Bispecific antibodies and their applications

Abstract

The present invention provides a bispecific antibody and application thereof. The bispecific antibody comprises an antigen binding domain that specifically binds to EpCAM and an antigen binding domain that specifically binds to CD3.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a bispecific antibody against EpCAM and CD3 and its application. Background Art

[0002] Bi-specific antibodies (BsAb), 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, bi-specific antibodies have many advantages: ① They can redirect specific immune effector cells to adjacent tumor cells to enhance tumor killing, which is not possible through combined 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 therapy, they can reduce the budget for development costs, clinical trials, and regulatory reviews; ④ Compared with single antibody drugs in combination therapy, they can simultaneously block two different pathways that play unique or overlapping functions in the pathogenesis.

[0003] Cancer and other diseases are caused by multiple factors, and there are many signal pathways in etiology. Immunotherapy with a single target cannot effectively kill the targeted cells. Patients treated with mAb 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 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

[0004] 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.

[0005] Specifically, the present invention relates to the following aspects:

[0006] 1. A bispecific antibody, characterized in that it comprises an antigen binding domain that specifically binds to EpCAM and an antigen binding domain that specifically binds to CD3,

[0007] The antigen binding domain that specifically binds to EpCAM is selected from the group consisting of:

[0008] 1) An antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof:

[0009] (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 14, and

[0010] (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 13,

[0011] 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

[0012] 2) an antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof:

[0013] (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 16, and

[0014] (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 15,

[0015] 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;

[0016] The antigen binding domain that specifically binds to CD3 is selected from the group consisting of:

[0017] 1) An antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof:

[0018] 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,

[0019] Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown in SEQ ID NO:44, the sequence of CDRH2 is shown in SEQ ID NO:45, and the sequence of CDRH3 is shown in SEQ ID NO:46, the sequence of CDRL1 is shown in SEQ ID NO:47, the sequence of CDRL2 is shown in SEQ ID NO:48, and the sequence of CDRL3 is shown in SEQ ID NO:49; or

[0020] 2) an antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof:

[0021] 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,

[0022] 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%, 99% identity, respectively.

[0023] 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):

[0024] (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

[0025] (ii) a heavy chain variable region set forth in SEQ ID NO: 16, and a light chain variable region set forth in SEQ ID NO: 15; and

[0026] The antigen binding domain that specifically binds to CD3 comprises the following heavy chain variable region and light chain variable region (or variants thereof):

[0027] (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

[0028] (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;

[0029] 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.

[0030] 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):

[0031] (i) a heavy chain variable region set forth in SEQ ID NO: 14, and a light chain variable region set forth in SEQ ID NO: 13; and

[0032] wherein the antigen binding domain that specifically binds to CD3 is selected from the group consisting of:

[0033] (1) ScFv shown in SEQ ID NO: 18 or a variant thereof,

[0034] (2) ScFv shown in SEQ ID NO: 19 or a variant thereof,

[0035] 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.

[0036] 3. The bispecific antibody of item 1 or 2, wherein the bispecific antibody comprises

[0037] (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 of the same; 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 any one of SEQ ID NOs: 2) and a first Fc fragment; preferably, the first Fc fragment comprises a hinge region (preferably a sequence as shown in any one of SEQ ID NOs: 3), CH2 (preferably a sequence as shown in any one of SEQ ID NOs: 6, 7, 66-71) and CH3a;

[0038] (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 the N-terminus to the 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, and the second Fc fragment comprises, from the N-terminus to the 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, and 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);

[0039] 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;

[0040] 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 in Table 6;

[0041] 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, which are replaced by an amino acid residue that has a positive charge under physiological conditions, and the other CH3 domain comprises one or more substitutions, which are replaced by 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 of 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, for example, one or more replacements in Table 7, 2) the replacement forms a disulfide bond between the heavy chain and the fusion peptide, for example, the replacement in Table 8, and / or 3) the replacement results in a significant decrease in the binding ability between Fc and protein A, for example, H435 and Y436 on a CH3 domain are replaced with arginine and phenylalanine, respectively, as shown in Table 9;

[0042] Preferably, wherein:

[0043] a) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a knob-hole structure;

[0044] b) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms an ionic bond;

[0045] c) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a disulfide bond; and / or

[0046] d) CH3b of the fusion peptide and CH3a of the heavy chain have substitutions that result in decreased ability to bind to protein A;

[0047] 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;

[0048] Preferably, the first Fc fragment and / or the second Fc fragment comprises a CH2 of a sequence selected from any one of SEQ ID Nos: 6, 7, 66-71 and / or a CH3 of a sequence selected from any one of SEQ ID Nos: 8, 9, 11, 12, 72-76;

[0049] Preferably, the sequences of CH3a and CH3b are selected from the group consisting of:

[0050] (1) one of the sequences is shown in SEQ ID NO: 8, and the other sequence is shown in SEQ ID NO: 11;

[0051] (2) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 12;

[0052] (3) one of the sequences is shown in SEQ ID NO: 72, and the other sequence is shown in SEQ ID NO: 74;

[0053] (4) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 75;

[0054] (5) one of the sequences is shown in SEQ ID NO: 73, and the other sequence is shown in SEQ ID NO: 76;

[0055] Preferably, the bispecific antibody is selected from the group consisting of:

[0056] (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; the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0057] (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; the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0058] (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; the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1;

[0059] (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; the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1;

[0060] (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; the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0061] (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; the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0062] (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;

[0063] (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;

[0064] (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;

[0065] (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;

[0066] (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; the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0067] (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; the light chain comprises SEQ ID NO: 13 and SEQ ID NO: 1, or consists of them.

[0068] 4. A nucleic acid composition comprising: a nucleic acid sequence encoding the bispecific antibody according to any one of items 1 to 3, preferably,

[0069] The nucleic acid composition comprises:

[0070] 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;

[0071] b) a second expression vector comprising a second nucleic acid encoding an antigen-binding domain or a fusion peptide that specifically binds to CD3 as defined in any one of items 1 to 3.

[0072] 5. An expression vector comprising the nucleic acid composition of item 4.

[0073] 6. A host cell comprising the expression vector of item 5.

[0074] 7. A pharmaceutical composition comprising the bispecific antibody of any one of items 1 to 3, and a pharmaceutical 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, 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., preferably, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form; more preferably, the dosage form of the pharmaceutical composition is an injection, including intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection, intracranial injection, or intracavity injection.

[0075] 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 marker, a detectable marker, such as a chemiluminescent labeling compound (e.g., luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts, and oxalate esters), or a fluorescent luminescent metal (e.g., 152Eu, or a lanthanide label).

[0076] 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.

[0077] 10. The bispecific antibody of any one of items 1-3, 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, 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 tumors, cervical cancer, or oral cancer.

[0078] 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 (such as 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 tumors, cervical cancer, or oral cancer.

[0079] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0080] The terms involved in the present invention have the conventional meanings understood by those skilled in the art. When 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 meanings.

[0081] Those of ordinary skill in the art will appreciate that the CDR region of an antibody is responsible for the binding specificity of the antibody to an antigen. In the case of known antibody heavy chain and light chain variable region sequences, there are currently several methods for determining the CDR region of an antibody, including Kabat, IMGT, Chothia and AbM numbering systems. However, the application of each definition of the CDR of an antibody or its variants will be within the scope of the terms defined and used herein. If the variable region amino acid sequence of the antibody is given, then those skilled in the art can usually determine a specific CDR, without relying on any experimental data outside the sequence itself.

[0082] 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 and 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 biological activity that binds to an antigen. Examples of this include, but are not limited to, a complementarity determining region (CDR) of a heavy chain or light chain or its ligand binding portion, a heavy chain or light chain variable region, a heavy chain 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 mouse, 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 parts, can be prepared using DNA recombinant techniques well known in the art. The immunoglobulin molecules or antibody molecules of the present application may 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.

[0083] 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'), diabody, disulfide-stabilized diabody (ds-Diabody), ScFv multimers (such as ScFv dimers, ScFv trimers), multispecific antibodies formed by 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 a parent antibody or parent antibody fragment. The term "antibody fragment" includes aptamers, 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. Typically, 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.

[0084] "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 linked with 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 VH to the C-terminus of VL, and vice versa. The protein retains the properties of the original immunoglobulin, except that the constant region has been removed and a linker has been introduced. ScFv molecules are known in the art, such as those described in U.S. Pat. No. 5,892,019.

[0085] The antigen binding domain that binds to EpCAM and CD3 is Fab, or ScFv, or 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 guide 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 heavy and light chains containing variable regions, as long as the variable region is identical to the variable region of the heavy and light chains of the antibodies of the present invention or at least 90% homology, preferably at least 95% homology, and optimally 96%, 97%, 98% or more than 99% homology. Therefore, the present invention includes those 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 more than 96%, 97%, 98% or 99%) homology with the CDRs of the present invention.

[0086] The present invention also includes fragments, variants, derivatives and analogs of the antibodies. Antibodies, antigen-binding fragments, 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 antibodies, or chimeric antibodies, antibodies grafted and / or modified by CDR, single-chain antibodies (e.g., ScFv), double-chain antibodies, antigen epitope binding fragments, e.g., 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 fragment, antigen-binding fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide 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) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6×His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0087] The antibody of the present invention refers to a polypeptide having human EpCAM and CD3 binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substitution is made with amino acids with similar or similar properties, the function of the protein is generally not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus generally does not change 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.

[0088] The antibody of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide 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) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0089] "Conservative amino acid substitutions" are those 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). Therefore, 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 may be replaced by a structurally similar string of amino acids that differ in sequence and / or in the composition of the side chain family.

[0090] 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.

[0091]

[0092]

[0093] In some embodiments, the conservative substitution is preferably a substitution 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 is replaced by Gly or by Ser; Arg is replaced by Lys; Asn is replaced by Gln or by His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or by Pro; His is replaced by Asn or by Gln; Ile is replaced by Leu or by Val; Leu is replaced by Ile or by Val; Lys is replaced by Arg, replaced by Gln or by Glu; Met is replaced by Leu, replaced by Tyr or by Ile; Phe is replaced by Met, replaced by Leu or by Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and / or Phe is replaced by Val, replaced by Ile or by Leu.

[0095] Fc amino acid numbering follows the Kabat numbering. "Kabat numbering" refers to the numbering system described by Kabat et al., which is recorded in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The 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 are the third constant region CH3 domain of the heavy chain.

[0102] The antibodies can be modified to improve heterodimer pairing efficiency. For example, in some 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 the wild-type antibody fragment, which form a knob-into-hole structure 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 (Y1) 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-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, and 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 residue can be arginine (R), histidine (H) or lysine (K). In another aspect, the negatively charged amino acid residue can be aspartic acid (D) or glutamic acid (E). Amino acid residues that can be replaced include, but are not limited to, D356, L368, K392, D399 and K409.

[0107] Table 7. Combinations of CH3 amino acid substitutions 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 D356K D399K K392D K409D 2 L368R D399K K392D K409D 3 L368K D399K K392D K409D 4 L368R D399K K409D 5 L368K D399K 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, so that the heterodimer and homodimer had different protein A binding activities, making it easy to separate the two during affinity chromatography.

[0113] Table 9. One 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] 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 difference in size can be used to facilitate separation of heterodimers and homodimers.

[0118] 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 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 the antibody, which is called a fusion peptide. This fusion peptide is also referred to as a "single-chain unit" throughout the present application.

[0119] 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, which 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 are different 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 have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the starting sequence. In addition, nucleotide or amino acid substitutions, deletions, or insertions may also be performed to make conservative substitutions or changes in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence from a specified protein may 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, a polypeptide or amino acid sequence from a specified 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.

[0120] 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 the composition to be detected (for example, 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 the inserted sequence, such as green fluorescent protein (GFP) and the like. The label itself can be detected (for example, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze a chemical change of a substrate compound or composition, which change 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 be detected only or quantified. The reaction that is only detected generally includes a reaction that can only confirm its presence, wherein the reaction that can be quantified generally includes a reaction with a quantifiable value (for example, that can be reported by a number) such as intensity, polarization and / or other properties. In luminescent or fluorescent assays, the detectable reaction can be directly using a luminophore or fluorescent group associated with a component of the assay that actually involves binding, or indirectly using a luminophore or fluorescent group linked to another (eg, a reporter molecule or indicator) component.

[0121] In some embodiments, the antibodies of the invention can be combined with therapeutic agents (such as chemotherapeutic agents, such as cisplatin, carboplatin), prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents or PEG. The antibodies of the 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 agents or diagnostic agents, cytotoxic agents, which can be drugs or toxins, ultrasound enhancers, non-radioactive labels, combinations thereof and other such components known in the art.

[0122] In certain embodiments, antigen-binding polypeptides include amino acid sequences or one or more groups that are not usually combined with antibodies. For example, the single-chain Fv antibody fragment of the present application may include a flexible linker sequence, or may be modified to add a functional group (e.g., polyethylene glycol (PEG), drugs, toxins or markers). The antibody of the present application, its variant or derivative include modified derivatives, that is, any type of molecule is covalently connected to the antibody and the covalent connection will not prevent the antibody from binding to the antigen epitope. In addition, the antibody may include one or more non-classical amino acids.

[0123] It should be noted that non-definite quantitative entity definitions 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, non-definite quantitative definitions, the terms "one or more" and "at least one" are used interchangeably herein.

[0124] As used herein, the term "treatment" refers to both therapeutic treatment and preventive or prophylactic measures, in which an undesirable physiological change or disease, such as the development of cancer, is prevented or slowed down (mitigated) 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., so that it does not worsen) of the disease state, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total), whether or not detectable. "Treatment" may also refer to prolonging survival compared to the expected survival if not receiving treatment. Those conditions in need of treatment include those already having the condition or symptom as well as those susceptible to having the condition or symptom or those in which the condition or symptom is to be prevented.

[0125] The term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, domestic animals, farm animals, zoos, playgrounds, 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, etc.), and the like.

[0126] 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 associated with such diseases or disorders, such as diseases associated with 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.

[0127] The antibodies of the present application or their variants or derivatives can be used to treat, prevent, diagnose and / or predict 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, nausea and vomiting fluid), such as EpCAM-positive tumors.

[0128] The method of administering the antibody, its variant or derivative includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antibody or composition can be administered by any convenient route, such as by infusion or large dose injection, absorbed by the epithelium or mucosa and the inner layer of the skin (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Therefore, the pharmaceutical composition containing the antibody of the present application can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, topically (such as through powders, ointments, drops or transdermal patches), buccally or as an oral or nasal spray. The term "parenteral" used herein refers to a 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 polypeptide or composition of the present application locally to the area in need of treatment, which may be achieved, for example but not limited to, by local perfusion during surgery, by topical application, for example in combination 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 a membrane or fiber. Preferably, when administering the protein (including antibodies) of the present application, care must be taken to use materials that do not absorb the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 .Schematic diagram of the YBODY antibody structure.

[0130] Figure 2 Detection of the binding effect of HCT116 and Jurkat cells mediated by bispecific antibodies. A: Negative control flow cytometric plot of HCT116+Jurkat without antibody, where Q1 is CFSE-stained Jurkat cells, Q2 is co-bound Jurkat and HCT116 cells, Q3 is PKH26-stained HCT116 cells, and Q4 is unstained cells; B: Flow cytometric plot of the HCT116+Jurkat+M701A 10μg / ml experimental group, the meaning of each quadrant is the same as before; C: Concentration gradient curves of HCT116 and Jurkat cell binding mediated by different antibodies.

[0131] Figure 3 . Detection of biological activity of bispecific antibodies (reporter gene system).

[0132] Figure 4 Bispecific antibody-mediated killing in vitro detection. 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.

[0133] Figure 5 . In vivo efficacy of bispecific antibodies in the HCT116 human colon cancer model. A: Growth changes of mouse tumor volume; B: Changes in mouse body weight.

[0134] Figure 6 .In vivo efficacy of the bispecific antibody in the OVCAR-3 human ovarian cancer model. A: Growth changes of mouse tumor volume; B: Changes in mouse body weight. DETAILED DESCRIPTION

[0135] The method and application of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

[0136] Example 1: Construction of expression vector for bispecific antibody

[0137] 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 an anti-EpCAM heavy chain and a 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 VL of anti-CD3 is connected to the hinge region and CH2 through a linker. The heavy chain Fc of the monovalent unit and the Fc of the single-chain unit (with human IgG heavy chain Fc as the skeleton) are modified by amino acid mutation, so that they are not easy to form homodimers (homodimer) but easy to form heterodimers (heterodimer). Using existing plasmids or synthetic gene fragments as templates, PCR and overlapping PCR are used to amplify each chain corresponding to the bispecific antibody, and each antibody chain is cloned into the pcDNA3.1 vector (Invitrogen) by enzyme cutting, connection or recombination. The specific sequence information of each antibody chain is shown in Table 1 and the sequence table.

[0138] Table 1. Amino acid sequence information corresponding to each antibody molecule

[0139]

[0140]

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

[0142] The plasmid was extracted according to the conventional plasmid extraction method and used for chemical transfection of CHO-S cells (from Gibco). The transfected cells were suspended and shaken 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 preliminary purified bispecific antibody was obtained by protein A affinity chromatography, and 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 corresponding to each protein was calculated. The expression amount of bispecific antibody molecules was 40 mg / L to 91 mg / L, and the initial purity was 45%-81%. The expression amount and initial purity of M701A, M701B, M701C, M701D, M701E, M701F, M701G, M701H, M701I, M701J and M701K were significantly better than M701. Next, the affinity sample was purified by cation exchange chromatography, and finally a bispecific antibody with a HPLC-SEC purity of >95% was obtained. The purification recovery rate of each bispecific antibody is shown in Table 2.

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

[0144] Antibody ID Expression HPLC-SEC Purification recovery rate M701 47.2mg / L 45.91％ 14％ M701A 90.8mg / L 81.12％ 57％ M701B 84.1mg / L 78.53％ 53％ M701C 54.4mg / L 64.54％ 32％ M701D 65.8mg / L 58.63％ 23％ M701E 49mg / L 55.76％ 22％ M701F 52.4mg / L 66.32％ 30％ M701G 41mg / L 70.76％ 25％ M701H 73.3mg / L 72.03％ 43％ M701I 80.6mg / L 73.21％ 45％ M701J 87.7mg / L 78.21％ 47％ M701K 81.3mg / L 74.50％ 41％

[0145] Example 3: Thermal stability test of bispecific antibodies

[0146] The purified samples of each bispecific antibody were diluted to 0.5 mg / ml with a buffer (25 mM citric acid + 50 mM NaCl, pH 60), dispensed into 1.5 mL EP tubes at 100 μL / tube, placed in a 40°C water bath for a 14-day thermal acceleration experiment, and the changes in purity and affinity were detected. The day of placing in a 40°C water bath was counted as D0, and the 14th day was recorded as D14.

[0147] Human EpCAM (SB, Cat: 10694-H08H) and human CD3 antigen (SB, Cat: CT038-H2508H) were fixed on the CM5 chip by amino coupling method, and the antigen coupling amount was 1500RU. When detecting the binding activity of the antigen end, the sample was diluted to the starting concentration with 1×HBS-EP+buffer, and then diluted to 4 concentrations by 2 times. The detection was performed from low concentration to high concentration on the machine, with a binding flow rate of 30μL / min, a binding time of 120s, and a dissociation time of 300s. The chip was regenerated with a pH1.5 Glycine solution, with a regeneration flow rate of 10μL / min and a regeneration time of 30s. After the detection, the result spectrum was fitted with the software Biacore T200 EvaluationSoftware in a 1∶1Binding fitting mode to obtain the dissociation equilibrium constant (KD).

[0148] 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 which 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.

[0149] Table 3. Purity and affinity testing of heat-accelerated samples of bispecific antibodies

[0150]

[0151] Note: d. indicates not detected.

[0152] Example 4: Detection of cell affinity of bispecific antibody EpCAM

[0153] The affinity of the antibody to human EpCAM on the cell surface was detected by FACS method using human colon cancer cell HCT116 (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) as the positive cell expressing human EpCAM on the cell membrane surface.

[0154] 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. After centrifugation at 350×g for 5 min, the supernatant was removed. The dual antibody was diluted to 1000 nM with buffer, and then diluted in multiples, and then added to a 96-well plate at 50 μL / well. After resuspending, it was incubated in the dark for 1 hour. After centrifugation, the supernatant was removed, washed twice with buffer, and then resuspended in the diluted PE-labeled anti-human IgGFc antibody (Biolegend, 409304), incubated in the dark for 30 min, washed twice with buffer, and then resuspended in 100 μL buffer. The flow cytometer (BD Accuri TM C6) Test on the computer.

[0155] Each bispecific antibody has a significant binding effect with HCT116 cells, among which M700 is an anti-EpCAM monoclonal antibody control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26), and the specific EC50 values ​​are shown in Table 4.

[0156] Table 4. Binding ability of bispecific antibodies to HCT116 cells

[0157] Antibody ID 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

[0158] Example 5: Bispecific Antibody CD3 Affinity Detection (Biacore)

[0159] The human CD3 antigen (SB, Cat: CT038-H2508H) was fixed on the CM5 chip by amino coupling method, and the antigen coupling amount was 1500RU. When detecting the binding activity of the CD3 antigen end, the sample was diluted to the starting concentration with 1×HBS-EP+buffer, and then diluted to 4 concentrations by 2 times. The detection was performed from low concentration to high concentration on the machine, with a binding flow rate of 30μL / min, a binding time of 120s, and a dissociation time of 300s; the chip was regenerated with a pH1.5 Glycine solution, with a regeneration flow rate of 10μL / min and a regeneration time of 30s. After the detection, the result spectrum was fitted with the software Biacore T200 Evaluation Software in a 1:1Binding fitting mode to obtain the dissociation equilibrium constant (KD). As shown in Table 5, the series of bispecific antibodies all have binding effects with 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 2827nM, 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 combination of different anti-EpCAM antibodies and different anti-CD3 antibodies to form bispecific antibodies has no regularity in the expression of affinity.

[0160] Table 5. Biacore detection of the binding ability of each bispecific antibody to human CD3 antigen

[0161] Antibody ID M701 M701A M701B M701H M701I M701J M701K Kd(nM) 25.44 21.15 28.27 95.26 40.03 23.45 28.78

[0162] Example 6: Bispecific antibody-mediated cell bridging

[0163] 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 divided into 1:1 ratio (1×10 5 HCT116 cells: 1×10 5Jurkat cells) were added with gradient dilutions of the test antibodies 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 used 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 ). After incubation for 1 hour, the cells were washed and resuspended, and flow cytometry (BD Accuri TM C6) On-machine detection, CFSE and PKH26 double-positive cells are HCT116 and Jurkat cells bridged by antibodies.

[0164] The results are as follows Figure 2 As shown, when Jurkat and HCT116 cells were mixed in a ratio of 1:1, after adding hIgG, M700, M100, and Mco101 for 1 hour, HCT116 and Jurkat cells were not bridged; after adding M701A and M701B, the bridged cells accounted for about 40% of the total PKH26-positive cells, the activity of M701A and M701B was equivalent, and the cell interaction mediated by the bispecific antibody showed a positive dose-effect relationship with the antibody concentration.

[0165] Example 7: Detection of Biological Activity of Bispecific Antibodies (Reporter Gene Method)

[0166] Jurkat-CD3-NFAT-RE-Luc cells (Promega) were used to detect the biological activity of 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 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) and cultured at 4×10 4 10 cells / well were added to a 96-well all-white culture plate and placed in a 37°C, 5% CO2 incubator for overnight culture 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 taken out and the cells were blown off to prepare a single cell suspension. According to the effector-target ratio E:T=1.5:1, 40 μL, i.e. 6×104 Plate the plates in 96-well culture plates at 37°C and 5% CO2 for 6 hours, add 80 μL of Bio-Glo luciferase detection solution to each well, incubate at room temperature for 15 minutes away from light, place in a multi-function reader, and read the luminescence value using chemiluminescence.

[0167] 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.

[0168] Example 8: In vitro killing assay mediated by bispecific antibodies

[0169] 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 cells (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) to obtain a 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 to collect the cells, 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 added to a 96-well plate 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 in a 96-well plate (2×10 cells / well). 5 The cell culture plate was placed in a cell culture incubator for 72 h, and the cells were digested into a single cell suspension and then a 1 μg / mL final concentration of 1 μg / mL propidium bromide (PI) solution was added. After incubation for 10 min, the cells were analyzed by flow cytometry (BD AccuriTM C6) The cells were tested and the percentage of CFSE+PI+ double positive cells to CFSE+ positive cells was analyzed.

[0170] like Figure 4 As shown in A and 4B, M701A only has a killing effect on B16-EpCAM cells expressing EpCAM, but not 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 had significant killing effects on HCT116 cells, with killing EC50 ranging from 78.14 to 25.43 ng / ml, and there was no significant difference between the killing level of M701J and M701A. The killing activity of the above bispecific antibodies was significantly stronger than that of M701 (EC50 of M701 was 69.17 ng / ml).

[0171] Example 9: In vivo efficacy of bispecific antibodies in the HCT116 human colon cancer xenograft model

[0172] A sufficient amount of HCT116 cells and effector CIK cells (cytokine-induced killer, cytokine-induced killer cells, are a group of heterogeneous cells obtained by 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 / cell) and CIK (2×10 6cells / mouse), inoculation volume 0.1ml / mouse, to establish a human colon cancer HCT116 xenograft tumor model. Drug administration began 1h after inoculation. The experiment was divided into test drug M701A2mg / kg group, M701A 1mg / kg group, M7011mg / kg group, monoclonal antibody control M700 2mg / kg group and vehicle control group (normal saline), with 8 mice in each group. The drug was administered by tail vein injection 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 and the complete tumor regression rate, and the safety was evaluated based on the changes in animal weight and death.

[0173] Tumor size calculation formula: Tumor volume (mm 3 )=05×(tumor long diameter×tumor short diameter 2 ).

[0174] 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 the treatment group; C TV : Mean tumor volume of vehicle control group).

[0175] 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%.

[0176] like Figure 5 As shown in A, the test drug M701A (2 mg / kg, 1 mg / kg) showed significant tumor inhibition effects on the 30th day after drug withdrawal in the treatment group (i.e., the 33rd day after inoculation), and the relative tumor inhibition rates TGI (%) were 100% and 9382%, respectively. There were statistically significant differences relative to the vehicle control group (p values ​​were both <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 were 1 mg / kg). As shown in Figure 5 As shown in B, no animals lost weight during the treatment and no signs of drug toxicity were observed.

[0177] In the same tumor model, M701B, M701J and M701K showed similar tumor inhibition effects as M701A at the same dose, and there was no decrease in body weight.

[0178] Example 10: In vivo efficacy of bispecific antibodies in the OVCAR-3 human ovarian cancer xenograft model

[0179] 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 / cell) and CIK (1×10 7 cells / mouse), inoculation volume 0.2ml / mouse, Matrigel gel content 50% (0.1ml / mouse), and human ovarian cancer OVCAR-3 heterotopic transplant tumor model was established. Drug treatment was given 1h after inoculation. The experiment was divided into test drug M701A (5mg / kg), CD3 end isotype control Mco101 (5mg / kg), monoclonal antibody control M700 (5mg / 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 weight and death.

[0180] like Figure 6 As shown in A, the test drug M701A (5 mg / kg) showed significant tumor inhibition effect on the 44th day after drug withdrawal in the treatment group (i.e., the 48th day after inoculation), and the relative tumor inhibition rate TGI (%) was 98.97%. There were statistically significant differences relative to the vehicle control group (p values ​​were all <0.001), and the complete tumor regression rate in the M701A (5 mg / kg) group was 875%. The efficacy of this group was significantly better than 5 mg / kg of M700 (TGI = 70.42%) (p value <0.001) and 5 mg / kg of Mcol01 (TGI = 68.87%) (p value <0.001). Figure 6 As shown in B, no animals lost weight during the treatment and no signs of drug toxicity were observed.

[0181] In the same tumor model, M701B, M701J and M701K showed similar tumor inhibition effects as M701A at the same dose, and there was no decrease in body weight.

[0182] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0183] Sequence Listing

[0184]

[0185]

[0186]

[0187]

[0188] SEQUENCE LISTING <110> Wuhan Youzhiyou Biopharmaceutical Co., Ltd. <120> Bispecific antibodies and their applications <130> IP210557 <160> 76 <170> PatentIn version 3.5 <210> 1 <211> 107 <212> PRT <213> ˹ <220> <223> ELECTRICAL APPLICATIONS <400> 1 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 2 <211> 98 <212> PRT <213> ˹ <220> <223> 㶨 CH1 İ <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 3 <211> 15 <212> PRT <213> ˹ <220> <223> İ <400> 3 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> ˹ <220> <223> 1 İ <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 5 <211> 4 <212> PRT <213> ˹ <220> <223> 2 İ <400> 5 Gly Ala Ala Ala 1 <210> 6 <211> 110 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 6 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 7 <211> 109 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 7 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Ala Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 100 105 <210> 8 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 8 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 9 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 9 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 10 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 10 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 11 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 ĵİ <400> 11 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Arg Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe 50 55 60 Phe Leu Ala Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 12 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 12 Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 13 <211> 113 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ İ <400> 13 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 14 <211> 120 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ İ <400> 14 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20 25 30 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40 45 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 15 <211> 109 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ İ <400> 15 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Ile Ile 35 40 45 Tyr Gly Ala Ser Thr Thr Ala Ser Gly Ile Pro Ala Arg Phe Ser Ala 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 115 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ İ <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Leu Trp Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 17 <211> 241 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 130 135 140 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 145 150 155 160 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 165 170 175 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 180 185 190 Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 195 200 205 Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215 220 Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn 225 230 235 240 Arg <210> 18 <211> 240 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 18 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile 130 135 140 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 165 170 175 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro 180 185 190 Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 195 200 205 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215 220 Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 225 230 235 240 <210> 19 <211> 249 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185 190 Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Val Glu Ile Lys 245 <210> 20 <211> 241 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser 145 150 155 160 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala 165 170 175 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 180 185 190 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile 195 200 205 Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215 220 Ser Ser Asn Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235 240 Arg <210> 21 <211> 247 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Thr Ser Gly Gly Arg Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Arg Gln Tyr Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Pro Asn Ser Val 130 135 140 Ser Thr Ser Leu Gly Ser Thr Val Lys Leu Ser Cys Thr Leu Ser Ser 145 150 155 160 Gly Asn Ile Glu Asn Asn Tyr Val His Trp Tyr Gln Leu Tyr Glu Gly 165 170 175 Arg Ser Pro Thr Thr Met Ile Tyr Asp Asp Asp Lys Arg Pro Asp Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala 195 200 205 Phe Leu Thr Ile His Asn Val Ala Ile Glu Asp Glu Ala Ile Tyr Phe 210 215 220 Cys His Ser Tyr Val Ser Ser Phe Asn Val Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Thr Val Leu Arg Gln Pro 245 <210> 22 <211> 242 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 22 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Met Gly Tyr Trp His Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 145 150 155 160 Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 165 170 175 Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro 180 185 190 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 195 200 205 Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg 210 215 220 Ser Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg <210> 23 <211> 242 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg <210> 24 <211> 244 <212> PRT <213> ˹ <220> <223> -CD3 ScFv İ <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 130 135 140 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys 225 230 235 240 Val Glu Ile Lys <210> 25 <211> 220 <212> PRT <213> ˹ <220> <223> M700 <400> 25 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 26 <211> 450 <212> PRT <213> ˹ <220> <223> M700 <400> 26 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20 25 30 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40 45 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 27 <211> 213 <212> PRT <213> ˹ <220> <223> M100 <400> 27 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 28 <211> 449 <212> PRT <213> ˹ <220> <223> M100 <400> 28 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 29 <211> 219 <212> PRT <213> ˹ <220> <223> Mco101 <400> 29 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 30 <211> 448 <212> PRT <213> ˹ <220> <223> Mco101 <400> 30 Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85 90 95 Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 31 <211> 476 <212> PRT <213> ˹ <220> <223> Mco101 ĵ <400> 31 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile 130 135 140 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 165 170 175 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro 180 185 190 Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 195 200 205 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215 220 Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 225 230 235 240 Gly Ala Ala Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Arg Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser 420 425 430 Phe Phe Leu Ala Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 32 <211> 17 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRL1 <400> 32 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 33 <211> 7 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRL2 <400> 33 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 34 <211> 9 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRL3 <400> 34 Gln Asn Asp Tyr Ser Tyr Pro Leu Thr 1 5 <210> 35 <211> 10 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRH1 <400> 35 Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly 1 5 10 <210> 36 <211> 17 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRH2 <400> 36 Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 37 <211> 10 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRH3 <400> 37 Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr 1 5 10 <210> 38 <211> 11 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRL1 <400> 38 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 39 <211> 7 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRL2 <400> 39 Gly Ala Ser Thr Thr Ala Ser 1 5 <210> 40 <211> 11 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRL3 <400> 40 Gln Gln Tyr Asn Asn Trp Pro Pro Ala Tyr Thr 1 5 10 <210> 41 <211> 5 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRH1 <400> 41 Ser Tyr Ala Ile Ser 1 5 <210> 42 <211> 17 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRH2 <400> 42 Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 43 <211> 6 <212> PRT <213> ˹ <220> <223> -EpCAM ɱ CDRH3 <400> 43 Gly Leu Leu Trp Asn Tyr 1 5 <210> 44 <211> 10 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRH1 <400> 44 Gly Tyr Thr Phe Thr Arg Tyr Thr Met His 1 5 10 <210> 45 <211> 17 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRH2 <400> 45 Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 46 <211> 10 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRH3 <400> 46 Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10 <210> 47 <211> 10 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRL1 <400> 47 Arg Ala Ser Ser Ser Val Ser Tyr Met Asn 1 5 10 <210> 48 <211> 8 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRL2 <400> 48 Asp Thr Ser Lys Val Ala Ser Gly 1 5 <210> 49 <211> 9 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRL3 <400> 49 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <210> 50 <211> 119 <212> PRT <213> ˹ <220> <223> -CD3 ScFv ɱ <400> 50 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 51 <211> 106 <212> PRT <213> ˹ <220> <223> -CD3 ScFv ɱ <400> 51 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 52 <211> 10 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRH1 <400> 52 Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn 1 5 10 <210> 53 <211> 19 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRH2 <400> 53 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 54 <211> 14 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRH3 <400> 54 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 55 <211> 14 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRL1 <400> 55 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 56 <211> 7 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRL2 <400> 56 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 57 <211> 9 <212> PRT <213> ˹ <220> <223> -CD3 ScFv CDRL3 <400> 57 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 58 <211> 125 <212> PRT <213> ˹ <220> <223> -CD3 ScFv ɱ <400> 58 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 59 <211> 109 <212> PRT <213> ˹ <220> <223> -CD3 ScFv <400> 59 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 60 <211> 106 <212> PRT <213> ˹ <220> <223> 㶨 CL İ <400> 60 Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 61 <211> 106 <212> PRT <213> ˹ <220> <223> 㶨 CL İ <400> 61 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 62 <211> 106 <212> PRT <213> ˹ <220> <223> 㶨 CL İ <400> 62 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Ala Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 63 <211> 106 <212> PRT <213> ˹ <220> <223> 㶨 CL İ <400> 63 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Lys Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Asn Thr Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 100 105 <210> 64 <211> 106 <212> PRT <213> ˹ <220> <223> 㶨 CL İ <400> 64 Gly Gln Pro Lys Ala Ala Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Lys Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Ala Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 65 <211> 106 <212> PRT <213> ˹ <220> <223> 㶨 CL İ <400> 65 Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 66 <211> 110 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 66 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 67 <211> 110 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 67 Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 68 <211> 110 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 68 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 69 <211> 110 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 69 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 70 <211> 110 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 70 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 71 <211> 109 <212> PRT <213> ˹ <220> <223> 㶨 CH2 İ <400> 71 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 100 105 <210> 72 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 72 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 73 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 73 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 74 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 74 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 75 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 75 Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 76 <211> 107 <212> PRT <213> ˹ <220> <223> 㶨 CH3 İ <400> 76 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105

Claims

1. A bispecific antibody, characterized in that It consists of a light chain-heavy chain pair that specifically binds to EpCAM and a fusion peptide that specifically binds to CD3, wherein The light chain-heavy chain pair consists of a light chain and a heavy chain; the light chain consists of a light chain variable region and a light chain constant region, and the heavy chain consists of a heavy chain variable region, CH1 and a first Fc fragment; the first Fc fragment consists of a hinge region, CH2 and CH3a; the antigen binding domain that specifically binds to EpCAM in the light chain-heavy chain pair comprises the following CDRs: CDRH1-H3 in the heavy chain variable region and CDRL1-L3 contained in the light chain variable region, wherein the sequence of CDRH1 is shown in SEQ ID NO:35, the sequence of CDRH2 is shown in SEQ ID NO:36, the sequence of CDRH3 is shown in SEQ ID NO:37, the sequence of CDRL1 is shown in SEQ ID NO:32, the sequence of CDRL2 is shown in SEQ ID NO:33, and the sequence of CDRL3 is shown in SEQ ID NO:34; The fusion peptide is composed of a ScFv that specifically binds to CD3, a connecting peptide and a second Fc fragment; the ScFv is composed of a heavy chain variable region, a connecting peptide and a light chain variable region in sequence from the N-terminus to the C-terminus, the second Fc fragment is composed of a hinge region, CH2 and CH3b in sequence from the N-terminus to the C-terminus, and the C-terminus of the light chain variable region is connected to the hinge region of the second Fc fragment through a connecting peptide; the antigen binding domain that specifically binds to CD3 in the fusion peptide comprises the following CDRs: (1) CDRH1-H3 in the heavy chain variable region and CDRL1-L3 in the light chain variable region, wherein the sequence of CDRH1 is shown in SEQ ID NO:44, the sequence of CDRH2 is shown in SEQ ID NO:45, and the sequence of CDRH3 is shown in SEQ ID NO:46; the sequence of CDRL1 is shown in SEQ ID NO:47, the sequence of CDRL2 is shown in SEQ ID NO:48, and the sequence of CDRL3 is shown in SEQ ID NO:49; or (2) CDRH1-H3 in the heavy chain variable region and CDRL1-L3 in the light chain variable region, wherein the sequence of CDRH1 is shown in SEQ ID NO:52, the sequence of CDRH2 is shown in SEQ ID NO:53, the sequence of CDRH3 is shown in SEQ ID NO:54, the sequence of CDRL1 is shown in SEQ ID NO:55, the sequence of CDRL2 is shown in SEQ ID NO:56, and the sequence of CDRL3 is shown in SEQ ID NO:

57.

2. The bispecific antibody of claim 1, wherein the antigen-binding domain that specifically binds to EpCAM comprises the heavy chain variable region shown in SEQ ID NO: 14, and the light chain variable region shown in SEQ ID NO: 13; and the antigen-binding domain that specifically binds to CD3 comprises: (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 shown in SEQ ID NO:58 and the light chain variable region shown in SEQ ID NO:

59.

3. The bispecific antibody of claim 1, wherein the antigen-binding domain that specifically binds to EpCAM comprises the heavy chain variable region shown in SEQ ID NO: 14, and the light chain variable region shown in SEQ ID NO: 13; and the antigen-binding domain that specifically binds to CD3 comprises: (1) ScFv shown in SEQ ID NO: 18, or (2) ScFv shown in SEQ ID NO:

19.

4. The bispecific antibody of claim 1, wherein the first Fc fragment and the second Fc fragment are human IgG Fc fragments.

5. The bispecific antibody of claim 1, wherein the first Fc fragment and / or the second Fc fragment comprises at least one combination of the following substitutions compared to the wild-type antibody: (1) One CH3 substitution is T366W, and the other CH3 substitution is Y407A; (2) One CH3 substitution is T366W, and the other CH3 substitution is Y407V; (3) One CH3 substitution is T366Y, and the other CH3 substitution is Y407A; (4) One CH3 substitution is T366Y, and the other CH3 substitution is Y407V; (5) One CH3 substitution is T366W, and the other CH3 substitution is T366S, L368A, Y407V; (6) One CH3 substitution is D356K, D399K, and the other CH3 substitution is K392D, K409D; (7) One CH3 substitution is L368R, D399K, and the other CH3 substitution is K392D, K409D; (8) One CH3 substitution is L368K, D399K, and the other CH3 substitution is K392D, K409D; (9) One CH3 substitution is L368R, D399K, and the other CH3 substitution is K409D; (10) One CH3 substitution is L368K, D399K, and the other CH3 substitution is K409D; (11) One CH3 substitution is L368R, and the other CH3 substitution is K409D; (12) One CH3 substitution is L368K, and the other CH3 substitution is K409D; (13) One CH3 substitution is S354C, and the other CH3 substitution is Y349C; (14) One of the substitutions on CH3 is H435R, Y436F.

6. The bispecific antibody of claim 1, wherein: (a) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a knob-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 ability to protein A.

7. The bispecific antibody of claim 1, wherein the connecting peptide in the ScFv comprises the sequence of SEQ ID No: 4; the connecting peptide between the C-terminus of the light chain variable region and the hinge region of the second Fc fragment comprises the sequence of SEQ ID No: 5; the CH1 comprises the sequence of SEQ ID No: 2; and / or the light chain constant region comprises a sequence selected from any one of SEQ ID Nos: 1, 60-65.

8. The bispecific antibody of claim 1, wherein the first Fc fragment and / or the second Fc fragment comprises a hinge region of the sequence of SEQ ID No: 3 and a CH2 of the sequence selected from any one of SEQ ID Nos: 6, 7, 66-71.

9. The bispecific antibody of claim 1, wherein 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; and (5) One of the sequences is shown as SEQ ID NO:73, and the other sequence is shown as SEQ ID NO:

76.

10. The bispecific antibody of claim 1, wherein the bispecific antibody is selected from the group consisting of: (1) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:1; (2) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:1; (3) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:1; (4) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:1; (5) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:1; (6) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:1; (7) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:1; and (8) It consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide 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 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 consists of SEQ ID NO:13 and SEQ ID NO:

1.

11. A nucleic acid composition comprising: a nucleic acid sequence encoding the bispecific antibody according to any one of claims 1 to 10.

12. The nucleic acid composition of claim 11, comprising: (a) a first expression vector comprising a first nucleic acid encoding 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 a fusion peptide that specifically binds to CD3 as defined in any one of claims 1 to 3.

13. An expression vector comprising the nucleic acid composition of claim 11 or 12.

14. A host cell comprising the expression vector of claim 13.

15. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-10, and a pharmaceutically acceptable carrier, and optionally, a drug for treating cancer and / or malignant effusion caused by cancer, wherein the cancer is an EpCAM-positive tumor, and the EpCAM-positive tumor is colorectal cancer, gastric cancer, breast cancer, ovarian cancer, pancreatic cancer, or lung cancer.

16. The pharmaceutical composition according to claim 15, wherein the dosage form comprises an enteral dosage form or a parenteral dosage form.

17. The pharmaceutical composition according to claim 15 or 16, which is in the form of an injection, including intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, intratumor injection, intracavitary injection, or intracranial injection.

18. A kit comprising the bispecific antibody of any one of claims 1 to 10, the pharmaceutical composition of any one of claims 15 to 17, and optionally, a drug for treating cancer and / or malignant effusion caused by cancer, wherein the cancer is an EpCAM-positive tumor, and the EpCAM-positive tumor is colorectal cancer, gastric cancer, breast cancer, ovarian cancer, pancreatic cancer, or lung cancer.

19. Use of the bispecific antibody according to any one of claims 1 to 10 and the pharmaceutical composition according to any one of claims 15 to 17 in the preparation of a drug or a kit for treating cancer and / or malignant effusion caused by cancer, wherein the cancer is an EpCAM-positive tumor, and the EpCAM-positive tumor is colorectal cancer, gastric cancer, breast cancer, ovarian cancer, pancreatic cancer, or lung cancer.

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