Bispecific antibodies and their applications

Abstract

The present invention provides a bispecific antibody and uses thereof, the bispecific antibody comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3.

Description

[Technical Field]

[0001] This invention relates to the field of immunology. Specifically, it relates to bispecific antibodies against EpCAM and CD3 and their applications. [Background technology]

[0002] Bispecific antibodies (BsAbs), also known as dual-target antibodies, can simultaneously recognize and bind to two different antigens or epitopes, blocking two different signaling pathways to perform their function. Compared to monoclonal antibodies (mAbs) that recognize a single antigen, biantibodies offer several advantages: (1) They can redirect specific immune effector cells to neighboring tumor cells to enhance tumor killing, which cannot be achieved by combining mAb therapeutic strategies; (2) They increase binding specificity through the interaction of two different cell surface antigens; (3) They reduce development costs, clinical trial budgets, and regulatory review budgets compared to single-antibody drug discovery in combination therapy; and (4) They can simultaneously block two different pathways that exert unique or overlapping functions in the pathogenesis, compared to single-antibody drugs in combination therapy.

[0003] Cancer and other diseases are both caused by multiple factors, and pathogenically, they involve many signaling pathways, making single-target immunotherapy ineffective in killing target cells. Patients treated with mAb therapy may develop drug resistance or become unresponsive to treatment. Therefore, biantibodies have already become a major treatment option for many diseases, including cancer, inflammation, viral infections, and autoimmune diseases. However, biantibodies do not exist in nature and must be realized through recombinant DNA, cell fusion, or chemical bonding technologies. While recombinant DNA technology is currently the most widely used technology for BsAb production, many obstacles remain, such as difficulty in BsAb expression, low yield, difficulty in purification, and poor stability. Therefore, it is essential to establish novel bispecific antibodies to overcome these obstacles and to set up corresponding immunotoxic animal models. This invention provides a novel bispecific antibody and describes its pharmacodynamic study methods and results. [Overview of the project] [Problems that the invention aims to solve]

[0004] This invention aims to develop a novel bispecific antibody and its applications, characterized by comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3. [Means for solving the problem]

[0005] Specifically, the present invention relates to several aspects as follows:

[0006] 1. A bispecific antibody comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3, Here, the antigen-binding domain that specifically binds to EpCAM is selected from the following group: 1) Antigen-binding domains that specifically bind to EpCAM, including the following CDRs or their variants: (i) CDRH1, CDRH2 and CDRH3 included in the heavy chain variable region shown in SEQ ID NO:14, (ii) CDRL1, CDRL2 and CDRL3 included in the light chain variable region shown in SEQ ID NO:13 Preferably, according to the Kabat sequence number 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, the sequence of CDRH3 is shown as SEQ ID NO:37, or 2) Antigen-binding domains that specifically bind to EpCAM, including the following CDRs or their variants: (i) CDRH1, CDRH2 and CDRH3 included in the heavy chain variable region shown in SEQ ID NO:16, (ii) CDRL1, CDRL2 and CDRL3 included in the light chain variable region shown in SEQ ID NO:15 Preferably, according to the Kabat sequence number system and the CDR definition system, the sequence of CDRL1 is shown as SEQ ID NO:38, the sequence of CDRL2 is shown as SEQ ID NO:39, the sequence of CDRL3 is shown as SEQ ID NO:40, the sequence of CDRH1 is shown as SEQ ID NO:41, the sequence of CDRH2 is shown as SEQ ID NO:42, and the sequence of CDRH3 is shown as SEQ ID NO:43. The antigen-binding domain that specifically binds to the aforementioned CD3 is selected from the following group: 1) Antigen-binding domains that specifically bind to CD3, including the following CDRs or their variants: CDRH1, CDRH2 and CDRH3 included in the heavy chain variable region shown in SEQ ID NO:50, and CDRL1, CDRL2 and CDRL3 included in the light chain variable region shown in SEQ ID NO:51, Preferably, according to the Kabat sequence number system, the sequence of CDRH1 is shown as SEQ ID NO:44, the sequence of CDRH2 is shown as SEQ ID NO:45, the sequence of CDRH3 is shown as SEQ ID NO:46, the sequence of CDRL1 is shown as SEQ ID NO:47, the sequence of CDRL2 is shown as SEQ ID NO:48, the sequence of CDRL3 is shown as SEQ ID NO:49, or 2) Antigen-binding domains that specifically bind to CD3, including the following CDRs or their variants: CDRH1, CDRH2 and CDRH3 included in the heavy chain variable region shown in SEQ ID NO:58, and CDRL1, CDRL2 and CDRL3 included in the light chain variable region shown in SEQ ID NO:59, Preferably, according to the Kabat sequence number 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 and the corresponding CDRs each have a difference of 3, 2, or 1 amino acid, or each has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity. This is a bispecific antibody characterized by these characteristics.

[0007] 2. The antigen-binding domain that specifically binds to EpCAM includes the following heavy chain variable region and light chain variable region (or variant thereof): (i) Heavy chain variable region shown in SEQ ID NO:14, light chain variable region shown in SEQ ID NO:13, or (ii) Heavy chain variable region shown in SEQ ID NO:16, light chain variable region shown in SEQ ID NO:15, and Here, the antigen-binding domain that specifically binds to CD3 includes the following heavy chain variable region and light chain variable region (or its variant): (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) Heavy chain variable region shown in SEQ ID NO: 58 and light chain variable region shown in SEQ ID NO: 59, Preferably, the antigen-binding domain that specifically binds to EpCAM is in the form of a Fab fragment, and the antigen-binding domain that specifically binds to CD3 is in the form of an ScFv. Preferably, the antigen-binding domain that specifically binds to EpCAM includes the following heavy chain variable region and light chain variable region (or variant thereof): (i) Heavy chain variable region shown in SEQ ID NO:14, light chain variable region shown in SEQ ID NO:13, and Here, the antigen-binding domain that specifically binds to the CD3 is selected from the following group: (1) ScFv or its variants shown in SEQ ID NO:18, (2) ScFv or its variants shown in SEQ ID NO:19, The bispecific antibody according to item 1, wherein the mutant and the corresponding variable region or ScFv each have a difference of 3, 2, or 1 amino acids, or each have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity, respectively.

[0008] 3. The bispecific antibody comprises the following: (1) A light-heavy chain pair that specifically binds to EpCAM, wherein the light-heavy chain pair includes or consists of a light chain and a heavy chain, where the light chain includes a light chain variable region and a light chain constant region (preferably the sequence is shown in any one of SEQ ID NO: 1, 60-65), and the heavy chain includes a heavy chain variable region, CH1 (preferably the sequence is shown in SEQ ID NO: 2), and a first Fc fragment, preferably the first Fc fragment includes a hinge region (preferably the sequence is shown in SEQ ID NO: 3), CH2 (preferably the sequence is shown in any one of SEQ ID NO: 6, 7, 66-71), and CH3a. (2) A fusion peptide that specifically binds to CD3, wherein the fusion peptide comprises or consists of an ScFv that specifically binds to CD3 and a second Fc fragment, preferably the ScFv comprises a heavy chain variable region, a linking peptide (preferably the sequence shown in SEQ ID NO: 4) and a light chain variable region sequentially from the N end to the C end, the second Fc fragment comprises a hinge region (preferably the sequence shown in SEQ ID NO: 3), CH2 (preferably the sequence shown in any one of SEQ ID NO: 6, 7, or 66-71) and CH3b sequentially from the N end to the C end, preferably the C end of the light chain variable region and the hinge region of the second Fc fragment are linked by a linking peptide (preferably the sequence shown in SEQ ID NO: 5), Preferably, the first Fc fragment and the second Fc fragment are human or humanized Fc fragments, such as human IgG Fc fragments, such as IgG1, IgG2, IgG3, IgG4, and IgG5 Fc fragments. Preferably, compared to the wild-type antibody, the first Fc fragment and / or the second Fc fragment include one or more substitutions that form a mortar-and-pestle structure between the heavy chain and the fusion peptide, for example, T366 on one CH3 domain is substituted with a relatively large amino acid residue such as tyrosine (Y) or tryptophan (W), and Y407 on the other CH3 domain is substituted with a relatively small amino acid residue such as threonine (T), alanine (A), or valine (V), including, for example, one or more substitutions in Table 6. Preferably, the first Fc fragment and / or the second Fc fragment contains one or more substitutions, 1) the substitution forms a salt bridge pair between the heavy chain and the fusion peptide. For example, one CH3 domain contains one or more substitutions and is substituted by an amino acid residue having a positive charge under physiological conditions, and another CH3 domain contains one or more substitutions and is substituted by an amino acid residue having a negative charge under one or more physiological conditions. For example, the amino acid residue having a positive charge is arginine (R), histidine (H) or lysine (K). For example, the amino acid residue having a negative charge is aspartic acid (D) or glutamic acid (E). For example, the substituted amino acid residue contains one or more of D356, L368, K392, D399 and K409. For example, it is one or more substitutions in Table 7. 2) The substitution forms a disulfide bond between the heavy chain and the fusion peptide. For example, it is the substitution in Table 8, and / or 3) The substitution significantly reduces the binding ability between Fc and protein A. For example, H435 and Y436 on one CH3 domain are substituted with arginine and phenylalanine, respectively, as shown in Table 9. Preferably, here: a) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a mortise-and-tenon 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 a decrease in the binding ability to protein A. Preferably, CH1 contains the sequence of SEQ ID No: 2, and / or CL contains the sequence selected from any one of SEQ ID Nos: 1, 60 - 65. Preferably, the first Fc fragment and / or the second Fc fragment contains CH2 of the sequence selected from any one of SEQ ID Nos: 6, 7, 66 - 71 and / or CH3 of the sequence selected from any one of SEQ ID Nos: 8, 9, 11, 12, 72 - 76. Preferably, the sequences of CH3a and CH3b are selected from the group consisting of: (1) One of the sequences is shown in SEQ ID NO:8 and the other sequence is shown in SEQ ID NO:11; (2) One of the sequences is shown in SEQ ID NO:9 and the other sequence is shown in SEQ ID NO:12; (3) One of the sequences is shown in SEQ ID NO:72 and the other sequence is shown in SEQ ID NO:74; (4) One of the sequences is shown in SEQ ID NO:9 and the other sequence is shown in SEQ ID NO:75; (5) One of the sequences is shown in SEQ ID NO:73 and the other sequence is shown in SEQ ID NO:76. Preferably, the bispecific antibody is selected from the group consisting of: (1) It comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (2) It comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (3) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1. (4) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1. (5) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1. (6) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1. (7) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1. (8) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1. (9) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1. (10) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1. (11) It comprises or consists of a fusion peptide, a heavy chain and a light chain, where the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1. (12) The bispecific antibody according to item 1 or 2, comprising or consisting of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1.

[0009] 4. A nucleic acid composition comprising a nucleic acid sequence encoding a bispecific antibody as described in any one of items 1 to 3, preferably, nucleic acid composition, 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, b) A nucleic acid composition comprising a second expression vector comprising a second nucleic acid encoding an antigen-binding domain or fusion peptide that specifically binds to CD3 as defined in any one of items 1 to 3.

[0010] 5. An expression vector comprising the nucleic acid composition of item 4.

[0011] 6. Host cells containing the expression vector specified in item 5.

[0012] 7. A bispecific antibody as described in any one of items 1 to 3, a pharmaceutically acceptable carrier, and optionally, cancer (EpCAM-positive tumors, e.g., colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal pellucida, cutaneous carcinoma, basal cell carcinoma, sarcoma, nasal glioma, craniopharyngotoma, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer) A pharmaceutical composition comprising a drug (e.g., a small molecule drug or a high molecular weight drug) for treating malignant ascites, malignant fluid retention, malignant pleural effusion, etc., wherein the dosage form of the pharmaceutical composition comprises a gastrointestinal dosage form or an extra-gastrointestinal dosage form, and more preferably the dosage form of the pharmaceutical composition comprises an injectable preparation including intravenous injection, intravenous drip infusion, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

[0013] 8. A conjugate or fusion protein comprising a bispecific antibody as described in any one of items 1 to 3, preferably comprising a substance A conjugated or fused with the bispecific antibody, wherein substance A is a therapeutic agent, a drug precursor, a protein (e.g., an enzyme), a virus, a lipid, a biological response modifier (e.g., an immunomodulator), a PEG, a hormone, an oligonucleotide, a diagnostic agent, It may be a drug or a toxin. cytotoxicity Agent, super Sound wave enhancers, non-radioactive markers —, for example Chemiluminescent marker compounds (e.g., luminol, isoluminol, thermoactive acridinium esters, imidazole, acridinium salts, oxalic acid esters) detection markers such as , or fluorescent metals (e.g., 152Eu, or lanthanide markers) Selected from the group consisting of , conjugate or fusion protein.

[0014] 9. A kit comprising a bispecific antibody as described in any one of paragraphs 1 to 3, and optionally a drug for treating cancer (EpCAM-positive tumors, e.g., colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal pellucida, cutaneous carcinoma, basal cell carcinoma, sarcoma, nasal glioma, craniopharyngotoma, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer, etc.) and / or malignant ascites, malignant fluid accumulation, malignant pleural effusion, etc. (e.g., small molecule drug or large molecule drug).

[0015] 10. Uses of the bispecific antibody described in any one of paragraphs 1 to 3 for the treatment of cancer, or in the manufacture of therapeutic drugs or kits for cancer and / or malignant ascites, malignant fluid, malignant pleural effusion, etc., wherein the cancer, for example, EpCAM-positive tumors are, for example, colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal pellucida, cutaneous carcinoma, basal cell carcinoma, sarcoma, nasal glioma, craniopharyngotoma, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer.

[0016] 11. A method for treating cancer and / or malignant ascites, malignant fluid, malignant pleural effusion, comprising administering to a subject a therapeutically effective dose of any one of items 1 to 3, wherein the cancer EpCAM-positive tumor is, for example, colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal pellucida, cutaneous carcinoma, basal cell carcinoma, sarcoma, nasal glioma, craniopharyngotoma, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumor, cervical cancer, or oral cancer.

[0017] Within the scope of the present invention, it should be understood that the above technical features of the present invention and the technical features specifically described below (for example, in the examples) can be combined to form novel or preferred technical solutions. Due to space limitations, each will not be repeated here.

[0018] The terms used herein have the ordinary meanings that are understood by those skilled in the art. The definitions of terms used herein are, where permitted and in the art, used to include all meanings when a term has more than one definition.

[0019] Those skilled in the art will understand that the CDR region of an antibody is responsible for its binding specificity to an antigen. Given that the antibody heavy chain and light chain variable region sequences are known, there are now several methods for determining the antibody CDR region, including Kabat, IMGT, Chothia, and the AbM numbering system. However, each application of the definition of CDR for various antibodies or their variants falls within the scope of the terms defined and used herein. Given the variable region amino acid sequence of the antibody, those skilled in the art can usually determine a specific CDR without relying on any experimental data other than the sequence itself.

[0020] As used herein, “antibody” or “antigen-binding fragment” means a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The term “antibody” is used broadly and includes immunoglobulins or antibody molecules, said antibody molecules including monoclonal or polyclonal human, humanized, complexed and chimeric antibodies, as well as antibody fragments. Thus, the term “antibody” includes any protein or peptide containing a specific molecule, said specific molecule including at least a portion of an immunoglobulin molecule that has biological activity to bind to an antigen. Examples of such situations include, but are not limited to, the complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding portion, the heavy or light chain variable region, the heavy or light chain constant region, the framework (FR) region or any portion thereof, or at least a portion of the binding protein. In the present invention, antibodies include mouse, chimeric, humanized or whole-human antibodies prepared using techniques familiar to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies containing human and non-human portions, can be prepared using DNA recombination techniques well known in the art. The immunoglobulin molecule or antibody molecule of this application may be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or a subclass.

[0021] The terms “antibody fragment” or “antigen-binding fragment” include, but are 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 antibody dsFv (dsFv-dsFv'), double-chain antibody (Diabody), disulfide-stabilized double-chain antibody (ds-Diabody), ScFv multimers (ScFv dimer, ScFv trimer, etc.), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, nanoantibodies, single-domain antibodies (sdab), domain antibodies, bivalent domain antibodies, or any other antibody fragments that bind to an antigen but do not contain a complete antibody structure. Regardless of structure, an antigen-binding fragment comprises a polypeptide or polypeptide complex capable of binding to the same antigen that binds to the parent antibody or parent antibody fragment. The term "antibody fragment" includes aptamers, aptamer enantiomers (spiegelmers), and diabodies. The term "antibody fragment" also includes synthetic or genetically modified proteins that, like antibodies, can bind to specific antigens and form complexes. Generally, an antibody fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and optimally at least about 100 consecutive amino acids.

[0022] A "single-stranded 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 some embodiments, these regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker may be glycine-rich to be flexible, and may contain serine or threonine to be soluble, and the N-terminus of VH may be linked to the C-terminus of VL, and vice versa. The protein retains the properties of the original immunoglobulin simply by removing the constant region and introducing the linker. ScFv molecules are known in the art, as described in U.S. Patent No. 5,892,019.

[0023] The antigen-binding domain that binds EpCAM to CD3 is Fab, or ScFv, or a non-covalent bond (Fv) between the heavy chain variable region (VH) and the light chain variable region (VL). Any of the antibodies or polypeptides described above may further comprise additional polypeptides, such as an antibody N-terminal signal peptide for inducing secretion, or other heterologous polypeptides described herein, such as a 6×His tag, for purification. The present invention includes not only complete antibodies but also antibody fragments having immunoactivity or fusion proteins formed with antibodies and other sequences. The present invention further provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention comprises any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain and light chain containing a variable region, as long as the variable region is identical to or at least 90% homology, preferably at least 95% homology, and optimally 96%, 97%, 98%, or 99% or more homology with the variable regions of the heavy chain and light chain of the antibody of the present invention. Therefore, the present invention includes molecules having a light chain and heavy chain variable region of a monoclonal antibody having a CDR, insofar as the CDR has 90% or more homology (preferably 95% or more, optimally 96%, 97%, 98%, or 99% or more) with the CDR of the present invention.

[0024] The present invention further includes fragments, variants, derivatives, and analogues of the antibodies. The antibodies, antigen-binding fragments, variants, or derivatives of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies, etc.), human antibodies, animal-derived antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, CDR grafted and / or modified antibodies, 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-bonded Fv (dsFv), fragments containing a VL domain or VH domain, fragments produced from Fab expression libraries, and anti-idiotic (anti-Id) antibodies. The antibody fragments, antigen-binding fragments, derivatives, or analogues of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted (where such substituted amino acid residues may or may not be encoded by codons), or (ii) polypeptides having substituents in one or more amino acid residues, or (iii) polypeptides formed by the fusion of a mature polypeptide with another compound (a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by the fusion of an additional amino acid sequence to such polypeptide sequence (e.g., a preamble sequence or secretory sequence, or a sequence or proprotein sequence for purifying such polypeptide, or a fusion protein with a 6×His tag). According to the teachings herein, these fragments, derivatives, and analogues are within the scope well known to those skilled in the art.

[0025] The antibody of the present invention refers to a polypeptide containing the above-mentioned CDR region that has binding activity between human EpCAM and CD3. The term further includes variant forms of polypeptides containing the above-mentioned CDR region that have the same function as the antibody of the present invention. These variant forms include, but are not limited to, the deletion, insertion and / or substitution of one or more (generally 1 to 50, preferably 1 to 30, more preferably 1 to 20, and optimally 1 to 10) amino acids, and the addition of one or more (generally up to 20, more preferably up to 10, and more preferably up to 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable performance usually does not change the function of the protein. Also, for example, the addition of one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention. The variant forms of the polypeptide include homologous sequences, conserved variants, equipotential variants, native variants, induced variants, proteins encoded by DNA capable of hybridizing with the coding DNA of the antibody of the present invention under high or low strict conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0026] The antibodies of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, (ii) polypeptides having substituents in one or more amino acid residues, (iii) polypeptides formed by fusing a mature polypeptide with another compound (a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to such a polypeptide sequence (e.g., a preamble sequence or secretory sequence, or a sequence or proprotein sequence for purifying such polypeptides, or a fusion protein with a 6×His tag). According to the teachings herein, these fragments, derivatives and analogues are within the scope well known to those skilled in the art.

[0027] "Conservative amino acid substitution" means that one of the amino acid residues is replaced by an amino acid residue having a similar side chain. The family of amino acid residues having similar side chains is defined in this art and includes 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable that non-essential amino acid residues of immunoglobulin polypeptides are replaced by other amino acid residues derived from the same side chain family. In some other embodiments, the amino acid sequence may be substituted with a structurally similar amino acid sequence, the latter differing in order and / or side chain family composition.

[0028] The following table shows non-restrictive examples of conservative amino acid substitutions, where a similarity score of 0 or greater indicates the presence of a conservative substitution between the two amino acids. [Table 1] TIFF0007868145000002.tif31169

[0029] In some embodiments, the conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar 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, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0030] Particularly preferred conservative substitutions are as follows: Substitute Ala with Gly or Ser; Substitute Arg with Lys; Substitute Asn with Gln or His; Substitute Asp with Glu; Substitute Cys with Ser; Substitute Gln with Asn; Substitute Glu with Asp; Substitute Gly with Ala or Pro; Substitute His with Asn or Gln; Substitute Ile with Leu or Val; Leu Replace with Ile or Val; replace Lys with Arg; replace with Gln or Glu; replace Met with Leu; replace with Tyr or Ile; replace Phe with Met; replace with Leu or Tyr; replace Ser with Thr; replace Thr with Ser; replace Trp with Tyr; replace Tyr with Trp; and / or replace Phe with Val; replace with Ile or Leu.

[0031] The Fc amino acid number follows the Kabat numbering system. The "Kabat numbering system" is a numbering system described by Kabat et al., and its details are documented in the U.S. Department of Health and Public Services' "Sequence of Proteins of Immunological Interest" (1983). The specific numbers are shown in the following table: [Table 2]

[0032] Here, The amino acids 221-227 form the hinge domain. The amino acids at positions 228-340 form the CH2 domain, which is the second constant region of the heavy chain. The amino acids 341-447 form the third constant region of the heavy chain, the CH3 domain.

[0033] Antibodies may be modified to improve heterodimer pairing efficiency. For example, in some embodiments, the Fc fragment of the monovalent unit heavy chain and / or the Fc fragment of the fusion peptide may contain one or more substitutions compared to the wild-type antibody fragment, with these substitutions forming a pestle-into-holes structure pair. Pestle-into-holes structures are known in this 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).

[0034] In one embodiment, T366 on one CH3 domain is substituted with a relatively large amino acid residue such as tyrosine (Y) or tryptophan (W). Then, Y407 on another CH3 domain may be substituted with a relatively small amino acid residue such as threonine (T), alanine (A), or valine (V). [Table 3]

[0035] In one embodiment, one of the CH3 domains includes one or more substitutions of an amino acid residue that has a positive charge under physiological conditions, and another CH3 domain includes one or more substitutions of one or more amino acid residues that have a negative charge under physiological conditions. In one embodiment, the positively charged amino acid residue may be arginine (R), histidine (H), or lysine (K). In another embodiment, the negatively charged amino acid residue may be aspartic acid (D) or glutamic acid (E). The amino acid residues that may be substituted include, but are not limited to, D356, L368, K392, D399, and K409. [Table 4]

[0036] In one embodiment, S354 on one CH3 domain is substituted with cysteine, and Y349 on another CH3 domain is also substituted with cysteine, and the two substituted residues form a disulfide bond. [Table 5]

[0037] In one embodiment, H435 and Y436 on a single CH3 domain are substituted with arginine and phenylalanine, respectively. This substitution significantly reduces the binding ability between Fc and protein A, thereby giving heterodimers and homodimers different protein A binding activities, allowing for easy separation of the two during affinity chromatography. [Table 6]

[0038] In one preferred example of the present invention, the CH3 amino acid sequence of Fc that forms a heterodimer is shown in the following table: [Table 7] TIFF0007868145000009.tif230169 TIFF0007868145000010.tif55169

[0039] One embodiment of this application provides a heterodimer antibody comprising two different antigen-binding polypeptide units. In some embodiments, the heterodimer differs in size from the corresponding homodimer, and this size difference may be utilized to facilitate the separation of the heterodimer from the homodimer.

[0040] In some embodiments, as shown in Figure 1, one of these two antigen-binding polypeptide units contains a light-heavy chain pair similar to that of a wild-type antibody. Throughout this application, this unit is also referred to as a “monovalent unit.” In some embodiments, as shown in Figure 1, the other antigen-binding polypeptide unit contains a single-chain variable fragment (ScFv). Such an ScFv can be fused to the N-terminus of a constant fragment (Fc) of an antibody, called a fusion peptide. Throughout this application, this fusion peptide is also referred to as a “single-chain unit.”

[0041] Any of the antibodies or polypeptides described herein may further comprise additional polypeptides, such as the encoded polypeptides described herein, signal peptides for the constant region of the antibody to guide secretion, or other heterologous polypeptides described herein. The antibodies described herein may be modified so that their amino acid sequences differ from those of naturally occurring conjugated polypeptides. For example, a polypeptide or amino acid sequence derived from a particular protein may be similar to the start sequence, for example, having a certain percentage of identity with the start sequence, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the start sequence. Furthermore, nucleotide or amino acid substitutions, deletions, or insertions may be made to conservative substitutions or modifications in the “non-essential” amino acid region. For example, a polypeptide or amino acid sequence derived from a particular protein may be identical to the starting sequence except for one or more independent amino acid substitutions, insertions, or deletions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more independent amino acid substitutions, insertions, or deletions. In a particular example, the polypeptide or amino acid sequence derived from a particular 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.

[0042] As used herein, the term “detectable tag” means a compound or composition that is directly or indirectly detectable, conjugating directly or indirectly to a composition to be detected (e.g., a polynucleotide or a protein such as an antibody) in order to obtain a “tagged” composition. The term further provides sequences that conjugate to the polynucleotide, which provide a signal such as green fluorescent protein (GFP) upon expression of the insertion sequence. The tag itself (e.g., a radioisotope tag or a fluorescent tag) can be detected, or, in the case of an enzyme tag, can catalyze a chemical change in a substrate compound or composition, and the modification can be detected. The tag can be used for small-scale detection or is more suitable for high-throughput screening. Similarly, suitable tags include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). The tag may be detectable only or quantifiable. A detection-only reaction generally includes a reaction that merely confirms its presence, where a quantifiable reaction generally includes a reaction that has quantifiable (e.g., digitally reportable) values ​​such as intensity, polarization, and / or other properties. In emission or fluorescence analysis, detectable reactions can be achieved by directly using a luminescent or fluorescent group that is actually bound to the analyte, or by indirectly using a luminescent or fluorescent group bound to another component (e.g., a reporter molecule or therapeutic agent).

[0043] In some embodiments, the antibodies of the present invention can be conjugated to therapeutic agents (e.g., chemotherapy drugs such as cisplatin and carboplatin), drug precursors, peptides, proteins, enzymes, viruses, lipids, bioresponse modifiers, drugs, or PEG. The antibodies of the present invention can be conjugated to therapeutic agents containing detectable markers such as radioactive markers, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic agents or diagnostic agents, cytotoxic substances, etc., which may be drugs or toxins, ultrasound enhancers, non-radioactive markers, combinations thereof, and other such components known in the art.

[0044] In some embodiments, the antigen-binding polypeptide comprises an amino acid sequence or one or more groups that do not normally bind to an antibody. For example, the single-chain Fv antibody fragment of this application may comprise a flexible linker sequence or a functional group that may be modified to be added (e.g., polyethylene glycol (PEG), a drug, a toxin, or a marker). The antibody, its variants, or derivatives of this application comprise modified derivatives, i.e., any type of molecule is covalently bound to the antibody, and such covalent bond does not inhibit the antibody's binding to the antigen epitope. The antibody may comprise one or more non-classical amino acids.

[0045] It should be noted that the implicit limitation of an entity means one or more such entities; for example, “multifunctional antibody” should be understood to mean 11 or more multifunctional antibodies. Similarly, the terms “one or more” and “at least one,” when limited by an implicit number, are used interchangeably herein.

[0046] As used herein, the term “treatment” means therapeutic treatment and preventive or preventive measures taken in a subject to prevent or delay (mitigate) an undesirable physiological change or disease, such as the progression of cancer. Beneficial or necessary clinical outcomes include, but are not limited to, alleviating symptoms, reducing the severity of the disease, stabilizing the disease state (e.g., preventing exacerbation), delaying or slowing the progression of the disease, improving or alleviating the disease state, and (partially or completely) alleviating the disease, whether detectable or not. “Treatment” may also mean that survival can be extended compared to the expected survival time without treatment. Situations requiring treatment include cases where the disease or symptoms are already present, or where the disease or symptoms are likely to develop, or where the disease or symptoms can be prevented.

[0047] The term "subject," "individual," "animal," "patient," or "mammal" refers to any subject requiring diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammalian subjects include humans, domesticated animals, farm animals, zoo animals, exercise dwellers, or animals such as dogs, cats, guinea pigs, rabbits, rats, mice, rodents, horses, cattle, dairy cows, primates (e.g., humans such as crab-eating macaques, macaques, baboons, monkeys, chimpanzees, etc.).

[0048] The antigen-binding polypeptides, variants, or derivatives described herein can be used in several therapeutic and diagnostic methods related to cancer or infectious diseases. This application also relates to antibody-based therapies, which include administering the bispecific antibodies of this application to patients such as animals, mammals, and humans to treat one or more of the diseases or conditions described herein. The therapeutic agents of this application include, but are not limited to, the antibodies of this application (including their variants and derivatives described herein) and nucleic acids, or polynucleotides encoding the antibodies of this application (including their variants and derivatives described herein). The antibodies of this application can also be used to treat, suppress, or prevent diseases, diseases, or conditions, including malignant diseases, pathologies, or conditions associated with such diseases or pathologies, such as diseases related to immune responses. In some embodiments, the antibodies of the present invention can be used as immunosuppressants. In some embodiments, the antibodies of the present invention can be used to treat autoimmune diseases. The antigen-binding polypeptides, variants, or derivatives of this application are used to inhibit the growth, progression, and / or metastasis of cancer, particularly those described in the paragraphs above or below.

[0049] The antibodies of this 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 (e.g., malignant ascites, malignant pleural effusion, malignant cardiac effusion), such as EpCAM-positive tumors.

[0050] Methods of administering antibodies, their variants, or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antibody or composition can be administered via any convenient route, e.g., by injection or large-volume infusion, absorbed into the endodermal layers of the skin (e.g., oral mucosa, rectal and intestinal mucosa) via epithelium or mucosa, and administered together with other bioactive agents. Accordingly, the antibody-containing pharmaceutical compositions of this application can be administered orally, rectally, non-intestinally, intracerebrally, intravaginally, intraperitoneally, topically (e.g., powders, ointments, drops, transdermal patches), or as oral or nasal sprays. As used herein, the term "non-intestinal" means administration patterns including injection and infusion into the venous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular regions. Administration may be systemic or topical. Furthermore, it is necessary to administer the antigen-binding polypeptide or composition of this application topically to the area requiring treatment, which can be achieved, for example, by local perfusion during surgery, topical application, combination with postoperative wound dressings, injection, catheter, suppository, or implant, which is a porous, non-porous, or gel-like material including a membrane or fiber. Preferably, when administering the protein (including the antibody) of this application, care should be taken to use a material that does not absorb the protein. [Brief explanation of the drawing]

[0051] [Figure 1] This is a schematic diagram of the YBODY antibody structure. [Figure 2] This is the detection of HCT116-Jurkat cell binding via bispecific antibodies. A: Negative control flowgraph without HCT116+Jurkat 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: Flowgraph of the HCT116+Jurkat+M701A 10 μg / ml experimental group, with the characteristic meaning of each quadrant being the same as before; C: Concentration gradient curves of HCT116-Jurkat cell binding via different antibodies. [Figure 3] This involves the detection of the biological activity of bispecific antibodies (using reporter gene lines). [Figure 4] This is the detection of in vitro toxicity mediated by bispecific antibodies. A: In vitro toxicity of B16-EpCAM by M701A; B: In vitro toxicity of B16 by M701A; C: In vitro toxicity of HCT116 by M701A; D: In vitro toxicity of OVCAR-3 by M701A; E: In vitro toxicity of CHO-K1-huEpCAM by different bispecific antibodies; F: In vitro toxicity of HCT116 by different bispecific antibodies. [Figure 5] This is the in vivo efficacy of the bispecific antibody HCT116 in a human-derived colon cancer model. A: Changes in tumor volume growth in mice; B: Changes in body weight in mice. [Figure 6] This is the in vivo efficacy of a bispecific antibody in a human-derived ovarian cancer model of OVCAR-3. A: Changes in tumor volume growth in mice; B: Changes in body weight in mice. [Modes for carrying out the invention]

[0052] The methods and applications of the present invention are described below in reference to the accompanying drawings, but the examples are for illustrative purposes only and not to limit the scope of the invention. Those skilled in the art can make several simple inferences or substitutions without departing from the concept of the present invention, all of which should be considered to fall within the scope of the protection of the present invention.

[0053] Example 1: Construction of an expression vector for a bispecific antibody The bispecific antibody structure targeting EpCAM and CD3 includes an anti-EpCAM binding region and an anti-CD3 binding region. The monovalent unit consists of a pair of anti-EpCAM heavy and light chains, while the single-chain unit is the ScFv-Fc form of anti-CD3, defined as a YBODY structure (Figure 1), where the VL of anti-CD3 is linked to the hinge region and CH2 via a linker. Here, the heavy chain Fc of the monovalent unit and the Fc of the single-chain unit (with human IgG heavy chain Fc as its backbone) have been modified with amino acid mutations to make them less likely to form homodimers and more likely to form heterodimers. Using existing plasmids or synthetic gene fragments as templates, each chain corresponding to the bispecific antibody was amplified by PCR and duplicate PCR, and each antibody chain was cloned into the pcDNA3.1 vector (Invitrogen) by enzyme linkage or recombination. Specific sequence information for each antibody chain is shown in Table 1 and the sequence listing. [Table 8]

[0054] Example 2: Expression and purification of bispecific antibodies Plasmids were extracted using conventional plasmid extraction methods and used for chemical transfection of CHO-S cells (derived from Gibco). Transfected cells were cultured in suspension on a 5% CO2 shaking platform at 37°C for 7-10 days. The supernatant was collected by centrifugation at 3000g and filtered through a 0.22μm filtration membrane. Preliminarily purified bispecific antibodies were obtained by protein A affinity chromatography. The concentration of purified proteins was measured by UV absorbance at 280nm and the corresponding quenching coefficient. Antibody purity was tested by high-performance size exclusion chromatography (HPLC-SEC), and the corresponding expression levels of each protein were calculated. The expression levels of the bispecific antibody molecules ranged from 40 mg / L to 91 mg / L, with initial purities ranging from 45% to 81%. The expression levels and initial purities of M701A, M701B, M701C, M701D, M701E, M701F, M701G, M701H, M701I, M701J, and M701K were all clearly superior to those of M701. Next, affinity samples were purified by cation exchange chromatography to obtain bispecific antibodies with HPLC-SEC purity >95%. The purification and recovery rates of each bispecific antibody are shown in Table 2. [Table 9]

[0055] Example 3: Detection of thermal stability of bispecific antibodies Purified samples of each bispecific antibody were diluted to 0.5 mg / ml in buffer (25 mM citrate + 50 mM NaCl, pH 6.0), dispensed at 100 μL / tube into 1.5 mL EP tubes, and subjected to a 14-day thermal acceleration experiment in a 40°C water bath to detect changes in purity and affinity. The sample was counted as D0 on the day it was placed in the 40°C water bath, and as D14 on the 14th day.

[0056] An amino coupling method was employed to immobilize human EpCAM (SB, Cat: 10694-H08H) and human CD3 antigen (SB, Cat: CT038-H2508H) on CM5 chips. The antigen coupling amount was 1500 RU. When detecting antigen end-binding activity, the sample was diluted to the starting concentration with 1 × HBS-EP+ buffer, and then diluted to four concentrations using a 2x gradient. The samples were then extruded and detected from low to high concentrations. The binding flow rate was 30 μL / min, the binding time was 120 s, and the dissociation time was 300 s. A regenerated chip with a pH 1.5 glycine solution was used, with a regeneration flow rate of 10 μL / min and a regeneration time of 30 s. After detection was complete, data fitting was performed on the resulting spectrum using the Biacore T200 Evaluation Software with a 1:1 binding fitting method to obtain the dissociation equilibrium constant (KD).

[0057] As shown in Table 3, the purity decrease of M701A, M701B, M701H, M701I, M701J, and M701K on the 14th day of thermal acceleration was less than 5%, and among them, the purity change of M701A, M701B, M701J, and M701K was less than 2%, and the affinity at both ends remained basically unchanged, explaining that M701A, M701B, M701J, and M701K have good thermal stability. [Table 10]

[0058] Example 4: Affinity detection of bispecific antibody-terminal cells of EpCAM cells Using FACS, human colon cancer cells HCT116 (Shanghai Institute of Biosciences, Chinese Academy of Sciences) were selected as positive cells expressing human EpCAM on their cell membrane surface, and the affinity between the antibody and human EpCAM on the cell surface was detected.

[0059] HCT116 cells were collected by centrifugation, resuspended in buffer (PBS + 1% FBS), and 2 × 10⁶ cells were collected. 5Cells were added to a 96-well plate at a rate of 1 cell / well, with 50 μL per well. The cells were then centrifuged at 350 × g for 5 min, and the supernatant was removed. The bivalent antibody was diluted to 1000 nM in buffer, then gradient diluted, and added to a 96-well plate at a rate of 50 μL / well. After resuspending, the cells were incubated away from light for 1 hour, centrifuged, the supernatant was removed, washed twice with buffer, and then resuspended in diluted PE-labeled anti-human IgG Fc antibody (Biolegend, 409304). The cells were incubated away from light for 30 min, washed twice with buffer, and then resuspended in 100 μL of buffer. Flow cytometry (BD Accuri) was performed. TM It was detected by moving to C6).

[0060] Each bispecific antibody showed clear binding activity to HCT116 cells, where M700 was an anti-EpCAM terminal mab (mAb) control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26). The specific EC50 values ​​are shown in Table 4. [Table 11]

[0061] Example 5: Detection of CD3-terminal affinity of bispecific antibodies (Biacore) An amino coupling method was used to immobilize human CD3 antigen (SB, Cat: CT038-H2508H) on a CM5 chip. The antigen coupling amount was 1500 RU. When detecting CD3 antigen end-binding activity, the sample was diluted to the starting concentration with 1 × HBS-EP + buffer, and then diluted to four concentrations using a 2x gradient. The samples were then extruded and detected from low to high concentrations. The binding flow rate was 30 μL / min, the binding time was 120 s, and the dissociation time was 300 s. A regenerated chip with a pH 1.5 glycine solution was used, with a regeneration flow rate of 10 μL / min and a regeneration time of 30 s. After detection, data fitting was performed on the result spectrum using the Biacore T200 Evaluation Software with a 1:1 binding fitting method to obtain the dissociation equilibrium constant (KD). As shown in Table 5, all of the series bispecific antibodies exhibit binding activity to human CD3 antigen. Here, the variable region sequences of the anti-EpCAM antibodies M701A and M701B are the same (SEQ ID NO: 13 and SEQ ID NO: 14), and the variable region sequences of the anti-CD3 antibodies are SEQ ID NO: 18 and SEQ ID NO: 19, respectively, with corresponding affinities of 21.15 nM and 28.27 nM, respectively. However, the variable region sequences of the anti-EpCAM antibodies are of different types (SEQ ID NO: 15 and SEQ ID NO: 16), and the variable region sequences of the anti-CD3 antibodies are also SEQ ID NO: 18 and SEQ ID NO: 19, with corresponding affinities of the bispecific antibodies M701H and M701I being 95.26 nM and 40.03 nM, respectively. This explains that when different anti-EpCAM antibodies and different anti-CD3 antibodies are combined to form bispecific antibodies, there is no regularity in the expression of affinity. [Table 12]

[0062] Example 6: Cell bridging action mediated by bispecific antibodies EpCAM-positive cell line HCT116 was stained with PKH26, and CD3-positive cell line Jurkat (Shanghai Institute of Biosciences, Chinese Academy of Sciences) was stained with CFSE. After staining, the cells were divided into a 1:1 ratio (1 × 10⁻¹⁶). 5Individual HCT116 cells: 1 × 10 5 The antibodies to be tested, serially diluted in individual Jurkat cells, were added and mixed, and incubated. Here, M700 was used as the anti-EpCAM mab control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26), M100 as the anti-CD3 mab control (light chain SEQ ID NO: 27 and heavy chain SEQ ID NO: 28), and Mco101 as the CD3 terminal isotype control for the bispecific antibody (light chain SEQ ID NO: 29, heavy chain SEQ ID NO: 30 and single-chain SEQ ID NO: 31; structure is the same as in Figure 1). After incubation for 1 hour, washing and resuspending, flow cytometry (BD Accuri) was performed. TM Cells detected by extrusion into C6) and found to be double-positive for CFSE and PKH26 were antibody-crosslinked HCT116 and Jurkat cells.

[0063] As shown in Figure 2, when Jurkat and HCT116 cells were mixed in a 1:1 ratio and hIgG, M700, M100, and Mco101 were added and allowed to react for 1 hour, HCT116 and Jurkat cells were not crosslinked. After adding M701A and M701B, crosslinked cells accounted for approximately 40% of the total number of PKH26-positive cells, indicating that the activity of M701A and M701B was considerable, and the cell interaction and antibody concentration mediated by bispecific antibodies showed a positive quantitative effect relationship.

[0064] Example 7: Detection of biological activity of bispecific antibodies (reporter gene method) The biological activity of bispecific antibodies was detected using Jurkat-CD3-NFAT-RE-Luc cells (Promega). CHO-K1 cells were transfected with a pLV-puro (Inovogen Tech.Co., cat.No.VL3001) vector containing DNA encoding the human EpCAM gene (NCBI Sequence ID: NM_002354.3) to obtain the cell line CHO-K1-huEpCAM, which stably expresses human EpCAM. CHO-K1-huEpCAM cells were collected as target cells, resuspended in buffer (PBS + 1% FBS), and measured in 4 × 10⁶ units.4 The cells were added to a 96-well white culture plate at 10¢ / well, incubated overnight in a 5% CO2 incubator at 37°C for 18-24 hours, the culture medium was removed from the plate, and 40 μL of antibody dilution was added per well. Jurkat-CD3-NFAT-RE-Luc cells, i.e., effector cells, were isolated, the cells were blown away to prepare a single-cell suspension, and 40 μL per well, i.e., 6 × 10¹⁶ cells, were added to the 96-well white culture plate at an effect target ratio E:T = 1.5:1. 4 The cells were placed in plates one per well, and the all-white 96-well culture plates were placed at 37°C and incubated in a 5% CO2 incubator for 6 hours. 80 μL of Bio-Glo luciferase detection solution was added to each well, and the plates were incubated at room temperature for 15 minutes, away from light. The plates were then placed on a multifunction reader, and the luminescence values ​​were read by chemiluminescence.

[0065] As shown in Figure 3, in the reporter gene evaluation system, the bispecific antibodies M701, M701A, M701B, M701H, M701I, M701J, and M701K all showed biological activity, and the activity of M701A, M701J, and M701K was stronger than that of M701.

[0066] Example 8: In vitro detection of lethality via bispecific antibodies 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 bispecific antibodies. The pLV-puro (Inovogen Tech.Co., cat.No.VL3001) vector containing DNA encoding the human EpCAM gene (NCBI accession number: NM_002354.3) was transfected into mouse melanoma cells B16 cells (Shanghai Institute of Life Sciences, Chinese Academy of Sciences) to obtain a cell line B16-EpCAM that stably expresses human EpCAM. Here, B16 was used as a negative cell that does not express EpCAM. Other target cells expressing EpCAM include human colon cancer cells HCT116 (Shanghai Institute of Life Sciences, 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 into single-cell suspensions with pancreatin, collected by centrifugation at 300 g for 5 min, and then 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 with a Vi-cell cell counter and added to a 96-well plate according to the experimental design, with 2×10 4 cells / 100 μL per well. Antibodies at the corresponding concentrations were added at 50 μL / well. hPBMCs were counted with a Cellometer cell counter and added to the 96-well plate (2×10 5 cells / 50 μL, effector-to-target ratio 10:1). The cell culture plate was cultured in a cell incubator for 72 h. After digesting the cells into single-cell suspensions, they were added to a propidium iodide (PI) solution with a final concentration of 1 μg / mL and incubated for 10 min. Then, they were analyzed by flow cytometry (BD Accuri TM C6), and the percentage of CFSE+PI+ double-positive cells among CFSE+ positive cells was analyzed.

[0067] As shown in Figures 4A and 4B, M701A was lethal only to B16-EpCAM cells expressing EpCAM, and not to B16 cells without EpCAM expression. The control antibody Mco101 was lethal to all cells, demonstrating the targeted nature of bispecific antibody activity. As shown in Figures 4C and 4D, M701A showed clear lethal activity against HCT116 and OVCAR-3 cells, and was stronger than that of the control Mco101. As shown in Figure 4E, the bispecific antibodies M701A, M701B, M701H, and M701I all showed clear lethal activity against CHO-K1-huEpCAM cells, with the effects of M701A and M701B being significantly stronger than those of M701H and M701I. As shown in Figure 4F, the bispecific antibodies M701J and M701K exhibited clear killing activity against HCT116 cells. The EC50 values ​​for killing cells were 7.814-25.43 ng / ml for both antibodies, and the killing level of M701J was not significantly different from that of M701A. The killing activity of the above bispecific antibodies was clearly stronger than that of M701 (EC50 of M701 was 69.17 ng / ml).

[0068] Example 9: In vivo efficacy of bispecific antibody in a HCT116 human-derived colon cancer xenograft model. Sufficient amounts of HCT116 cells and effector cells (CIK cells, which are cytokine-induced killer cells, are a population of heterogeneous cells obtained after co-culturing human peripheral blood mononuclear cells in vitro with multiple cytokines for a certain period of time. These cells express two types of membrane protein molecules, CD3+ and CD56+, and are therefore also called NK cell-like T lymphocytes) were cultured according to the culture conditions, and the cells were collected and counted. Pre-mixed HCT116 cells (2 × 10⁶) were placed on the right dorsal side of each mouse. 6 Cells / animal) and CIK (2 × 10 6A xenograft tumor model of human-derived colon cancer (HCT116) was established by inoculating mice with cells (0.1 ml / mice) in a single dose. Drug therapy was started 1 hour after inoculation. The experiment was divided into groups of 8 mice: M701A 2 mg / kg, M701A 1 mg / kg, M701 1 mg / kg, mab control M700 2 mg / kg, and solvent control (physiological saline). Intravenous administration was performed three times on days 0, 2, and 4 after inoculation. Therapeutic efficacy was evaluated based on the relative tumor suppression rate and complete tumor regression rate, and safety was evaluated based on changes in animal body weight and mortality.

[0069] Tumor size calculation formula: Tumor volume (mm) 3 ) = 0.5 × (Tumor diameter × Tumor diameter) 2 ).

[0070] Relative tumor suppression rate (TGI) (%): TGI = 1 - T / C (%). T and C are the tumor volume (TV) at a specific time point in the treatment group and control group, respectively. The formula is as follows: T / C% = T TV / C TV ×100%(T TV : Mean tumor volume of the treatment group; C TV (Mean tumor volume in the solvent control group).

[0071] Complete regression rate: Tumor volume of 63 mm during or after treatment. 3 It is defined as being less than [a certain value]. Complete tumor eradication rate (%) = Number of animals that achieved complete eradication / Total number of animals in that group × 100%.

[0072] As shown in Figure 5A, the study drug M701A (2 mg / kg, 1 mg / kg) showed a significant tumor suppressive effect in the treatment group 30 days after drug discontinuation (i.e., 33 days after inoculation), with relative tumor suppression rates (TGI) of 100% and 93.82%, respectively. The relative solvent control group showed a statistically significant difference (p-value <0.001 in both cases), and all tumors in the M701A (2 mg / kg) group reached the standard for complete elimination. The efficacy of M701A at both doses was clearly superior to that of M700 at 2 mg / kg (p-value <0.001), and the efficacy of M701A at the same dose was clearly superior to that of M701 (both 1 mg / kg). As shown in Figure 5B, there was no weight loss in the animals during the treatment process, and no manifestations of drug toxicity were observed.

[0073] In the same tumor model, M701B, M701J, and M701K showed similar tumor-suppressing effects to M701A at the same dose, and did not cause weight loss.

[0074] Example 10: In vivo efficacy of a bispecific antibody in a xenograft tumor model of OVCAR-3 human-derived ovarian cancer. A sufficient amount of OVCAR-3 cells and effector cells CIK were cultured according to the culture conditions, and the cells were collected and counted. Pre-mixed OVCAR-3 cells (1 × 10⁶) were placed on the right dorsal side of each mouse. 7 Cells / animal) and CIK (1 × 10⁻¹⁰ 7 A human-derived ovarian cancer (OVCAR-3) atypia tumor model was established by inoculating cells (0.2 ml / mice) with a Matrigel gel content of 50% (0.1 ml / mouse). Treatment was administered 1 hour after inoculation. The experiment was divided into groups: the investigational drug M701A (5 mg / kg), the CD3 terminal isotype control Mco101 (5 mg / kg), the mab control M700 (5 mg / kg), and the solvent control group (physiological saline). Each group consisted of 8 mice. Intravenous administration was performed three times on days 0, 2, and 4 after inoculation. Therapeutic efficacy was evaluated based on the relative tumor suppression rate (TGI) and the complete tumor regression rate, and safety was evaluated based on changes in animal body weight and mortality.

[0075] As shown in Figure 6A, the test drug M701A (5 mg / kg) showed a significant tumor suppressive effect in the treatment group 44 days after drug discontinuation (i.e., 48 days after inoculation), with a relative tumor suppression rate (TGI) of 98.97%. The relative solvent control groups all showed statistically significant differences (p-values ​​<0.001 in all cases), and the complete tumor elimination rate in the M701A (5 mg / kg) group was 87.5%. The efficacy of this group was significantly superior to that of M700 (5 mg / kg, TGI = 70.42%) (p-value <0.001) and Mco101 (5 mg / kg, TGI = 68.87%) (p-value <0.001). As shown in Figure 6B, there was no weight loss in the animals during the treatment process, and no indication of drug toxicity was observed.

[0076] In the same tumor model, M701B, M701J, and M701K showed similar tumor-suppressing effects to M701A at the same dose, and did not cause weight loss.

[0077] All documents referenced in this invention are cited by reference in this application, and each document is cited by reference individually. Furthermore, after reading the above teachings of this invention, those skilled in the art should understand that various changes or modifications can be made to the invention, and these equivalent forms are also within the scope defined by the claims appended to this application. [Table 13] TIFF0007868145000017.tif236169 TIFF0007868145000018.tif236169 TIFF0007868145000019.tif245169 TIFF0007868145000020.tif245169 TIFF0007868145000021.tif245169 TIFF0007868145000022.tif245169 TIFF0007868145000023.tif136169 Table 14 TIFF0007868145000025.tif245169 TIFF0007868145000026.tif119169

Claims

1. A bispecific antibody comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3, Here, the antigen-binding domain that specifically binds to the EpCAM comprises a light chain and a heavy chain, or a light chain-heavy chain pair comprising them, wherein the light chain comprises the light chain variable region shown in SEQ ID NO: 13 or a variant thereof having at least 90% identity with SEQ ID NO: 13, and the light chain constant region shown in SEQ ID NO: 1, wherein the heavy chain comprises the heavy chain variable region shown in SEQ ID NO: 14 or a variant thereof having at least 90% identity with SEQ ID NO: 14, CH1 shown in SEQ ID NO: 2, and a first Fc fragment, wherein the first Fc fragment comprises the hinge region shown in SEQ ID NO: 3, CH2 shown in SEQ ID NO: 6, and CH3a shown in SEQ ID NO:

8. The antigen-binding domains that specifically bind to EpCAM include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3: According to the Kabat sequence number system, the sequence of CDRL1 is shown in SEQ ID NO: 32, the sequence of CDRL2 is shown in SEQ ID NO: 33, the sequence of CDRL3 is shown in SEQ ID NO: 34, the sequence of CDRH1 is shown in SEQ ID NO: 35, the sequence of CDRH2 is shown in SEQ ID NO: 36, and the sequence of CDRH3 is shown in SEQ ID NO:

37. The antigen-binding domain that specifically binds to CD3 comprises an ScFv that specifically binds to CD3 as shown in SEQ ID NO: 18 and a second Fc fragment, or a fusion peptide comprising them, wherein the ScFv comprises, sequentially from the N-end to the C-end, the heavy chain variable region shown in SEQ ID NO: 50 or a variant thereof having at least 90% identity with SEQ ID NO: 50, a linked peptide shown in SEQ ID NO: 4, and the light chain variable region shown in SEQ ID NO: 51 or a variant thereof having at least 90% identity with SEQ ID NO: 51, wherein the second Fc fragment comprises, sequentially from the N-end to the C-end, the hinge region shown in SEQ ID NO: 3, CH2 shown in SEQ ID NO: 6, and CH3b shown in SEQ ID NO: 11, and the C-end of the light chain variable region and the hinge region of the second Fc fragment are connected by SEQ It is linked by the linked peptide shown in ID NO:

5. 1) The antigen-binding domains that specifically bind to CD3 include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3: According to the Kabat sequence number system, the sequence of CDRH1 is shown as SEQ ID NO: 44, the sequence of CDRH2 is shown as SEQ ID NO: 45, the sequence of CDRH3 is shown as SEQ ID NO: 46, the sequence of CDRL1 is shown as SEQ ID NO: 47, the sequence of CDRL2 is shown as SEQ ID NO: 48, and the sequence of CDRL3 is shown as SEQ ID NO:

49. Bispecific antibodies.

2. A nucleic acid composition comprising a nucleic acid sequence encoding the bispecific antibody described in claim 1.

3. A pharmaceutical composition for treating cancer, malignant ascites, malignant effusion, or malignant pleural effusion, comprising the bispecific antibody described in claim 1 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition further contains a drug, or The cancer is an EpCAM-positive tumor, or The pharmaceutical composition according to claim 3, wherein the dosage form of the pharmaceutical composition is a gastrointestinal dosage form or an extra-gastrointestinal dosage form.

5. If the cancer is colorectal cancer, stomach cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal pellucida, dermatocarcinoma, basal cell carcinoma, sarcoma, nasal glioma, craniopharyngocarcinoma, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumor, cervical cancer, or oral cancer, The pharmaceutical composition according to claim 4, wherein the dosage form of the pharmaceutical composition is an injectable preparation including intravenous injection, intravenous drip infusion, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

6. A conjugate or fusion protein comprising a bispecific antibody as described in claim 1, and a substance A conjugated or fused with the bispecific antibody, wherein substance A is selected from the group consisting of therapeutic agents, drug precursors, proteins, viruses, lipids, bioresponse modifiers, PEG, hormones, oligonucleotides, diagnostic agents, cytotoxic agents which may be drugs or toxins, ultrasound enhancers, non-radioactive markers, detection markers, or fluorescent metals.

7. A kit comprising the bispecific antibody described in claim 1.

8. A kit according to claim 7, further comprising a drug for treating cancer, malignant ascites, malignant fluid retention, or malignant pleural effusion, wherein the cancer is the cancer described in claim 5.

9. A bispecific antibody according to claim 1 for the treatment of cancer, malignant ascites, malignant fluid retention, or malignant pleural effusion, wherein the cancer is the cancer described in claim 5.

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