Bispecific antibody prodrug and use thereof

Abstract

A bispecific antibody prodrug and the use thereof. The bispecific antibody prodrug comprises a structural unit 1, a structural unit 2 and a structural unit 3, wherein the structural unit 1 is an anti-CD3 antibody, the structural unit 2 is an antibody against a tumor cell surface antigen, and the structural unit 3 is a targeting peptide for tumor cells.

Description

Bispecific antibody prodrugs and their applications Technical Field

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

[0002] Bifunctional antibodies, also known as bispecific antibodies (BsAb), are specific drugs that simultaneously target two different antigens and can be produced by immunosorting and purification. In addition, they can also be obtained through genetic engineering, which enables the design and production of many different forms of bispecific antibody fragments. Genetic engineering has certain advantages in terms of flexibility in terms of binding site optimization, synthetic form considerations, and yield. Because this form is similar to naturally occurring IgG, it has advantages in antibody engineering, expression, and purification, and has been proven to be one of the ideal forms of bifunctional antibodies. BsAb is widely used in biomedicine, especially in immunotherapy for tumors.

[0003] Currently, a focus of immunotherapy research is how to use BsAb-mediated cytotoxicity to kill tumor cells. BsAb can be designed to simultaneously target tumor cells and effector cells and activate effector cells to kill tumor cells. The role of the immune system, especially T cell-mediated cytotoxicity, in tumor control is well-established, and increasing evidence indicates that T cells play a primary role in controlling tumor growth and survival in cancer patients. Over the past few decades, treatments for various types of tumors have aimed to reshape and improve the function and activity of T cells, giving them stronger and longer-lasting anti-tumor properties. Among them, T cell-bridging Bispecific T-cell Engager (BiTE): a bispecific antibody composed of a T cell binding domain (such as CD3) and a tumor binding domain (mainly tumor-associated antigens, TAAs), allows T cells to establish a physical connection with tumor cells, which can promote cytotoxic T cells to kill tumors. 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.

[0004] Despite the successful application of blinatumomab in B-cell acute lymphoblastic leukemia (ALL), BiTEs have had a less-than-optimal response in solid tumors, encountering similar challenges as cell-based therapies. This may be due to the limited penetration of BiTEs into tumor tissues and the numerous immunosuppressive factors in the tumor microenvironment. Furthermore, the T cell binding domain-mediated activation of T cells in non-tumor tissues can lead to severe side effects and the generation of a cytokine storm.

[0005] The in vivo efficacy of BiTEs largely depends on the specificity of tumor-associated antigens (TAAs), but they may also be expressed on non-tumor tissues, such as epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (Her2). Existing BiTEs are designed by combining antibodies targeting antigens that are also expressed in peripheral tissues with CD3. However, the effect of such a combination is that the CD3 complex can still be cross-linked by this tumor-associated antigen in peripheral non-tumor tissues, resulting in non-tumor but targeted toxicity, mainly manifested as cytokine storm and peripheral tissue damage. These problems seriously limit the ideal therapeutic outcomes of BiTEs. Summary of the Invention

[0006] 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.

[0007] Specifically, the present invention provides the following aspects:

[0008] 1. A bispecific antibody prodrug comprising structural unit 1, structural unit 2, and structural unit 3, wherein structural unit 1 is an anti-CD3 antibody, structural unit 2 is an anti-tumor cell surface antigen antibody, and structural unit 3 is a targeting peptide for tumor cells, wherein the antibodies of structural unit 1 and structural unit 2 are in Fab or scFv form;

[0009] The structural unit 3 is connected to the N-terminus of the structural unit 1 via a linker, and the linker is selected from the group consisting of:

[0010] (1) Linker 1, which is a pH-sensitive linker,

[0011] (2) Linker 2, which is a cleavable linker,

[0012] (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 sequence of Linker 3 is as shown in SEQ ID NO: 13 or 14. Preferably, the bispecific antibody prodrug is in the form of mRNA or Cart.

[0013] 2. The bispecific antibody prodrug according to item 1, wherein the bispecific antibody prodrug further comprises a structural unit 4, wherein the structural unit 4 is an antibody Fc fragment or human serum albumin,

[0014] 1) When the structural unit 4 is an Fc fragment of an antibody:

[0015] 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 prodrug;

[0016] 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;

[0017] II) When the structural unit 4 is human serum albumin:

[0018] The bispecific antibody prodrug is as follows from the N-terminus to the C-terminus:

[0019] a) Structural unit 2-Structural unit 4-Structural unit 1-Structural unit 3,

[0020] b) structural unit 3-structural unit 4-structural unit 1-structural unit 2,

[0021] c) structural unit 2 - structural unit 1 - structural unit 4 - structural unit 3, or

[0022] d) Structural unit 3 - structural unit 1 - structural unit 4 - structural unit 2.

[0023] 3. The bispecific antibody prodrug according to item 1, 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.

[0024] 4. The bispecific antibody prodrug according to any one of claims 1 to 2, wherein the structural unit 1, binding unit 2 and binding unit 4 constitute a Y-shaped structure of an IgG-type antibody, wherein the structural unit 1 is an anti-CD3 antibody in the Fab form, the structural unit 2 is an anti-tumor cell surface antigen antibody in the scFv form, and the structural unit 4 is an Fc fragment.

[0025] Structural unit 3 is an anti-tumor cell surface antigen antibody in Fab form, wherein the C-terminus of structural unit 3 is connected to the N-terminus of structural unit 1 via a cleavable linker, wherein the tumor cell surface antigens of structural units 2 and 3 are the same or different, preferably the same.

[0026] 5. The bispecific antibody prodrug according to any one of items 1 to 4, wherein the sequence of the anti-CD3 antibody is selected from the group consisting of:

[0027] (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;

[0028] (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;

[0029] (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;

[0030] (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;

[0031] (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

[0032] (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.

[0033] 6. The bispecific antibody prodrug according to any one of items 1 to 5, wherein the pH-sensitive linker is selected from HA, or the cleavable linker is selected from the cleavage site of MMP, uPA or CD13,

[0034] Preferably, the MMP is selected from the group consisting of:

[0035] (1) any one of SEQ ID NOs: 6-8 and 83-94, or

[0036] (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

[0037] (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.

[0038] 7. The bispecific antibody prodrug according to any one of items 1 to 6, 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).

[0039] 8. The bispecific antibody prodrug according to any one of items 1 to 7, wherein the tumor cell surface antigen targeted by structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by structural unit 2 is PDL1, and the tumor cell surface antigen targeted by structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by structural unit 2 is PDL1, and the tumor cell surface antigen targeted by structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by structural unit 2 is MSLN, and the tumor cell surface antigen targeted by structural unit 3 is MSLN; or wherein the tumor cell surface antigen targeted by structural unit 2 is FAP, and the tumor cell surface antigen targeted by structural unit 3 is FAP, preferably, the LCDR1 sequence of the structural unit 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 the structural unit 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 HCDR2 sequence is shown in SEQ ID NO:81, and the HCDR3 sequence is shown in SEQ ID NO:82; or the LCDR1 sequence of the structural unit 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 HCDR2 sequence is shown in SEQ ID NO:81, and the HCDR3 sequence is shown in SEQ ID NO:82; the LCDR1 sequence of the structural unit 3 is shown in SEQ ID NO: 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.

[0040] 9. The bispecific antibody prodrug according to any one of items 1 to 7, wherein

[0041] 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;

[0042] The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2, and the structural unit 3 is RGD;

[0043] 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;

[0044] The tumor cell surface antigen targeted by structural unit 2 is MSLN, and the structural unit 3 is CNGRC.

[0045] 10. The bispecific antibody prodrug according to any one of items 1 to 9, wherein the Fc fragment is an Fc fragment of IgG1, IgG2, IgG3 or IgG4, preferably the Fc fragment is a humanized Fc fragment.

[0046] 11. The bispecific antibody prodrug according to any one of items 1 to 10, 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,

[0047] 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:

[0048] L234A and L235A,

[0049] L234A and G237A,

[0050] L235A and G237A,

[0051] or

[0052] L234A, L235A, and G237A;

[0053] 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:

[0054] 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.

[0055] 12. The bispecific antibody prodrug according to any one of items 1 to 11, wherein the Fc fragment is selected from SEQ ID NO: 19 and SEQ ID NO: 20.

[0056] 13. The bispecific antibody prodrug according to any one of items 1 to 12, wherein when the structural unit 4 is human serum albumin, it is the human serum albumin of ACCESSION: P02768.

[0057] 14. The bispecific antibody prodrug according to any one of items 1 to 13, wherein the structural unit 2 is selected from SEQ ID NO: 17 or SEQ ID NO: 18, and / or the structural unit 3 is selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 33 or SEQ ID NO: 34,

[0058] Preferably, the bispecific antibody prodrug is selected from the group consisting of:

[0059] (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,

[0060] (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,

[0061] (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,

[0062] (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,

[0063] (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

[0064] (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.

[0065] 15. The bispecific antibody prodrug according to any one of items 1 to 14, wherein the sequence of the bispecific antibody prodrug is selected from the group consisting of:

[0066] (1) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23,

[0067] (2) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 25,

[0068] (3) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27,

[0069] (4) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 29,

[0070] (5) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 31,

[0071] (6) A bispecific antibody prodrug comprising SEQ ID NO: 22 and a sequence selected from any one of SEQ ID NOs: 95-109, or consisting of SEQ ID NO: 22 and a sequence selected from any one of SEQ ID NOs: 95-109,

[0072] (7) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 22, SEQ ID NO: 107, and SEQ ID NO: 108, and

[0073] (8) A bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 22, SEQ ID NO: 107, and SEQ ID NO: 109,

[0074] (9) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 136, and SEQ ID NO: 137, wherein sequence A is selected from the group consisting of SEQ ID NOs: 4-14, 83-94, and 110-117;

[0075] (10) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 138, and SEQ ID NO: 139, wherein sequence A is selected from the group consisting of SEQ ID NOs: 4-14, 83-94, and 110-117.

[0076] (11) A bispecific antibody prodrug comprising a sequence represented by SEQ ID NO: 144, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 136, and SEQ ID NO: 137, or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 136, and SEQ ID NO: 137, wherein sequence A is selected from the group consisting of sequences represented by SEQ ID NOs: 4-14, 83-94, and 110-117;

[0077] (12) A bispecific antibody prodrug comprising a sequence represented by SEQ ID NO: 144, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 138, and SEQ ID NO: 139, or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 138, and SEQ ID NO: 139, wherein sequence A is selected from the group consisting of sequences represented by SEQ ID NOs: 4-14, 83-94, and 110-117;

[0078] (13) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 119 and SEQ ID NO: 127,

[0079] (14) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 121 and SEQ ID NO: 127,

[0080] (15) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 120, and SEQ ID NO: 128,

[0081] (16) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 118, and SEQ ID NO: 124,

[0082] (17) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 122, and SEQ ID NO: 127,

[0083] (18) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 134, and SEQ ID NO: 135,

[0084] (19) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 131 and SEQ ID NO: 132,

[0085] (20) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 123, and SEQ ID NO: 129,

[0086] (21) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 121 and SEQ ID NO: 126,

[0087] (22) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 122, and SEQ ID NO: 125,

[0088] (23) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 134, and SEQ ID NO: 135.

[0089] 16. A biomaterial selected from the group consisting of:

[0090] (1) a polynucleotide encoding the bispecific antibody prodrug according to any one of items 1 to 15,

[0091] (2) A carrier comprising the bispecific antibody prodrug according to any one of items 1 to 15, and

[0092] (3) A host cell comprising the bispecific antibody prodrug according to any one of items 1 to 15.

[0093] 17. A conjugate comprising the bispecific antibody prodrug of any one of items 1 to 15 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.

[0094] 18. A pharmaceutical composition comprising the bispecific antibody prodrug of any one of items 1 to 15 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is preferably 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.

[0095] 19. A kit comprising the bispecific antibody prodrug according to any one of items 1 to 15 and a therapeutic agent for treating tumors, preferably the therapeutic agent for treating tumors is selected from a chemotherapeutic drug, a targeted therapeutic agent, a T cell expressing a chimeric antigen receptor, or an angiogenesis inhibitor.

[0096] 20. Use of the bispecific antibody prodrug according to any one of items 1 to 15 in treating tumors, or in the preparation of a medicament for treating tumors.

[0097] 21. The use described in item 20, wherein the tumor is selected from gastric cancer, pancreatic cancer, lung cancer, colon cancer, melanoma, and microglioma.

[0098] definition

[0099] 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.

[0100] 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.

[0101] 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.

[0102] "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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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. Description of the drawings:

[0107] Figure 1. Schematic diagram of several structures of bispecific antibody prodrugs that bind to the same tumor surface-specific antigen.

[0108] Figure 2. Schematic diagrams of several structures of bispecific antibody prodrugs that bind to different tumor surface-specific antigens.

[0109] Figure 3. Schematic diagrams of several structures of bispecific antibody prodrugs, including a tumor-targeting peptide and an antibody that binds to a tumor-specific antigen on the surface.

[0110] Figure 4. Schematic diagrams of several structures of bispecific antibody prodrugs, including a tumor-targeting peptide and an antibody that binds to a tumor-specific antigen on the surface in tandem.

[0111] Figure 5. SEC-HPLC analysis of the purity of the bispecific antibody prodrug.

[0112] Figure 6. Flow cytometry was used to detect the binding ability of bispecific antibody prodrugs 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.

[0113] Figure 7. SDS-PAGE electrophoresis analysis of the efficiency and results of fusion protein digestion by MMP14. The bispecific antibody prodrug used was PDL1×CLDN-MMP(double)-CD3(UCHT1), with the structure 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) after overnight digestion with MMP14, and lane 3 shows the electrophoresis of PDL1×CD3(UCHT1) alone, with the structure shown in Figure 1A.

[0114] 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 CD3 KI 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 is PDL1×CD3(UCHT1) with the structure shown in Figure 2A.

[0115] Figure 9. Compared with bispecific antibodies, bispecific antibody prodrugs 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.

[0116] 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, the bispecific antibody prodrug with the structure of PDL1×CLDN-MMP(double)-CD3(OKT3) 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.

[0117] Figure 11. SDS-PAGE images of bispecific antibody prodrugs cleaved or not by MMP14, wherein Figure 11A shows an SDS PAGE image of PDL1×CLDN-MMP(double)-CD3(OKT3) with a structure as 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 an SDS PAGE image of PDL1×targeting peptide-HA-MMP(double)-CD3(OKT3) with a structure as shown in Figure 3C after cleavage (+) or not (-) by MMP14, wherein lanes 3 and 4 from the left are irrelevant samples.

[0118] Figure 12. PBMC activation by bispecific antibody prodrugs after MMP14 cleavage or not, wherein Figure 12A shows the PBMC activation by PDL1×CLDN-MMP(double)-CD3(OKT3) with the structure shown in Figure 2D with or without MMP14 cleavage; Figure 12B shows the PBMC activation by PDL1×NGR-HA-MMP(double)-CD3(OKT3) with the structure shown in Figure 3C with or without MMP14 cleavage.

[0119] Figure 13. Bispecific antibody prodrug structure represented as anti-TAA x anti-TAA-cleavable linker-anti-CD3.

[0120] Figure 14. Binding of bispecific antibody prodrugs (with cleavable sites of varying lengths in the middle) to hCD3 KI mouse spleen cells after MMP cleavage (Figure 14A, labeled NC after the figure legend) or cleavage (Figure 14B, labeled C after the figure legend). PC refers to CLDN×CD3 (Fab), without a TAA antibody at the N-terminus of the CD3 antibody; the structural unit between VH and VL represents a cleavable linker. For example, VH 8aa VL 6aa indicates that the anti-CD3 VH and anti-CLDN VH are connected by a bispecific antibody prodrug with an 8-amino acid cleavable linker, and the anti-CD3 VL and anti-CLDN VL are connected by a 6-amino acid cleavable linker, and so on.

[0121] Figure 15. PBMC activation assay using a bispecific antibody prodrug. T cell surface activation markers were detected after 24 hours of co-culture of SNU1-CLDN and PBMC. The labeling for each group follows the nomenclature shown in Figure 14 . The bispecific antibody used in Figure 15A is CLDN x CLDN-MMP (cleavable linker)-CD3 (SP34), with the structure shown in Figure 14A . The bispecific antibody used in Figure 15B is CLDN x CLDN-MMP (cleavable linker)-CD3 (UCHT1), with the structure shown in Figure 14A .

[0122] Figure 16. Antitumor efficacy and peripheral toxicity of bispecific antibody prodrugs in experimental animals. Figure 16A shows the animal experiment workflow: Rag1 knockout mice were transplanted with 2e6 hCD3 KI mouse spleen cells per mouse one day in advance. Each mouse was subcutaneously inoculated with 1e6 MC38-CLDN tumor cells on day 0, followed by an intraperitoneal injection of 100 pmol of drug on day 14. Figure 16B shows tumor growth after intraperitoneal injection of each drug group. Figure 16C shows the release of the cytokine IFN-γ in peripheral blood after tumor inoculation and intraperitoneal injection of each drug group on the second day. DETAILED DESCRIPTION

[0123] 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.

[0124] experimental animals

[0125] 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.

[0126] cell lines

[0127] 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.

[0128] 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).

[0129] Biostatistical analysis

[0130] 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.

[0131] Example 1. Construction, expression and purification of fusion protein

[0132] 1. Construction of BiTE:

[0133] To design a highly effective and low-toxic bispecific antibody prodrug fusion protein for tumors, a T-cell bridged 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:

[0134] i. non-cleavable linkers (e.g. GS of different lengths (SEQ ID NO: 1-3, 24, 28, 30, 32), FIG. 1B ),

[0135] 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 ),

[0136] iii. a cleavable linker (e.g., a polypeptide substrate cleavable by an MMP (SEQ ID NO: 6-9, FIG. 1D ),

[0137] iv. pH-sensitive linkers formed in series (e.g., multiple HA linkers (SEQ ID NO: 10-12, FIG. 1E ), or

[0138] 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 ),

[0139] 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 .

[0140] 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 ).

[0141] 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.

[0142] 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.

[0143] 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 peptides; Figure 4B shows that based on Figure 2D, anti-TAA is connected to tumor cell targeting peptides; Figure 4C shows that based on Figure 2E, anti-TAA is connected to tumor cell targeting peptides; Figure 4D shows that based on Figure 2F, anti-TAA is connected to tumor cell targeting peptides; Figure 4E shows that based on Figure 2G, anti-TAA is connected to tumor cell targeting peptides.

[0144] The sequences used in the examples are as follows:

[0145] 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.

[0146] The commercialized Atezolizumab sequence (SEQ ID NO: 17) was used as the anti-PDL1 antibody.

[0147] The anti-CLDN18.2 sequence is the sequence shown in SEQ ID NO: 18.

[0148] 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.

[0149] Each antibody was linked via different linkers and constructed into the expression vector pEE6.4 plasmid (available from LifeScience Market), using a signal peptide (SEQ ID NO: 21, SEQ ID NO: 47 is also available). 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.

[0150] 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.

[0151] 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.

[0152] Construct the following expression vector:

[0153] (1)PEE6.4-HindⅢ-IgK(signal)-anti-Claudin 18.2-Fck-EcoRI

[0154] (2)PEE6.4-HindⅢ-IgK(signal)-anti-CD3(UCHT1)-Fch-EcoRI

[0155] (3) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-Linker 1-anti-CD3-Fch-EcoRI

[0156] (4) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-Linker 2-anti-CD3-Fch-EcoRI

[0157] (5) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 2×2-anti-CD3-Fch-EcoRI

[0158] (6) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 3+2-anti-CD3-Fch-EcoRI

[0159] (7) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 3×2-anti-CD3-Fch-EcoRI

[0160] (8) PEE6.4-HindⅢ-IgK (signal)-anti-Claudin 18.2-linker 4×2-anti-CD3-Fch-EcoRI

[0161] (9)PEE6.4-HindⅢ-IgK(signal)-anti-PDL1-Fck-EcoRI

[0162] (10)PEE6.4-HindⅢ-IgK(signal)-anti-CD3(OKT3)-Fch-EcoRI

[0163] (11) PEE6.4-HindⅢ-IgK(signal)-NGR-HA-Linker 2x2-Anti-CD3(OKT3)-Fch-EcoRI

[0164] Among them, HindⅢ and EcoRI are restriction enzyme cleavage sites; Fck represents the inactivated Fc region (knob, SEQ ID NO: 19), and Fch represents the inactivated Fc region (hole, SEQ ID NO: 20). The connecting sequences between the fragments are:

[0165] (1) Linker 1: G4S: GGGGS (SEQ ID NO: 32)

[0166] (2) Linker 2: MMP: MMP: any one of SEQ ID NOs: 6-8, linker 2×2 represents any two-by-two combination of SEQ ID NOs: 6-8,

[0167] (3) Linker 3: HA: NQHTIDLADS (SEQ ID NO: 10)

[0168] (4) Linker 4: H(MMP)A: NQHIANPVDS (SEQ ID NO: 13)

[0169] The obtained fusion proteins are as follows:

[0170] Anti-CLDN18.2 x 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;

[0171] 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;

[0172] Anti-PDL1 x 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, CD3-Fc (Hole) shown in SEQ ID NO: 27;

[0173] Anti-PDL1 x 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;

[0174] 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.

[0175] 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.

[0176] In the following examples, MMP (cleavable linker) refers to a linker shown in one of SEQ ID NOs: 110-117.

[0177] In the following examples and figures, CLDN represents a domain that binds to CLDN18.2, and CD3 represents a domain that binds to CD3.

[0178] 1.2 Transient transfection to quickly express the target protein:

[0179] 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.

[0180] 1.3 SEC-HPLC identification

[0181] Taking the anti-CLDN18.2 x anti-CLDN18.2-MMP (double) - anti-CD3 (UCHT1) shown in FIG1D and the anti-CLDN18.2 x anti-CD3 (UCHT1) shown in FIG1A as examples, first prepare the mobile phase: 500 ml 1 M 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).

[0182] Example 2. In vitro biological activity study of fusion protein

[0183] 2.1 Detection of fusion protein binding ability to antigen

[0184] The binding properties of the purified bispecific antibody prodrugs 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).

[0185] (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.

[0186] (2) Protein sample preparation: Each protein sample was diluted to a maximum concentration of 400 nmol / L according to the equimolar mass, and then diluted 10-fold. The tubes were labeled and arranged in the following order: 1:10, 1:100, 1:100, 1:10000, 1:100000. 450 μl of FACS buffer was added to each tube, and 50 μl of the highest concentration protein sample was transferred to the 1:10 dilution tube. Mix thoroughly using a pipette only. 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.

[0187] (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.

[0188] (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.

[0189] (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.

[0190] 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.

[0191] 2.2 MMP14 in vitro enzyme cleavage assay

[0192] The protein samples were cleaved in vitro using the enzyme MMP14 to verify the cleavage effect.

[0193] 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. The 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.

[0194] 2.3 Experimental study on the recovery of MMP14 activity after in vitro enzymatic cleavage

[0195] 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.

[0196] CD3 + Cell sorting: using MojoSort TMHuman CD3 T 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.

[0197] Pour off and collect the liquid, and adjust the cell concentration to 3 × 10 6 / mL.

[0198] (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.

[0199] (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.

[0200] (3) Commercial fluorescent antibody-labeled protein sample: Add 150 μL 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 (0.5 μL antibody is added to 50 μL FACS and mixed thoroughly). Incubate at 4°C in the dark for 30 minutes.

[0201] (4) Flow cytometry: Add 150 μL of FACS buffer to each well, mix thoroughly, 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.

[0202] (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 / mL. 100uL was added to each well of a round-bottom 96-well plate.

[0203] (6) Prepare protein samples of different concentrations and whether they were cleaved or not using RPMI1640 as described in 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.

[0204] (7) After incubation, the cells were centrifuged at 1600 rpm for 5 minutes, and the supernatant was collected for measurement of 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 (+MMP14), the biological activity of the fusion protein CD3 antibody in the structure of PDL1×CLDN-MMP(double)-CD3(UCHT1) shown in Figure 2D can be well restored, and can be restored to the effect of PDL1×CD3(UCHT1) shown in the structure of Figure 1A (Figure 8). The above results indicate that the activity of CD3 in the antibody prodrug can be well blocked by anti-CLDN18.2, and its activity on T cells can be effectively restored after MMP cleavage.

[0205] Example 3. BiTE prodrugs exhibit better anti-tumor effects than BiTEs

[0206] To determine whether the TAA×TAA / targeting peptide-CD3 (i.e., bispecific antibody prodrug) we designed and produced 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.

[0207] 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 3The 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.

[0208] 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.

[0209] Example 4. BiTE prodrugs exhibit lower peripheral toxicity than BiTEs

[0210] Because human CD3 KI mice are less susceptible to peripheral toxicity, we used hPBMC-humanized (hu-PBMC) NSG mice as an in vivo mouse model. These mice were generated by infusion of purified human PBMCs from healthy donors, and using adult human peripheral blood mononuclear cells has the fastest engraftment rate and enables short-term studies of robust effector and memory T cell function.

[0211] We selected A549 cells overexpressing human CLDN18.2 protein (P56856) by lentiviral infection as the tumor model for this study and inoculated 1*10 6A549-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)]), PDL1 x 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 Immunoprecipitation. TM The Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences) was used to detect the levels of inflammatory cytokines in serum, 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 with the PBS control group, the PDL1×CD3(OKT3) construct shown in Figure 2A showed high levels of 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, indicating that PDL1×CLDN-MMP(double)-CD3(OKT3) has lower peripheral toxicity.

[0212] Example 5. Activity of BiTE prodrugs can be restored after enzymatic cleavage

[0213] OKT3 (SEQ ID NO: 16) and UCHT1 (SEQ ID NO: 15) are widely used monoclonal antibodies against CD3. In the structure of the bispecific antibody prodrug, we also used OKT3 as the core. According to the method of Example 1, we gene synthesized and produced the 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.

[0214] 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 prodrug forms could be completely cleaved, Figure 11A (PDL1×CLDN-MMP(double)-CD3) and Figure 11B (PDL1×NGR-HA-MMP(double)-CD3).

[0215] By repeating the in vitro PBMC activation experiment 2.3 after enzymatic cleavage of Example 2, it can be seen that both the antibody prodrug of 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.

[0216] Example 7: In vitro functional assay of bispecific antibody prodrugs

[0217] 7.1 Detection of the Binding Ability of Bispecific Antibody Prodrugs to the Antigen hCD3

[0218] According to the structure shown in FIG13 , taking CLDN x CLDN-MMP (cleavable linker)-CD3 (SP34) as an example, the sequence combination is as follows:

[0219] VH 8aa VL 6aa: SEQ ID NOs: 22, 119 and 127.

[0220] VH 6aa TA VL 6aa: SEQ ID NOs: 22, 121 and 127.

[0221] VH 6aa VL 6aa TA: SEQ ID NO: 22, 120 and 128.

[0222] VH 10aa VL 10aa: SEQ ID NOs: 22, 118 and 124.

[0223] PC: SEQ ID NO: 22, 136 and 137.

[0224] As described in Example 2.1, the target cells were spleen cells from hCD3 KI mice. Figure 14A shows that different prodrug molecules showed little binding to hCD3 KI mouse spleen cells without MMP cleavage. As described in Example 2.2, using the MMP cleavage method, Figure 14B shows that different prodrug molecules recovered different lengths of MMP (cleavable linker) proteins after MMP cleavage to varying degrees.

[0225] 7.2 Effects of Bispecific Antibody Prodrugs on T Cell Function (Activation Markers)

[0226] As described in Example 2.3, the bispecific antibody used in FIG15A is the CLDN x CLDN-MMP (cleavable linker)-CD3 (SP34) structure shown in FIG14A , and the sequence combination is as follows:

[0227] PC: SEQ ID NO: 22, 136 and 137

[0228] VH 10aa VL 10aa: SEQ ID NO: 22, 118 and 124

[0229] VH 8aa VL 6aa: SEQ ID NO: 22, 119 and 127

[0230] VH 6aa TA VL 6aa: SEQ ID NO: 22, 121 and 127

[0231] VH 5aa VL 6aa: SEQ ID NOs: 22, 122, and 127

[0232] VH G4S VL G4S: SEQ ID NOs: 22, 134 and 135

[0233] After co-culture of SNU1-CLDN cells (SNU1 cells (from ATCC) overexpressing Claudin18.2) and PBMC for 24 hours, the proportion of cells positive for T cell surface activation markers CD25 and CD69 was detected. The results showed that the shorter the linker, the weaker the activation.

[0234] As described in Example 2.3, the bispecific antibody used in FIG15B is the CLDN x CLDN-MMP (cleavable linker)-CD3 (UCHT1) structure shown in FIG14A , and the sequence combination is as follows:

[0235] PC: SEQ ID NO: 22, 138 and 139

[0236] VH 10aa VL 10aa: SEQ ID NO: 22, 131 and 132

[0237] After co-culture of SNU1-CLDN cells and PBMCs for 24 hours, the proportion of cells positive for T cell surface activation markers CD25 and CD69 was detected. The results showed that the T cell activation ability of the bispecific antibody could be completely restored after MMP enzymatic cleavage.

[0238] Example 8: Anti-tumor effect of bispecific antibody prodrug

[0239] According to the structure shown in FIG13 , taking CLDN x CLDN-MMP (cleavable linker)-CD3 (SP34) as an example, the sequence combination is as follows:

[0240] PC: SEQ ID NO: 22, 136 and 137

[0241] VH 10aa VL 10aa: SEQ ID NO: 22, 118 and 124

[0242] LSGKD (antibody containing linker LSGKD): SEQ ID NO: 22, 123 and 129

[0243] VH 6aa TA VL 7aa: SEQ ID NO: 22, 121 and 126

[0244] VH 5aa TA VL 8aa: SEQ ID NO: 22, 122 and 125

[0245] VH G4S VL G4S: SEQ ID NOs: 22, 134 and 135

[0246] To determine whether systemic administration can effectively fight tumors while reducing peripheral toxicity, Figure 16A shows the process of tumor inoculation and treatment. MC38-CLDN (MC38 cells overexpressing Claudin18.2) was inoculated into hCD3 humanized mice (hCD3EDG) and treated with a lower dose via systemic administration. The experimental results are shown in Figure 16B:

[0247] hCD3EDG mice (n=5 / group) were subcutaneously inoculated with 1×10 6 MC38-CLDN cells. On day 14, the tumor grew to 240 mm 3 The mice were randomly divided into groups at 40 days of age. On day 14, the mice were administered at an equimolar mass of 100 pmol / mouse. The calculated protein was dissolved in 200 ul of PBS and then intraperitoneally injected. The PBS control group and several groups of proteins with different linkers were treated. The length (a), width (b), and height (c) of the tumor were measured using a vernier caliper. The mouse tumor volume = a×b×c / 2. The tumor size was measured twice a week, and the mouse tumor growth curve was recorded. The tumor growth curve showed that the anti-tumor effect was weaker when the intermediate linker was non-cleavable, but the anti-tumor effect was stronger when the intermediate linker was cleavable (Figure 16B).

[0248] We collected peripheral blood 24 hours after intraperitoneal injection and measured serum IFN-γ levels. The results showed that the bispecific antibody prodrug significantly reduced peripheral toxicity, and shorter linkers had lower peripheral toxicity (Figure 16C). Combining peripheral toxicity with tumor efficacy, we found that shorter, cleavable linkers had lower peripheral toxicity and better anti-tumor efficacy.

[0249] Sequence Listing

[0250] G4S2:GGGGSGGGGS (SEQ ID NO: 1)

[0251] G4S4:GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:2)

[0252] G4S3:GGGGSGGGGSGGGGS(SEQ ID NO:3)

[0253] G4S5:GGGGSGGGGSGGGGGSGGGGSGGGGS(SEQ ID NO:24)

[0254] (GGS)2:GGSGGS (SEQ ID NO:28)

[0255] (GGS)3:GGSGGSGGS(SEQ ID NO:30)

[0256] HA-MMP: NQHTIDLADSSGFIANPVTA (SEQ ID NO:4)

[0257] MMP-HA: SGFIANPVTANQHTIDLADS (SEQ ID NO:5)

[0258] MMP high: SGRSPAIFTA (SEQ ID NO:6)

[0259] MMP medium: SGFIANPVTA (SEQ ID NO:7)

[0260] MMP low: SGRSENIRTA (SEQ ID NO:8)

[0261] MMP(Double): SGRSENIRTASGRSPAIFTA (SEQ ID NO:9)

[0262] HA: NQHTIDLADS (SEQ ID NO:10)

[0263] HA2: NQHTIDLADSNQHTIDLADS (SEQ ID NO:11)

[0264] HA n : (NQHTIDLADS) n (SEQ ID NO:12) n = 3

[0265] H(MMP)A: NQHIANPVDS (SEQ ID NO:13)

[0266] H(MMP)A2: NQHIANPVDSNQHIANPVDS (SEQ ID NO:14)

[0267] Anti-CD3 (single-chain, clone: UCHT1, VL-VH)

[0268] Anti-CD3 (single-chain, clone: OKT3, VL-VH)

[0269] Anti-PDL1 (single-chain, Atezolizumab, VL-VH)

[0270] The CDR1 sequence of PDL1 VL is QDVSTA (SEQ ID NO:73)

[0271] The CDR2 sequence of PDL1 VL is SAS

[0272] The CDR3 sequence of PDL1 VL is QQYLYHPATF (SEQ ID NO: 74)

[0273] The CDR1 sequence of PDL1 VH is GFTFSDSW (SEQ ID NO: 75)

[0274] The CDR2 sequence of PDL1 VH is ISPYGGST (SEQ ID NO: 76)

[0275] The CDR3 sequence of PDL1 VH is ARRHWPGGFDYW (SEQ ID NO: 77)

[0276] Anti-CLDN18.2(single-chain,VL-VH)

[0277] The CDR1 sequence of CLDN VL is QSLLNSGNQKNY (SEQ ID NO: 78)

[0278] The CDR2 sequence of CLDN VL is WAS

[0279] The CDR3 sequence of CLDN VL is QNDYFYPFTF (SEQ ID NO: 79)

[0280] The CDR1 sequence of CLDN VH is GYTFTSYW (SEQ ID NO: 80)

[0281] The CDR2 sequence of CLDN VH is IYPSDSYT (SEQ ID NO: 81)

[0282] The CDR3 sequence of CLDN VH is ARVYYGNSFAYW (SEQ ID NO: 82)

[0283] Inactivation Fc region(Knob)

[0284] Inactivation Fc region(Hole)

[0285] IgGκ:METDTLLLWVLLLWVPGSTG (SEQ ID NO:21)

[0286] CLDN×CD3(UCHT1) [Anti-CLDN single-chain (VL-VH) and CD3 single-chain (VL-VH)]

[0287] CLDN×CLDN-MMP(double)-CD3(UCHT1) [Anti-CLDN single-chain (VL-VH) and CD3 single-chain (VL-VH)]

[0288] PDL1×CD3(OKT3) [Anti-PDL1 single-chain (VL-VH) and CD3 single-chain (VL-VH)]

[0289] PDL1×CLDN-MMP(double)-CD3(OKT3) [Anti-PDL1 single-chain (VL-VH) and CD3 single-chain (VL-VH)]

[0290] PDL1×NGR-HA-MMP-CD(OKT3) [Anti-PDL1 single-chain (VL-VH) and CD3 single-chain (VL-VH)]

[0291] G4S: GGGGS (SEQ ID NO:32)

[0292] NGR: GGGGSCNGRC (SEQ ID NO:33)

[0293] NGR2: GGGGSCNGRCGGGGSGGGGSCNGRC (SEQ ID NO:34)

[0294] Human CD3 Clone: OKT3

[0295] VL

[0296] The CDR1 sequence of OKT3 VL is SSVSY (SEQ ID NO:48)

[0297] The CDR2 sequence of OKT3 VL is DTS

[0298] The CDR3 sequence of OKT3 VL is QQWSSNPFTF (SEQ ID NO: 49)

[0299] VH:

[0300] The CDR1 sequence of OKT3 VH is GYTFTRYT (SEQ ID NO: 50)

[0301] The CDR2 sequence of OKT3 VH is INPSRGYT (SEQ ID NO: 51)

[0302] The CDR3 sequence of OKT3 VH is ARYYDDHYCLDYW (SEQ ID NO: 52)

[0303] Human CD3 Clone:L2K

[0304] VL:

[0305] VH:

[0306] Human CD3 Clone:UCHT1

[0307] VL:

[0308] The CDR1 sequence of UCHT1 VL is QDIRNY (SEQ ID NO: 53)

[0309] The CDR2 sequence of UCHT1 VL is YTS

[0310] The CDR3 sequence of UCHT1 VL is QQGNTLPWTF (SEQ ID NO: 54)

[0311] VH:

[0312] The CDR1 sequence of UCHT1 VH is GYSFTGYT (SEQ ID NO: 55)

[0313] The CDR2 sequence of UCHT1 VH is INPYKGVS (SEQ ID NO: 56)

[0314] The CDR3 sequence of UCHT1 VH is ARSGYYGDSDWYFDV (SEQ ID NO: 57)

[0315] Human / Murine CD3 Clone:SP34

[0316] VL:

[0317] The CDR1 sequence of SP34 VL is TGAVTTSNY (SEQ ID NO: 58)

[0318] The CDR2 sequence of SP34 VL is GTN

[0319] The CDR3 sequence of SP34 VL is ALWYSNLWVF (SEQ ID NO: 59)

[0320] VH:

[0321] The CDR1 sequence of SP34 VH is GETFVTYA (SEQ ID NO: 60)

[0322] The CDR2 sequence of SP34 VH is IRSKYNNYAT (SEQ ID NO: 61)

[0323] The CDR3 sequence of SP34 VH is VRHGNFGNSYVSWFAYW (SEQ ID NO: 62)

[0324] Murine CD3 Clone:17A2

[0325] VL:

[0326] The CDR1 sequence of 17A2 VL is QNVINY (SEQ ID NO: 63)

[0327] The CDR2 sequence of 17A2 VL is YAS

[0328] The CDR3 sequence of 17A2 VL is QRIYNSPWTF (SEQ ID NO: 64)

[0329] VH:

[0330] The CDR1 sequence of 17A2 VH is GFTFSNFP (SEQ ID NO: 65)

[0331] The CDR2 sequence of 17A2 VH is ISSGGGGT (SEQ ID NO: 66)

[0332] The CDR3 sequence of 17A2 VH is VRPQGGFASW (SEQ ID NO: 67)

[0333] Murine CD3 Clone:2C11

[0334] VL:

[0335] The CDR1 sequence of 2C11 VL is QDISNY (SEQ ID NO: 68)

[0336] The CDR2 sequence of 2C11 VL is YTN

[0337] The CDR3 sequence of 2C11 VL is QQYYNYPWTF (SEQ ID NO: 69)

[0338] VH:

[0339] The CDR1 sequence of 2C11 VH is GFTFSGYG (SEQ ID NO: 70)

[0340] The CDR2 sequence of 2C11 VH is ITSSSINI (SEQ ID NO: 71)

[0341] The CDR3 sequence of 2C11 VH is ARFDWDKNYW (SEQ ID NO: 72)

[0342] signal peptide

[0343] Cleavable linker

[0344] HA-MMP(Double)-Anti-CD3(OKT3)-Fc(Hole)

[0345] HA-MMP-Anti-CD3(OKT3)-Fc(Hole)

[0346] NGR-HA-MMP(Double)-Anti-CD3(OKT3)-Fc(Hole)

[0347] CLDN-MMP-Anti-CD3(UCHT1 VH VL)-Fc(Hole)

[0348] CLDN-MMP(Double)-Anti-CD3(UCHT1 VH VL)-Fc(Hole)

[0349] CLDN-HA-MMP-Anti-CD3(UCHT1)-Fc(Hole)

[0350] CLDN-[H(MMP)A]*2-Anti-CD3(UCHT1)-Fc(Hole)

[0351] CLDN-MMP-Anti-CD3(UCHT1)-Fc(Hole)

[0352] CLDN-(G4S)*4-Anti-CD3(UCHT1)-Fc(Hole)

[0353] CLDN-(G4S)*2-Anti-CD3(UCHT1)-Fc(Hole)

[0354] CLDN-(MMP)*2-Anti-CD3(UC 2G4S HT1)-Fc(Hole)

[0355] CLDN-[H(MMP)A]-Anti-CD3(UCHT1)-Fc(Hole)

[0356] Anti-CLDN(VL-CL)

[0357] Anti-CLDN(VH-CH)-MMP(Double)-Anti-CD3-Fc(Hole)QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARVYYGNSFAYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVSGRSENIRTASGRSPAIFTAMDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:108, where the underlined sequence represents Anti-CLDN(VH-CH))

[0358] Anti-CLDN(VH-CH)-MMP-Anti-CD3-Fc(Hole)QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARVYYGNSFAYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVSGRSENIRTAMDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:109, where the underlined sequence represents Anti-CLDN(VH-CH))

[0359] The cleavable linkers in the examples are as follows:

[0360] MMP 10AA: GSWLPSSITA (SEQ ID NO:110)

[0361] MMP 8AA: WLPSSITA (SEQ ID NO:111)

[0362] MMP 7AA: LPSSITA (SEQ ID NO:112)

[0363] MMP 6AA:WLPSSI(SEQ ID NO:113)

[0364] MMP 6AA(TA):PSSITA(SEQ ID NO:114)

[0365] MMP 5AA:PSSIT(SEQ ID NO:115)

[0366] LSGKD(SEQ ID NO:116)

[0367] LSGK(SEQ ID NO:117)

[0368] Anti-CLDN(VH)-MMP(10AA)-Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0369] Anti-CLDN(VH)-MMP(8AA)-Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0370] Anti-CLDN(VH)-MMP(6AA)-Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0371] Anti-CLDN(VH)-MMP(6AA,TA)-Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0372] Anti-CLDN(VH)-MMP(5AA)-Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0373] Anti-CLDN(VH)-LSGKD-Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0374] Anti-CLDN(VL)-MMP(10AA)-Anti-CD3(SP34-VL)-Fc(Hole)

[0375] Anti-CLDN(VL)-MMP(8AA)-Anti-CD3(SP34-VL)-Fc(Hole)

[0376] Anti-CLDN(VL)-MMP(7AA)-Anti-CD3(SP34-VL)-Fc(Hole)

[0377] Anti-CLDN(VL)-MMP(6AA)-Anti-CD3(SP34-VL)-Fc(Hole)

[0378] Anti-CLDN(VL)-MMP(6AA,TA)-Anti-CD3(SP34-VL)-Fc(Hole)

[0379] Anti-CLDN(VL)-LSGDK-Anti-CD3(SP34-VL)-Fc(Hole)

[0380] Anti-CLDN(VH)-MMP(10AA)-Anti-CD3(UCHT1-VH-CH)-Fc(Hole)

[0381] Anti-CLDN(VL)-MMP(10AA)-Anti-CD3(UCHT1-VL)-Fc(Hole)

[0382] Anti-CLDN(VH)-G4S-Anti-CD3(UCHT1-VH-CH)-Fc(Hole)

[0383] Anti-CLDN(VL)-G4S-Anti-CD3(UCHT1-VL)-Fc(Hole)

[0384] Anti-CLDN(VH)-G4S-Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0385] Anti-CLDN(VL)-G4S-Anti-CD3(SP34-VL)-Fc(Hole)

[0386] Anti-CD3(SP34-VH-CH)-Fc(Hole)

[0387] Anti-CD3(SP34-VL)-Fc(Hole)

[0388] Anti-CD3(UCHT1-VH-CH)-Fc(Hole)

[0389] Anti-CD3(UCHT1-VH-CH)-Fc(Hole)

[0390] Anti-CLDN(VL)

[0391] Anti-CLDN(VH)

[0392] Anti-MSLN(VL)

[0393] Anti-MSLN(VH)

[0394] MLSN-Fc(Knob)

Claims

1. A bispecific antibody prodrug comprising structural unit 1, structural unit 2, and structural unit 3, wherein structural unit 1 is an anti-CD3 antibody, structural unit 2 is an anti-tumor cell surface antigen antibody, and structural unit 3 is a targeting peptide for tumor cells, wherein the antibodies of structural unit 1 and structural unit 2 are in Fab or scFv form; The structural unit 3 is connected to the N-terminus of the structural unit 1 via 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 sequence of Linker 3 is as shown in SEQ ID NO: 13 or 14. Preferably, the bispecific antibody prodrug is in the form of a protein drug, mRNA or Car T.

2. The bispecific antibody prodrug according to claim 1, wherein the bispecific antibody prodrug further comprises a structural unit 4, wherein 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 prodrug; 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 prodrug is as follows from the N-terminus to the 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.

3. The bispecific antibody prodrug according to claim 1, 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.

4. The bispecific antibody prodrug according to any one of claims 1 to 2, wherein the structural unit 1, binding unit 2 and binding unit 4 constitute a Y-shaped structure of an IgG antibody, wherein the structural unit 1 is an anti-CD3 antibody in Fab form, the structural unit 2 is an anti-tumor cell surface antigen antibody in scFv form, and the structural unit 4 is an Fc fragment. Structural unit 3 is an anti-tumor cell surface antigen antibody in Fab form, wherein the C-terminus of structural unit 3 is connected to the N-terminus of structural unit 1 via a cleavable linker, wherein the tumor cell surface antigens of structural units 2 and 3 are the same or different, preferably the same.

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

6. The bispecific antibody prodrug according to any one of claims 1 to 5, 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.

7. The bispecific antibody prodrug according to any one of claims 1 to 6, 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).

8. The bispecific antibody prodrug according to any one of claims 1 to 7, wherein the tumor cell surface antigen targeted by structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by structural unit 2 is Claudin18.2, and the tumor cell surface antigen targeted by structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by structural unit 2 is PDL1, and the tumor cell surface antigen targeted by structural unit 3 is Claudin18.2; wherein the tumor cell surface antigen targeted by structural unit 2 is PDL1, and the tumor cell surface antigen targeted by structural unit 3 is PDL1; wherein the tumor cell surface antigen targeted by structural unit 2 is MSLN, and the tumor cell surface antigen targeted by structural unit 3 is MSLN; or wherein the tumor cell surface antigen targeted by structural unit 2 is FAP, and the tumor cell surface antigen targeted by structural unit 3 is FAP, preferably, the LCDR1 sequence of the structural unit 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 the structural unit 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 HCDR2 sequence is shown in SEQ ID NO:81, and the HCDR3 sequence is shown in SEQ ID NO:82; or the LCDR1 sequence of the 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; the LCDR1 sequence of the structural unit 3 is shown in SEQ ID NO: 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.

9. The bispecific antibody prodrug according to any one of claims 1 to 7, wherein The tumor cell surface antigen targeted by the structural unit 2 is Claudin18.2 as shown in ACCESSION: P56856, and the structural unit 3 is Claudin18.2 or CNGRC as shown in ACCESSION: P56856; 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. 10 . The bispecific antibody prodrug according to claim 1 , wherein the Fc fragment is an Fc fragment of IgG1, IgG2, IgG3 or IgG4, preferably a humanized Fc fragment.

11. The bispecific antibody prodrug according to any one of claims 1 to 10, 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, 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. 12 . The bispecific antibody prodrug according to claim 1 , wherein the Fc fragment is selected from SEQ ID NO: 19 and SEQ ID NO:

20. 13 . The bispecific antibody prodrug according to claim 1 , wherein when the structural unit 4 is human serum albumin, it is the human serum albumin of ACCESSION: P02768.

14. The bispecific antibody prodrug according to any one of claims 1 to 13, wherein the structural unit 2 is selected from SEQ ID NO: 17 or SEQ ID NO: 18, and / or the structural unit 3 is selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 33 or SEQ ID NO: 34, Preferably, the bispecific antibody prodrug 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.

15. The bispecific antibody prodrug according to any one of claims 1 to 14, wherein the sequence of the bispecific antibody prodrug is selected from the group consisting of: (1) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23, (2) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 25, (3) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27, (4) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 29, (5) a bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 31, (6) A bispecific antibody prodrug comprising SEQ ID NO: 22 and a sequence selected from any one of SEQ ID NOs: 95-109, or consisting of SEQ ID NO: 22 and a sequence selected from any one of SEQ ID NOs: 95-109, (7) A bispecific antibody prodrug comprising or consisting of the sequences shown in SEQ ID NO: 22, SEQ ID NO: 107, and SEQ ID NO: 108, (8) A bispecific antibody prodrug 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: 140, SEQ ID NO: 141, SEQ ID NO: 136, and SEQ ID NO: 137, wherein sequence A is a sequence selected from the group consisting of SEQ ID NOs: 4-14, 83-94, and 110-117; (10) A bispecific antibody prodrug comprising or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 138, or SEQ ID NO: 139, wherein sequence A is selected from the group consisting of SEQ ID NOs: 4-14, 83-94, and 110-117. (11) A bispecific antibody prodrug comprising a sequence represented by SEQ ID NO: 144, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 136, and SEQ ID NO: 137, or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 136, and SEQ ID NO: 137, wherein sequence A is selected from the group consisting of sequences represented by SEQ ID NOs: 4-14, 83-94, and 110-117; (12) A bispecific antibody prodrug comprising a sequence represented by SEQ ID NO: 144, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 138, and SEQ ID NO: 139, or consisting of a sequence represented by SEQ ID NO: 22, sequence A, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 138, and SEQ ID NO: 139, wherein sequence A is selected from the group consisting of sequences represented by SEQ ID NOs: 4-14, 83-94, and 110-117; (13) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 119 and SEQ ID NO: 127, (14) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 121 and SEQ ID NO: 127, (15) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 120, and SEQ ID NO: 128, (16) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 118, and SEQ ID NO: 124, (17) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 122, and SEQ ID NO: 127, (18) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 134, and SEQ ID NO: 135, (19) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 131 and SEQ ID NO: 132, (20) A bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 123, and SEQ ID NO: 129, (21) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 121 and SEQ ID NO: 126, (22) a bispecific antibody prodrug comprising or consisting of SEQ ID NO: 22, SEQ ID NO: 122, and SEQ ID NO: 125, (23) A bispecific antibody prodrug comprising SEQ ID NO: 22, SEQ ID NO: 134 and SEQ ID NO: 135, or consisting of SEQ ID NO: 22, SEQ ID NO: 134 and SEQ ID NO:

135.

16. A biomaterial selected from the group consisting of: (1) a polynucleotide encoding the bispecific antibody prodrug according to any one of claims 1 to 15, (2) a carrier comprising the bispecific antibody prodrug according to any one of claims 1 to 15, and (3) A host cell comprising the bispecific antibody prodrug according to any one of claims 1 to 15.

17. A conjugate comprising the bispecific antibody prodrug of any one of claims 1 to 15 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.

18. A pharmaceutical composition comprising the bispecific antibody prodrug of any one of claims 1 to 15 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is preferably 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.

19. A kit comprising the bispecific antibody prodrug according to any one of claims 1 to 15 and a therapeutic agent for treating tumors, preferably the therapeutic agent for treating tumors is selected from a chemotherapeutic agent, a targeted therapeutic agent, a T cell expressing a chimeric antigen receptor, or an angiogenesis inhibitor.

20. Use of the bispecific antibody prodrug according to any one of claims 1 to 15 in treating tumors, or in preparing a medicament for treating tumors.

21. The use according to claim 20, wherein the tumor is selected from gastric cancer, pancreatic cancer, lung cancer, colon cancer, melanoma, and microglioma.

Images