Bispecific antibodies and uses thereof

Abstract

The invention relates to a bispecific antibody and application thereof. The bispecific antibody comprises (i) CDRH1, CDRH2 and CDRH3 contained in a heavy chain variable region as shown in SEQ ID NO: 14 or 16, and (ii) CDRL1, CDRL2 and CDRL3 contained in a light chain variable region as shown in SEQ ID NO: 13 or 15; the heavy chain variable region is shown as SEQ ID NO: 50 or 58, the CDRH1, the CDRH2 and the CDRH3 are contained in the heavy chain variable region, and the CDRL1, the CDRL2 and the CDRL3 are contained in the light chain variable region, wherein the heavy chain variable region is shown as SEQ ID NO: 51 or SEQ ID NO: 59, and the light chain variable region is shown as SEQ ID NO: 51 or SEQ ID NO: 59.

Description

[0001] This application is application number: 2021801041721, application date: 2021-11-19, and the patent name is: Divisional application for bispecific antibodies and their applications. Technical Field

[0002] The present invention relates to the technical field of immunology, and in particular to bispecific antibodies against EpCAM and CD3 and their applications. Background Art

[0003] Bispecific antibodies (BsAbs), also known as dual-targeting antibodies, can simultaneously recognize and bind to two different antigens or epitopes and block two different signaling pathways to exert their effects. Compared with monoclonal antibodies (mAbs) that recognize a single antigen, BsAbs offer several advantages: ① They can redirect specific immune effector cells to adjacent tumor cells to enhance tumor killing, which is not possible with combination mAb treatment strategies; ② They increase binding specificity through the interaction of two different cell surface antigens; ③ Compared with the development of single antibody drugs in combination therapies, they can reduce development costs, clinical trials, and regulatory review budgets; and ④ Compared with single antibody drugs in combination therapies, they can simultaneously block two different pathways that play unique or overlapping roles in pathogenesis.

[0004] Cancer and other diseases are caused by multiple factors, and there are many signaling pathways in the etiology. Single-target immunotherapy cannot effectively kill the targeted cells. Patients who receive mAb treatment may develop drug resistance or no response to treatment. Therefore, bispecific antibodies have become the main choice for the treatment of many diseases such as cancer, inflammation, viral infection and autoimmune diseases. However, bispecific antibodies do not exist in the natural environment and need to be achieved through recombinant DNA, cell fusion or chemical binding technology. Among them, recombinant DNA technology is currently the most commonly used technology for preparing BsAb, but there are still many obstacles such as difficulty in expressing BsAb, low yield, difficulty in purification, and poor stability. Therefore, it is very necessary to construct a new bispecific antibody that can overcome the above obstacles and establish a corresponding immune killing animal model. The present invention provides a new bispecific antibody and describes the research method and results of its pharmacodynamics. Summary of the Invention

[0005] The present invention develops a novel bispecific antibody characterized by comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3; and uses thereof.

[0006] Specifically, the present invention relates to the following aspects:

[0007] 1. A bispecific antibody comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3,

[0008] The antigen-binding domain that specifically binds to EpCAM is selected from the group consisting of:

[0009] 1) An antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof:

[0010] (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 14, and

[0011] (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 13,

[0012] Preferably, according to the Kabat sequence numbering system, the sequence of CDRL1 is shown as SEQ ID NO:32, the sequence of CDRL2 is shown as SEQ ID NO:33, the sequence of CDRL3 is shown as SEQ ID NO:34, the sequence of CDRH1 is shown as SEQ ID NO:35, the sequence of CDRH2 is shown as SEQ ID NO:36, and the sequence of CDRH3 is shown as SEQ ID NO:37; or

[0013] 2) an antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof:

[0014] (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 16, and

[0015] (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 15,

[0016] Preferably, according to the Kabat sequence numbering system and CDR definition system, the sequence of CDRL1 is shown as SEQ ID NO:38, the sequence of CDRL2 is shown as SEQ ID NO:39, the sequence of CDRL3 is shown as SEQ ID NO:40, the sequence of CDRH1 is shown as SEQ ID NO:41, the sequence of CDRH2 is shown as SEQ ID NO:42, and the sequence of CDRH3 is shown as SEQ ID NO:43;

[0017] The antigen-binding domain that specifically binds to CD3 is selected from the group consisting of:

[0018] 1) An antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof:

[0019] CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:50, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:51,

[0020] Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:44, the sequence of CDRH2 is shown as SEQ ID NO:45, and the sequence of CDRH3 is shown as SEQ ID NO:46, the sequence of CDRL1 is shown as SEQ ID NO:47, the sequence of CDRL2 is shown as SEQ ID NO:48, and the sequence of CDRL3 is shown as SEQ ID NO:49; or

[0021] 2) an antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof:

[0022] CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:58, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:59,

[0023] Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:52, the sequence of CDRH2 is shown as SEQ ID NO:53, and the sequence of CDRH3 is shown as SEQ ID NO:54, the sequence of CDRL1 is shown as SEQ ID NO:55, the sequence of CDRL2 is shown as SEQ ID NO:56, and the sequence of CDRL3 is shown as SEQ ID NO:57; wherein the variants of the CDRs have 3, 2 or 1 amino acid differences with the corresponding CDRs, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, respectively.

[0024] 2. The bispecific antibody of item 1, wherein the antigen-binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof):

[0025] (i) the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region set forth in SEQ ID NO: 13; or

[0026] (ii) the heavy chain variable region set forth in SEQ ID NO: 16, and the light chain variable region set forth in SEQ ID NO: 15; and

[0027] The antigen-binding domain that specifically binds to CD3 comprises the following heavy chain variable region and light chain variable region (or variants thereof):

[0028] (1) the heavy chain variable region shown in SEQ ID NO: 50 and the light chain variable region shown in SEQ ID NO: 51, or

[0029] (2) the heavy chain variable region set forth in SEQ ID NO:58 and the light chain variable region set forth in SEQ ID NO:59;

[0030] Preferably, the antigen-binding domain that specifically binds to EpCAM is in the form of a Fab fragment, and the antigen-binding domain that specifically binds to CD3 is in the form of a ScFv.

[0031] Preferably, the antigen binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof):

[0032] (i) the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region set forth in SEQ ID NO: 13; and

[0033] wherein the antigen binding domain that specifically binds to CD3 is selected from the group consisting of:

[0034] (1) ScFv shown in SEQ ID NO: 18 or a variant thereof,

[0035] (2) ScFv shown in SEQ ID NO: 19 or a variant thereof,

[0036] wherein the variant has 3, 2 or 1 amino acid differences with the corresponding variable region or ScFv, respectively, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively.

[0037] 3. The bispecific antibody of item 1 or 2, wherein the bispecific antibody comprises

[0038] (1) A light chain-heavy chain pair that specifically binds to EpCAM, the light chain-heavy chain pair comprising a light chain and a heavy chain, or consisting thereof; wherein the light chain comprises a light chain variable region and a light chain constant region (preferably a sequence as shown in any one of SEQ ID NOs: 1 and 60-65), and the heavy chain comprises a heavy chain variable region, CH1 (preferably a sequence as shown in SEQ ID NO: 2), and a first Fc fragment; preferably, the first Fc fragment comprises a hinge region (preferably a sequence as shown in SEQ ID NO: 3), CH2 (preferably a sequence as shown in any one of SEQ ID NOs: 6, 7, 66-71), and CH3a;

[0039] (2) a fusion peptide that specifically binds to CD3, the fusion peptide comprising or consisting of a ScFv that specifically binds to CD3 and a second Fc fragment; preferably, the ScFv comprises, from N-terminus to C-terminus, a heavy chain variable region, a connecting peptide (preferably a sequence as shown in SEQ ID NO: 4), and a light chain variable region; the second Fc fragment comprises, from N-terminus to C-terminus, a hinge region (preferably a sequence as shown in SEQ ID NO: 3), CH2 (preferably a sequence as shown in any one of SEQ ID NOs: 6, 7, 66-71), and CH3b; preferably, the C-terminus of the light chain variable region is connected to the hinge region of the second Fc fragment via a connecting peptide (preferably a sequence as shown in SEQ ID NO: 5);

[0040] Preferably, the first Fc fragment and the second Fc fragment are human or humanized Fc fragments, such as human IgG Fc fragments, such as IgG1, IgG2, IgG3, IgG4, IgG5 Fc fragments;

[0041] Preferably, compared to the wild-type antibody, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions that form a knob-and-hole pairing between the heavy chain and the fusion peptide, for example, T366 on one CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W), and Y407 on the other CH3 domain is replaced by a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V), for example, comprising one or more substitutions listed in Table 6;

[0042] Preferably, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions, 1) the substitutions form a salt bridge pairing between the heavy chain and the fusion peptide, for example, one CH3 domain comprises one or more substitutions, substituted with an amino acid residue that has a positive charge under physiological conditions, and the other CH3 domain comprises one or more substitutions, substituted with one or more amino acid residues that have a negative charge under physiological conditions, for example, the positively charged amino acid residue is arginine (R), histidine (H) or lysine (K), for example, the negatively charged amino acid residue is arginine (R), histidine (H) or lysine (K). The amino acid residue in the heavy chain may be aspartic acid (D) or glutamic acid (E), for example, the replaced amino acid residues include one or more of D356, L368, K392, D399 and K409, such as one or more replacements in Table 7, 2) the replacement forms a disulfide bond between the heavy chain and the fusion peptide, such as the replacements in Table 8, and / or 3) the replacement results in a significant decrease in the binding ability between Fc and protein A, such as H435 and Y436 on a CH3 domain are replaced with arginine and phenylalanine, respectively, as shown in Table 9;

[0043] Preferably, wherein:

[0044] a) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a knob-and-hole structure;

[0045] b) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms an ionic bond;

[0046] c) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a disulfide bond; and / or

[0047] d) CH3b of the fusion peptide and CH3a of the heavy chain have substitutions that result in decreased binding to protein A;

[0048] Preferably, CH1 comprises the sequence of SEQ ID No: 2; and / or CL comprises a sequence selected from any one of SEQ ID Nos: 1, 60-65;

[0049] Preferably, the first Fc fragment and / or the second Fc fragment comprises a CH2 of any one of SEQ ID Nos: 6, 7, 66-71 and / or a CH3 of any one of SEQ ID Nos: 8, 9, 11, 12, 72-76;

[0050] Preferably, the sequences of CH3a and CH3b are selected from the group consisting of:

[0051] (1) one of the sequences is shown in SEQ ID NO:8, and the other sequence is shown in SEQ ID NO:11;

[0052] (2) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 12;

[0053] (3) one of the sequences is shown in SEQ ID NO:72, and the other sequence is shown in SEQ ID NO:74;

[0054] (4) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 75;

[0055] (5) one of the sequences is shown in SEQ ID NO:73, and the other sequence is shown in SEQ ID NO:76;

[0056] Preferably, the bispecific antibody is selected from the group consisting of:

[0057] (1) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0058] (2) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0059] (3) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1;

[0060] (4) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1;

[0061] (5) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0062] (6) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0063] (7) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1.

[0064] (8) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1.

[0065] (9) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1.

[0066] (10) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1.

[0067] (11) comprising or consisting of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0068] (12) It comprises a fusion peptide, a heavy chain and a light chain, or consists of them; wherein the fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, or consists of them; the heavy chain comprises SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, or consists of them; and the light chain comprises SEQ ID NO:13 and SEQ ID NO:1, or consists of them.

[0069] 4. A nucleic acid composition comprising: a nucleic acid sequence encoding the bispecific antibody according to any one of items 1 to 3, preferably,

[0070] The nucleic acid composition comprises:

[0071] a) a first expression vector comprising a first nucleic acid encoding an antigen-binding domain or a light chain-heavy chain pair that specifically binds to EpCAM as defined in any one of items 1 to 3;

[0072] b) a second expression vector comprising a second nucleic acid encoding the antigen-binding domain or fusion peptide that specifically binds to CD3 as defined in any one of items 1 to 3.

[0073] 5. An expression vector comprising the nucleic acid composition of item 4.

[0074] 6. A host cell comprising the expression vector of item 5.

[0075] 7. A pharmaceutical composition comprising the bispecific antibody of any one of items 1 to 3, a pharmaceutically acceptable carrier, and optionally, a drug (such as a small molecule drug or a macromolecule drug) for treating cancer (EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, cutaneous squamous cell carcinoma, cutaneous basal cell carcinoma, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer, etc.) and / or malignant ascites, malignant effusion, or malignant pleural effusion, etc. Preferably, the pharmaceutical composition is in the form of an enteral or parenteral dosage form; more preferably, the pharmaceutical composition is in the form of an injection, such as intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

[0076] 8. A conjugate or fusion protein comprising the bispecific antibody of any one of items 1 to 3, preferably comprising a substance A conjugated or fused to the bispecific antibody, wherein the substance A is selected from a therapeutic agent, a drug precursor, a protein (e.g., an enzyme), a virus, a lipid, a biological response modifier (e.g., an immunomodulator), PEG, a hormone, an oligonucleotide, a diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancer, a non-radioactive label, a detectable label, such as a chemiluminescent labeling compound (e.g., luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts, and oxalate esters), or a fluorescent metal (e.g., 152Eu, or a lanthanide label).

[0077] 9. A kit comprising the bispecific antibody of any one of items 1 to 3, and optionally, a drug (such as a small molecule drug or a macromolecule drug) for treating cancer (EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, skin squamous cell carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer, etc.) and / or malignant ascites, malignant effusion, malignant pleural effusion, etc.

[0078] 10. The bispecific antibody of any one of items 1 to 3, for use in treating cancer, or in the preparation of a medicament or kit for treating cancer and / or malignant ascites, malignant effusion, malignant pleural effusion, etc., wherein the cancer is, for example, an EpCAM-positive tumor, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, squamous cell carcinoma of the skin, basal cell carcinoma of the skin, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck cancer, cervical cancer, or oral cancer.

[0079] 11. A method for treating cancer and / or malignant ascites, malignant effusion, or malignant pleural effusion, comprising administering to a subject a therapeutically effective amount of the bispecific antibody of any one of items 1-3, wherein the cancer is an EpCAM-positive tumor, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, squamous cell carcinoma of the skin, basal cell carcinoma of the skin, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck cancer, cervical cancer, or oral cancer.

[0080] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0081] The terms used in the present invention have their conventional meanings as understood by those skilled in the art. Where a term has two or more definitions as used and / or accepted in the art, the definition of the term used herein is intended to include all of the meanings.

[0082] It will be appreciated by those of ordinary skill in the art that the CDR region of an antibody is responsible for the binding specificity of the antibody to the antigen. Given the sequences of the heavy and light chain variable regions of known antibodies, there are currently several methods for determining the CDR regions of antibodies, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, each application of the definition of the CDR of an antibody or its variants will fall within the scope of the terms defined and used herein. Given the variable region amino acid sequence of the antibody, a person skilled in the art can typically determine a specific CDR without relying on any experimental data outside the sequence itself.

[0083] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including monoclonal or polyclonal human, humanized, composite and chimeric antibodies, as well as antibody fragments. Therefore, the term "antibody" includes any protein or peptide containing a specific molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of this include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein. In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art. The immunoglobulin molecules or antibody molecules of the present application can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0084] The term "antibody fragment" or "antigen-binding fragment" includes, but is not limited to, F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, dAb, Fab / c, complementarity determining region (CDR) fragments, single-chain Fvs (ScFv), disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), diabodies, disulfide-stabilized diabodies (ds-Diabodies), ScFv multimers (such as ScFv dimers, ScFv trimers), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, nanobodies, single domain antibodies (sdab), domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. Regardless of the structure, an antigen-binding fragment includes any polypeptide or polypeptide complex that is capable of binding to the same antigen as the parent antibody or parent antibody fragment. The term "antibody fragment" includes aptamers, aptamer enantiomers (spiegelmers), and diabodies. The term "antibody fragment" also includes any synthetic or genetically modified protein that, like an antibody, can bind to a specific antigen to form a complex. Generally, an antibody fragment has at least about 50 consecutive amino acids of an antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0085] "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.

[0086] The antigen binding domain that binds to EpCAM and CD3 is a Fab, or ScFv, or a non-covalent pairing (Fv) between the heavy chain variable region (VH) and the light chain variable region (VL). Any of the above antibodies or polypeptides may also include additional polypeptides, for example, a signal peptide at the N-terminus of the antibody, which is used to direct secretion, or other heterologous polypeptides as described herein, such as a 6×His tag for purification. The present invention includes not only complete antibodies, but also antibody fragments with immunological activity or fusion proteins formed by antibodies and other sequences. The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain and a light chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the variable region of the heavy chain and light chain of the antibody of the present invention, preferably at least 95% homologous, and most preferably 96%, 97%, 98% or more than 99% homologous. Therefore, the present invention includes molecules having monoclonal antibody light chain and heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably 96%, 97%, 98% or 99% or more) homology with the CDRs of the present invention.

[0087] The present invention also includes fragments, variants, derivatives and analogs of the antibody. The antibody, Fab, their variants or derivatives of the present application, include but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, etc.), human antibodies, animal-derived antibodies, humanized antibodies, primatized (primatized) antibodies or chimeric antibodies, CDR grafted and / or modified antibodies, single-chain antibodies (for example, ScFv), double-chain antibodies, epitope binding fragments, for example, Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (ScFv), single-chain antibodies, disulfide-linked Fv (dsFv), fragments comprising VL domains or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. The antibody fragments, antigen-binding fragments, derivatives, or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, a sequence for purifying the polypeptide, a proprotein sequence, or a fusion protein formed with a 6×His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0088] The antibodies of the present invention refer to polypeptides that have binding activity to human EpCAM and CD3 and include the above-mentioned CDR regions. The term also includes variant forms of polypeptides that have the same function as the antibodies of the present invention and include the above-mentioned CDR regions. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not alter the function of the protein. For another example, addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention. Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0089] The antibodies of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence for purifying the polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0090] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues of immunoglobulin polypeptides are preferably replaced by other amino acid residues from the same side chain family. In other embodiments, a string of amino acids can be replaced by a structurally similar string of amino acids that differ in sequence and / or in the composition of the side chain family.

[0091] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids.

[0092] C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0093] In some embodiments, the conservative substitutions are preferably substitutions in which an amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0094] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0095] Fc amino acid numbering follows the Kabat numbering system. "Kabat numbering" refers to the numbering system described by Kabat et al., as described in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Specific numbering is shown in the table below:

[0096] Fc amino acid numbering based on the Kabat numbering system

[0097]

[0098] in,

[0099] Amino acids 221-227 are the hinge domain.

[0100] Amino acids 228-340 are the second constant region CH2 domain of the heavy chain,

[0101] Amino acids 341-447 constitute the third constant region CH3 domain of the heavy chain.

[0102] Antibodies can be modified to improve heterodimer pairing efficiency. For example, in certain aspects, the Fc fragment of the heavy chain of the monovalent unit and / or the Fc fragment of the fusion peptide can contain one or more substitutions compared to a wild-type antibody fragment, which form a knob-into-hole pair. Knob-into-hole configurations are known in the art. See, for example, Ridgway et al., “'Knob-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization,” Protein Engineering 9(7):617-21 (1996).

[0103] In one aspect, T366 on one CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W). Then, Y407 on the other CH3 domain can be replaced by a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V).

[0104] Table 6. Combinations of Fc amino acid substitutions form knob-in-hole pairs between monovalent units and single-chain units to improve heterodimer pairing efficiency.

[0105] Combination number A replacement on CH3 Replacement on another CH3 1 T366W Y407A 2 T366W Y407V 3 T366Y Y407A 4 T366Y Y407V 5 T366W T366S, L368A, Y407V

[0106] In one aspect, one of the CH3 domains contains one or more substitutions with an amino acid residue that has a positive charge under physiological conditions, while the other CH3 domain contains one or more substitutions with one or more amino acid residues that have a negative charge under physiological conditions. In one aspect, the positively charged amino acid residues can be arginine (R), histidine (H), or lysine (K). In another aspect, the negatively charged amino acid residues can be aspartic acid (D) or glutamic acid (E). Amino acid residues that can be substituted include, but are not limited to, D356, L368, K392, D399, and K409.

[0107] Table 7. Combinations of CH3 amino acid substitutions that form ionic bonds between monovalent units and single-chain units to improve heterodimer pairing efficiency

[0108] Combination number A replacement on CH3 Replacement on another CH3 1 D356KD399K K392DK409D 2 L368RD399K K392DK409D 3 L368KD399K K392DK409D 4 L368RD399K K409D 5 L368KD399K K409D 6 L368R K409D 7 L368K K409D

[0109] On one hand, S354 on one CH3 domain is replaced by cysteine, and Y349 on the other CH3 domain is also replaced by cysteine, and the residues at the two replaced positions form a disulfide bond.

[0110] Table 8. Combinations of CH3 amino acid substitutions to form disulfide bonds between monovalent units and single-chain units to improve heterodimer pairing efficiency

[0111] Combination number A replacement on CH3 Replacement on another CH3 1 S354C Y349C

[0112] On the one hand, H435 and Y436 on a CH3 domain were replaced with arginine and phenylalanine, respectively. This replacement resulted in a significant decrease in the binding ability between Fc and protein A, resulting in different protein A binding activities between heterodimers and homodimers, making it easy to separate the two during affinity chromatography.

[0113] Table 9. A CH3 amino acid substitution results in decreased binding to Protein A.

[0114] Combination number Replacement on CH3 1 H435R,Y436F

[0115] In a preferred embodiment of the present invention, the CH3 amino acid sequence of the Fc forming the heterodimer is shown in the following table:

[0116]

[0117]

[0118] One embodiment of the present application provides a heterodimeric antibody comprising two different antigen-binding polypeptide units. In some aspects, the heterodimer is different in size from its corresponding homodimer, and the size difference can be used to facilitate separation of the heterodimer and homodimer.

[0119] In some aspects, such as Figure 1 In one embodiment, one of the two antigen-binding polypeptide units comprises a light chain-heavy chain pair similar to that of a wild-type antibody. Throughout this application, this unit is also referred to as a "monovalent unit". In certain aspects, such as Figure 1 In the present invention, the other antigen-binding polypeptide unit comprises a single-chain variable fragment (ScFv). Such a ScFv can be fused to the N-terminus of the constant fragment (Fc) of an antibody, and is referred to as a fusion peptide. This fusion peptide is also referred to as a "single-chain unit" throughout this application.

[0120] Any of the above antibodies or polypeptides may also include additional polypeptides, for example, encoded polypeptides as described herein, signal peptides from antibody constant regions that are used to direct secretion, or other heterologous polypeptides as described herein. The antibodies described herein may be modified so that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity with the starting sequence, for example, it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence. In addition, nucleotide or amino acid substitutions, deletions, or insertions may be made to make conservative substitutions or changes in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence from a designated protein can be identical to the starting sequence except for one or more independent amino acid substitutions, insertions, or deletions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more independent amino acid substitutions, insertions, or deletions. In certain embodiments, the polypeptide or amino acid sequence from a designated protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 independent amino acid substitutions, insertions, or deletions relative to the starting sequence.

[0121] 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.

[0122] In some embodiments, the antibodies of the present invention can be conjugated to therapeutic agents (e.g., chemotherapeutic agents such as cisplatin, carboplatin), prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG. The antibodies of the present invention can be linked to or fused to therapeutic agents, which can include detectable labels, such as radioactive labels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents, which can be drugs or toxins, ultrasound enhancement agents, non-radioactive labels, combinations thereof, and other such compositions known in the art.

[0123] In certain embodiments, antigen-binding polypeptides include amino acid sequences or one or more groups that are not usually bound to antibodies. For example, the single-chain Fv antibody fragment of the present application may include a flexible linker sequence, or may be modified to include a functional group (for example, polyethylene glycol (PEG), drugs, toxins, or markers) added. The antibody of the present application, its variant or derivative include modified derivatives, that is, any type of molecule is covalently attached to the antibody and the covalent attachment will not prevent the antibody from being bound to the antigenic epitope. In addition, the antibody may include one or more non-classical amino acids.

[0124] It should be noted that the definition of an entity without a clear number should refer to one or more (kinds) of the entity; for example, "multifunctional antibody" should be understood to mean one or more (kinds) of multifunctional antibodies. Similarly, the terms "one or more" and "at least one" without a clear number of definitions are used interchangeably herein.

[0125] As used herein, the term "treat" refers to both therapeutic treatment and prophylactic or preventative measures, in which an undesirable physiological change or disease, such as the development of cancer, is prevented or slowed down in a subject. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (e.g., preventing it from worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether or not detectable. "Treatment" may also refer to prolonging survival compared to the expected survival if not receiving treatment. Conditions in need of treatment include those who already have the condition or symptom as well as those who are susceptible to having the condition or symptom or those in which the condition or symptom is to be prevented.

[0126] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domestic animals, farm animals, zoos, sports farms, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, rats, horses, cattle, cows, primates (e.g., humans, monkeys such as cynomolgus monkeys, macaques, baboons, and chimpanzees), and the like.

[0127] As described herein, the antigen-binding polypeptides, variants or derivatives of the present application can be used in certain treatments and diagnostic methods related to cancer or infectious diseases. The application also relates to antibody-based treatments, which include administering the bispecific antibodies of the present application to patients, such as animals, mammals and humans, for the treatment of one or more diseases or conditions described herein. The therapeutic drugs of the present application include, but are not limited to, antibodies of the present application (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding antibodies of the present application (including their variants and derivatives as described herein). The antibodies of the present application can also be used to treat, suppress or prevent diseases, disorders or conditions, including malignant diseases, disorders, or conditions related to such diseases or disorders, such as diseases related to immune responses. In some embodiments, the antibodies of the present invention can be used as immunosuppressants. In some embodiments, the antibodies of the present invention can be used to treat autoimmune diseases. The antigen-binding polypeptides of the present application, their variants or derivatives are used to suppress the growth, development and / or metastasis of cancer, particularly those listed above or listed in the following paragraphs.

[0128] The antibodies of the present application or their variants or derivatives can be used to treat, prevent, diagnose and / or prognose other diseases or conditions associated with increased cell survival, including but not limited to cancer or tumors, including the development and / or metastasis of malignant tumors, and related diseases (such as malignant ascites, malignant pleural effusion, and effusion), such as EpCAM-positive tumors.

[0129] The method of administering antibody, its variant or derivative includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral route.The antibody or composition can be administered by any convenient route, for example, by infusion or bolus injection, absorbed by epithelium or mucosa and skin inner layer (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents.Therefore, the pharmaceutical composition containing antibody of the present application can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, topically (such as through powder, ointment, drops or transdermal patch), buccal administration or as oral or nasal spray.Term " parenteral " used herein refers to the mode of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.Administration can be systemic or topical. It may also be desirable to administer the antigen-binding polypeptides or compositions of the present invention locally to the area in need of treatment, which can be achieved by, for example, but not limited to, local infusion during surgery, topical application, such as in conjunction with a postoperative wound dressing, by injection, by catheter, by suppository, or by implant, wherein the implant is a porous, non-porous, or gel-like material, including membranes or fibers. Preferably, when administering the proteins (including antibodies) of the present invention, care must be taken to use materials that do not absorb the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 .Schematic diagram of the YBODY antibody structure.

[0131] Figure 2 Bispecific antibody-mediated binding assay for HCT116 and Jurkat cells. A: Flow cytometric plot of the negative control HCT116+Jurkat assay without antibody. Q1 represents CFSE-stained Jurkat cells, Q2 represents co-bound Jurkat and HCT116 cells, Q3 represents PKH26-stained HCT116 cells, and Q4 represents unstained cells. B: Flow cytometric plot of the HCT116+Jurkat+M701A 10 μg / ml experimental group. The quadrants represent the same meanings as above. C: Concentration gradient curves of HCT116 and Jurkat cell binding mediated by different antibodies.

[0132] Figure 3 . Detection of biological activity of bispecific antibodies (reporter gene system).

[0133] Figure 4 Bispecific antibody-mediated killing assay in vitro. A: In vitro killing of B16-EpCAM by M701A; B: In vitro killing of B16 by M701A; C: In vitro killing of HCT116 by M701A; D: In vitro killing of OVCAR-3 by M701A; E: In vitro killing of CHO-K1-huEpCAM by different bispecific antibodies; F: In vitro killing of HCT116 by different bispecific antibodies.

[0134] Figure 5 In vivo efficacy of the bispecific antibody in the HCT116 human colon cancer model. A: Changes in mouse tumor volume; B: Changes in mouse body weight.

[0135] Figure 6 In vivo efficacy of the bispecific antibody in the OVCAR-3 human ovarian cancer model. A: Changes in mouse tumor volume; B: Changes in mouse body weight. DETAILED DESCRIPTION

[0136] The following describes the method and application of the present invention in conjunction with the accompanying drawings. The examples provided are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. A person skilled in the art would be able to make several simple deductions or substitutions without departing from the scope of the present invention, all of which should be considered to fall within the scope of protection of the present invention.

[0137] Example 1: Construction of expression vector for bispecific antibodies

[0138] The bispecific antibody structure targeting EpCAM and CD3 includes an anti-EpCAM binding region and an anti-CD3 binding region. The monovalent unit is a pair formed by the anti-EpCAM heavy chain and the light chain, and the single-chain unit is an anti-CD3 ScFv-Fc form, which is defined as a YBODY structure (e.g. Figure 1 ), wherein the anti-CD3 VL is connected to the hinge region and CH2 via a linker. The heavy chain Fc of the monovalent unit and the Fc of the single-chain unit (based on the human IgG heavy chain Fc as the backbone) undergo amino acid mutations to render them less likely to form homodimers and more likely to form heterodimers. Using existing plasmids or synthetic gene fragments as templates, the corresponding chains of the bispecific antibody are amplified by PCR and overlapping PCR. Each antibody chain is then cloned into the pcDNA3.1 vector (Invitrogen) by enzyme ligation or recombination. The specific sequence information of each antibody chain is shown in Table 1 and the sequence listing.

[0139] Table 1. Amino acid sequence information corresponding to each antibody molecule

[0140]

[0141] Example 2: Expression and purification of bispecific antibodies

[0142] The plasmid was extracted according to conventional plasmid extraction methods and used for chemical transfection of CHO-S cells (from Gibco). The transfected cells were suspended and cultured in a shaker at 37°C and 5% CO2 for 7-10 days. The supernatant was harvested by centrifugation at 3000×g and filtered with a 0.22μm filter membrane. The preliminarily purified bispecific antibody was obtained by protein A affinity chromatography. The concentration of the purified protein was determined by UV absorbance at 280nm and the corresponding extinction coefficient. The antibody purity was tested by high-performance size exclusion chromatography (HPLC-SEC), and the expression level of each protein was calculated. The expression levels of bispecific antibodies ranged from 40 mg / L to 91 mg / L, with initial purities ranging from 45% to 81%. M701A, M701B, M701C, M701D, M701E, M701F, M701G, M701H, M701I, M701J, and M701K all significantly outperformed M701 in both expression and initial purity. The affinity sample was then purified by cation exchange chromatography, ultimately yielding bispecific antibodies with HPLC-SEC purities exceeding 95%. The purification recoveries for each bispecific antibody are shown in Table 2.

[0143] Table 2. Expression level and initial HPLC-SEC purity of bispecific antibodies

[0144]

[0145]

[0146] Example 3: Thermal stability test of bispecific antibodies

[0147] Purified samples of each bispecific antibody were diluted to 0.5 mg / mL in buffer (25 mM citric acid + 50 mM NaCl, pH 6.0) and aliquoted into 1.5 mL EP tubes at 100 μL / tube. The tubes were then placed in a 40°C water bath for 14 days for a thermal acceleration test, and changes in purity and affinity were measured. The day of placement in the 40°C water bath was designated D0, and the 14th day was designated D14.

[0148] Human EpCAM (SB, Cat: 10694-H08H) and human CD3 antigen (SB, Cat: CT038-H2508H) were immobilized on a CM5 chip using the amino coupling method. The antigen coupling capacity was 1500 RU. To test the antigen-binding activity, the sample was diluted to the starting concentration with 1× HBS-EP+buffer and then serially diluted to four concentrations, with the assay performed from low to high concentrations. The binding flow rate was 30 μL / min, the association time was 120 s, and the dissociation time was 300 s. The chip was regenerated with a pH 1.5 glycocine solution at a regeneration flow rate of 10 μL / min and a regeneration time of 30 s. After the assay, the resulting spectra were fitted using the Biacore T200 Evaluation Software using a 1:1 binding fit to calculate the dissociation equilibrium constant (KD).

[0149] The results are shown in Table 3. The purity of M701A, M701B, M701H, M701I, M701J, and M701K decreased by less than 5% on the 14th day of thermal acceleration. Among them, the purity of M701A, M701B, M701J, and M701K changed by less than 2%, and the affinity at both ends remained basically unchanged, indicating that M701A, M701B, M701J, and M701K have good thermal stability.

[0150] Table 3. Purity and affinity testing of bispecific antibody heat-accelerated samples

[0151]

[0152] Note: nd means not detected.

[0153] Example 4: Detection of cell affinity of bispecific antibody EpCAM

[0154] The affinity of the antibody to human EpCAM on the cell surface was detected using the FACS method, using human colon cancer cell HCT116 (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) as the positive cells expressing human EpCAM on the cell membrane surface.

[0155] HCT116 cells were collected by centrifugation and resuspended in buffer (PBS + 1% FBS) at a concentration of 2 × 10 5 Cells / well were added to a 96-well plate, 50 μL per well. The supernatant was removed after centrifugation at 350×g for 5 minutes. The double antibody was diluted to 1000 nM with buffer and serially diluted, and then added to a 96-well plate at 50 μL / well. After resuspending, the plate was incubated in the dark for 1 hour. After centrifugation, the supernatant was removed, the plate was washed twice with buffer and then resuspended in diluted PE-labeled anti-human IgG Fc antibody (Biolegend, 409304), incubated in the dark for 30 minutes, washed twice with buffer and then resuspended in 100 μL buffer. The cells were analyzed by flow cytometry (BD Accuri). TM C6) Computer testing.

[0156] All bispecific antibodies had significant binding effects on HCT116 cells, among which M700 was an anti-EpCAM monoclonal antibody control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26). The specific EC50 values ​​are shown in Table 4.

[0157] Table 4. Binding ability of bispecific antibodies to HCT116 cells

[0158] Antibody No. M700 M701 M701A M701B M701H M701I M701J M701K EC50 (nM) 1.782 10.501 3.565 3.719 7.151 7.086 3.779 5.652

[0159] Example 5: Bispecific Antibody CD3 Affinity Detection (Biacore)

[0160] Human CD3 antigen (SB, Cat: CT038-H2508H) was immobilized on a CM5 chip using the amino coupling method. The antigen coupling capacity was 1500 RU. To test the CD3 antigen binding activity, the sample was diluted to the starting concentration with 1× HBS-EP+buffer and then serially diluted to four concentrations, with the assay performed from low to high concentrations. The binding flow rate was 30 μL / min, the association time was 120 s, and the dissociation time was 300 s. The chip was regenerated using Glycine solution at pH 1.5 at a flow rate of 10 μL / min and a regeneration time of 30 s. After the assay, the resulting data were fitted using Biacore T200 Evaluation Software using a 1:1 binding fit to obtain the dissociation equilibrium constant (KD). As shown in Table 5, all bispecific antibodies in the series bound to the human CD3 antigen. Among them, the anti-EpCAM antibody variable region sequences of M701A and M701B are the same (SEQ ID NO: 13 and SEQ ID NO: 14), and the anti-CD3 antibody variable region sequences are SEQ ID NO: 18 and SEQ ID NO: 19, respectively, with corresponding affinities of 21.15nM and 28.27nM, respectively. However, the anti-EpCAM antibody variable region sequence becomes another (SEQ ID NO: 15 and SEQ ID NO: 16), and the anti-CD3 antibody variable region sequence is still SEQ ID NO: 18 and SEQ ID NO: 19, and the corresponding bispecific antibodies M701H and M701I have affinities of 95.26nM and 40.03nM, respectively. This shows that the affinity of bispecific antibodies formed by combining different anti-EpCAM antibodies and different anti-CD3 antibodies is not regular.

[0161] Table 5. Biacore detection of the binding ability of each bispecific antibody to human CD3 antigen

[0162] Antibody No. M701 M701A M701B M701H M701I M701J M701K Kd(nM) 25.44 21.15 28.27 95.26 40.03 23.45 28.78

[0163] Example 6: Bispecific Antibody-Mediated Cell Bridging

[0164] EpCAM-positive cell line HCT116 was stained with PKH26, and CD3-positive cell line Jurkat (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) was stained with CFSE. The stained cells were plated at a ratio of 1:1 (1×10 5 HCT116 cells: 1×10 5Jurkat cells) were added with serially diluted antibodies to be tested and mixed and incubated, wherein M700 was an anti-EpCAM monoclonal antibody control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26), M100 was an anti-CD3 monoclonal antibody control (light chain SEQ ID NO: 27 and heavy chain SEQ ID NO: 28), Mco101 was an anti-luciferase and anti-CD3 bispecific antibody, and served as the CD3 end isotype control of the bispecific antibody (light chain SEQ ID NO: 29, heavy chain SEQ ID NO: 30 and single chain SEQ ID NO: 31, with the same structure). Figure 1 ). Incubate for 1 hour, wash and resuspend, and analyze by flow cytometry (BD Accuri TM C6) On-chip detection shows that CFSE and PKH26 double-positive cells are HCT116 and Jurkat cells bridged by antibodies.

[0165] The results are as follows Figure 2 As shown, when Jurkat and HCT116 cells were mixed in a 1:1 ratio and treated with hIgG, M700, M100, and Mco101 for 1 hour, no bridge was observed between the HCT116 and Jurkat cells. However, after adding M701A and M701B, the bridged cells accounted for approximately 40% of the total PKH26-positive cells. The activity of M701A and M701B was comparable, and the bispecific antibody-mediated cell interaction showed a positive dose-response relationship with the antibody concentration.

[0166] Example 7: Detection of Biological Activity of Bispecific Antibodies (Reporter Gene Method)

[0167] The biological activity of the bispecific antibody was detected using Jurkat-CD3-NFAT-RE-Luc cells (Promega). The pLV-puro (Inovogen Tech. Co., cat. No. VL3001) vector containing DNA encoding the human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into CHO-K1 cells to obtain the cell line CHO-K1-huEpCAM stably expressing human EpCAM. CHO-K1-huEpCAM was collected as target cells and resuspended in buffer (PBS + 1% FBS). The cells were cultured at 4×10 4 Cells / well were added to a 96-well white culture plate and cultured overnight at 37°C in a 5% CO2 incubator for 18 to 24 hours. The culture medium in the plate was removed and 40 μL of antibody diluent was added to each well. Jurkat-CD3-NFAT-RE-Luc cells, i.e., effector cells, were removed and dispersed to prepare a single cell suspension. At an effector-target ratio of E:T = 1.5:1, 40 μL of antibody diluent, i.e., 6×104 Plate the plate with 96 wells per well, place the all-white 96-well culture plate in a 37°C, 5% CO2 incubator for 6 hours, add 80 μL of Bio-Glo luciferase detection solution to each well, incubate at room temperature for 15 minutes in the dark, place the plate in a multi-function reader, and read the luminescence value using chemiluminescence.

[0168] The results are as follows Figure 3 As shown, in this reporter gene evaluation system, the bispecific antibodies M701, M701A, M701B, M701H, M701I, M701J, and M701K all exhibited biological activity, and the activities of M701A, M701J, and M701K were stronger than those of M701.

[0169] Example 8: In vitro killing assay mediated by bispecific antibodies

[0170] The isolated PBMCs were used as effector cells and EpCAM-expressing cells were used as target cells to detect the in vitro killing effect mediated by the bispecific antibody. The pLV-puro (Inovogen Tech. Co., cat. No. VL3001) vector containing DNA encoding the human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into mouse melanoma cells B16 (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) to obtain the cell line B16-EpCAM that stably expresses human EpCAM, where B16 was used as a negative cell that does not express EpCAM. Other EpCAM-expressing target cells include human colon cancer cells HCT116 (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences), human ovarian cancer cells OVCAR-3 (CCTCC, China Center for Type Culture Collection), and CHO-K1-huEpCAM. The cells were digested with trypsin to form a single-cell suspension, centrifuged at 300 g for 5 min, and stained with 5 μM 5,6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (37°C, 15 min). After washing twice with complete medium, the cells were counted on a Vi-cell cell counter and then plated into 96-well plates according to the experimental design, with 2 × 10 cells per well. 4 Cells / 100 μL. Add the corresponding concentration of antibody at 50 μL / well, count the hPBMCs on a Cellometer cell counter, and place them into a 96-well plate (2×10 cells per well). 5 The cell culture plate was placed in a cell culture incubator and cultured for 72 h. After the cells were digested into a single cell suspension, a propidium bromide (PI) solution with a final concentration of 1 μg / mL was added. After incubation for 10 min, the cells were analyzed by flow cytometry (BD Accuri).TM C6) The cells were tested and the percentage of CFSE+PI+ double-positive cells to CFSE+ positive cells was analyzed.

[0171] like Figure 4 As shown in A and 4B, M701A only has a killing effect on B16-EpCAM cells expressing EpCAM, but has no killing effect on B16 cells without EpCAM expression, while the control antibody Mco101 has no killing effect on any cells, indicating that the bispecific antibody has a targeted effect. Figure 4 As shown in C and 4D, M701A had a significant killing effect on HCT116 and OVCAR-3 cells, and was stronger than the control Mco101. Figure 4 As shown in Figure E, the bispecific antibodies M701A, M701B, M701H, and M701I all had significant killing effects on CHO-K1-huEpCAM cells, and the effects of M701A and M701B were significantly stronger than those of M701H and M701I. Figure 4 As shown in Figure F, both bispecific antibodies M701J and M701K showed significant cytotoxicity against HCT116 cells, with EC50 values ​​ranging from 7.814 to 25.43 ng / ml. The cytotoxicity of M701J was not significantly different from that of M701A. The cytotoxicity of these bispecific antibodies was significantly stronger than that of M701 (EC50 of M701 was 69.17 ng / ml).

[0172] Example 9: In vivo efficacy of bispecific antibodies in the HCT116 human colon cancer xenograft model

[0173] A sufficient amount of HCT116 cells and effector cells CIK (cytokine-induced killer, cytokine-induced killer cells, is a group of heterogeneous cells obtained by co-culturing human peripheral blood mononuclear cells with multiple cytokines in vitro for a period of time. Because this type of cell expresses two membrane protein molecules, CD3+ and CD56+, it is also called NK cell-like T lymphocytes) were cultured according to the culture conditions, and the cells were collected and counted. The pre-mixed HCT116 cells (2×10 6 cells / mouse) and CIK (2×10 6cells / mouse), with an inoculation volume of 0.1 ml / mouse, to establish a human colon cancer HCT116 xenograft tumor model. Treatment began 1 hour after inoculation. The experiment was divided into a test drug M701A 2 mg / kg group, an M701A 1 mg / kg group, an M701 1 mg / kg group, a monoclonal antibody control M700 2 mg / kg group, and a vehicle control group (normal saline), with 8 mice in each group. The drug was administered by tail vein injection on days 0, 2, and 4 after inoculation, for a total of three doses. The efficacy was evaluated based on the relative tumor inhibition rate and the complete tumor regression rate, and the safety was evaluated based on the changes in animal body weight and death.

[0174] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0175] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T and C are the tumor volumes (TV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T / C% = T TV / C TV ×100%(T TV : Average tumor volume of treatment group; C TV : mean tumor volume of vehicle control group).

[0176] Complete tumor regression rate: defined as the tumor volume being less than 63 mm during or after treatment. 3 Complete tumor regression rate (%) = number of animals achieving complete regression in a group / total number of animals in the group × 100%.

[0177] like Figure 5 As shown in A, the test drug M701A (2 mg / kg, 1 mg / kg) showed significant tumor inhibition effects in the treatment group on the 30th day after drug withdrawal (i.e., the 33rd day after inoculation), with relative tumor inhibition rates TGI (%) of 100% and 93.82%, respectively. There were statistically significant differences relative to the vehicle control group (p values ​​< 0.001), and all tumors in the M701A (2 mg / kg) group in both groups reached the standard of complete regression. The efficacy of M701A at these two doses was significantly better than that of M700 at 2 mg / kg (p value < 0.001), and the efficacy of M701A at the same dose was significantly better than that of M701 (both 1 mg / kg). As shown in Figure 5 As shown in B, no animals lost weight during the treatment and no drug toxicity was observed.

[0178] In the same tumor model, M701B, M701J and M701K showed similar tumor inhibitory effects as M701A at the same dose, without any decrease in body weight.

[0179] Example 10: In vivo efficacy of bispecific antibodies in the OVCAR-3 human ovarian cancer xenograft model

[0180] A sufficient amount of OVCAR-3 cells and effector CIK cells were cultured according to the culture conditions, and the cells were collected and counted. The pre-mixed OVCAR-3 cells (1×10 7 cells / mouse) and CIK (1×10 7 cells / mouse), inoculation volume 0.2 ml / mouse, Matrigel gel content 50% (0.1 ml / mouse), to establish a human ovarian cancer OVCAR-3 heterotopic transplant tumor model. Treatment was given 1 hour after inoculation. The experiment was divided into the test drug M701A (5 mg / kg), CD3 end isotype control Mco101 (5 mg / kg), monoclonal antibody control M700 (5 mg / kg) group and solvent control group (normal saline), with 8 mice in each group. The drug was injected into the tail vein, and the drug was given on the 0th, 2nd and 4th days after inoculation, for a total of three times. The efficacy was evaluated based on the relative tumor inhibition rate (TGI) and the complete tumor regression rate, and the safety was evaluated based on the changes in animal body weight and death.

[0181] like Figure 6 As shown in A, the test drug M701A (5 mg / kg) showed a significant tumor inhibition effect in the treatment group on the 44th day after drug withdrawal (i.e., the 48th day after inoculation), with a relative tumor inhibition rate TGI (%) of 98.97%. There were statistically significant differences relative to the vehicle control group (p values ​​< 0.001). The complete tumor regression rate in the M701A (5 mg / kg) group was 87.5%, and the efficacy of this group was significantly better than that of 5 mg / kg M700 (TGI = 70.42%) (p value < 0.001) and 5 mg / kg Mco101 (TGI = 68.87%) (p value < 0.001). Figure 6 As shown in B, no animals lost weight during the treatment and no drug toxicity was observed.

[0182] In the same tumor model, M701B, M701J and M701K showed similar tumor inhibitory effects as M701A at the same dose, without any decrease in body weight.

[0183] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0184] Sequence Listing

[0185]

[0186]

[0187]

[0188]

[0189]

Claims

1. A bispecific antibody, characterized in that comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3, The antigen-binding domain that specifically binds to EpCAM is selected from the group consisting of: 1) An antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 13, Preferably, according to the Kabat sequence numbering system, the sequence of CDRL1 is shown as SEQ ID NO:32, the sequence of CDRL2 is shown as SEQ ID NO:33, the sequence of CDRL3 is shown as SEQ ID NO:34, the sequence of CDRH1 is shown as SEQ ID NO:35, the sequence of CDRH2 is shown as SEQ ID NO:36, and the sequence of CDRH3 is shown as SEQ ID NO:37; or 2) an antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 15, Preferably, according to the Kabat sequence numbering system and CDR definition system, the sequence of CDRL1 is shown as SEQ ID NO:38, the sequence of CDRL2 is shown as SEQ ID NO:39, the sequence of CDRL3 is shown as SEQ ID NO:40, the sequence of CDRH1 is shown as SEQ ID NO:41, the sequence of CDRH2 is shown as SEQ ID NO:42, and the sequence of CDRH3 is shown as SEQ ID NO:43; The antigen binding domain that specifically binds to CD3 is selected from the group consisting of: 2) an antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof: CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:50, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:51, Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:44, the sequence of CDRH2 is shown as SEQ ID NO:45, and the sequence of CDRH3 is shown as SEQ ID NO:46, the sequence of CDRL1 is shown as SEQ ID NO:47, the sequence of CDRL2 is shown as SEQ ID NO:48, and the sequence of CDRL3 is shown as SEQ ID NO:49; or 2) an antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof: CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:58, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:59, Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:52, the sequence of CDRH2 is shown as SEQ ID NO:53, and the sequence of CDRH3 is shown as SEQ ID NO:54, the sequence of CDRL1 is shown as SEQ ID NO:55, the sequence of CDRL2 is shown as SEQ ID NO:56, and the sequence of CDRL3 is shown as SEQ ID NO:57; wherein the variant CDRs have 3, 2 or 1 amino acid differences with the corresponding CDRs, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, respectively, Preferably, the antigen-binding domain that specifically binds to EpCAM is in the form of a Fab fragment, and the antigen-binding domain that specifically binds to CD3 is in the form of an ScFv.

2. The bispecific antibody of claim 1, wherein the antigen-binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof): (i) the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region set forth in SEQ ID NO: 13; or (ii) the heavy chain variable region set forth in SEQ ID NO: 16, and the light chain variable region set forth in SEQ ID NO: 15; and The antigen-binding domain that specifically binds to CD3 comprises the following heavy chain variable region and light chain variable region (or variants thereof): (1) the heavy chain variable region shown in SEQ ID NO: 50 and the light chain variable region shown in SEQ ID NO: 51, or (2) the heavy chain variable region set forth in SEQ ID NO:58 and the light chain variable region set forth in SEQ ID NO:59; Preferably, The antigen-binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof): (i) the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region set forth in SEQ ID NO: 13; and wherein the antigen binding domain that specifically binds to CD3 is selected from the group consisting of: (1) ScFv shown in SEQ ID NO: 18 or a variant thereof, (2) ScFv shown in SEQ ID NO: 19 or a variant thereof, wherein the variant has 3, 2 or 1 amino acid differences with the corresponding variable region or ScFv, respectively, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively.

3. The bispecific antibody of claim 1 or 2, wherein the bispecific antibody comprises (1) A light chain-heavy chain pair that specifically binds to EpCAM, the light chain-heavy chain pair comprising a light chain and a heavy chain, or consisting thereof; wherein the light chain comprises a light chain variable region and a light chain constant region (preferably a sequence as shown in any one of SEQ ID NOs: 1 and 60-65), and the heavy chain comprises a heavy chain variable region, CH1 (preferably a sequence as shown in SEQ ID NO: 2), and a first Fc fragment; preferably, the first Fc fragment comprises a hinge region (preferably a sequence as shown in SEQ ID NO: 3), CH2 (preferably a sequence as shown in any one of SEQ ID NOs: 6, 7, 66-71), and CH3a; (2) a fusion peptide that specifically binds to CD3, the fusion peptide comprising or consisting of a ScFv that specifically binds to CD3 and a second Fc fragment; preferably, the ScFv comprises, from N-terminus to C-terminus, a heavy chain variable region, a connecting peptide (preferably a sequence as shown in SEQ ID NO: 4), and a light chain variable region; the second Fc fragment comprises, from N-terminus to C-terminus, a hinge region (preferably a sequence as shown in SEQ ID NO: 3), CH2 (preferably a sequence as shown in any one of SEQ ID NOs: 6, 7, 66-71), and CH3b; preferably, the C-terminus of the light chain variable region is connected to the hinge region of the second Fc fragment via a connecting peptide (preferably a sequence as shown in SEQ ID NO: 5); Preferably, the first Fc fragment and the second Fc fragment are human or humanized Fc fragments, such as human IgG Fc fragments, such as IgG1, IgG2, IgG3, IgG4, IgG5 Fc fragments; Preferably, compared to the wild-type antibody, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions that form a knob-and-hole pairing between the heavy chain and the fusion peptide, for example, T366 on one CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W), and Y407 on the other CH3 domain is replaced by a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V), for example, comprising one or more substitutions listed in Table 6; Preferably, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions, 1) the substitutions form a salt bridge pairing between the heavy chain and the fusion peptide, for example, one CH3 domain comprises one or more substitutions, substituted with an amino acid residue that has a positive charge under physiological conditions, and the other CH3 domain comprises one or more substitutions, substituted with one or more amino acid residues that have a negative charge under physiological conditions, for example, the positively charged amino acid residue is arginine (R), histidine (H) or lysine (K), for example, the negatively charged amino acid residue is arginine (R), histidine (H) or lysine (K). The amino acid residue in the heavy chain may be aspartic acid (D) or glutamic acid (E), for example, the replaced amino acid residues include one or more of D356, L368, K392, D399 and K409, such as one or more replacements in Table 7, 2) the replacement forms a disulfide bond between the heavy chain and the fusion peptide, such as the replacements in Table 8, and / or 3) the replacement results in a significant decrease in the binding ability between Fc and protein A, such as H435 and Y436 on a CH3 domain are replaced with arginine and phenylalanine, respectively, as shown in Table 9; Preferably, wherein: a) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a knob-and-hole structure; b) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms an ionic bond; c) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a disulfide bond; and / or d) CH3b of the fusion peptide and CH3a of the heavy chain have substitutions that result in decreased binding to protein A; Preferably, CH1 comprises the sequence of SEQ ID No: 2; and / or CL comprises a sequence selected from any one of SEQ ID Nos: 1, 60-65; Preferably, the first Fc fragment and / or the second Fc fragment comprises a CH2 of any one of SEQ ID Nos: 6, 7, 66-71 and / or a CH3 of any one of SEQ ID Nos: 8, 9, 11, 12, 72-76; Preferably, the sequences of CH3a and CH3b are selected from the group consisting of: (1) one of the sequences is shown in SEQ ID NO:8, and the other sequence is shown in SEQ ID NO:11; (2) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 12; (3) one of the sequences is shown in SEQ ID NO:72, and the other sequence is shown in SEQ ID NO:74; (4) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 75; (5) one of the sequences is shown in SEQ ID NO:73, and the other sequence is shown in SEQ ID NO:76; Preferably, the bispecific antibody is selected from the group consisting of: (1) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (2) it comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (3) it comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1; (4) it comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1; (5) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (6) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (7) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (8) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (9) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1; (10) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1; (11) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (12) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises, or consists of, SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9; the heavy chain comprises, or consists of, SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12; and the light chain comprises, or consists of, SEQ ID NO:13 and SEQ ID NO:

1.

4. A nucleic acid composition comprising: a nucleic acid sequence encoding the bispecific antibody according to any one of claims 1 to 3, preferably, The nucleic acid composition comprises: a) a first expression vector comprising a first nucleic acid encoding an antigen-binding domain or a light chain-heavy chain pair that specifically binds to EpCAM as defined in any one of claims 1 to 3; b) a second expression vector comprising a second nucleic acid encoding the antigen-binding domain or fusion peptide that specifically binds to CD3 as defined in any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid composition of claim 4. A host cell comprising the expression vector of claim 5 .

7. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 3, a pharmaceutically acceptable carrier, and optionally, a drug (such as a small molecule drug or a macromolecule drug) for treating cancer (EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, cutaneous squamous cell carcinoma, cutaneous basal cell carcinoma, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer, etc.) and / or malignant ascites, malignant effusion, malignant pleural effusion, etc. Preferably, the pharmaceutical composition is in the form of an enteral or parenteral dosage form; more preferably, the pharmaceutical composition is in the form of an injection, such as intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

8. A conjugate or fusion protein comprising the bispecific antibody of any one of claims 1 to 3, preferably comprising a substance A conjugated or fused to the bispecific antibody, wherein the substance A is selected from a therapeutic agent, a drug precursor, a protein (e.g., an enzyme), a virus, a lipid, a biological response modifier (e.g., an immunomodulator), PEG, a hormone, an oligonucleotide, a diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancer, a non-radioactive label, a detectable label, such as a chemiluminescent labeling compound (e.g., luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts, and oxalate esters), or a fluorescent metal (e.g., 152Eu, or a lanthanide label).

9. A kit comprising the bispecific antibody of any one of claims 1 to 3, and optionally, a drug (such as a small molecule drug or a macromolecule drug) for treating cancer (EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, skin squamous cell carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer, etc.) and / or malignant ascites, malignant effusion, malignant pleural effusion, etc.

10. The bispecific antibody of any one of claims 1 to 3 for use in treating cancer, or in the preparation of a medicament or kit for treating cancer and / or malignant ascites, malignant effusion, malignant pleural effusion, etc., wherein the cancer is, for example, an EpCAM-positive tumor, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, squamous cell carcinoma of the skin, basal cell carcinoma of the skin, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck cancer, cervical cancer, or oral cancer.

11. A method for treating cancer and / or malignant ascites, malignant effusion, or malignant pleural effusion, comprising administering to a subject a therapeutically effective amount of the bispecific antibody of any one of claims 1 to 3, wherein the cancer is an EpCAM-positive tumor, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, squamous cell carcinoma of the skin, basal cell carcinoma of the skin, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck cancer, cervical cancer, or oral cancer.

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