chains, respectively (wherein the positions of the cytokine and receptor are interchangeable); Figure 18H shows that the cytokine is fused at the C-terminus of the anti-TAA light and heavy chains (it can also be fused on the other side of the C-terminus).

[0195] FIG19 shows the in vivo anti-tumor effect of the IL2 fusion protein of the present invention.

[0196] FIG20 shows the in vivo anti-tumor effect of the fusion protein of IL2 and IL2 receptor Ra in a PBMC humanized model.

[0197] FIG21 shows the in vivo anti-tumor effect of the IL15 fusion protein of the present invention.

[0198] FIG. 22 illustrates the in vivo anti-tumor effect of an IL15 fusion protein without structural unit 3 according to the present invention.

[0199] FIG. 23 shows the in vivo anti-tumor effect of the IL21 fusion protein of the present invention ( FIG. 23A ) and the cytokine levels in peripheral serum ( FIG. 23B ).

[0200] FIG24 shows the in vivo anti-tumor effect of the exemplary IL4 fusion protein of the present invention ( FIG24A ) and the cytokine levels in peripheral serum ( FIG24B ).

[0201] FIG. 25 shows the binding ability of the IL10 fusion proteins with the structures shown in FIG. 13A and FIG. 13C to PBMCs in accordance with the present invention.

[0202] FIG26 shows the in vivo anti-tumor effect of the IL10 fusion protein shown in FIG13A and FIG13C ( FIG26A ) and the cytokine levels in peripheral serum ( FIG26B ) in an example of the present invention. DETAILED DESCRIPTION

[0203] The present invention is further described in detail by the following examples. It will be understood by those skilled in the art that the present invention is not limited by the following. The scope of the present invention is defined by the claims. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field (for example, refer to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press) or according to the product instructions. If the manufacturer of the reagents or instruments used is not specified, they are conventional products that can be purchased on the market.

[0204] experimental animals

[0205] Human CD3 KI (hCD3EDG) mice were purchased from Jicui Yaokang. NSG-SGM3 mice were imported from JAX and bred in-house. Wild-type C57BL / 6 mice were purchased from the Weitonglihua Laboratory Animal Center in Beijing, China. Unless otherwise specified, all experiments used mice aged 8–10 weeks. Mice were maintained in a specific pathogen-free (SPF) barrier environment. Animal husbandry and experimental procedures complied with the regulations of the Changping Laboratory Animal Care Committee.

[0206] cell lines

[0207] FreeStyle™ 293F cell line (Invitrogen) is a suspension cell line derived from the HEK293 cell line. It is cultured in SMM293-TII medium and used for transient transfection to express fusion proteins.

[0208] A549 is a human non-small cell lung cancer cell line, and MC38 is a mouse colorectal cancer cell line with a C57 background. Both were purchased from ATCC. A549-CLDN and MC38-CLDN cell lines (i.e., CLDN-overexpressing cell lines) were established by lentiviral infection with cells overexpressing mouse CLDN18.2 protein (P56856) and cultured in complete DMEM medium (supplemented with 10% inactivated fetal bovine serum, 2 mmol / l L-glutamine, 0.1 mmol / l non-essential amino acids, 100 U penicillin, and 100 μg / ml streptomycin).

[0209] Biostatistical analysis

[0210] All data were analyzed using GraphPad Prism statistical software. Mouse survival curves were analyzed using one-way ANOVA, and other data were analyzed using two-tailed t-tests.

[0211] Example 1. Construction, expression and purification of fusion protein

[0212] 1. Construction of BiTE:

[0213] To design a highly effective and low-toxic bispecific antibody fusion protein for tumors, a T cell-bridging bispecific T-cell engager (BiTE) was used as the basis (Figure 1A). The activity of the CD3 antibody (anti-CD3) was masked using an antibody that binds to a tumor surface-specific antigen (anti-TAA) or a tumor cell-targeting peptide. The anti-TAA (or tumor cell-targeting peptide) and anti-CD3 sequences were linked together using different intermediate linkers selected from the group consisting of:

[0214] i. non-cleavable linkers (e.g. GS of different lengths (SEQ ID NO: 1-3, 24, 28, 30, 32), FIG. 1B ),

[0215] ii. a linker formed by connecting a cleavable linker and a pH-sensitive linker in series (e.g., a polypeptide substrate cleavable by an MMP and an HA linker, SEQ ID NO: 4-5, FIG. 1C ),

[0216] iii. a cleavable linker (e.g., a polypeptide substrate cleavable by an MMP (SEQ ID NO: 6-9, FIG. 1D ),

[0217] iv. pH-sensitive linkers formed in series (e.g., multiple HA linkers (SEQ ID NO: 10-12, FIG. 1E ), or

[0218] v. inserting a cleavable linker into the middle position of the pH-sensitive linker (e.g., a polypeptide substrate sequence that can be cleaved by MMP and an HA linker are placed alternately (SEQ ID NO: 13-14, FIG. 1F ),

[0219] vi. At one end of the Fc (SEQ ID NO: 19-20), the anti-TAA and anti-CD3 sequences were linked together in a homologous (Homo) format using a cleavable linker, as shown in FIG1G .

[0220] vii. In the absence of Fc (SEQ ID NOs: 19-20), structural units 1 and 3 are linked together via a cleavable linker (e.g., a polypeptide substrate cleavable by an MMP (SEQ ID NOs: 6-9), as shown in FIG1H ).

[0221] Figure 2 shows the BiTE structure, wherein Figure 2A shows a general BiTE structure, in which there is a CD3-binding domain and a TAA-binding domain; Figure 2B shows that based on the structure of Figure 2A, the CD3-binding domain is connected to an anti-TAA (or tumor cell targeting peptide) through a non-cleavable linker; Figure 2C shows that based on the structure of Figure 2A, the CD3-binding domain is connected to an anti-TAA (or tumor cell targeting peptide) through a cleavable linker and a pH-sensitive linker; Figure 2D shows that based on the structure of Figure 2A, the CD3-binding domain is connected to an anti-TAA (or tumor cell targeting peptide) through a cleavable linker. 2E shows that, based on the structure of FIG2A , the CD3-binding domain is connected to the anti-TAA (or tumor cell targeting peptide) through a pH-sensitive linker; FIG2F shows that, based on the structure of FIG2A , the CD3-binding domain is connected to the anti-TAA (or tumor cell targeting peptide) by an alternating placement of a cleavable linker and a pH-sensitive linker; FIG2G shows that, based on the structure of FIG2D , there is no Fc, and the CD3-binding domain is connected to the anti-TAA (or tumor cell targeting peptide) through a cleavable linker and a pH-sensitive linker.

[0222] Figure 3 shows that two different tumor antigens are bound in BiTE, and the one used to shield the anti-CD3 activity is a tumor cell targeting peptide (e.g., NGR, EphA2, EphA4), and the other non-shielding function is anti-TAA. Figure 3A shows that on the basic BiTE structure, the CD3-binding domain is connected to the structural unit 3 through a non-cleavable linker; Figure 3B shows that on the basic BiTE structure, the CD3-binding domain is connected to the structural unit 3 in series through a cleavable linker and a pH-sensitive linker; Figure 3C shows that on the basic BiTE structure, the CD3-binding domain is connected to the structural unit 3 through a cleavable linker; Figure 3D shows that on the basic BiTE structure, the CD3-binding domain is connected to the structural unit 3 through a pH-sensitive linker; Figure 3E shows that on the basic BiTE structure, the CD3-binding domain is connected to the structural unit 3 through a cleavable linker and a pH-sensitive linker in an alternating manner; Figure 3F shows that, based on the structure of Figure 3A, there is no Fc, and the CD3-binding domain is connected to the structural unit 3 through a cleavable linker and a pH-sensitive linker.

[0223] Figure 4 shows that the same tumor antigen is bound in BiTEs, and tumor targeting peptides and anti-TAA are connected in series to shield anti-CD3 activity. Figure 4A shows that based on Figure 2C, anti-TAA is connected to tumor cell targeting peptide; Figure 4B shows that based on Figure 2D, anti-TAA is connected to tumor cell targeting peptide; Figure 4C shows that based on Figure 2E, anti-TAA is connected to tumor cell targeting peptide; Figure 4D shows that based on Figure 2F, anti-TAA is connected to tumor cell targeting peptide; Figure 4E shows that based on Figure 2G, anti-TAA is connected to tumor cell targeting peptide.

[0224] The sequences used in the examples are as follows:

[0225] The sequence of the anti-CD3 antibody was selected from the UCHT1 (SEQ ID NO: 15) or OKT3 clone (SEQ ID NO: 16) that can activate CD3 signaling.

[0226] The commercialized Atezolizumab sequence (SEQ ID NO: 17) was used as the anti-PDL1 antibody.

[0227] The anti-CLDN18.2 sequence is the sequence shown in SEQ ID NO: 18.

[0228] The human IgG1 sequences lacking ADCC function and having “Knob” and “Hole” mutations are shown in SEQ ID NO: 19 and SEQ ID NO: 20.

[0229] Each antibody was linked via different linkers and constructed into the expression vector pEE6.4 plasmid (purchased from LifeScience Market), using a signal peptide (SEQ ID NO: 21, SEQ ID NO: 47 can also be used). Host cells containing the vector were constructed by transient transfection of 293F cells, cultured, and the supernatant collected. Finally, the protein was purified by affinity chromatography using a Protein A / G column and stored in aliquots at -80°C.

[0230] For the expression of heterodimeric proteins, two expression vector plasmids with the same molar ratio need to be transferred. Monomers expressed in cells can also spontaneously form heterodimers.

[0231] Specifically, expression plasmids of various fusion proteins were genetically synthesized on the PEE6.4 vector, and then the expression supernatants of various proteins were obtained by transient transfection into 293F cells. Finally, various proteins were purified by Protein A affinity chromatography column.

[0232] Construct the following expression vector:

[0233] (1)PEE6.4-HindⅢ-IgK(signal)-anti-Claudin 18.2-Fck-EcoRI

[0234] (2)PEE6.4-HindⅢ-IgK(signal)-anti-CD3(UCHT1)-Fch-EcoRI

[0235] (3) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-Linker 1-anti-CD3-Fch-EcoRI

[0236] (4) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-Linker 2-anti-CD3-Fch-EcoRI

[0237] (5) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 2×2-anti-CD3-Fch-EcoRI

[0238] (6) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 3+2-anti-CD3-Fch-EcoRI

[0239] (7) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 3×2-anti-CD3-Fch-EcoRI

[0240] (8) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 4×2-anti-CD3-Fch-EcoRI

[0241] (9)PEE6.4-HindⅢ-IgK(signal)-anti-PDL1-Fck-EcoRI

[0242] (10)PEE6.4-HindⅢ-IgK(signal)-anti-CD3(OKT3)-Fch-EcoRI

[0243] (11) PEE6.4-HindⅢ-IgK(signal)-NGR-HA-Linker 2x2-Anti-CD3(OKT3)-Fch-EcoRI

[0244] (12) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 2x2-anti-CD3 (UCHT1)-Fch-mutIL2-EcoRI

[0245] Among them, HindⅢ and EcoRI are restriction enzyme cleavage sites; Fck represents the inactivated Fc region (Knob, SEQ ID NO: 19), Fch represents the inactivated Fc region (hole, SEQ ID NO: 20), and mutIL2 represents the mutated IL2 (SEQ ID NO: 110).

[0246] The connection sequences between the fragments are:

[0247] (1) Linker 1: G4S: GGGGS (SEQ ID NO: 32)

[0248] (2) Linker 2: MMP: any one of SEQ ID NOs: 6-8, linker 2×2 represents any two-by-two combination of SEQ ID NOs: 6-8,

[0249] (3) Linker 3: HA: NQHTIDLADS (SEQ ID NO: 10)

[0250] (4) Linker 4: H(MMP14)A: NQHIANPVDS (SEQ ID NO: 13)

[0251] The obtained fusion proteins are as follows:

[0252] Anti-CLDN18.2×anti-CD3 (UCHT1) shown in Figure 1A, i.e., CLDN×CD3 [anti-CLDN single-chain (VL-VH) and CD3 single-chain (VL-VH)], CLDN-Fc (Knob) shown in SEQ ID NO: 22, CD3-Fc (Hole) shown in SEQ ID NO: 23;

[0253] Anti-CLDN18.2 x anti-CLDN18.2-MMP (double) - anti-CD3 (UCHT1) shown in Figure 1D, i.e., CLDN × CLDN-MMP (double) -CD3 (UCHT1) [anti-CLDN single-chain (VL-VH) and CD3 single-chain (VL-VH)], CLDN-Fc (Knob) shown in SEQ ID NO: 22, and CLDN-MMP (double) -CD3-Fc (Hole) shown in SEQ ID NO: 25;

[0254] Anti-PDL1×anti-CD3 (OKT3) shown in Figure 2A, i.e., PDL1×CD3 (OKT3) [anti-PDL1 single-chain (VL-VH) and CD3 single-chain (VL-VH)], PDL1-Fc (Knob) shown in SEQ ID NO: 26, and CD3-Fc (Hole) shown in SEQ ID NO: 27;

[0255] Anti-PDL1×anti-CLDN18.2-MMP(double)-anti-CD3(OKT3) shown in Figure 2D, i.e., PDL1×CLDN-MMP(double)-CD3(OKT3) [anti-PDL1 single-chain(VL-VH)and CD3 single-chain(VL-VH)], PDL1-Fc(Knob) shown in SEQ ID NO:26, CLDN-MMP(double)-CD3-Fc(Hole) shown in SEQ ID NO:29;

[0256] PDL1×NGR-HA-MMP(double)-CD3(OKT3) shown in Figure 3B, i.e., PDL1×NGR-HA-MMP(double)-CD3(OKT3) [anti-PDL1 single-chain(VL-VH)and CD3 single-chain(VL-VH)], PDL1-Fc(Knob) shown in SEQ ID NO:26, and NGR-HA-MMP(double)-CD3-Fc(Hole) shown in SEQ ID NO:31.

[0257] In the Examples, MMP (double) represents a linker combination of SEQ ID NO: 8 and SEQ ID NO: 6, and the sequence is shown in SEQ ID NO: 9.

[0258] 1.2 Transient transfection to quickly express the target protein:

[0259] Resuscitate Freestyle 293F cells as standard. Add the plasmid / PEI mixture to the cell suspension, transfect as standard, collect the supernatant, and purify the target protein using Protein A.

[0260] 1.3 SEC-HPLC identification

[0261] Taking the anti-CLDN18.2xanti-CLDN18.2-MMP (double)-anti-CD3 (UCHT1) shown in FIG1D and the anti-CLDN18.2xanti-CD3 (UCHT1) shown in FIG1A as examples, first prepare the mobile phase: 500 ml 1M Tris-HCl (pH 6.8), and insert the analytical column (Thermo MAbPac SEC-1, 5 μm The analytical column (4 x 300 mm) was installed on a Waters ACQUITY Premier UPLC instrument, with PDA detection at UV 280 nm, a flow rate of 0.15 ml / min, and a 30-minute separation. Data were acquired, processed, and exported using Empower software. The SEC-HPLC identification spectrum is shown in Figure 5 . The HPLC peak profile showed that the anti-CLDN18.2 x anti-CLDN18.2-MMP (double)-anti-CD3 (UCHT1) and anti-CLDN18.2 x anti-CD3 (UCHT1) proteins were essentially free of contaminants (Figure 5).

[0262] Example 2. In vitro biological activity study of fusion protein

[0263] 2.1 Detection of fusion protein binding ability to antigen

[0264] The binding properties of the purified bispecific antibodies were analyzed using flow cytometry. The results are shown in Figure 6 (the bispecific antibodies used were PDL1×CD3(UCHT1) with the structure shown in Figure 1A, PDL1×CLDN-MMP(double)-CD3(UCHT1) with the structure shown in Figure 2A, CLDN×CD3(UCHT1) with the structure shown in Figure 1A, CLDN×CLDN-MMP(double)-CD3(UCHT1) with the structure shown in Figure 1D, and PDL1×CLDN-MMP(double)-CD3(UCHT1) with the structure shown in Figure 2D).

[0265] (1) Target cell preparation: MC38 cells overexpressing Claundin 18.2 (ACCESSION: P56856) were selected as target cells for the binding test. The cells were cultured in DMEM containing 10% FBS (Fetal Bovine Serum) and 1% P / S (Penicillin, Streptomycin) for more than three generations. When used, the cells were resuspended and the concentration was adjusted to 5×10 6 / ml.

[0266] (2) Protein sample preparation: Each protein sample was diluted 10-fold in a 10-fold gradient, with the highest concentration set at 400 nmol / L. Label the tubes and arrange them in the following order: 1:10, 1:100, 1:100, 1:10000, 1:100000. Add 450 μl of FACS buffer to each tube, transfer 50 μl of the highest concentration protein sample to the 1:10 dilution tube, and mix thoroughly using a pipette. Continue the serial dilution by transferring 50 μl from the 1:10 tube to the 1:100 tube, and then to the 1:100000 tube.

[0267] (3) Incubation of cells and samples: Add 100 μl of the prepared cell suspension to each well of a round-bottom 96-well plate, i.e., 5 × 10 5 After centrifugation at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells with the prepared protein sample, make duplicate wells, and incubate at 4°C in the dark for 30 minutes.

[0268] (4) Commercial fluorescent antibody (Biolegend) conjugated to labeled samples: Add 150 μl of FACS buffer to a 96-well plate, mix thoroughly, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and repeat twice. Add 0.5 μl of commercial anti-human Fc-APC / PE per well (Biolegend, 0.5 μl antibody added to 50 μl FACS, mix thoroughly). Incubate at 4°C in the dark for 30 minutes.

[0269] (5) Flow cytometry: Add 150 μl of FACS buffer to each well, mix thoroughly, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and repeat twice. Resuspend the cells in 150-200 μl of FACS buffer and transfer to the flow cytometer.

[0270] The results in Figure 6 show that CLDN×CLDN-MMP(double)-CD3(UCHT1) with the structure shown in Figure 1D has the highest binding ability to the MC38-CLDN cell line, followed by PDL1×CLDN-MMP(double)-CD3(UCHT1) with the structure shown in Figure 2D), while PDL1×CD3(UCHT1) with the structure shown in Figure 1A has no binding to this cell line at all.

[0271] 2.2 MMP14 in vitro enzyme cleavage assay

[0272] The protein samples were cleaved in vitro using the enzyme MMP14 to verify the cleavage effect.

[0273] First, recombinant human MMP14 (R&D, Sciences) at a concentration of 80 μg / ml was incubated with 1.72 μg / ml rhFurin (Roche) in activation buffer (50 mM Tris, 1 mM CaCl2, 0.5% (w / v) Brij 35, pH 9.0, filtered through a 0.22 μm filter) for 2 hours. Protein samples and activated MMP14 were added to the assay buffer at a molar ratio of 5:1 and incubated for 24 hours. The protein concentration in the enzyme digestion system was 100 μg / ml. The activation buffer consisted of 50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 8.5, and filtered through a 0.22 μm filter. Proteins were then analyzed by SDS-PAGE, and relative gel band intensities were analyzed to quantify the amount of cleavage in each protein / MMP14 mixture. The SDS-PAGE electrophoresis pattern is shown in Figure 7. The results in FIG7 show that PDL1×CLDN-MMP(double)-CD3(UCHT1) with the structure shown in FIG2D can be completely cleaved by MMP enzyme.

[0274] 2.3 Experimental study on the recovery of MMP14 activity after in vitro enzymatic cleavage

[0275] Following the method described in the in vitro MMP14 enzyme cleavage assay in 2.2, we performed an in vitro enzyme cleavage experiment on PDL1×CLDN-MMP(double)-CD3(UCHT1) with the structure shown in Figure 2D, and set up a control with the same incubation conditions but without MMP14 enzyme. The SDS-PAGE electrophoresis identification pattern is shown in Figure 7, and the results of the binding ability recovery test after enzyme cleavage are shown in Figure 8.

[0276] CD3 + Cell sorting: using MojoSort TM Human CD3T Cell Isolation Kit (Biolegend) was used for sorting. According to the instructions, the materials in the kit were provided, and MojoSort TM Buffer was used to adjust the cell concentration to 1 x 10 8 / mL. Add 100 μL of biotin-antibody cocktail. Mix well and incubate on ice for 15 minutes. Resuspend the beads by vortexing at maximum speed, touching down 5 times. Add 10 μL of streptavidin nanobeads. Mix well and incubate on ice for 15 minutes. Add 2.5 mL of MojoSort TM buffer and place the tube in the magnet for 5 min.

[0277] Pour off and collect the liquid, and adjust the cell concentration to 3 × 10 6 / ml.

[0278] (1) Protein sample preparation: Each protein sample was set to a maximum concentration of 200 nmol / L according to the equimolar mass (the protein sample after enzyme digestion was still calculated according to the original molar mass), and then diluted 10-fold in a gradient and mixed thoroughly with a pipette.

[0279] (2) Incubation of cells and protein samples: Add 100 μl of the prepared cell suspension to each well of a round-bottom 96-well plate, i.e., 3×10 5 After centrifugation at 1600 rpm for 5 minutes, discard the supernatant, resuspend the cells with the prepared protein sample, make duplicate wells, and incubate at 4°C in the dark for 30 minutes.

[0280] (3) Commercial fluorescent antibody-labeled protein sample: Add 150 μl of FACS buffer to a 96-well plate, mix thoroughly, and centrifuge at 1600 rpm for 5 minutes. Discard the supernatant and repeat twice. Add 0.5 μl of commercial anti-human Fc-APC / PE per well (add 0.5 μl of antibody to 50 μl of FACS buffer and mix thoroughly). Incubate at 4°C in the dark for 30 minutes.

[0281] (4) Flow cytometry: Add 150 μl of FACS buffer to each well, mix thoroughly, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and repeat twice. Resuspend the cells in 150-200 μl of FACS buffer and transfer to the flow cytometer.

[0282] (5) To detect T cell activation in vitro, purified human PBMCs were resuspended in culture medium (RPMI1640 with 10% FBS and supplemented with 1% P / S and 1% glutamine) (Thermo Fisher Scientific) to adjust the concentration to 5×10 6 100 μl of the solution was added to each well of a round-bottom 96-well plate.

[0283] (6) Prepare protein samples of different concentrations and whether they were cleaved or not using RPMI1640 as described in 2.2-2. Add the prepared protein samples to each well in turn and repeat the wells. Incubate in a tissue culture incubator for 48 hours.

[0284] (7) After the incubation, the cells were centrifuged at 1600 rpm for 5 minutes, and the supernatant was collected for measuring cytokine release or discarded. The cells were stained with fluorescently labeled antibodies for T cell markers (CD3, CD4, CD8, etc.) and activation markers (CD69, CD25, etc.), detected by flow cytometry, and then analyzed using FlowJo software (FlowJo v10) and plotted as shown in Figure 8. The results in Figure 8 show that after in vitro enzyme cleavage, the biological activity of the fusion protein CD3 antibody in the structure of PDL1×CLDN-MMP (double)-CD3 (UCHT1) as shown in Figure 2D can be well restored, and can be restored to the effect of PDL1×CD3 (UCHT1) as shown in Figure 1A (Figure 8). The above results show that the activity of CD3 in the antibody can be well blocked by anti-CLDN18.2, and its activity on T cells can be effectively restored after MMP cleavage.

[0285] Example 3. New BiTEs exhibit better anti-tumor effects than traditional BiTEs

[0286] To determine whether the TAA×TAA / targeting peptide-CD3 designed and produced by us can effectively fight tumors when administered systemically and to compare its therapeutic effect with that of traditional bispecific antibodies, we inoculated MC38-CLDN (MC38 cells overexpressing Claudin 18.2) into hCD3 humanized mice (hCD3EDG) and treated the mice with a lower dose by systemic administration.

[0287] hCD3EDG mice (n=5 / group) were subcutaneously inoculated with 1×10 6 MC38-CLDN cells. On day 11, the tumor grew to 80-120 mm 3 The mice were randomly divided into groups and administered with PBS at 200 pmol / mouse on days 11 and 14. The mice were treated with PDL1×CD3(UCHT1) (non-targeted) as shown in FIG2A , PDL1×CLDN-MMP(double)-CD3(UCHT1) (targetable to the tumor microenvironment, non-targeted after MMP cleavage) as shown in FIG2D , CLDN×CD3(UCHT1) as shown in FIG1A , and CLDN×CLDN-MMP(double)-CD3(UCHT1) as shown in FIG1D . The tumor volumes of the tumor-bearing mice were recorded.

[0288] The experimental results are shown in Figure 9: The CLDN×CD3 group was able to inhibit tumor growth, but only 40% of the mice experienced tumor volume reduction and rapid rebound. The CLDN×CLDN-MMP(double)-CD3 group observed significant anti-tumor activity, with tumor volume reduction in 80% of the mice and complete tumor regression in 10% of the mice, but rebound also occurred later. Under low-dose treatment, the anti-tumor activity of the CLDN×CLDN-MMP(double)-CD3 group was significantly superior to the CLDN×CD3 group, and no significant weight loss was observed in any treatment group. This result indicates that CLDN×CLDN-MMP(double)-CD3 can effectively eliminate tumors and has a better tumor clearance effect than traditional bispecific antibodies.

[0289] Example 4. New BiTEs exhibit lower peripheral toxicity than traditional BiTEs

[0290] Because human CD3 KI mice are not very sensitive to peripheral toxicity, we used hPBMC humanized (hu-PBMC) NSG mice as an in vivo mouse model. These mice were generated by injecting purified human PBMCs from healthy donors. They have the fastest engraftment rate using adult peripheral blood mononuclear cells and enable short-term studies of robust effector and memory T cell function.

[0291] We selected A549 cells overexpressing human CLDN18.2 protein (P56856) by lentiviral infection as the tumor model for this study and inoculated 1*10 6 A549-CLDN cells were grown, which was counted as day 0. When the tumor grew to day 22, PBMCs were transplanted by tail vein injection. Two days later, the mice were randomly divided into groups and intraperitoneally injected with 100pnol of PDL1 x CD3 (PDL1×CD3(OKT3)[anti-PDL1 single-chain(VL-VH)and CD3 single-chain(VL-VH)]), PDL1x CLDN-CD3 (PDL1×CLDN-MMP(double)-CD3(OKT3)[anti-PDL1 single-chain(VL-VH)and CD3 single-chain(VL-VH)]). After 24 hours, venous blood was collected from the tail tip of the mice and allowed to stand until the serum on it precipitated. The blood was centrifuged at 10,000 rpm for 10 minutes, and the clear serum was transferred to a new EP tube. The blood was then purified using BD Fermentation Immunofluorescence ... TMThe Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences) was used to measure serum inflammatory cytokine levels, and the data were analyzed using a flow cytometry system and BD CBA analysis software. The results, as shown in Figure 10, show that compared to the PBS control group, the PDL1×CD3(OKT3) construct shown in Figure 2A showed high cytokine expression in peripheral serum, while the PDL1×CLDN-MMP(double)-CD3(OKT3) construct shown in Figure 2D showed a significant decrease in cytokine expression in peripheral serum.

[0292] Example 5. The activity of the new BiTE can be restored after cleavage

[0293] OKT3 (SEQ ID NO: 16) and UCHT1 (SEQ ID NO: 15) are widely used monoclonal antibodies against CD3. In the structure of bispecific antibodies, we also used OKT3 as the core. According to the method of Example 1, we gene synthesized and produced PDL1×CLDN-MMP(double)-CD3 (PDL1×CLDN-MMP(double)-CD3(OKT3)[anti-PDL1 single-chain(VL-VH)and CD3 single-chain(VL-VH)]) and PDL1×NGR-HA-MMP(double)-CD3 (PDL1×NGR-HA-MMP(double)-CD3(OKT3)[anti-PDL1 single-chain(VL-VH)and CD3 single-chain(VL-VH)]) proteins, respectively.

[0294] First, the cleavage activity of the two proteins was tested in vitro by repeating the MMP enzymatic cleavage method 2.2 of Example 2. The results are shown in Figure 11. SDS PAGE results showed that both antibody forms could be completely cleaved, Figure 11A (PDL1×CLDN-MMP(double)-CD3) and Figure 11B (PDL1×NGR-HA-MMP(double)-CD3).

[0295] By repeating the in vitro PBMC activation experiment 2.3 after enzymatic cleavage of Example 2, it can be seen that both the double antibody obtained by using the antibody against the tumor surface specific antigen (Figure 12A, PDL1×CLDN-MMP(double)-CD3) and the targeting peptide (Figure 12B, PDL1×NGR-HA-MMP(double)-CD3) can effectively reduce the activity of CD3, and the activity of CD3 is partially restored after enzymatic cleavage.

[0296] Example 6: Preparation of bispecific antibody fusion cytokine

[0297] Taking the bispecific antibody fused to the IL-2 mutant cytokine (SEQ ID NO: 110) as an example, the construction and expression purification process are described in detail. The same method is used for the expression and purification of other proteins in the present invention. First, the fusion protein is constructed on the pEE6.4 expression vector, and then the host cell containing the vector is constructed by transient transfection of 293F cells. For the expression of heterodimeric proteins, two expression vector plasmids with the same molar ratio need to be transferred. The monomers expressed in the cells can also spontaneously form heterodimers. The host cells are cultured and the cell supernatant is collected. Finally, the protein is purified by affinity chromatography on a ProteinA / G column. The specific steps are as follows:

[0298] Construction of expression vector

[0299] (1) Anti-CLDN18.2 single chain (VL-VH)

[0300] (2) Anti-CD3 single chain (VL-VH, (UCHT1)) and mutant IL2 (F42A, R38L, i.e., mut IL2)

[0301] (3) Anti-PDL1 x Anti-CD3 (OKT3)

[0302] The fusion protein was expressed by transient transfection of 293F cells:

[0303] Anti-CLDN18.2 x anti-CLDN18.2-MMP (double) -anti-CD3 (UCHT1) -MutIL2 shown in Figure 14A, i.e., CLDN × CLDN-MMP (double) -CD3 (UCHT1) [anti-CLDN single-chain (VL-VH) and CD3 single-chain (VL-VH) -mutIL2], CLDN-Fc (Knob) shown in SEQ ID NO: 22, CLDN-MMP (double) -CD3-Fc (Hole) shown in SEQ ID NO: 25, and mutIL2 shown in SEQ ID NO: 110.

[0304] Example 7: Study on the biological activity of fusion protein in vitro

[0305] Detection of the ability of fusion protein to bind to antigen.

[0306] As described in Example 2.1, the bispecific antibodies used were PDL1×CD3(OKT3) with a structure as shown in Figure 2A , CLDN×CLDN-MMP(double)-CD3(UCHT1) with a structure as shown in Figure 1D , and PDL1×CLDN-MMP(double)-CD3(UCHT1)-mutIL2 with a structure as shown in Figure 14A . Flow cytometry was used to detect the ability of the anti-CLDN18.2 antibody in the fusion protein to bind to its antigen, and the analysis results are shown in Figure 16 .

[0307] The results showed that anti-CLDN18.2 x anti-CLDN18.2-MMP (double) - anti-CD3 (UCHT1) -mutIL2 had a slightly higher binding ability to the CLDN18.2 antigen on the surface of MC38 cells than anti-CLDN18.2 x anti-CD3 (UCHT1), while anti-PDL1 x anti-CD3 (OKT3) had no binding to this cell line at all ( Figure 16 ).

[0308] Example 8: Anti-tumor effect of fusion protein

[0309] The structure of anti-CLDN18.2 is shown in FIG14A .

[0310] Taking x anti-CLDN18.2-MMP (double) -anti-CD3 (UCHT1) -mutIL2 and anti-CLDN18.2 x anti-CLDN18.2-MMP (double) -anti-CD3 (UCHT1) -IL15Ra as examples (wherein the sequence of CLDN18.2 is shown in SEQ ID NO: 22, the sequence of mutIL2 is shown in SEQ ID NO: 111, and the sequence of IL15Ra is shown in SEQ ID NO: 134), it was determined whether they could effectively fight tumors when administered systemically and whether they were superior to the therapeutic effect of a single bispecific antibody. MC38-CLDN (MC38 cells overexpressing Claudin 18.2) was inoculated into hCD3 humanized mice (hCD3EDG) and the mice were treated with a lower dose by systemic administration. The experimental results are shown in Figure 17:

[0311] hCD3EDG mice (n=5 / group) were subcutaneously inoculated with 1×10 6 MC38-CLDN cells. On day 13, the tumor grew to 220 mm 3The mice were randomly divided into groups and administered at an equimolar mass of 200 pmol / mouse on days 13 and 16. The calculated protein was dissolved in 200 μl of PBS and then intraperitoneally injected. The PBS control group, CLDN×CLDN MMP(double)-CD3(UCHT1), CLDN×CLDN-MMP(double)-CD3(UCHT1)-mut IL2, and CLDN×CLDN-MMP(double)-CD3(UCHT1)-IL15Ra were treated. Tumor length (a), width (b), and height (c) were measured using a vernier caliper. Tumor volume (a×b×c / 2) was measured twice weekly, and tumor growth curves were recorded. The tumor growth curves showed that the bispecific antibody fused to the cytokine had a superior anti-tumor effect ( Figure 17 ).

[0312] Example 9: Anti-tumor effect of IL2 fusion protein

[0313] Using the structures of CLDN x CDLN-CD3, CLDN-wtIL2 x CDLN-CD3, CLDN-mutIL2 x CDLN-CD3, and CLDN-mutIL2 x CD3, as shown in Figure 18A , we determined whether systemic administration of these fusion proteins effectively combats tumors and whether they surpass the therapeutic efficacy of a bispecific antibody alone. The anti-tumor efficacy of these novel fusion proteins was tested as described in Example 8. The results demonstrated that bispecific antibodies carrying either wild-type or mutant IL2 exhibited enhanced anti-tumor efficacy ( Figure 19 ).

[0314] As described in Example 4, we inoculated CFPAC1 (purchased from ATCC) tumor cells into NCG systemic immunodeficient mice and waited for the tumor to grow to 100 mm. 3 3e6 human PBMCs were then transplanted into the mice via the tail vein. Five days later, 200 pmol of fusion protein drugs (CLDN x CD3 and CLDN xRa-IL2-CD3 (see Figure 18G for structure)) were injected intraperitoneally into the mice, and tumor growth was measured ( Figure 20 ). The results showed that the bispecific antibody had better anti-tumor effects after carrying wild-type IL2 and IL2 receptor Ra ( Figure 20 ).

[0315] The sequences used in Example 9 are as follows:

[0316] Example 10: Anti-tumor effect of IL15 fusion protein

[0317] Using the structures of CLDN-hIL15 x CDLN-MMP-CD3, shown in Figure 18A, and CLDN-hIL15Ra x CDLN-MMP-CD3-hIL15, shown in Figure 18D, as examples, we determined whether these fusion proteins, when administered systemically, were effective against tumors and superior to the therapeutic efficacy of the bispecific antibody alone. The anti-tumor effects of these novel fusion proteins were tested as described in Example 8. The results demonstrated that bispecific antibody prodrugs carrying wild-type IL15 or IL15 and IL15Ra exhibited enhanced anti-tumor efficacy ( Figure 21 ). Furthermore, the anti-tumor efficacy of a bispecific antibody directly fused to IL15 (CLDN-hIL15 x CD3), shown in Figure 18H, was also enhanced ( Figure 22 ).

[0318] The sequences used in Example 10 are as follows:

[0319] Example 11: Anti-tumor effects and peripheral toxicity of IL21 fusion protein

[0320] IL21 was directly fused to the bispecific antibody using CLDN-IL21xCD3, as shown in FIG18H . The anti-tumor effect of the new fusion protein was tested as described in Example 8. We found that although the anti-tumor effect was not improved after fusion with IL21 ( FIG23A ), the levels of peripheral cytokines IFN and TNF were significantly reduced ( FIG23B ), demonstrating that it can reduce the peripheral toxicity caused by BiTE.

[0321] The sequences used in Example 11 are as follows:

[0322] Example 12: Anti-tumor effects and peripheral toxicity of IL4 fusion protein

[0323] CLDN x IL4-CD3, with the structure shown in Figure 13A , was fused to the N-terminus of CD3 via a cleavable linker based on a bispecific antibody. The anti-tumor effect of the new fusion protein was tested as described in Example 8. We found that although the anti-tumor effect was not improved after fusion with IL4 ( Figure 24A ), the levels of peripheral cytokines IFN and TNF were significantly reduced ( Figure 24B ), demonstrating that it can reduce the peripheral toxicity caused by BiTE.

[0324] The sequences used in Example 12 are as follows:

[0325] Example 13: Anti-tumor effect and peripheral toxicity of IL10 fusion protein

[0326] As shown in Figures 13A and 13C, two IL10 molecules were fused in series to the N-terminus of an anti-CD3 antibody sequence, connected by either a cleavable linker (MMP) or a non-cleavable linker (i.e., G4S or (G4S)3) of varying lengths. The different fusion proteins were incubated with PBMCs in vitro for half an hour at varying concentration gradients. The cells were then stained with an anti-hFc-APC secondary antibody (using the same procedure as in Example 2.1). The mean fluorescence intensity of each group was then measured by upflow cytometry. The results, shown in Figure 25, demonstrate that the fusion of two IL10 molecules significantly impacts CD3 affinity.

[0327] We then tested the anti-tumor effect of the IL10 fusion protein as described in Example 8. The results showed that the anti-tumor effect of the fusion bispecific antibody was significantly improved after fusion with IL10 ( Figure 26A ), and the secretion of peripheral cytokines was also reduced ( Figure 26B ), thereby reducing toxicity and increasing efficacy.

[0328] The sequences used in Example 13 are as follows:

[0329] Sequence Listing

[0330] G4S2:GGGGSGGGGS (SEQ ID NO: 1)

[0331] G4S4:GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:2)

[0332] G4S3:GGGGGSGGGGSGGGGS(SEQ ID NO:3)

[0333] G4S5:GGGGSGGGGSGGGGGSGGGGSGGGGS(SEQ ID NO:24)

[0334] (GGS)2:GGSGGS (SEQ ID NO:28)

[0335] (GGS)3:GGSGGSGGS(SEQ ID NO:30)

[0336] HA-MMP:NQHTIDLADSSGFIANPVTA(SEQ ID NO:4)

[0337] MMP-HA:SGFIANPVTANQHTIDLADS(SEQ ID NO:5)

[0338] MMP high:SGRSPAIFTA(SEQ ID NO:6)

[0339] MMP medium:SGFIANPVTA(SEQ ID NO:7)

[0340] MMP low:SGRSENIRTA(SEQ ID NO:8)

[0341] MMP(Double):SGRSENIRTASGRSPAIFTA(SEQ ID NO:9)

[0342] HA:NQHTIDLADS (SEQ ID NO: 10)

[0343] HA2:NQHTIDLADSNQHTIDLADS(SEQ ID NO:11)

[0344] HA n :(NQHTIDLADS) n (SEQ ID NO: 12) n=3

[0345] H(MMP)A:NQHIANPVDS(SEQ ID NO:13)

[0346] H(MMP)A2:NQHIANPVDSNQHIANPVDS(SEQ ID NO:14)

[0347] Anti-CD3(single-chain,clone:UCHT1,VL-VH)

[0348] Anti-CD3(single-chain,clone:OKT3,VL-VH)

[0349] Anti-PDL1 (single-chain, Atezolizumab, VL-VH)

[0350] The CDR1 sequence of PDL1 VL is QDVSTA (SEQ ID NO: 73)

[0351] The CDR2 sequence of PDL1 VL is SAS

[0352] The CDR3 sequence of PDL1 VL is QQYLYHPATF (SEQ ID NO: 74)

[0353] The CDR1 sequence of PDL1 VH is GFTFSDSW (SEQ ID NO: 75)

[0354] The CDR2 sequence of PDL1 VH is ISPYGGST (SEQ ID NO: 76)

[0355] The CDR3 sequence of PDL1 VH is ARRHWPGGFDYW (SEQ ID NO: 77)

[0356] Anti-CLDN18.2(single-chain,VL-VH)

[0357] The CDR1 sequence of CLDN VL is QSLLNSGNQKNY (SEQ ID NO: 78)

[0358] The CDR2 sequence of CLDN VL is WAS

[0359] The CDR3 sequence of CLDN VL is QNDYFYPFTF (SEQ ID NO: 79)

[0360] The CDR1 sequence of CLDN VH is GYTFTSYW (SEQ ID NO: 80)

[0361] The CDR2 sequence of CLDN VH is IYPSDSYT (SEQ ID NO: 81)

[0362] The CDR3 sequence of CLDN VH is ARVYYGNSFAYW (SEQ ID NO: 82)

[0363] Inactivation Fc region(Knob)

[0364] Inactivation Fc region(Hole)

[0365] IgGκ:METDTLLLWVLLLWVPGSTG (SEQ ID NO:21)

[0366] CLDN×CD3(UCHT1) [anti-CLDN single-chain(VL-VH) and CD3 single-chain(VL-VH)]

[0367] CLDN×CLDN-MMP(double)-CD3(UCHT1)[anti-CLDN single-chain(VL-VH) and CD3 single-chain(VL-VH)]

[0368] PDL1×CD3(OKT3)[anti-PDL1 single-chain(VL-VH) and CD3 single-chain(VL-VH)]

[0369] PDL1×CLDN-MMP(double)-CD3(OKT3)[anti-PDL1 single-chain(VL-VH) and CD3 single-chain(VL-VH)]

[0370] PDL1×NGR-HA-MMP(double)-CD3(OKT3)[anti-PDL1 single-chain(VL-VH)and CD3 single-chain(VL-VH)]

[0371] G4S:GGGGS (SEQ ID NO:32)

[0372] NGR:GGGGSCNGRC (SEQ ID NO:33)

[0373] NGR2:GGGGSCNGRCGGGGSGGGGGSCNGRC(SEQ ID NO:34)

[0374] Human CD3 Clone:OKT3

[0375] VL

[0376] The CDR1 sequence of OKT3 VL is SSVSY (SEQ ID NO: 48)

[0377] The CDR2 sequence of OKT3 VL is DTS

[0378] The CDR3 sequence of OKT3 VL is QQWSSNPFTF (SEQ ID NO: 49)

[0379] VH:

[0380] The CDR1 sequence of OKT3 VH is GYTFTRYT (SEQ ID NO: 50)

[0381] The CDR2 sequence of OKT3 VH is INPSRGYT (SEQ ID NO: 51)

[0382] The CDR3 sequence of OKT3 VH is ARYYDDHYCLDYW (SEQ ID NO: 52)

[0383] Human CD3 Clone:L2K

[0384] VL:

[0385] VH:

[0386] Human CD3 Clone:UCHT1

[0387] VL:

[0388] The CDR1 sequence of UCHT1 VL is QDIRNY (SEQ ID NO: 53)

[0389] The CDR2 sequence of UCHT1 VL is YTS

[0390] The CDR3 sequence of UCHT1 VL is QQGNTLPWTF (SEQ ID NO: 54)

[0391] VH:

[0392] The CDR1 sequence of UCHT1 VH is GYSFTGYT (SEQ ID NO: 55)

[0393] The CDR2 sequence of UCHT1 VH is INPYKGVS (SEQ ID NO: 56)

[0394] The CDR3 sequence of UCHT1 VH is ARSGYYGDSDWYFDV (SEQ ID NO: 57)

[0395] Murine CD3 Clone:SP34

[0396] VL:

[0397] The CDR1 sequence of SP34 VL is TGAVTTSNY (SEQ ID NO: 58)

[0398] The CDR2 sequence of SP34 VL is GTN

[0399] The CDR3 sequence of SP34 VL is ALWYSNLWVF (SEQ ID NO: 59)

[0400] VH:

[0401] The CDR1 sequence of SP34 VH is GETFVTYA (SEQ ID NO: 60)

[0402] The CDR2 sequence of SP34 VH is IRSKYNNYAT (SEQ ID NO: 61)

[0403] The CDR3 sequence of SP34 VH is VRHGNFGNSYVSWFAYW (SEQ ID NO: 62)

[0404] Murine CD3 Clone:17A2

[0405] VL:

[0406] The CDR1 sequence of 17A2 VL is QNVINY (SEQ ID NO: 63)

[0407] The CDR2 sequence of 17A2 VL is YAS

[0408] The CDR3 sequence of 17A2 VL is QRIYNSPWTF (SEQ ID NO: 64)

[0409] VH:

[0410] The CDR1 sequence of 17A2 VH is GFTFSNFP (SEQ ID NO: 65)

[0411] The CDR2 sequence of 17A2 VH is ISSGGGGT (SEQ ID NO: 66)

[0412] The CDR3 sequence of 17A2 VH is VRPQGGFASW (SEQ ID NO: 67)

[0413] Murine CD3 Clone:2C11

[0414] VL:

[0415] The CDR1 sequence of 2C11 VL is QDISNY (SEQ ID NO: 68)

[0416] The CDR2 sequence of 2C11 VL is YTN

[0417] The CDR3 sequence of 2C11 VL is QQYYNYPWTF (SEQ ID NO: 69)

[0418] VH:

[0419] The CDR1 sequence of 2C11 VH is GFTFSGYG (SEQ ID NO: 70)

[0420] The CDR2 sequence of 2C11 VH is ITSSSINI (SEQ ID NO: 71)

[0421] The CDR3 sequence of 2C11 VH is ARFDWDKNYW (SEQ ID NO: 72)

[0422] signal peptide

[0423] MYRMQLLSCIALSLALVTNS, SEQ ID NO:47

[0424] Cleavable linker

[0425] MPYDLYHP, (SEQ ID NO: 83)

[0426] DSGGFMLT, (SEQ ID NO: 84)

[0427] HEQLTV, (SEQ ID NO: 85)

[0428] RAAAVKSP, (SEQ ID NO: 86)

[0429] VPLSLY, (SEQ ID NO: 87)

[0430] ISSGLLSGRS, (SEQ ID NO: 88)

[0431] DLLAVVAAS, (SEQ ID NO: 89)

[0432] LSGSDN, (SEQ ID NO: 90)

[0433] SGSDN, (SEQ ID NO: 91)

[0434] HPVGLLAR, (SEQ ID NO:92)

[0435] LSGRSDNH, (SEQ ID NO: 93)

[0436] VPLSLYSG, (SEQ ID NO:94)

[0437] HA-MMP(double)-Anti-CD3(OKT3)-Fc(Hole)

[0438] HA-MMP-Anti-CD3(OKT3)-Fc(Hole)

[0439] NGR-HA-MMP(Double)-Anti-CD3(OKT3)-Fc(Hole)

[0440] CLDN-MMP-Anti-CD3(UCHT1 VH VL)-Fc(Hole)

[0441] CLDN-MMP(Double)-Anti-CD3(UCHT1 VH VL)-Fc(Hole)

[0442] CLDN-HA-MMP-Anti-CD3(UCHT1)-Fc(Hole)

[0443] CLDN-[H(MMP)A]*2-Anti-CD3(UCHT1)-Fc(Hole)

[0444] CLDN-MMP-Anti-CD3(UCHT1)-Fc(Hole)

[0445] CLDN-(G4S)4-Anti-CD3(UCHT1)-Fc(Hole)

[0446] CLDN-(G4S)2-Anti-CD3(UCHT1)-Fc(Hole)

[0447] CLDN-(MMP)2-Anti-CD3(UCHT1)-Fc(Hole)

[0448] CLDN-[H(MMP)A]-Anti-CD3(UCHT1)-Fc(Hole)

[0449] Anti-CLDN (VL-CL)

[0450] Anti-CLDN(VH-CH)-MMP(Double)-Anti-CD3-Fc(Hole)

[0451]

[0452] (SEQ ID NO: 108, wherein the underlined sequence represents Anti-CLDN (VH-CH))

[0453] Anti-CLDN(VH-CH)-MMP-Anti-CD3-Fc(Hole)

[0454]

[0455]

[0456] (SEQ ID NO: 109, wherein the underlined sequence represents Anti-CLDN (VH-CH))

[0457] Mutated IL2 (F42A, R38L):

[0458] CLDN-MMP(double)-CD3-mutIL2[anti-CLDN single-chain(VL-VH)and CD3single-chain(VL-VH)and mut IL2(F42A,R38L)]

[0459] Anti-CLDN-Fc(Knob)-IL10

[0460]

[0461]

[0462] (SEQ ID NO: 112, wherein the underline indicates the sequence of IL10)

[0463] Anti-CLDN-MMP(Double)-Anti-CD3-Fc(Hole)-IL10

[0464]

[0465] (SEQ ID NO: 113, wherein the underline indicates the sequence of IL10)

[0466] Anti-CLDN-MMP-Anti-CD3-Fc(Hole)-IL10

[0467]

[0468]

[0469] (SEQ ID NO: 114, wherein the underline indicates the sequence of IL10)

[0470] Anti-CLDN-G4S-Anti-CD3-Fc(Hole)-IL10

[0471]

[0472] (SEQ ID NO: 115, wherein the underline indicates the sequence of IL10)

[0473] IL10-Anti-CD3-Fc(Hole)

[0474]

[0475] (SEQ ID NO: 116, wherein the underline indicates the sequence of IL10)

[0476] Anti-CLDN(VL-CL)-IL10

[0477]

[0478] (SEQ ID NO: 117, wherein the underline indicates the sequence of IL10)

[0479] Anti-CLDN(VH-CH)-MMP(Double)-Anti-CD3-Fc(Hole)-IL10

[0480]

[0481] (SEQ ID NO: 118, wherein the underline indicates the sequence of anti-CLDN (VH-CH), and the italic indicates the sequence of IL-10)

[0482] Anti-CLDN(VH-CH)-MMP-Anti-CD3-Fc(Hole)-IL10

[0483]

[0484]

[0485] (SEQ ID NO: 119, wherein the underline indicates the sequence of IL10 and the italics indicate the sequence of anti-CLDN (VH-CH))

[0486] Anti-CLDN-Fc(Knob)-Pro-IL2

[0487]

[0488] (SEQ ID NO: 120, wherein the underline indicates the sequence of Pro-IL-2)

[0489] Anti-CLDN-Fc(Knob)-mutIL2

[0490]

[0491] (SEQ ID NO: 121, wherein the underline indicates the sequence of mutIL2, R38L / F42A)

[0492] Anti-CLDN-Fc(Knob)-mutIL2

[0493]

[0494] (SEQ ID NO: 122, wherein the underline indicates the sequence of mutIL2, R38E)

[0495] Anti-CLDN-Fc(Knob)-mutIL2

[0496]

[0497] (SEQ ID NO: 123, wherein the underline indicates the sequence of mutIL2, K35E)

[0498] Anti-CLDN-Fc(Knob)-mutIL2

[0499]

[0500]

[0501] (SEQ ID NO: 124, wherein the underlined sequence indicates the mutIL2 sequence, K35E / R38E)

[0502] Anti-CLDN-Fc(Knob)-mutIL2

[0503]

[0504] (SEQ ID NO: 125, wherein the underlined sequence represents mutIL2, K35A / R38E)

[0505] Anti-CLDN-MMP(Double)-Anti-CD3-Fc(Hole)-mutIL2

[0506]

[0507] (SEQ ID NO: 126, wherein the underlined sequence represents mutIL2, K35A / R38E)

[0508] Anti-CLDN-MMP-Anti-CD3-Fc(Hole)-mutIL2

[0509]

[0510]

[0511] (SEQ ID NO: 127, wherein the underlined sequence indicates the mutIL2 sequence, K35E / R38E)

[0512] Anti-CLDN-G4S-Anti-CD3-Fc(Hole)-mutIL2

[0513]

[0514] (SEQ ID NO: 128, wherein the underlined sequence indicates the mutIL2 sequence, K35E / R38E)

[0515] Anti-CLDN-Fc(Knob)-IL21

[0516]

[0517]

[0518] (SEQ ID NO: 129, wherein the underline indicates the sequence of IL21)

[0519] Anti-CLDN(VL-CL)-IL21

[0520]

[0521] (SEQ ID NO: 130, wherein the underline indicates the sequence of IL21)

[0522] Anti-CLDN-MMP(Double)-Anti-CD3-Fc(Hole)-IL21

[0523]

[0524] (SEQ ID NO: 131, wherein the underline indicates the sequence of IL21)

[0525] Anti-CLDN-MMP-Anti-CD3-Fc(Hole)-IL21

[0526]

[0527]

[0528] (SEQ ID NO: 132, wherein the underline indicates the sequence of IL21)

[0529] Mouse IL15Ra complex:

[0530] (SEQ ID NO: 133, wherein the bold sequence is the sequence of IL15 and the italic sequence is the IL15 receptor sequence)

[0531] CLDN-MMP(double)-CD3-Fc(Hole)-mouse IL15 Ra

[0532]

[0533] (SEQ ID NO: 134, wherein the sequences in bold and underlined are MMP sites, the bold sequence is the IL15 sequence, and the italic sequence is the IL15 receptor sequence)

[0534] Mouse IL15:

[0535] Mouse IL15 receptor:

[0536] IL-15 human:

[0537] human IL15 receptor:

[0538] CLDN-MMP(double)-CD3-Fc(Hole)-human IL15 Ra

[0539]

[0540] (SEQ ID NO: 139, wherein the bold sequence is the sequence of IL15 and the italic sequence is the IL15 receptor sequence)

[0541] CLDN-MMP(double)-CD3-Fc(Hole)-human IL15

[0542]

[0543]

[0544] (SEQ ID NO: 140, wherein the bold sequence is the sequence of IL15)

[0545] Anti-CLDN-Fc(Knob)-IL15 Ra

[0546]

[0547] (SEQ ID NO: 141, wherein the bold sequence is the sequence of IL15 and the italic sequence is the IL15 receptor sequence)

[0548] Anti-CLDN-Fc(Knob)-IL15

[0549]

[0550] (SEQ ID NO: 142, wherein the bold sequence is the sequence of IL15)

[0551] IL15-Anti-CD3-Fc(Hole)

[0552]

[0553] (SEQ ID NO: 143, wherein the bold sequence is the sequence of IL15)

[0554] Anti-CLDN(VL-CL)-IL15

[0555]

[0556] (SEQ ID NO: 144, wherein the bold sequence is the sequence of IL15)

[0557] Anti-CLDN(VL-CL)-IL15 Ra

[0558]

[0559] (SEQ ID NO: 145, wherein the bold sequence is the sequence of IL15 and the italic sequence is the IL15 receptor sequence)

[0560] Human IL15 Ra complex sequence

[0561] IL-15 human mutant E87C:

[0562] IL-15 human mutant L45C:

[0563] Cleavable connectors:

[0564] MMP 10AA:GSWLPSSITA(SEQ ID NO:149)

[0565] MMP 8AA:WLPSSITA(SEQ ID NO:150)

[0566] MMP 7AA:LPSSITA(SEQ ID NO:151)

[0567] MMP 6AA:WLPSSI(SEQ ID NO:152)

[0568] MMP 6AA(TA):PSSITA(SEQ ID NO:153)

[0569] MMP 5AA:PSSIT(SEQ ID NO:154)

[0570] LSGKD(SEQ ID NO:155)

[0571] MLSN-Fc(Knob)

[0572] Anti-CD3(SP34-scFv)-Fc(Hole)

[0573] Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0574] Anti-CD3(SP34-VL-CL)

[0575] Anti-CD3(UCHT1-VH-CH)-Fc(Hole)

[0576] Anti-CD3(UCHT1-VL-CL)

[0577] Anti-CLDN(VL)

[0578] Anti-CLDN(VH)

[0579] Anti-MSLN(VL)

[0580] Anti-MSLN(VH)

[0581] IL2

[0582] IL2Ra

[0583] IL2Rb

[0584] Pro-IL2(MMP)

[0585] Pro-IL2(G4S)

[0586] mutIL2 R38E

[0587] mutIL2 K35E

[0588] mutIL2 K35E / R38E

[0589] mutIL2 K35A / R38E

[0590] mutIL2 K35E / R38E

[0591] mutIL2 K35E / R38E / K43E

[0592] mutIL2 K35E / R38E / K43E / N88E

[0593] hIL15 E46K

[0594] hIL21

[0595] mIL21

[0596] mIL4

[0597] hIL4

[0598] IL10

[0599] 2xIL10

[0600] CLDN-hIL15Ra

[0601] CLDN-MMP-CD3-hIL15

Claims

1. A fusion protein comprising structural unit A and structural unit B, wherein structural unit A is an IgG bispecific antibody, the bispecific antibody comprising structural unit 1 and structural unit 2, wherein structural unit 1 is an anti-CD3 antibody, and structural unit 2 is an anti-tumor cell surface antigen antibody, wherein the antibodies of structural unit 1 and structural unit 2 are in Fab or scFv form, and the structural unit B is one or more (preferably 2) cytokines or cytokine prodrugs, and the structural unit B is connected to the N-terminus and / or C-terminus of the bispecific antibody via a cleavable linker or a non-cleavable linker, wherein the cytokine prodrug is an antibody or receptor of the cytokine connected to the cytokine via a cleavable linker. Preferably, the fusion protein exists in the form of a protein drug, mRNA or Car T.

2. The fusion protein according to claim 1, wherein when the structural units 1 and 2 are both in the form of scFv, the structural unit B is located at the N-terminus or C-terminus of the structural unit 1; When the structural unit 1 and / or structural unit 2 is in the form of scFv and / or Fab, the bispecific antibody further comprises a structural unit 4, which is an antibody Fc fragment or human serum albumin. 1) When the structural unit 4 is an Fc fragment of an antibody: The structural unit 1 and the structural unit 2 are both located at the N-terminus of the bispecific antibody; the structural unit 4 is connected to the C-terminus of the structural unit 1 and the structural unit 2 respectively; The structural unit B is located at the N-terminus of the structural unit 1 and / or structural unit 2 or at the C-terminus of the structural unit 4, and the structural unit B is one or two cytokines or cytokine prodrugs; II) When the structural unit 4 is human serum albumin: The bispecific antibody is as follows from N-terminus to C-terminus: a) Structural unit 2-Structural unit 4-Structural unit 1, b) Structural unit 4-structural unit 1-structural unit 2, c) Structural unit 2 - Structural unit 1 - Structural unit 4, or d) Structural unit 1-structural unit 4-structural unit 2.

3. The fusion protein according to claim 1, wherein the structural unit A further comprises a structural unit 4, the structural unit 4 is an Fc fragment of an antibody, the structural unit 1 and the structural unit 2 are in the form of scFv, and the structural unit 2 is located at the N-terminus of the structural unit 1, the C-terminus of the structural unit 1 is connected to one chain of the Fc fragment, and the structural unit B is connected to the other chain of the Fc fragment, preferably the structural unit B is one or two cytokines or cytokine prodrugs.

4. The fusion protein according to claim 1 or 2, wherein the structural unit A further comprises structural unit 3 and / or structural unit 4, The structural unit 3 is a tumor cell targeting peptide, which is connected to the N-terminus of the structural unit 1 through a linker, and the linker is selected from the group consisting of: (2) Linker 1, which is a pH-sensitive linker, (2) Linker 2, which is a cleavable linker, (3) Linker 3, which is a combination of a pH-sensitive linker and a cleavable linker, wherein the cleavable linker and the pH-sensitive linker are sequentially connected (wherein the cleavable linker is located at the N-terminus or the C-terminus), or the Linker 3 sequence is as shown in SEQ ID NO: 13 or 14, The structural unit 4 is an antibody Fc fragment or human serum albumin, 1) When the structural unit 4 is an Fc fragment of an antibody: The structural unit 1, structural unit 2 and structural unit 3 are simultaneously located at the N-terminus or C-terminus of the bispecific antibody; The structural unit 4 is connected to the C-terminal of the structural unit 1 and the structural unit 2, respectively, and the N-terminal of the structural unit 1 is connected to the structural unit 3, or the structural unit 4 is connected to the N-terminal of the structural unit 1 and the structural unit 2, respectively, and the C-terminal of the structural unit 1 is connected to the structural unit 3; II) When the structural unit 4 is human serum albumin: The bispecific antibody is as follows from N-terminus to C-terminus: a) Structural unit 2-Structural unit 4-Structural unit 1-Structural unit 3, b) structural unit 3-structural unit 4-structural unit 1-structural unit 2, c) structural unit 2 - structural unit 1 - structural unit 4 - structural unit 3, or d) Structural unit 3 - structural unit 1 - structural unit 4 - structural unit 2.

5. The fusion protein according to claim 4, wherein (1) the bispecific antibody is composed of structural units 1-4, the structural units 1-3 are located at the N-terminus of the bispecific antibody, the structural unit 4 is located at the C-terminus of the bispecific antibody, the structural unit 3 is connected to the N-terminus of the structural unit 1 through a linker, the structural units 1 and 3 are in the form of Fab, the structural unit 2 is in the form of scFv, and the structural unit 4 is the Fc fragment of the antibody, wherein the cytokine is connected to the C-terminus of the light chain variable region of the structural unit 1 or to the C-terminus of any chain of the structural unit 4 via a cleavable linker or a non-cleavable linker; (2) The bispecific antibody is composed of structural unit 1, structural unit 2 and structural unit 4, wherein the structural units 1 and 2 are located at the N-terminus of the bispecific antibody, the structural unit 4 is located at the C-terminus of the bispecific antibody, the structural unit 1 is in the form of Fab, the structural unit 2 is in the form of scFv, and the structural unit 4 is the Fc fragment of the antibody. The cytokine is connected to the N-terminus of one chain of the structural unit 1 through a cleavable linker or a non-cleavable linker, and the cytokine receptor is connected to the N-terminus of the other chain of the structural unit 1 through a cleavable linker or a non-cleavable linker.

6. The fusion protein according to claim 5, wherein when the cytokine is linked to the C-terminus of the light chain variable region of the structural unit 1, the other end of the cytokine is further linked to a cytokine receptor via a cleavable or non-cleavable linker; or When the cytokine is linked to the C-terminus of any chain of the structural unit 4, the structural unit B further comprises the cytokine receptor, wherein the cytokine receptor is linked to the C-terminus of the other chain of the structural unit 4 via a cleavable or non-cleavable linker, or the cytokine receptor is linked to the other end of the cytokine via a cleavable or non-cleavable linker.

7. The fusion protein according to claim 4, wherein the structural unit 3 is selected from an anti-tumor cell surface antigen antibody, NGR, CNGRC, RGD, MT1-AF7p, YSA or TNYL. When the structural unit 3 is an anti-tumor cell surface antigen antibody, it is the same as or different from the tumor cell surface antigen targeted by the structural unit 2, and is in the form of Fab or scFv.

8. The fusion protein according to claim 4 or 7, wherein the structural unit B is located at the C-terminus of the structural unit A, and the structural unit B is one or two cytokines or cytokine prodrugs.

9. The fusion protein according to claim 1 or 2, wherein the cytokine is selected from the wild type or mutant thereof of IL2 (such as ACCESSION P60568), IL15 (such as ACCESSION P40933), IL-21 (such as ACCESSION Q9HBE4-1 or Q9HBE4-2), IL-10 (such as ACCESSION P22301), or IL-4 (such as ACCESSION P05112).

10. The fusion protein according to claim 5 or 6, wherein the cytokine is a wild type or mutant of IL2 or IL15, preferably, the sequence of IL2 is as shown in ACCESSION P60568, the sequence of IL15 is as shown in SEQ ID NO: 135 or 137, the sequence of the IL2 mutant is as shown in SEQ ID NO: 110, 171-177, and the sequence of the IL15 mutant is as shown in SEQ ID NO: 147, 148, 178. Preferably, when the structural unit B comprises a cytokine receptor, the sequence of the IL15 receptor is as shown in SEQ ID NO: 136 or 138, the IL15-IL15 receptor complex is selected from the sequence shown in SEQ ID NO: 133 or 146, and the sequence of the IL2 receptor is as shown in SEQ ID NO: 167 or 168.

11. The fusion protein according to any one of claims 1 to 10, wherein the sequence of the anti-CD3 antibody is selected from the group consisting of: (1) OKT3, wherein the CDR1 sequence of its VL is shown in SEQ ID NO:48, the CDR2 sequence of its VL is DTS, the CDR3 sequence of its VL is shown in SEQ ID NO:49, the CDR1 sequence of its VH is shown in SEQ ID NO:50, the CDR2 sequence of its VH is shown in SEQ ID NO:51, and the CDR3 sequence of its VH is shown in SEQ ID NO:52; preferably, the amino acid sequence of its VL is shown in SEQ ID NO:35, and the amino acid sequence of its VH is shown in SEQ ID NO:36; (2) L2K, whose VL amino acid sequence is shown in SEQ ID NO: 37, and whose VH amino acid sequence is shown in SEQ ID NO: 38; (3) UCHT1, wherein the CDR1 sequence of its VL is shown in SEQ ID NO: 53, the CDR2 sequence of its VL is YTS, the CDR3 sequence of its VL is shown in SEQ ID NO: 54, the CDR1 sequence of its VH is shown in SEQ ID NO: 55, the CDR2 sequence of its VH is shown in SEQ ID NO: 56, and the CDR3 sequence of its VH is shown in SEQ ID NO: 57; preferably, the amino acid sequence of its VL is shown in SEQ ID NO: 39, and the amino acid sequence of its VH is shown in SEQ ID NO: 40; (4) SP34, wherein the CDR1 sequence of its VL is shown in SEQ ID NO: 58, the CDR2 sequence of its VL is GTN, the CDR3 sequence of its VL is shown in SEQ ID NO: 59, the CDR1 sequence of its VH is shown in SEQ ID NO: 60, the CDR2 sequence of its VH is shown in SEQ ID NO: 61, and the CDR3 sequence of its VH is shown in SEQ ID NO: 62; preferably, the amino acid sequence of its VL is shown in SEQ ID NO: 41, and the amino acid sequence of its VH is shown in SEQ ID NO: 42; (5) 17A2, wherein the CDR1 sequence of its VL is shown in SEQ ID NO:63, the CDR2 sequence of its VL is YAS, the CDR3 sequence of its VL is shown in SEQ ID NO:64, the CDR1 sequence of its VH is shown in SEQ ID NO:65, the CDR2 sequence of its VH is shown in SEQ ID NO:66, and the CDR3 sequence of its VH is shown in SEQ ID NO:67; preferably, the amino acid sequence of its VL is shown in SEQ ID NO:43, and the amino acid sequence of its VH is shown in SEQ ID NO:44; and (6) 145-2C11, the CDR1 sequence of its VL is shown in SEQ ID NO:68, the CDR2 sequence of its VL is YTN, the CDR3 sequence of its VL is shown in SEQ ID NO:69, the CDR1 sequence of its VH is shown in SEQ ID NO:70, the CDR2 sequence of its VH is shown in SEQ ID NO:71, and the CDR3 sequence of its VH is shown in SEQ ID NO:72; preferably, the amino acid sequence of its VL is shown in SEQ ID NO:45, and the amino acid sequence of its VH is shown in SEQ ID NO:

46.

12. The fusion protein according to any one of claims 4 to 10, wherein the pH-sensitive linker is selected from HA, or the cleavable linker is selected from the cleavage site of MMP, uPA or CD13, Preferably, the MMP is selected from the group consisting of: (1) any one of SEQ ID NOs: 6-8 and 83-94, or (2) any two combinations of any one sequence selected from SEQ ID NOs: 6-8 and any one sequence selected from SEQ ID NOs: 83-94, or (3) Any one sequence selected from SEQ ID NOs: 1-3, 24, 28, 30 and 32 in combination with any one sequence selected from SEQ ID NOs: 6-8 and 83-94.

13. The fusion protein according to any one of claims 1 to 12, wherein the tumor cell surface antigen is selected from Claudin18.2 (such as ACCESSION: P56856), PDL1 (such as ACCESSION: Q9NZQ7), CD139 (such as ACCESSION: P15144), MSLN (such as ACCESSION: Q12321), GPC3 (such as ACCESSION: Q8CFZ4), Her29r (such as ACCESSION: P04626), EGFR (such as ACCESSION: P00533), GD2 (such as ACCESSION: Q9UI17), Trop2 (such as ACCESSION: P09758), CD19 (such as ACCESSION: P15391), CD20 (such as ACCESSION: P11836), Her3 ACCESSION: P21860, FolR1 (such as ACCESSION: P15328), Nectin4 (such as ACCESSION: Q96NY8), CD276 (such as ACCESSION: Q5ZPR3), CD30 (such as ACCESSION: P28908), CD70 (such as ACCESSION: P32970), BCMA (such as ACCESSION: Q02223), CD22 (such as ACCESSION: P20273), CD33 (such as ACCESSION: P20138), GPRC5D (such as ACCESSION: Q9NZD1), HLADR (such as ACCESSION: Q29744), VTCN1 (such as ACCESSION: Q7Z7D3), CD79B (such as ACCESSION: P40295), CDH3 A group consisting of ACCESSION:P22223, DR5ACCESSION:P21918, MUC1 (such as ACCESSION:P15941), NT5E (such as ACCESSION:P21589), FAP (such as ACCESSION:Q12884) and PSMA (such as ACCESSION:Q04609).

14. The fusion protein according to any one of claims 4 to 13, wherein the tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by the structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by the structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by the structural unit 2 is PDL1, and the tumor cell surface antigen targeted by the structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by the structural unit 2 is PDL1, and the tumor cell surface antigen targeted by the structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by the structural unit 2 is MSLN, and the tumor cell surface antigen targeted by the structural unit 3 is MSLN; or wherein the tumor cell surface antigen targeted by the structural unit 2 is FAP, and the tumor cell surface antigen targeted by the structural unit 3 is FAP, preferably, the LCDR1 sequence of the structural unit 2 or 3 targeting the tumor cell surface antigen PDL1 is as shown in SEQ ID NO:73, the LCDR2 sequence is SAS, the LCDR3 sequence is shown in SEQ ID NO:74, the HCDR1 sequence is shown in SEQ ID NO:75, the HCDR2 sequence is shown in SEQ ID NO:76, and the HCDR3 sequence is shown in SEQ ID NO:

77. The LCDR1 sequence of structural unit 2 or 3 for the tumor cell surface antigen Claudin18.2 is shown in SEQ ID NO:78, the LDR2 sequence is WAS, the LCDR3 sequence is shown in SEQ ID NO:79, the HCDR1 sequence is shown in SEQ ID NO:80, the VCDR2 sequence is shown in SEQ ID NO:81, and the HCDR3 sequence is shown in SEQ ID NO:82; or the LCDR1 sequence of structural unit 2 is shown in SEQ ID NO:78, the LCDR2 sequence is WAS, the LCDR3 sequence is shown in SEQ ID NO:79, the HCDR1 sequence is shown in SEQ ID NO:80, the HCDR2 sequence is shown in SEQ ID NO:81, and the HCDR3 sequence is shown in SEQ ID NO:

82. NO:82, the LCDR1 sequence of the structural unit 3 is shown in SEQ ID NO:73, the LCDR2 sequence is SAS, the LCDR3 sequence is shown in SEQ ID NO:74, the HCDR1 sequence is shown in SEQ ID NO:75, the HCDR2 sequence is shown in SEQ ID NO:76, and the HCDR3 sequence is shown in SEQ ID NO:

77.

15. The fusion protein according to any one of claims 4 to 13, wherein The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2 shown in ACCESSION: P56856, and the structural unit 3 is CNGRC; The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2, and the structural unit 3 is RGD; The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2 shown in ACCESSION: P56856, and the structural unit 3 is TNYL; The tumor cell surface antigen targeted by structural unit 2 is MSLN, and the structural unit 3 is CNGRC. The fusion protein according to any one of claims 4 to 15 , wherein the Fc fragment is an Fc fragment of IgG1, IgG2, IgG3 or IgG4, preferably the Fc fragment is a humanized Fc fragment.

17. The fusion protein according to any one of claims 4 to 16, wherein the heavy chain constant region of the Fc fragment adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834, Preferably, according to the EU numbering system, based on Ig gamma-1 chain C region, ACCESSION: P01857, the heavy chain constant region of the Fc fragment has the following mutations at positions 234, 235 and / or 237: L234A and L235A, L234A and G237A, L235A and G237A, or L234A, L235A, and G237A; Preferably, according to the EU numbering system, the heavy chain constant region of the Fc fragment further has one or more mutations selected from the following: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, H268A, E318A, and K320A.

18. The fusion protein according to any one of claims 4 to 17, wherein the Fc fragment is selected from SEQ ID NO: 19 and SEQ ID NO:

20.

19. The fusion protein according to any one of claims 4 to 18, wherein when the structural unit 4 is human serum albumin, it is the human serum albumin of ACCESSION: P02768.

20. The fusion protein according to any one of claims 4 to 19, wherein the structural unit 2 is selected from SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 156 and / or the structural unit 3 is selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 156, SEQ ID NO: 33 or SEQ ID NO: 34, Preferably, the bispecific antibody is selected from the group consisting of: (1) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 17 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 18, (2) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 18 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 17, (3) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 17 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 33, (4) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 18 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 33, (5) the structural unit 2 is selected from the sequence shown in SEQ ID NO: 18 and the structural unit 3 is selected from the sequence shown in SEQ ID NO: 34, and (6) The structural unit 2 is selected from the sequence shown in SEQ ID NO: 17 and the structural unit 3 is selected from the sequence shown in SEQ ID NO:

34.

21. The fusion protein according to any one of claims 4 to 20, wherein the sequence of the bispecific antibody is selected from the group consisting of: (1) A bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23, (2) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 25, (3) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27, (4) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 29, (5) A bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 31, (6) A bispecific antibody comprising or consisting of SEQ ID NO: 22 and a sequence selected from any one of SEQ ID NOs: 95-109, (7) a bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22, SEQ ID NO: 107 and SEQ ID NO: 108, and (8) A bispecific antibody comprising or consisting of the sequences shown in SEQ ID NO: 22, SEQ ID NO: 107, and SEQ ID NO: 109, (9) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 162, and SEQ ID NO: 163, wherein sequence A is selected from any one of SEQ ID NOs: 4-14, 83-94, or 149-155; (10) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, wherein sequence A is a sequence selected from any one of SEQ ID NOs: 4-14, 83-94, or 149-155; (11) A bispecific antibody prodrug comprising a sequence represented by SEQ ID NO: 156, sequence A, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, and SEQ ID NO: 165, or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, and SEQ ID NO: 165, wherein sequence A is a sequence selected from any one of SEQ ID NOs: 4-14, 83-94, or 149-155, (12) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 156, sequence A, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, and SEQ ID NO: 165, wherein sequence A is selected from any one of SEQ ID NOs: 4-14 or 83-94 and 149-155, Preferably, the fusion protein is selected from the group consisting of: (1) A fusion protein comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26 or 156 and a sequence selected from the group consisting of SEQ ID NO: 111; (2) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112 and SEQ ID NO: 113; (3) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112 and SEQ ID NO: 114; (4) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112 and SEQ ID NO: 115; (5) a fusion protein comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26 or 156 and a sequence selected from the group consisting of SEQ ID NO: 116; (6) a fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 117, and SEQ ID NO: 108, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 117, and SEQ ID NO: 108; (7) a fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112, SEQ ID NO: 118, and SEQ ID NO: 107; (8) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 112, SEQ ID NO: 119, and SEQ ID NO: 107; (9) A fusion protein comprising or consisting of a sequence selected from any one of SEQ ID NOs: 120-125 and a sequence selected from any one of SEQ ID NOs: 25, 29, or 95-109; (10) A fusion protein comprising a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 126-128, or consisting of a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 126-128; (11) A fusion protein comprising or consisting of SEQ ID NO: 129 and a sequence selected from any one of SEQ ID NOs: 23, 25, 27, 29, 95-109, or 157; (12) A fusion protein comprising or consisting of SEQ ID NO: 130, SEQ ID NO: 109, and a sequence selected from SEQ ID NO: 22, 26, or 156; (13) A fusion protein comprising or consisting of SEQ ID NO: 131 and a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156; (14) A fusion protein comprising or consisting of SEQ ID NO: 132 and a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156; (15) A fusion protein comprising a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 134, 139, 140, or 143, or a fusion protein comprising a sequence selected from any one of SEQ ID NOs: 22, 26, or 156 and a sequence selected from any one of SEQ ID NOs: 134, 139, 140, or 143; (16) A fusion protein comprising or consisting of SEQ ID NO: 141 and a sequence selected from any one of SEQ ID NOs: 25, 29, 95-109, or 157; (17) A fusion protein comprising or consisting of SEQ ID NO: 142 and a sequence selected from any one of SEQ ID NOs: 25, 29, 95-109, or 157; (18) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 139 and SEQ ID NO: 141; (19) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 140 and SEQ ID NO: 142; (20) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 108, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 108; (21) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 109, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 144, and SEQ ID NO: 109; (22) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 145, and SEQ ID NO: 108, or consisting of a sequence selected from the group consisting of SEQ ID NO: 22, 26, or 156, SEQ ID NO: 145, and SEQ ID NO: 108; (23) A fusion protein comprising a sequence selected from the group consisting of SEQ ID NOs: 22, 26, and 156, SEQ ID NOs: 145, and SEQ ID NOs: 109, or consisting of a sequence selected from the group consisting of SEQ ID NOs: 22, 26, and 156, SEQ ID NOs: 145, and SEQ ID NOs: 109; (24) A fusion protein comprising or consisting of the sequences shown in SEQ ID NO: 185, SEQ ID NO: 186, and SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161; (25) A fusion protein comprising an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody is selected from the sequence shown in any one of SEQ ID NOs: 23, 25, 27, 29, 95-109 or 157, the anti-tumor cell surface antigen antibody is selected from the sequence shown in any one of SEQ ID NOs: 22, 26 or 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 146-148, 166 or 169-184, and the linker is selected from the sequence shown in any one of SEQ ID NOs: 1-14, 24, 28, 30, 32, 83-94 or 149-155, preferably, the anti-CD3 antibody is in the form of scFv, and the cytokine is located at the C-terminus of the Fc fragment; (26) A fusion protein comprising an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises SEQ ID NO: 158 and SEQ ID NO: 159, the anti-tumor cell surface antigen antibody is selected from the sequence shown in any one of SEQ ID NO: 22, 26, or 156, the cytokine is selected from the sequence shown in any one of SEQ ID NO: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NO: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and the cytokine and the cytokine receptor are located at the N-terminus of the anti-CD3 antibody; (27) A fusion protein comprising an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises SEQ ID NO: 160 and SEQ ID NO: 161, the anti-tumor cell surface antigen antibody is selected from the sequence shown in any one of SEQ ID NO: 22, 26, or 156, the cytokine is selected from the sequence shown in any one of SEQ ID NO: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NO: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and the cytokine and the cytokine receptor are located at the N-terminus of the anti-CD3 antibody; (28) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 162 and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NO: 110, 133, 135, 137, 146-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody or at the C-terminus of the Fc fragment; (29) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162 and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 146-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody; (30) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164 and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody; (31) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164 and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166 or 169-184, and the linker is selected from the sequence shown in SEQ ID NO: The sequence shown in any one of NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, and the cytokine is located at the C-terminus of the anti-CD3 antibody; (32) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 162, and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: The sequence shown in any one of 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody; wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody, (33) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 22, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody; (34) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody; (35) A fusion protein comprising an anti-CD3 antibody, two anti-tumor cell surface antigen antibodies, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, or consisting of an anti-CD3 antibody, an anti-tumor cell surface antigen antibody, an Fc fragment, a cytokine, a receptor for the cytokine, and a linker, wherein the anti-CD3 antibody comprises the sequences shown in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, and SEQ ID NO: 165, the anti-tumor cell surface antigen antibody comprises the sequence shown in SEQ ID NO: 156, the cytokine is selected from the sequence shown in any one of SEQ ID NOs: 110, 133, 135, 137, 147-148, 166, or 171-178, the receptor for the cytokine is selected from the sequence shown in SEQ ID NOs: 136, 138, 167, or 168, and the linker is selected from the sequence shown in SEQ ID NOs: NO: 1-14, 24, 28, 30, 32, 83-94 or 149-155, wherein the anti-CD3 antibody is in Fab form, and one of the anti-tumor cell surface antigen antibodies is in Fab form and is located at the N-terminus of the anti-CD3 antibody, wherein the cytokine and the cytokine receptor are located at the C-terminus of the Fc fragment, or at the C-terminus of the anti-CD3 antibody and the anti-tumor cell surface antigen antibody in scFv form, respectively; or wherein the cytokine and the cytokine receptor are linked together and the cytokine receptor is linked to the C-terminus of the Fc fragment or the C-terminus of the anti-CD3 antibody; (36) a fusion protein comprising SEQ ID NO: 22, SEQ ID NO: 160, and SEQ ID NO: 161; (37) A fusion protein comprising SEQ ID NO: 22, sequence a, and sequence b, wherein sequence a comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence b comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus; (38) A fusion protein comprising sequence a, sequence b, and sequence c, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3, and SEQ ID NO: 110 from the N-terminus to the C-terminus, sequence b comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence c comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus; (39) A fusion protein comprising sequence a, sequence b, and sequence c, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3, and SEQ ID NO: 176 from the N-terminus to the C-terminus, sequence b comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence c comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus; (40) a fusion protein comprising sequence a, SEQ ID NO: 160 and SEQ ID NO: 161, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 176 from the N-terminus to the C-terminus; (41) a fusion protein comprising SEQ ID NO: 22, sequence a and sequence b, wherein sequence a comprises SEQ ID NO: 167, SEQ ID NO: 149 and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence b comprises SEQ ID NO: 110, SEQ ID NO: 32 and SEQ ID NO: 161 from the N-terminus to the C-terminus; (42) a fusion protein comprising SEQ ID NO: 22, sequence a and sequence b, wherein sequence a comprises SEQ ID NO: 163, SEQ ID NO: 149 and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence b comprises SEQ ID NO: 162, SEQ ID NO: 149 and SEQ ID NO: 161 from the N-terminus to the C-terminus; (43) a fusion protein comprising SEQ ID NO: 185, SEQ ID NO: 186 and sequence a, wherein sequence a comprises SEQ ID NO: 162, SEQ ID NO: 149 and SEQ ID NO: 161 from the N-terminus to the C-terminus; (44) A fusion protein comprising sequence a, sequence b, and sequence c, wherein sequence a comprises SEQ ID NO: 22, SEQ ID NO: 3, and SEQ ID NO: 137 from the N-terminus to the C-terminus, sequence b comprises SEQ ID NO: 163, SEQ ID NO: 149, and SEQ ID NO: 160 from the N-terminus to the C-terminus, and sequence c comprises SEQ ID NO: 162, SEQ ID NO: 149, and SEQ ID NO: 161 from the N-terminus to the C-terminus; (45) a fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 23, SEQ ID NO: 3, and SEQ ID NO: 137 from the N-terminus to the C-terminus; (46) a fusion protein comprising SEQ ID NO: 22 and SEQ ID NO: 23; (47) a fusion protein comprising SEQ ID NO: 22 and SEQ ID NO: 157; (48) a fusion protein comprising SEQ ID NO: 129 and SEQ ID NO: 157; (49) a fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 181, SEQ ID NO: 32, SEQ ID NO: 149, and SEQ ID NO: 157 from the N-terminus to the C-terminus; (50) a fusion protein comprising SEQ ID NO: 22 and SEQ ID NO: 116; (51) A fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 184, SEQ ID NO: 3 and SEQ ID NO: 23 from the N-terminus to the C-terminus, or (52) A fusion protein comprising SEQ ID NO: 22 and sequence a, wherein sequence a comprises SEQ ID NO: 184, SEQ ID NO: 32, SEQ ID NO: 149 and SEQ ID NO: 23 from N-terminus to C-terminus.

22. A biomaterial selected from the group consisting of: (1) A polynucleotide encoding the fusion protein according to any one of claims 1 to 21, (2) A vector comprising the fusion protein according to any one of claims 1 to 21, and (3) A host cell comprising the fusion protein according to any one of claims 1 to 21.

23. A conjugate comprising the fusion protein of any one of claims 1 to 21 and a conjugated moiety, wherein the conjugated moiety is selected from a purification tag (such as a His tag, a streptavidin tag), a cytotoxic agent, a detectable label, a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

24. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 21 and a pharmaceutical carrier, preferably the pharmaceutical composition is in a dosage form suitable for oral administration to the gastrointestinal tract (GI), preferably, the dosage form is selected from tablets, capsules, pills, powders, granules, emulsions, microemulsions, solutions, suspensions, syrups and elixirs; or the drug is in a dosage form suitable for subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

25. A kit comprising the fusion protein according to any one of claims 1 to 21 and a therapeutic agent for treating tumors, preferably the therapeutic agent for treating tumors is selected from chemotherapeutics, targeted therapeutics, T cells expressing chimeric antigen receptors or angiogenesis inhibitors.

26. Use of the fusion protein according to any one of claims 1 to 21 in treating tumors, or in preparing a medicament for treating tumors.

27. The use according to claim 26, wherein the tumor is selected from gastric cancer, pancreatic cancer, lung cancer, colon cancer, melanoma, and microglioma.

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