EpCAM antibodies or antigen-binding portions thereof and uses thereof

Abstract

The invention provides an EpCAM antibody or an antigen binding part and application thereof, the EpCAM antibody or the antigen binding part comprises a light chain variable region VL and a heavy chain variable region VH, the VH comprises a first heavy chain complementarity determining region HCDR1, a second heavy chain complementarity determining region HCDR2 and a third heavy chain complementarity determining region HCDR3, and the amino acid sequence of the HCDR3 is shown as the following formula: X1, X2, X3, X4, X5, X6, X7, X8, X9, X10 and X11. Amino acid sites of the HCDR3 are mutated to improve the affinity and lethality of the EpCAM antibody or the antigen binding part thereof, so that the EpCAM antibody or the antigen binding part thereof has a killing effect on EpCAM positive cell strains or solid tumors, and autologous cells of a patient do not need to participate in construction.

Description

[0001] This application claims the priority of a Chinese patent application with an application date of November 29, 2023, an application number of 2023116250951, and an invention title of "EpCAM Antibody or Its Antigen-Binding Portion and Their Uses", the content of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the technical field of immunocyte therapy, and in particular to an EpCAM antibody or its antigen-binding portion and their uses, and specifically to an EpCAM antibody or its antigen-binding portion, nucleic acid molecule, chimeric antigen receptor, vector, immunocyte, pharmaceutical composition and uses. Background Art

[0003] Chimeric antigen receptor gene-modified T cells (CAR-T) target and bind active T cells with tumor-killing ability to specific structures on the surface of tumor cells through a specific CAR structure, thereby exerting the tumor cell-killing effect of T cells.

[0004] EpCAM is one of the main surface antigens of human colon cancer, a glycosylated type I membrane protein of 30 to 40 kDa, which can be highly expressed in various human epithelial tissues, cancers, as well as progenitor cells and stem cells, and thus has the potential to become an important target for colon cancer treatment.

[0005] In recent years, tumor CAR-T cell therapy has mainly focused on the treatment of hematological malignancies. CAR-T therapy for solid tumors still lacks highlights and there are still many problems. For example, since autologous T cells of patients are required for construction and it is customized, this characteristic makes the manufacturing process of CAR-T cells time-consuming and expensive in large-scale production.

[0006] Therefore, it is necessary to develop novel CAR allogeneic immunocytes targeting the EpCAM antigen to solve the above problems existing in the prior art. Summary of the Invention

[0007] The object of the present invention is to provide an EpCAM antibody or its antigen-binding portion, a nucleic acid molecule, a chimeric antigen receptor, a vector, an immune cell, a pharmaceutical composition and uses thereof. The EpCAM antibody or its antigen-binding portion comprises a light chain variable region VL and a heavy chain variable region VH. The VL comprises a first light chain complementary determining region LCDR1, a second light chain complementary determining region LCDR2 and a third light chain complementary determining region LCDR3. The VH comprises a first heavy chain complementary determining region HCDR1, a second heavy chain complementary determining region HCDR2 and a third heavy chain complementary determining region HCDR3. The amino acid sequence of the LCDR1 is as shown in SEQ ID NO. 1, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO. 2, the amino acid sequence of the LCDR3 is as shown in SEQ ID NO. 3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO. 4, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO. 5, and the amino acid sequence of the HCDR3 is shown by the following formula: X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 wherein X 1 is selected from R and A, X 2 is selected from S and A, X 3 is selected from P and A, X 4 is selected from Y and A, X 5 is selected from G and A, X 6 is selected from Y and A, X 7 is selected from D and A, X 8 is selected from E and A, X 9 is selected from Y and A, X 10 is selected from G and A, and X 11 is selected from L and A.

[0008] In a preferred embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 positions in X 1 -X 11 are not simultaneously A.

[0009] The beneficial effects of the present invention are as follows: By mutating the amino acid sites of the HCDR3, the affinity and killing ability of the EpCAM antibody or its antigen-binding portion are improved, so that the EpCAM antibody or its antigen-binding portion has a killing effect on EpCAM-positive cell lines or solid tumors, and there is no need to use autologous cells of the patient to participate in the construction.

[0010] Further, the amino acid sequence of the HCDR3 is as shown in any one of SEQ ID NO. 6 to SEQ ID NO. 16 and SEQ ID NO. 73.

[0011] Further, the amino acid sequence of the VH is as shown in any one of SEQ ID NO. 18 to SEQ ID NO. 28 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acids shown in any one of SEQ ID NO. 18 to SEQ ID NO. 28.

[0012] Further, the amino acid sequence of the VL is as shown in SEQ ID NO. 17 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acids shown in SEQ ID NO. 17.

[0013] Further, the EpCAM antibody or its antigen-binding portion is in the form of scFv, and the scFv contains an amino acid sequence as shown in any one of SEQ ID NO. 29 to SEQ ID NO. 39.

[0014] The present invention also provides a first nucleic acid molecule, which contains a polynucleotide encoding the EpCAM antibody or its antigen-binding portion.

[0015] The present invention also provides a chimeric antigen receptor, which contains an antigen-binding domain, a transmembrane domain, and a signal transduction domain. The antigen-binding domain contains the EpCAM antibody or its antigen-binding portion, and the chimeric antigen receptor contains an amino acid sequence as shown in any one of SEQ ID NO. 40 to SEQ ID NO. 50.

[0016] The present invention also provides a second nucleic acid molecule, which contains a polynucleotide encoding the chimeric antigen receptor. The second nucleic acid molecule may contain a nucleic acid sequence as shown in any one of SEQ ID NO. 51 to SEQ ID NO. 61.

[0017] The present invention further provides a vector, which comprises the second nucleic acid molecule.

[0018] The present invention further provides an immune cell, which comprises the chimeric antigen receptor, the second nucleic acid molecule, or the vector.

[0019] Furthermore, the immune cell includes any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages. In one embodiment, the immune cell is more sensitive to colon cancer, lung cancer, breast cancer, gastric cancer, gastric cancer lung metastasis, lung cancer gastric metastasis, breast cancer gastric metastasis, and / or gastric cancer breast metastasis comprising EpCAM gene mutations N120S, G222S, and / or S228Y.

[0020] The present invention further provides a pharmaceutical composition, which comprises the EpCAM antibody or its antigen-binding portion, the first nucleic acid molecule, the chimeric antigen receptor, the second nucleic acid molecule, the vector, or the immune cell.

[0021] The present invention further provides the use of the EpCAM antibody or its antigen-binding portion, the first nucleic acid molecule, the chimeric antigen receptor, the second nucleic acid molecule, the vector, or the immune cell in the preparation of a drug for treating a disease or disorder related to the expression of EpCAM.

[0022] Furthermore, the disease or disorder related to the expression of EpCAM includes cancer or malignancy.

[0023] Furthermore, the disease or disorder related to the expression of EpCAM includes hepatocellular carcinoma, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, head and neck cancer, squamous cell skin cancer, endometrial cancer of the uterine corpus, bladder cancer. The disease or disorder related to the expression of EpCAM is preferably colon cancer, lung cancer, breast cancer, gastric cancer, gastric cancer lung metastasis, lung cancer gastric metastasis, breast cancer gastric metastasis, and / or gastric cancer breast metastasis comprising EpCAM gene mutations N120S, G222S, and / or S228Y.

[0024] Furthermore, a method for preparing a cell expressing the chimeric antigen receptor described herein is provided, which includes introducing a vector into a cell, the vector comprising a polynucleotide encoding the chimeric antigen receptor.

[0025] Furthermore, the cell includes any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Schematic diagram of the flow cytometry analysis results of the cells obtained after stimulating and activating PBMC in the embodiments of the present invention; Figure 2 Schematic diagram of the flow cytometry analysis results of the cells obtained after stimulating, activating and removing T cells from PBMC in the embodiments of the present invention; Figure 3 Schematic diagram of the structure of the vector plasmid in the embodiments of the present invention; Figure 4 Expression of EpCAM in each cell in the experimental examples, comparative examples, blank example 1 and blank example 2 in the process of constructing EpCAM-CAR-NK cells (A0 sequence) and comparative CAR cells in the embodiments of the present invention; Figure 5 Schematic diagram of the results of the infection efficiency of EpCAM-CAR-NK-92MI cells (A0 sequence) in the embodiments of the present invention; Figure 6 Schematic diagram of the results of the EpCAM expression levels of breast cancer cell MDA-MB-231, human colon cancer cell RKO, human colon cancer cell SW480, colon cancer cell HCT116, and liver cancer cell HepG2 co-cultured with EpCAM-CAR-NK-92MI cells (A0 sequence) in the embodiments of the present invention; Figure 7 Schematic diagram of the results that +anti-EpCAM-CAR-NK-92MI cells (A0 sequence) in the embodiments of the present invention have a killing effect on HCT116 cells, with the abscissa in hours; Figure 8 Schematic diagram of the results that +anti-EpCAM-CAR-NK-92MI cells in the embodiments of the present invention have a killing effect on HCT116 cells, and the killing effect on HCT116 cells becomes stronger as the number of +anti-EpCAM-CAR-NK-92MI cells (A0 sequence) increases; Figure 9 Schematic diagram of the change in the expression level of IFN-γ in +NC CAR-NK-92MI cells and +anti-EpCAM-CAR-NK-92MI cells (A0 sequence) in the embodiments of the present invention; Figure 10 Schematic diagram of the change in the expression level of TNF-α in +NC CAR-NK-92MI cells and +anti-EpCAM-CAR-NK-92MI cells (A0 sequence) in the embodiments of the present invention; Figure 11 Schematic diagram of the change in the expression level of perforin in +NC CAR-NK-92MI cells and +anti-EpCAM-CAR-NK-92MI cells (A0 sequence) in the embodiments of the present invention; Figure 12 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A1-scFv in an embodiment of the present invention; Figure 13 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A2-scFv in an embodiment of the present invention; Figure 14 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A3-scFv in an embodiment of the present invention; Figure 15 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A4-scFv in an embodiment of the present invention; Figure 16 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A5-scFv in an embodiment of the present invention; Figure 17 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A6-scFv in an embodiment of the present invention; Figure 18 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A7-scFv in an embodiment of the present invention; Figure 19 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A21-CAR in an embodiment of the present invention.

[0027] Figure 20 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A22-CAR in an embodiment of the present invention; Figure 21 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A23-CAR in an embodiment of the present invention; Figure 22 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A24-CAR in an embodiment of the present invention; Figure 23 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A25-CAR in an embodiment of the present invention; Figure 24 Schematic diagram of flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A26-CAR in an embodiment of the present invention; Figure 25Schematic diagram of the flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A27-CAR in the embodiments of the present invention; Figure 26 Schematic diagram of the flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A28-CAR in the embodiments of the present invention; Figure 27 Schematic diagram of the flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A29-CAR in the embodiments of the present invention; Figure 28 Schematic diagram of the flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A30-CAR in the embodiments of the present invention; Figure 29 Schematic diagram of the flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing A31-CAR in the embodiments of the present invention; Figure 30 Schematic diagram of the results after co-culture of CAR T cells containing A1-scFv with HCT116 cells in the embodiments of the present invention; Figure 31 Schematic diagram of the results after co-culture of CAR T cells containing A2-scFv with HCT116 cells in the embodiments of the present invention; Figure 32 Schematic diagram of the results after co-culture of CAR T cells containing A3-scFv with HCT116 cells in the embodiments of the present invention; Figure 33 Schematic diagram of the results after co-culture of CAR T cells containing A4-scFv with HCT116 cells in the embodiments of the present invention; Figure 34 Schematic diagram of the results after co-culture of CAR T cells containing A5-scFv with HCT116 cells in the embodiments of the present invention; Figure 35 Schematic diagram of the results after co-culture of CAR T cells containing A6-scFv with HCT116 cells in the embodiments of the present invention; Figure 36 Schematic diagram of the results after co-culture of CAR T cells containing A7-scFv with HCT116 cells in the embodiments of the present invention; Figure 37 Schematic diagram of the results that EpCAM-CAR-NK-92MI cells based on A0 scFV have a killing effect on ovarian cancer RMG-I cells in the embodiments of the present invention; Figure 38Schematic diagram of the result that EpCAM-CAR-NK-92MI cells based on A0 scFV in the embodiment of the present invention have a killing effect on breast cancer MDA-MB-468 cells; Figure 39 Schematic diagram of the result that EpCAM-CAR-NK-92MI cells based on A0 scFV in the embodiment of the present invention have a killing effect on bladder cancer KU-19-19 cells.

[0028] Figure 40 Chimeric antigen receptor structure in the embodiment of the present invention. Detailed implementation manners

[0029] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains. The words such as "including" used herein mean that the elements or items appearing before this word cover the elements or items listed after this word and their equivalents, without excluding other elements or items.

[0030] The embodiments of the present invention provide an EpCAM antibody or its antigen-binding part and its uses, specifically relating to an EpCAM antibody or its antigen-binding part, nucleic acid molecule, chimeric antigen receptor, vector, immune cell, pharmaceutical composition and uses.

[0031] The "antigen-binding protein" described in the present invention includes a complete full-length antibody having an antigen-binding region and any fragment thereof in which the "antigen-binding portion" or "antigen-binding region" is retained, or a single-chain thereof such as a single-chain variable fragment (scFv), and also includes all recombinant forms of the antibody, such as an antibody expressed in a prokaryotic cell, an unglycosylated antibody, and an antibody fragment and derivative that binds to an antigen. Each heavy chain consists of a heavy-chain variable region (abbreviated as VH) and a heavy-chain constant region (abbreviated as CH). Each light chain consists of a light-chain variable region (abbreviated as VL) and a light-chain constant region (abbreviated as CL). VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0032] The "CAR" described in the present invention refers to: an extracellular antigen-binding region capable of binding an antigen, a hinge region, a transmembrane region, and an intracellular signaling region, where the intracellular signaling region refers to a protein that transmits information into the cell through a defined signaling pathway by generating a second messenger to regulate cell activity, or a protein that functions as an effector corresponding to such a messenger, and includes a primary signaling domain, and may further include a functional signaling domain (i.e., a co-stimulatory signaling domain) derived from a stimulatory molecule as defined below. The intracellular signaling region generates signals that can promote the immune effector function of the cell of the CAR. Examples of immune effector functions include, for example, cell lysis activity and helper activity, including cytokine secretion.

[0033] In the embodiments of the present invention, the antigen-binding molecule, scFv, antibody or fragment thereof directly blocks the binding site on the ligand or, in an indirect manner, such as by changing the structure or energy of the ligand, changes the binding ability of the ligand, or the antigen-binding molecule, scFv, antibody or fragment thereof prevents the protein bound thereto from exercising its biological function.

[0034] The "peptide", "polypeptide" and "protein" in the embodiments of the present invention are used interchangeably and refer to a compound containing amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids and there is no limit to the maximum number of amino acids that can include the sequence of the protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds.

[0035] "Specific binding" or "specifically bind to" in the embodiments of the present invention refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen.

[0036] "Nucleic acid" or "nucleic acid sequence" in the embodiments of the present invention refers to any molecule, preferably a polymeric molecule, containing ribonucleic acid, deoxyribonucleic acid or their analogue units. The nucleic acid can be single-stranded or double-stranded. The single-stranded nucleic acid can be a nucleic acid of one strand of denatured double-stranded DNA. Alternatively, the single-stranded nucleic acid can be a single-stranded nucleic acid not derived from any double-stranded DNA.

[0037] "Regulate" or "modulate" in the embodiments of the present invention generally includes the meanings of up-regulation or down-regulation in two different directions. In some cases, it can be understood as inhibition or enhancement, in some cases as reduction or increase, in some cases as decrease or increase, etc. The specific explanation is not limited and is understood and interpreted according to the actual application context. Exemplarily, in some embodiments, "regulating" the growth of tumor cells can be understood as inhibiting or enhancing the growth of tumor cells.

[0038] In the present invention: "CD3ζ" is defined as the protein provided by GenBank accession number BAG36664.1, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. "CD3ζ domain" is defined as the amino acid residues from the cytoplasmic domain of the ζ chain, which are sufficient to functionally transmit the initial signals required for T cell activation. On the one hand, the cytoplasmic domain of ζ contains residues 52 to 163 of GenBank accession number BAG36664.1, and its functional homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.

[0039] "CD28" is defined as the protein provided by GenBank accession number NP_006130.1, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. "CD28 signaling region" is defined as the amino acid residues from the cytoplasmic domain of CD28, which can transmit the co-stimulatory signals required for T cell activation; its sequence contains residues 180 to 220 of GenBank accession number NP_006130.1, and its functional homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. "CD28 hinge region" contains residues 114 to 152 of GenBank accession number NP_006130.1, and its functional homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. "CD28 transmembrane region" contains residues 153 to 179 of GenBank accession number NP_006130.1, and its functional homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.

[0040] The present invention provides an EpCAM antibody or an antigen-binding portion thereof, the EpCAM antibody or the antigen-binding portion thereof comprising a light-chain variable region VL and a heavy-chain variable region VH, the VL comprising a first light-chain complementarity-determining region LCDR1, a second light-chain complementarity-determining region LCDR2, and a third light-chain complementarity-determining region LCDR3, the VH comprising a first heavy-chain complementarity-determining region HCDR1, a second heavy-chain complementarity-determining region HCDR2, and a third heavy-chain complementarity-determining region HCDR3, the amino acid sequence of the LCDR1 being as shown in SEQ ID NO.1, the amino acid sequence of the LCDR2 being as shown in SEQ ID NO.2, the amino acid sequence of the LCDR3 being as shown in SEQ ID NO.3, the amino acid sequence of the HCDR1 being as shown in SEQ ID NO.4, the amino acid sequence of the HCDR2 being as shown in SEQ ID NO.5, and the amino acid sequence of the HCDR3 being shown by the following formula: X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 wherein X 1 is selected from R and A, X 2 is selected from S and A, X 3 is selected from P and A, X 4 is selected from Y and A, X 5 is selected from G and A, X 6 is selected from Y and A, X 7 is selected from D and A, X 8 is selected from E and A, X 9 is selected from Y and A, X 10 is selected from G and A, and X 11 is selected from L and A.

[0041] The present invention mutates the amino acid sites of the HCDR3 to improve the affinity and killing power of the EpCAM antibody or its antigen-binding portion, so that the EpCAM antibody or its antigen-binding portion has a killing effect on EpCAM-positive cell lines or solid tumors, and there is no need to use autologous cells of the patient to participate in the construction.

[0042] In some embodiments of the present invention, the amino acid sequence of the HCDR3 is any one of SEQ ID NO.6 to SEQ ID NO.16.

[0043] In some embodiments of the present invention, the amino acid sequence of the VH is as shown in any one of SEQ ID NOs. 18 to 28 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in any one of SEQ ID NOs. 18 to 28.

[0044] In some embodiments of the present invention, the amino acid sequence of the VL is as shown in SEQ ID NO. 17 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in SEQ ID NO. 17.

[0045] In some embodiments of the present invention, the EpCAM antibody or its antigen-binding portion is in the form of a scFv, and the scFv comprises an amino acid sequence as shown in any one of SEQ ID NOs. 29 to 39.

[0046] The present invention further provides a first nucleic acid molecule, which comprises a polynucleotide encoding the EpCAM antibody or its antigen-binding portion.

[0047] The present invention further provides a chimeric antigen receptor, which comprises an antigen-binding domain, a transmembrane domain, and a signal transduction domain. The antigen-binding domain comprises the EpCAM antibody or its antigen-binding portion, and the chimeric antigen receptor comprises an amino acid sequence as shown in any one of SEQ ID NOs. 40 to 50.

[0048] The present invention further provides a second nucleic acid molecule, which comprises a polynucleotide encoding the chimeric antigen receptor, and the second nucleic acid molecule comprises a nucleic acid sequence as shown in any one of SEQ ID NOs. 51 to 61.

[0049] The present invention further provides a vector, which comprises the second nucleic acid molecule.

[0050] The present invention further provides an immune cell, which comprises the chimeric antigen receptor, the second nucleic acid molecule, or the vector.

[0051] In some embodiments of the present invention, the immune cell includes any one or a combination of at least two of T cells, B cells, NK cells, mast cells, or macrophages.

[0052] The present invention further provides a pharmaceutical composition, which comprises the EpCAM antibody or its antigen-binding portion, the first nucleic acid molecule, the chimeric antigen receptor, the second nucleic acid molecule, the vector, or the immune cell.

[0053] The present invention further provides the use of the EpCAM antibody or its antigen-binding portion, the first nucleic acid molecule, the chimeric antigen receptor, the second nucleic acid molecule, the vector, or the immune cell in the preparation of a medicament for treating a disease or disorder associated with the expression of EpCAM.

[0054] In some embodiments of the present invention, the disease or disorder associated with the expression of EpCAM includes cancer or malignancy.

[0055] In some embodiments of the present invention, the disease or disorder associated with the expression of EpCAM includes hepatocellular carcinoma, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, head and neck cancer, squamous cell skin cancer, endometrial cancer of the uterine corpus, and bladder cancer.

[0056] In the present invention, the coding sequence of the CAR structure comprises 5'LTR-CMV-scfv-hinge-transmembrane-activation signal-WPRE-3'LTR, wherein the hinge comprises at least one of CD8, NKp46 TM+cyto, NKG2D TM+cyto, and NKp30 TM+cyto, the transmembrane comprises at least one of CD28, DAP10, DAP12, and 2B4, and the activation signal comprises at least one of CD3ζ, CD3ζ(I), P2A-IL15, and DAP12. Specifically, the CAR structure is any one of 5'LTR-CMV-scfv-CD8-CD28-CD3ζ-WPRE-3'LTR, 5'LTR-CMV-scfv-NKp46 TM+cyto-DAP10-CD3ζ-WPRE-3'LTR, 5'LTR-CMV-scfv-NKp46 TM+cyto-DAP12-CD3ζ(I)-WPRE-3'LTR, 5'LTR-CMV-scfv-NKp46 TM+cyto-DAP12-CD3ζ(I)-P2A-IL15-WPRE-3'LTR, 5'LTR-CMV-scfv-NKp46 TM+cyto-2B4-DAP12-WPRE-3'LTR, 5'LTR-CMV-scfv-NKG2D TM+cyto-DAP12-CD3ζ-WPRE-3'LTR, 5'LTR-CMV-scfv-NKG2D TM+cyto-2B4-DAP12-WPRE-3'LTR, 5'LTR-CMV-scfv-NKG2D TM+cyto-DAP12-CD3ζ(I)-P2A-IL15-WPRE-3'LTR, 5'LTR-CMV-scfv-NKp30 TM+cyto-DAP12-CD3ζ-WPRE-3'LTR, and 5'LTR-CMV-scfv-NKp30 TM+cyto-2B4-DAP12-WPRE-3'LTR.

[0057] In the present invention, the CAR structure may include scfv-hinge-transmembrane-activation signal, wherein the hinge includes at least one of CD8, NKp46TM+cyto, NKG2D TM+cyto, NKp30 TM+cyto, the transmembrane includes at least one of CD28, DAP10, DAP12, 2B4, and the activation signal includes at least one of CD3ζ, CD3ζ(I), P2A-IL15, DAP12. Specifically, the CAR structure is any one of scfv-CD8-CD28-CD3ζ, scfv-NKp46 TM+cyto-DAP10-CD3ζ, scfv-NKp46TM+cyto-DAP12-CD3ζ(I), scfv-NKp46 TM+cyto-DAP12-CD3ζ(I)-P2A-IL15, scfv-NKp46 TM+cyto-2B4-DAP12, scfv-NKG2D TM+cyto-DAP12-CD3ζ, scfv-NKG2D TM+cyto-2B4-DAP12, scfv-NKG2D TM+cyto-DAP12-CD3ζ(I)-P2A-IL15, scfv-NKp30 TM+cyto-DAP12-CD3ζ, scfv-NKp30 TM+cyto-2B4-DAP12.

[0058] In some embodiments of the present invention, the EpCAM-binding protein comprises a single-chain antibody, and the antibody fragment of the single-chain antibody is a single-chain variable region (scFv); the structure of the single-chain variable region is VH-(Linker)-VL or VL-linker-VH; the amino acid sequence of the linker is GGGGSGGGGSGGGGS.

[0059] Some embodiments of the present invention also provide a method for constructing a CAR cell comprising the EpCAM antibody or antigen-binding portion thereof, comprising the following steps: S1: Insert the sequence of the EpCAM antibody or its antigen-binding portion into the lentiviral vector pHBLV-CMV-MCS-EF1-Puro to obtain a vector plasmid; S2: Transduce the vector plasmid into T cells using a packaging plasmid for virus packaging to obtain a virus solution with a virus titer of 10 8-9 IFU / ml; S3: Transduce NK cells derived from human peripheral blood mononuclear cells and amplified and activated using a transfection mixture comprising the virus solution and polybrene, and control the multiplicity of infection to be 5-10 to obtain the CAR-NK cells targeting the EpCAM antigen.

[0060] In some embodiments of the present invention, in step S2, the step of transducing the vector plasmid into T cells using a packaging plasmid for virus packaging includes: S21: Mixing a transfection mixture composed of the vector plasmid, pSPAX2 plasmid, pMD2G plasmid, and transfection agent Lipofectamine with the T cells resuspended in complete DEME medium, and then culturing for 12 - 16 hours; S22: Replacing the medium in the culture obtained in step S21 with complete DEME medium containing antibiotics and sodium butyrate, then continuing to culture for 36 - 48 hours, and then collecting the supernatant and obtaining the virus solution from the supernatant.

[0061] In some embodiments of the present invention, in step S21, the pSPAX2 plasmid and pMD2G plasmid constitute the packaging plasmid.

[0062] In some embodiments of the present invention, in step S22, the antibiotic is streptomycin.

[0063] In some embodiments of the present invention, in step S21, controlling the molar ratio of the vector plasmid, pSPAX2 plasmid, and pMD2G plasmid to be 1:1:(1 - 2), and the mass ratio of the transfection agent Lipofectamine to the vector plasmid to be (4 - 6):1.

[0064] In some embodiments of the present invention, in step S22, in the complete DEME medium containing antibiotics and sodium butyrate, the volume content of the antibiotic is 0.5 mM - 2 mM, and the content of sodium butyrate is 0.5 mM - 2 mM.

[0065] In some embodiments of the present invention, in step S3, the step of transducing NK cells derived from human peripheral blood mononuclear cells and amplified and activated using a transfection mixture containing the virus solution and polybrene includes: S31: Resuspending a cell mixture composed of irradiated K562 mb - IL21 cells and the NK cells derived from human peripheral blood mononuclear cells and amplified and activated using serum - free RPMI medium containing rhlL - 2; S32: Mixing the transfection mixture containing the virus solution and polybrene with the resuspension obtained in step 31, culturing for 24 hours, and then replacing the medium with the serum - free RPMI medium containing rhlL - 2 and continuing to culture for 48 - 72 hours.

[0066] In some embodiments of the present invention, in step S31, the final concentration of rhlL-2 is 100-200 IU / mL, the number of irradiated K562 mb-IL21 cells is the same as the number of NK cells derived from human peripheral blood mononuclear cells and amplified and activated, and in the resuspension obtained through step S31, the final concentration of the NK cells derived from human peripheral blood mononuclear cells and amplified and activated is 0.5×10 6 cells / mL - 3×10 6 cells / mL.

[0067] In some embodiments of the present invention, in step S32, the final concentration of polybrene is 4-6 μg / mL.

[0068] Specifically, the sequences involved in the present invention are shown in Table 1.

[0069] Table 1 Sequences

[0070] Unless otherwise specified, the "cultivation" described in the embodiments of the present invention is carried out in a cell culture incubator at 37 °C with a carbon dioxide concentration of 5%.

[0071] The experimental methods whose operation methods are not specifically described in the embodiments of the present invention, such as double digestion methods, ligation and transformation, etc., are all conventional technical means for those skilled in the art. For specific operations, reference can be made to: Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Third Edition) (2001), Cold Spring Harbor Laboratory Press, or refer to the instructions provided by the manufacturer corresponding to the embodiments of the present invention.

[0072] For the data related to statistical analysis in each embodiment of the present invention, each experiment is repeated at least 3 times, and the experimental result data is statistically analyzed using GraphPad Prism 8.0 software. A two-tailed unpaired t-test is used to calculate the statistical difference between two groups of data, and ANOVA variance analysis is used to calculate the statistical difference for the comparison of differences among multiple groups of data. p < 0.05 is considered to have a statistical difference.

[0073] Example 1: Preparation method of NK cells derived from human peripheral blood mononuclear cells and amplified and activated The blood samples of healthy volunteers in this example are from Saili Biotechnology Co., Ltd., and the healthy volunteers are aware of the use of the blood samples and have signed an informed consent form.

[0074] Take 10 ml of the blood sample of healthy volunteers and dilute it with 20 mL of PBS. Slowly add the diluted blood to 10 ml of Ficoll-Paque TM PLUS reagent (from GE healthcare), and then centrifuge at 800 g centrifugal force at room temperature for 30 min. After centrifugation, remove the serum layer, and then aspirate the middle white flocculent cell layer (this layer is the PBMC layer) into a centrifuge tube. Add 30 mL of PBS to dilute the cell solution, then centrifuge at 400 g centrifugal force at room temperature for 15 min, discard the supernatant, resuspend the obtained precipitate with 1 mL of PBS, filter it through a 70-μm filter membrane, and then centrifuge the obtained resuspended solution at 1500 rmp for 5 min and discard the supernatant to obtain a cell precipitate. Resuspend the cell precipitate with complete RPMI medium (from Shanghai Yuanpei Biotechnology Co., Ltd.) plus 200 U / mL rhIL-2 (from Biolegend) to a concentration of 1×10 6After culturing for 2 hours at a density of [number] cells / mL, the cells were stimulated and activated using a human T cell activation / proliferation kit (from Miltenyi Biotec GmbH). The specific procedure is described in the accompanying documents of the kit. Anti-CD3 antibody was added to the mixture obtained after stimulation and activation to specifically bind to T cells. The resulting mixture was added to a magnetic separation column to remove the labeled T cells and the eluted material was collected; anti-CD16 and anti-CD56 were added to the reaction of the collected eluted material to specifically bind to NK cells to form a magnetic immune complex, which was then added to another magnetic separation column for elution so that the NK cells specifically binding to anti-CD16 and anti-CD56 remained in the column to obtain labeled NK cells. Then, the magnetic separation column was gently pressurized with PBS wash solution to elute and separate the NK cells.

[0075] The mixture obtained after stimulation and activation and the wash solution containing NK cells were respectively detected by flow cytometry to obtain Figure 1 the schematic diagram of the flow cytometry analysis results of the cells obtained after stimulation and activation as shown, and Figure 2 the schematic diagram of the flow cytometry analysis results of NK cells as shown. Referring to Figure 1 and Figure 2 the subsets of NK cells can be seen, proving that NK cells were successfully obtained from PBMC in Example 1.

[0076] Example 2: Treatment of K562-mb-IL21 cells K562-mb-IL21 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were used as feeder cells to promote the growth and proliferation of NK cells and inhibit the differentiation of NK cells to ensure the success of subsequent virus infection and transduction.

[0077] K562-mb-IL21 cells were resuspended in complete RPMI medium to a concentration of 0.5×10 6 cells / mL - 3×10 6 cells / mL and then cultured for 7 days, and the old complete RPMI medium was replaced with fresh complete RPMI medium every 2 - 3 days.

[0078] After the culture was completed, the mixture obtained by culturing was centrifuged and precipitated. The resulting precipitate was resuspended in complete RPMI medium and adjusted to a concentration of 0.5×10 6 cells / mL - 3×10 6 cells / mL. Then, the cells were irradiated with a gamma cell irradiator at 100 Gy for 30 minutes to obtain a suspension of irradiated K562-mb-IL21 cells.

[0079] Example 3: Preparation of vector plasmid This embodiment provides a specific implementation manner of step S1. The chimeric antigen receptor sequence was designed by the inventors and synthesized by Genewiz (Suzhou). The obtained anti-EpCAM-CAR sequence was inserted into the lentiviral vector pHBLV-CMV-MCS-EF1-Puro to obtain the vector plasmid as shown in Figure 3 as follows.

[0080] The lentiviral vector pHBLV-CMV-MCS-EF1-Puro is from Hanheng Biotechnology (Shanghai) Co., Ltd. The CAR sequence was provided by the applicant, Fudan University, and stored in the form of PUC19 plasmid. The specific preparation process of the vector plasmid was completed by Hanheng Biotechnology (Shanghai) Co., Ltd. Specifically, both pHBLV-CMV-MCS-EF1-Puro and the PUC19 plasmid containing the CAR sequence were double digested with EcoRI and NotI. The target bands were recovered using an agarose gel recovery kit according to the instructions, and the recovered fragments were ligated in a ProFlex PCR instrument at a molar ratio of CAR structure: pCDH-CMV-MCS-EF1-Puro = 5:1 and transformed at 16 °C for 4 h.

[0081] Example 4: Viral packaging of the vector plasmid and lentiviral infection of T cells Isolation of T cells from human blood: Take a blood sample from a healthy volunteer into a sterile 50 ml centrifuge tube, dilute it 2-fold with PBS, and then slowly add the diluted blood along the tube wall to the Ficoll reagent (diluent: Ficoll = 3:1). Centrifuge at 800 g for 30 min, and adjust the acceleration and deceleration of the centrifuge to 2 and 1 respectively. After centrifugation, aspirate the middle white flocculent cell layer into a 50 ml centrifuge tube. This layer is the human peripheral blood mononuclear cell (PBMC) layer. Subsequently, dilute the cell solution with an appropriate amount of PBS, centrifuge at 400 g for 15 min, discard the supernatant, resuspend the cells with EasySepTM Buffer, and isolate human T cells according to the steps provided by the Human T Cell Isolation Kit, and culture them in RPMI complete medium. This embodiment of the present invention provides a specific implementation manner of step S2. The vector plasmid is transduced into T cells using packaging plasmids for viral packaging to obtain a virus solution with a virus titer of 10 8- 9 IFU / ml. Among them, the packaging plasmids pSPAX2 plasmid and pMD2G plasmid are selected, and the specific synthesis steps are as follows: Packaging of lentivirus The lentiviral vector plasmid carries the GFP protein, and the transfection efficiency can be observed through a fluorescence microscope. It also has puromycin resistance, and positive cells can be screened with an appropriate concentration of puromycin.

[0082] 1. Uniformly seed T cells in good condition in a 6-well plate, with 2 mL of culture medium in each well; 2. Mix 5 μg of pMD.2G, 10 μg of psPAX2, and 10 μg of EpCAM CAR vector plasmid, and dilute with 250 μl of Opti-MEM medium, and mix well to obtain a mixed plasmid dilution; 3. Pipette 10 μl of Lipo-2000 gently and dilute it with 250 μl of Opti-MEM medium, and mix well to obtain a Lipo-2000 dilution; 4. Gently add the mixed plasmid dilution to the Lipo-2000 dilution, and incubate at room temperature for 15 minutes to obtain a final mixed solution; 5. Uniformly drip the final mixed solution into the wells of the 6-well plate containing T cells, and gently shake the plate; 6. Place the cells in the incubator and let them stand for 6 hours. After that, discard the culture medium and replace it with fresh medium; 7. Two days after transfection, collect the supernatant culture medium (containing lentivirus solution) to obtain a 48-hour lentivirus culture solution, and store the 48-hour lentivirus culture solution at 4 °C; 8. Add DMEM cell culture medium containing 1% serum to the cell culture plate. After 72 hours of transfection, collect the supernatant culture medium (containing lentivirus solution) to obtain a 72-hour lentivirus culture solution, and combine it with the 48-hour lentivirus culture solution to obtain a mixed lentivirus culture solution; 9. After centrifugation at 3500 rpm for 10 minutes, remove the T cells; Aliquot the virus supernatant, detect the virus titer of the virus supernatant, and finally store the virus supernatant at -80 °C. Among them, the virus titer of the virus supernatant is 10 8-9 IFU / ml, and the virus titer represents the number of virus particles with transduction function in each milliliter of virus solution.

[0083] Construction of EpCAM-CAR-T cells Viral transfection of the EpCAM gene: Use Hanheng Biotechnology to package the virus for transfection: Virus titer: 2.5×10 8 TU / ml. Take PBMC (CD3+ T cells) and place them in a 24-well plate for transfection. The transfection system is as follows:

[0084] After adding the reagents according to the system, centrifuge at 200 g at room temperature for 1 h, place it in the incubator for continuous culture. After 4 h, make up the liquid volume to 500 μl. The next day, centrifuge and change the liquid, and take the cells for flow cytometry to detect the expression of EpCAM1-CAR.

[0085] Example 5: Construction of CAR-NK cells targeting EpCAM antigen (abbreviated as EpCAM-CAR-NK cells) This example provides step S3: a step of transducing NK cells derived from human peripheral blood mononuclear cells provided in Example 1 and amplified and activated with a transfection mixture containing the virus solution and polyamide to obtain the CAR-NK cells targeting EpCAM antigen.

[0086] Construction of EpCAM-CAR-NK-92MI cells by lentiviral transfection (The amino acid sequence of A0-scFv is QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWIG NIYPSYIYTNYNQEFKD KVTLTVDESTSTAYMELSSLRSEDTAVYYCT RSPYGYDEYGL DYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTMTCKSSQSLLNSRNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNDYVYPLTFGQGTKLEIK) For the method of constructing CAR with scFv, refer to Figure 40 , and the CAR sequence refers to A0-CAR in Table 1 Specifically, the step of transducing NK cells derived from human peripheral blood mononuclear cells and amplified and activated with a transfection mixture of lentivirus and polyamine includes S31: Resuspend the cell mixture composed of irradiated K562-mb-IL21 in Example 2 and the NK cells derived from human peripheral blood mononuclear cells provided in Example 1 and amplified and activated with serum-free RPMI medium containing rhlL-2, wherein the final concentration of rhlL-2 is controlled to be 100 IU / mL, and the number of irradiated K562 mb-IL21 cells and NK cells derived from human peripheral blood mononuclear cells and amplified and activated is the same, and the final concentration is 0.5×10 6 cells / mL; S32: Mix the transfection mixture containing the virus solution and polyamine with the resuspension obtained in step 31 and culture for 24 hours, and then replace the medium with serum-free RPMI medium containing rhlL-2 and continue to culture for 48 - 72 hours. Specifically, add 200 mL of the resuspension obtained in step 31 to a 96-well U-bottom plate so that each well contains 1×10 5After culturing for 12 hours, centrifuge the 96-well U-bottom plate at 300 g for 10 minutes and remove the supernatant. Then, add serum-free RPMI medium, the virus solution of Example 4 (10 mL), and polybrene suspension to each well, and control the multiplicity of infection (MOI) to be 5 and the final concentration of polybrene to be 5 μg / mL. After adding, culture for 24 hours to complete virus infection. After 24 hours of culture, centrifuge the 96-well U-bottom plate at 300 g for 10 minutes and remove the supernatant. Then, add an equal amount of the serum-free RPMI medium containing rhlL-2 in step S31 and continue to culture for 3 days to complete transduction.

[0087] In step S31 of some embodiments, control the final concentration of rhlL-2 to be 100 - 200 IU / mL. The number of irradiated K562 mb-IL21 cells is the same as the number of NK cells derived from human peripheral blood mononuclear cells and amplified and activated, and the final concentration is 0.5×10 6 cells / mL - 3×10 6 cells / mL.

[0088] In step S32 of some embodiments, add 200 μL of the resuspension obtained in step S31 to the 96-well U-bottom plate so that each well contains 1×10 5 cells - 3×10 5 cells, and then culture for 12 - 36 hours; control the final concentration of polybrene to be 4 - 6 μg / mL; control MOI = 5 - 10; culture for 24 - 36 hours to complete virus infection; add an equal amount of the serum-free RPMI medium containing rhlL-2 in step S31 and continue to culture for 3 - 4 days to complete transduction.

[0089] Table 5 Transfection System

[0090] Note: The control virus is the GFP virus carrying an empty vector, purchased from Hanheng Biotechnology.

[0091] After adding reagents according to the system, centrifuge at 200 g for 1.5 h at room temperature. Place it in the incubator and continue to culture. After overnight, collect the cells in the 24-well plate, centrifuge at 1500 rpm for 5 min, add the virus to re-infect according to the method of the previous day, centrifuge at 200 g for 1.5 h at room temperature, and add the special complete medium to make up to 500 μL after overnight.

[0092] The difference between Blank Example 1 of this embodiment and the construction method of the above-mentioned EpCAM-CAR-NK-92MI lies in that: in step S32, PBS is used to replace the virus solution in equal amounts. The difference between Blank Example 2 of this embodiment and the construction method of the above-mentioned EpCAM-CAR-NK-92MI lies in that: in step S32, PBS is used to replace the virus solution and the polybrene suspension in equal amounts.

[0093] In this embodiment, flow cytometry was used to examine the EpCAM expression in each cell in the experimental examples, comparative examples, Blank Example 1, and Blank Example 2, and the Figure 4 result schematic diagram shown was obtained. Specifically, after washing and resuspending each cell with PBS respectively, 2 μL of biotinylated Protein L was added and incubated at 4°C for 45 minutes, then centrifuged at a centrifugal force of 800 g for 5 minutes. After that, the cells were resuspended with 100 μL of PBS, and 2 μL of streptavidin-PE was added and incubated in the dark at 4°C for 45 min. After the incubation, the cells were washed thoroughly with PBS and then resuspended with PBS. EpCAM protein was used as the primary antibody, and anti-EpCAM-FITC from Biolegend was used as the secondary antibody, and detected using a flow cytometer.

[0094] Flow cytometry (FACS) Collect the required cells. After counting, adjust the concentration to 1×10 6 / mL, and take about 100 - 200 μL of cells. At 4°C (replaced with 4-degree PBS, no need to cool down for centrifugation), centrifuge at 800 g for 5 min, and discard the supernatant. Wash the cells with 200 μL of PBS, centrifuge under the same conditions, and then resuspend the cells with 100 μL of PBS. Add 2 μL of biotinylated Protein L and incubate at 4°C for 45 min. After centrifuging under the same conditions, resuspend the cells with 100 μL of PBS, and add 2 μL of streptavidin-PE and 2 μL of anti-CD56-APC (invitrogen), in the dark, incubate at 4°C for 45 min. After completion, wash the cells with 500 μL of PBS as thoroughly as possible, then centrifuge under the same conditions, resuspend the cells with 200 μL of PBS, and then detect the CAR expression using a flow cytometer. Specifically, it is identified by flow cytometry, and the expression level is identified by gating. Refer to Figure 4 , A is Blank Example 1, B is Blank Example 2, C is the experimental example, D is Comparative Example 1, and the results show that the CAR of the embodiment was successfully expressed on CAR-NK.

[0095] The infection efficiency of the EpCAM-CAR-NK-92MI cells after lentiviral infection was detected. Refer to Figure 5 , and the infection efficiency of the EpCAM-CAR-NK-92MI cells was 69.96%.

[0096] Real-time cell analysis (RTCA) Prepare a tumor cell suspension with a certain concentration using complete DMEM medium. Add 50 μl of complete medium to the wells of E-plate 16, gently pat it into a semi-circular shape, place the E-Plate 16 on the RTCA Station, detect the baseline, ensure that the selected wells are in normal contact and the CI of all wells is lower than 0.063. Take out the E-Plate 16, add 50 μl of the well-mixed tumor cell suspension to the wells so that the number of HCT116 cells in each well is 8,000 cells / 100 μl (for the remaining cells, a cell gradient experiment needs to be done in advance to determine the appropriate cell density and serum concentration). Place the E-Plate 16 in the ultra-clean bench at room temperature for 30 min, and then place it on the RTCA Station in the incubator. After the system automatically scans, start step two. Pause after the tumor cells adhere to the wall (CI keeps rising and is at least 0.15 higher than the initial CI). Carefully discard the supernatant medium, add the control group of NK-92MI cells and CAR-NK-92MI with E:T ratios of 2:1, 4:1, and 8:1 respectively, and supplement with a certain amount of complete medium to make the liquid volume in each well the same, and continue to monitor for 60 h.

[0097] Example 6.1: Killing effect of EpCAM-CAR-NK-92MI cells on effector cells This example examined the relative expression levels of EpCAM in various tumor cells as Figure 6 shown. It can be seen from Figure 6 that the relative expression levels of EpCAM in human colon cancer cells SW480, colon cancer cells HCT116, and liver cancer cells HepG2 are significantly increased.

[0098] The EpCAM-CAR-NK-92MI cells obtained in Example 5 were co-cultured with breast cancer cells MDA-MB-231, human colon cancer cells RKO, human colon cancer cells SW480, colon cancer cells HCT116, and liver cancer cells HepG2 respectively, and the killing ability against relevant cancer cells was detected for various tumor cells.

[0099] The normal CAR-NK-92MI cells were denoted as +NC CAR-NK-92MI cells, while the EpCAM-CAR-NK-92MI cells were denoted as +anti-EpCAM-CAR-NK-92MI cells. The +NC CAR-NK-92MI cells were co-cultured with HCT116 cells, and at the same time, the +anti-EpCAM-CAR-NK-92MI cells were co-cultured with HCT116 cells. The HCT116 cells were cultured alone. After the culture was completed, the normalized cell index of HCT116 cells in the solution was calculated. Refer to Figure 7 , the +anti-EpCAM-CAR-NK-92MI cells had an in vitro killing effect on HCT116 cells.

[0100] The +NC CAR-NK-92MI cells and HCT116 cells were co-cultured at an effector-to-target ratio of 2:1, 4:1, and 8:1 for 12 hours. At the same time, the +anti-EpCAM-CAR-NK-92MI cells and HCT116 cells were co-cultured at an effector-to-target ratio of 2:1, 4:1, and 8:1 for 60 hours. The cell lysis of HCT116 cells was referred to Figure 8 , indicating that the +anti-EpCAM-CAR-NK-92MI cells had an in vitro killing effect on HCT116 cells, and with the increase in the number of +anti-EpCAM-CAR-NK-92MI cells, the killing effect on HCT116 cells became stronger; the changes in the expression levels of killing-related genes in the +NC CAR-NK-92MI cells and +anti-EpCAM-CAR-NK-92MI cells were detected. Refer to Figure 9 、 Figure 10 and Figure 11 , the contents of IFN-γ, TNF-α, and perforin increased with the increase in the number of +NC CAR-NK-92MI cells or +anti-EpCAM-CAR-NK-92MI cells, and when the effector-to-target ratio of the +anti-EpCAM-CAR-NK-92MI cells to HCT116 cells was 8:1, the contents of IFN-γ, TNF-α, and perforin were the highest. By consulting the literature, the literature related to CAR-T and CAR-NK all determined the killing ability on related cancer cells by detecting the changes in protein secretion through ELISA.

[0101] ELISA enzyme-linked immunosorbent assay The obtained mouse intestinal tissues were homogenized, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken. Blood was collected from the posterior orbital venous plexus of mice, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken. The obtained samples were subjected to subsequent protein expression detection according to the ELISA kit instructions. The main steps are as follows: (1) Preparation of standard: Quickly separate the standard sample tube, add the cytokine standard attached to the reconstitution kit according to the instructions, and vortex thoroughly to make the concentration of the reconstituted standard 4,000 pg / mL. Let the reconstituted standard stand at room temperature for 10 - 30 min. Then, pipette 200 μL of the cytokine standard with a concentration of 4,000 pg / mL from the mother liquor and add 200 μL of the standard diluent for dilution to obtain a cytokine solution with a concentration of 2,000 pg / mL, which serves as the highest concentration of the standard curve. Dilute it by a factor of 2 to 31.25 pg / mL.

[0102] (2) Washing: Add 200 μL of the 1× wash buffer attached to the kit into the strip of 8 - well tubes, let it stand at room temperature for 30 seconds and then discard, and pat dry as much as possible on the absorbent paper. After the plate washing is completed, the experiment needs to be carried out immediately without allowing the 8 - well tubes to air - dry.

[0103] (3) Addition of standard: Add 100 μL of the standard solution diluted by a factor of 2 into the standard wells. Add 100 μL of the standard diluent into the blank well as a blank control.

[0104] (4) Addition of sample: Add 80 μL of the 1× detection buffer and 20 μL of the sample solution to be tested into the sample wells.

[0105] (5) Addition of detection antibody: Add 50 μL of the antibody working solution to each well. The sample addition process needs to be completed within 10 min without interruption. After the sample addition is completed, incubate the system in the dark at room temperature for 2 h.

[0106] (6) Washing: Discard the original liquid in the wells and add 300 μL of the wash buffer to each well, wash 6 times. After each plate washing, pat dry the liquid as much as possible on the absorbent paper.

[0107] (7) Addition of enzyme and incubation: Add 100 μL of the streptavidin working solution to each well. Incubate at 200 rpm at room temperature in the dark for 40 min using a shaker.

[0108] (8) Washing: Discard the original liquid in the wells and add 300 μL of the wash buffer to each well, wash 6 times. After each plate washing, pat dry the liquid as much as possible on the absorbent paper.

[0109] (9) Addition of substrate: Add 100 μL of the substrate to the detection wells, incubate at 200 rpm at room temperature in the dark for 5 - 30 min using a shaker.

[0110] (10)Termination: Add 50 μL of termination solution to the detection well, and the color changes from blue to yellow. Within 30 min, use a microplate reader for detection. Measure the OD value at 450 nm and use 630 nm as the reference wavelength. Convert the OD value of the sample into the corresponding protein content according to the standard curve.

[0111] Example 6.2 EpCAM frameshift mutation and gene mutation on EpCAM CAR-T response This example investigated the differences in the killing of various tumor cells by EpCAM against CAR-T cells (A0-scFv) targeting the EpCAM antigen.

[0112] HEC59 cervical cancer, SNGM cervical cancer, TOV21G ovarian cancer, respectively contain frameshift mutations (Q201fs, Q201fs, P3fs) of the EpCAM gene

[0113] Colorectal cancer cell lines SNUC5 and SNU1040, pancreatic cancer cell line SUIT2, respectively contain mutations at the EpCAM (N120S, G222S, S228Y) gene locus

[0114] The EpCAM-CART cells obtained in Example 4 were co-cultured with colorectal cancer cell lines SNUC5 and SNU1040, pancreatic cancer cell line SUIT2, HEC59 cervical cancer, SNGM cervical cancer, and TOV21G ovarian cancer, respectively, and then the killing ability of various tumor cells against related cancer cells was detected.

[0115] Table 6

[0116] Example 7: Flow cytometry detection results of EpCAM-CAR-NK-92MI cells containing different scFVs Determination of the binding IC50 of affinity 1. Experimental design and sample preparation 1.1 Cell culture: CAR-NK cells: Culture CAR-NK cells in a medium (such as RPMI-1640 + supplements, such as 10% FBS, antibiotics). Tumor cells: Select appropriate tumor cell lines and culture them under suitable culture conditions to ensure that the tumor cells have sufficient proliferative vitality.

[0117] 1.2 Labeling cells: CAR-NK cell labeling: Use fluorescent dyes to label CAR-NK cells. Common dyes include CFSE (for tracking cells), or antibodies that directly label the CAR receptor. Tumor cell labeling: Label tumor cells with fluorescent dyes.

[0118] 2. Flow cytometry detection 2.1 Cell co-culture: Co-culture different numbers of CAR-NK cells with tumor cells. According to the experimental design, set multiple ratios (such as the ratio of CAR-NK:tumor cells), and the changes in these ratios help evaluate the affinity of CAR-NK cells. Sample cells at different time points (such as 1 hour, 4 hours, or 24 hours) and analyze the cell interactions in a flow cytometer.

[0119] 2.2 Fluorescent staining: After the co-culture, use antibodies to label specific markers on the surface of CAR-NK cells (such as CD56, CD16, etc.) or markers on the surface of tumor cells, and then detect the fluorescence intensity of these labels through a flow cytometer.

[0120] Measure the co-expression fluorescence intensity of CAR-NK cells and tumor cells through a flow cytometer to judge the binding situation between CAR-NK cells and tumor cells.

[0121] 3. Data analysis: Calculate the IC50 of the affinity 3.1 Set up experimental groups: You can set multiple groups according to different concentrations of CAR-NK cells and use flow cytometry data to evaluate the binding degree of CAR-NK cells and tumor cells at different concentrations. Set the ratio of CAR-NK:tumor cells to 1:1, 1:5, 1:10, etc., and evaluate the influence of the concentration gradient on the affinity.

[0122] 3.2 Analyze the data: Flow cytometry data usually gives the fluorescence intensity and the number of cells in each experimental group. Calculate the binding rate of CAR-NK cells and tumor cells in each experimental group (for example, reflect the affinity through the ratio of co-expressed fluorescent labels).

[0123] 3.3 Calculate the IC50: The IC50 refers to the concentration of CAR-NK cells that can inhibit 50% of the maximum binding. Calculate the IC50: According to different concentrations of CAR-NK cells, plot the relationship curve between the affinity (binding rate) and the concentration. According to the fitted curve, the IC50 corresponds to the concentration of CAR-NK cells that makes the affinity (binding rate) reach 50%. Flow cytometry was performed on EpCAM-CAR-NK-92MI cells containing A1-scFv, EpCAM-CAR-NK-92MI cells containing A2-scFv, EpCAM-CAR-NK-92MI cells containing A3-scFv, EpCAM-CAR-NK-92MI cells containing A4-scFv, EpCAM-CAR-NK-92MI cells containing A5-scFv, EpCAM-CAR-NK-92MI cells containing A6-scFv, EpCAM-CAR-NK-92MI cells containing A7-scFv, EpCAM-CAR-NK-92MI cells containing A8-scFv, EpCAM-CAR-NK-92MI cells containing A9-scFv, EpCAM-CAR-NK-92MI cells containing A10-scFv, and EpCAM-CAR-NK-92MI cells containing A11-scFv, respectively. The test results are respectively referred to Figure 12 , Figure 13 , Figure 14 , Figure 15 , Figure 16 , Figure 17 , Figure 18 . The difference between A1-scFv to A11-scFv and A0-scFv is the substitution of an alanine residue in HCDR3. The Binding IC50 of EpCAM-CAR-NK-92MI cells containing A0-scFv is 45.3 nM (nanomoles per liter).

[0124] Referring to Figure 12 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A1-scFv is 86.21 nanomoles per liter; referring to Figure 13 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A2-scFv is 51.06 nanomoles per liter; referring to Figure 14 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A3-scFv is 94.86 nanomoles per liter; referring to Figure 15 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A4-scFv is 70.61 nanomoles per liter; referring to Figure 16 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A5-scFv is 103.6 nanomoles per liter; referring to Figure 17, the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A6-scFv is 88.79 nanomoles per liter; reference Figure 18 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A7-scFv is 48.73 nanomoles per liter. The Binding IC50 of EpCAM-CAR-NK-92MI cells containing A8-scFv is 60 - 110 nM. The Binding IC50 of EpCAM-CAR-NK-92MI cells containing A9-scFv is <80 nM. The Binding IC50 of EpCAM-CAR-NK-92MI cells containing A10-scFv is <81 nM. The Binding IC50 of EpCAM-CAR-NK-92MI cells containing A11-scFv is 60 - 110 nM.

[0125] reference Figure 19 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A21-CAR is 49.02 nanomoles per liter; reference Figure 20 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A22-CAR is 48.41 nanomoles per liter; reference Figure 21 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A23-CAR is 48.32 nanomoles per liter; reference Figure 22 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A24-CAR is 56.05 nanomoles per liter; reference Figure 23 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A25-CAR is 24.51 nanomoles per liter; reference Figure 24 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A26-CAR is 68.18 nanomoles per liter; reference Figure 25 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A27-CAR is 54.91 nanomoles per liter; reference Figure 26 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A28-CAR is 31.32 nanomoles per liter; reference Figure 27 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A29-CAR is 48.33 nanomoles per liter; reference Figure 28, the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A30-CAR is 53.22 nanomoles per liter; refer to Figure 29 , the Binding IC50 of EpCAM-CAR-NK-92MI cells containing A31-CAR is 43.12 nanomoles per liter.

[0126] Example 8: Co-culture of EpCAM-CAR-T cells containing different scFvs with HCT116 cells In this example, RTCA (Real-Time Cell Analyzer) was used to measure the cell killing experiment: 100 μL of HCT116 cell suspension with a density of 5x10 4 cells / mL was seeded into a special well plate with an electronic chip, cultured in an incubator, and the cell proliferation and adhesion were measured by the RTCA instrument. After the cells were completely adhered, the successfully transfected CAR-T cells were added to each well at different ratios, and the proliferation of HCT116 cells was continuously measured.

[0127] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratio of normal T cells to HCT116 cells was 4:1, CAR T cells containing A1-scFv were co-cultured with HCT116 cells, the effector-to-target ratio of CAR T cells containing A1-scFv to HCT116 cells was 4:1, and the culture results were referred to Figure 30 , refer to Figure 30 It can be seen that CAR T cells containing A1-scFv have a killing effect on HCT116 cells.

[0128] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratio of normal T cells to HCT116 cells was 2:1, CAR T cells containing A2-scFv were co-cultured with HCT116 cells, the effector-to-target ratio of CAR T cells containing A2-scFv to HCT116 cells was 2:1, and the culture results were referred to Figure 31 , refer to Figure 31 It can be seen that CAR T cells containing A2-scFv have a killing effect on HCT116 cells.

[0129] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratio of normal T cells to HCT116 cells was 1:1, CAR T cells containing A3-scFv were co-cultured with HCT116 cells, the effector-to-target ratio of CAR T cells containing A3-scFv to HCT116 cells was 1:1, and the culture results were referred to Figure 32 , refer to Figure 32It can be seen that CAR T cells containing A3-scFv have a killing effect on HCT116 cells.

[0130] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratio of normal T cells to HCT116 cells was 1:5, CAR T cells containing A4-scFv were co-cultured with HCT116 cells, the effector-to-target ratio of CAR T cells containing A4-scFv to HCT116 cells was 1:5, and the culture results were referred to Figure 33 and Figure 33 It can be seen that CAR T cells containing A4-scFv have a killing effect on HCT116 cells.

[0131] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratio of normal T cells to HCT116 cells was 1:5, CAR T cells containing A5-scFv were co-cultured with HCT116 cells, the effector-to-target ratio of CAR T cells containing A5-scFv to HCT116 cells was 1:5, and the culture results were referred to Figure 34 and Figure 34 It can be seen that CAR T cells containing A5-scFv have a killing effect on HCT116 cells.

[0132] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratios of normal T cells to HCT116 cells were 1:5 and 1:10, CAR T cells containing A6-scFv were co-cultured with HCT116 cells, the effector-to-target ratios of CAR T cells containing A6-scFv to HCT116 cells were 4:1, 2:1 and 1:1, and the culture results were referred to Figure 35 and Figure 35 It can be seen that CAR T cells containing A6-scFv have a killing effect on HCT116 cells.

[0133] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratios of normal T cells to HCT116 cells were 1:5 and 1:10, CAR T cells containing A7-scFv were co-cultured with HCT116 cells, the effector-to-target ratios of CAR T cells containing A7-scFv to HCT116 cells were 4:1, 2:1 and 1:1, and the culture results were referred to Figure 36 and Figure 36 It can be seen that CAR T cells containing A7-scFv have a killing effect on HCT116 cells.

[0134] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratios of normal T cells to HCT116 cells were 1:5 and 1:10, CAR T cells containing A8-scFv were co-cultured with HCT116 cells, the effector-to-target ratios of CAR T cells containing A8-scFv to HCT116 cells were 4:1, 2:1 and 1:1, and CAR T cells containing A8-scFv had a killing effect on HCT116 cells.

[0135] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratios of normal T cells to HCT116 cells were 1:5 and 1:10, CAR T cells containing A9-scFv were co-cultured with HCT116 cells, the effector-to-target ratios of CAR T cells containing A9-scFv to HCT116 cells were 4:1, 2:1 and 1:1, and CAR T cells containing A9-scFv had a killing effect on HCT116 cells.

[0136] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratios of normal T cells to HCT116 cells were 1:5 and 1:10, CAR T cells containing A10-scFv were co-cultured with HCT116 cells, the effector-to-target ratios of CAR T cells containing A10-scFv to HCT116 cells were 4:1, 2:1 and 1:1, and CAR T cells containing A10-scFv had a killing effect on HCT116 cells.

[0137] HCT116 cells were cultured alone, normal T cells were co-cultured with HCT116 cells, the effector-to-target ratios of normal T cells to HCT116 cells were 1:5 and 1:10, CAR T cells containing A11-scFv were co-cultured with HCT116 cells, the effector-to-target ratios of CAR T cells containing A11-scFv to HCT116 cells were 4:1, 2:1 and 1:1, and CAR T cells containing A11-scFv had a killing effect on HCT116 cells.

[0138] CCK8 assay for cell killing experiment: 100 μL of HCT116 cell suspension with a density of 5×10 4 cells / mL was seeded into a 96-well plate and cultured in an incubator. After the cells were completely adherent, the supernatant was discarded. Transfected CAR-T cells were added to each well at different ratios, and 10 μL of CCK8 solution was added simultaneously. After gently mixing, the absorbance value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader after incubation for a period of time. Table 6 summarizes that CAR T cells containing any one of A1-scFv to A11-scFv had a killing effect on HCT116 cells.

[0139] Table 6

[0140] Example 9: Killing effect of EpCAM-CAR-NK-92MI cells on tumor cells The experimental group was: EpCAM-CAR-NK-92MI cells and ovarian cancer RMG-I cells were co-cultured at a cell number ratio of 2:1, 4:1, and 8:1, and then the number of ovarian cancer RMG-I cells was calculated; the control group was: the number of ovarian cancer RMG-I cells was calculated after the ovarian cancer RMG-I cells were cultured alone, referring to Figure 37 , EpCAM-CAR-NK-92MI cells have a killing effect on ovarian cancer RMG-I cells, and as the number of EpCAM-CAR-NK-92MI cells increases, the killing effect on ovarian cancer RMG-I cells increases.

[0141] The experimental group was: EpCAM-CAR-NK-92MI cells and breast cancer MDA-MB-468 cells were co-cultured at a cell number ratio of 2:1, 4:1, and 8:1, and then the number of breast cancer MDA-MB-468 cells was calculated; the control group was: the number of breast cancer MDA-MB-468 cells was calculated after the breast cancer MDA-MB-468 cells were cultured alone, referring to Figure 38 , EpCAM-CAR-NK-92MI cells have a killing effect on breast cancer MDA-MB-468 cells, and as the number of EpCAM-CAR-NK-92MI cells increases, the killing effect on breast cancer MDA-MB-468 cells increases.

[0142] The experimental group was: EpCAM-CAR-NK-92MI cells and bladder cancer KU-19-19 cells were co-cultured at a cell number ratio of 2:1, 4:1, and 8:1, and then the number of bladder cancer KU-19-19 cells was calculated; the control group was: the number of bladder cancer KU-19-19 cells was calculated after the bladder cancer KU-19-19 cells were cultured alone, referring to Figure 39 , EpCAM-CAR-NK-92MI cells have a killing effect on bladder cancer KU-19-19 cells, and as the number of EpCAM-CAR-NK-92MI cells increases, the killing effect on bladder cancer KU-19-19 cells increases.

[0143] Cell viability detection (1) Digest the tumor cells in the logarithmic growth phase with trypsin, resuspend them in RPMI-1640 complete medium, and plate 600-2500 cells / 150 μL of cell suspension per well in a 96-well plate based on the growth rate of different tumor cells. Ensure that the cell confluence is about 80% after about 5-6 days of cell growth. Then, culture them in a cell culture incubator for 5 h.

[0144] (2) After the cells adhered, add successfully transfected CAR-NK cells in different proportions to each well and add 10 μL CCK8 solution. Mix gently. After incubation for a period of time, use an ELISA reader to measure the absorbance at 450 nm.

[0145] (3) Prepare MTS solution: Mix MTS and PMS in 9 volumes of 1640 culture medium (containing 2% FBS) at a ratio of 1:20 for later use; discard the cell culture medium in the 96-well plate and invert it on absorbent paper to remove the residual liquid. Add 100 μL of the prepared MTS solution to each well and incubate in a 37°C cell culture incubator away from light. During this period, observe the color of the reaction supernatant. When it turns "light brown", use a Biotek microplate reader to read the value at a wavelength of 490 nm (the OD490 nm reading should be between 0.5 and 0.7).

[0146] (4) Data analysis and processing: the blank control well readings were subtracted from the obtained readings, and then the relative cell viability value was obtained according to the cell absorbance value / (average absorbance value of the negative control group × 100%). GraphPad Prism 8 software was used to obtain a scatter plot and fit the cell viability curve according to nonlinear regression, and the corresponding IC50 value was calculated. Table 7 shows the results of CAR-NK killing (E:T=4:1).

[0147] Table 7

[0148] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. An EpCAM antibody or an antigen-binding portion thereof, characterized in that It comprises a light chain variable region VL and a heavy chain variable region VH, wherein the VL comprises a first light chain complementary determining region LCDR1, a second light chain complementary determining region LCDR2 and a third light chain complementary determining region LCDR3, and the VH comprises a first heavy chain complementary determining region HCDR1, a second heavy chain complementary determining region HCDR2 and a third heavy chain complementary determining region HCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO. 1, the amino acid sequence of the LCDR2 is shown in SEQ ID NO. 2, the amino acid sequence of the LCDR3 is shown in SEQ ID NO. 3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO. 4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO. 5, and the amino acid sequence of the HCDR3 is shown in the following formula: X1X2X3X4X5X6X7X8X9X 10 X 11 in X1 is selected from R and A, X2 is selected from S and A, X3 is selected from P and A, X4 is selected from Y and A, X5 is selected from G and A, X6 is selected from Y and A, X7 is selected from D and A, X8 is selected from E and A, X9 is selected from Y and A, X 10 is selected from G and A, and X 11 Selected from L and A.

2. The EpCAM antibody or antigen-binding portion thereof according to claim 1, characterized in that The amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO. 6 to SEQ ID NO. 16 and SEQ ID NO.

73.

3. The EpCAM antibody or antigen-binding portion thereof according to claim 1, characterized in that The amino acid sequence of the VH is as shown in any one of SEQ ID NO. 18 to SEQ ID NO. 28 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in any one of SEQ ID NO. 18 to SEQ ID NO.

28.

4. The EpCAM antibody or antigen-binding portion thereof according to claim 1, characterized in that The amino acid sequence of VL is as shown in SEQ ID NO. 17 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in SEQ ID NO.

17.

5. The EpCAM antibody or antigen-binding portion thereof according to claim 1, characterized in that The EpCAM antibody or its antigen-binding portion is in the form of scFv, and the scFv comprises an amino acid sequence as shown in any one of SEQ ID NO. 29 to SEQ ID NO.

39.

6. A first nucleic acid molecule, characterized in that The first nucleic acid molecule comprises a polynucleotide encoding the EpCAM antibody or antigen-binding portion thereof according to any one of claims 1 to 5.

7. A chimeric antigen receptor, characterized in that The invention comprises an antigen binding domain, a transmembrane domain and a signal transduction domain, wherein the antigen binding domain comprises the EpCAM antibody or the antigen binding portion thereof according to any one of claims 1 to 5.

8. The chimeric antigen receptor according to claim 7, wherein the chimeric antigen receptor comprises an amino acid sequence as shown in any one of SEQ ID NO. 40 to SEQ ID NO.

50.

9. A second nucleic acid molecule, characterized in that The second nucleic acid molecule comprises a polynucleotide encoding the chimeric antigen receptor of claim 7 or 8.

10. The second nucleic acid molecule according to claim 9, wherein the second nucleic acid molecule comprises a nucleic acid sequence as shown in any one of SEQ ID NO. 51 to SEQ ID NO.

61.

11. A carrier, characterized in that Comprising the second nucleic acid molecule of claim 6 or claim 9 or 10.

12. An immune cell, characterized in that: Comprising the chimeric antigen receptor of claim 7 or 8, the second nucleic acid molecule of claim 9 or 10, or the vector of claim 11.

13. The immune cell according to claim 12, characterized in that The immune cells include any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages; Preferably, the immune cells are more sensitive to colon cancer, lung cancer, breast cancer, gastric cancer, gastric cancer lung metastasis, lung cancer gastric metastasis, breast cancer gastric metastasis, and / or gastric cancer breast metastasis containing EpCAM gene mutations N120S, G222S, and / or S228Y.

14. A pharmaceutical composition, characterized in that Comprising the EpCAM antibody or antigen binding portion thereof according to any one of claims 1 to 5, the first nucleic acid molecule according to claim 6, the chimeric antigen receptor according to claim 7 or 8, the second nucleic acid molecule according to claim 9 or 10, the vector according to claim 11, or the immune cell according to claim 12 or 13.

15. Use of the EpCAM antibody or antigen-binding portion thereof according to any one of claims 1 to 5, the first nucleic acid molecule according to claim 6, the chimeric antigen receptor according to claim 7 or 8, the second nucleic acid molecule according to claim 9 or 10, the vector according to claim 11, or the immune cell according to claim 12 or 13 in the preparation of a medicament for treating a disease or condition associated with the expression of EpCAM.

16. The use according to claim 15, characterized in that The disease or disorder associated with the expression of EpCAM includes cancer or malignant tumor.

17. The use according to claim 15, characterized in that The diseases or conditions associated with the expression of EpCAM include hepatocellular carcinoma, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, head and neck cancer, squamous cell skin cancer, uterine endometrial cancer, and bladder cancer; preferably colon cancer, lung cancer, breast cancer, gastric cancer, gastric cancer lung metastasis, lung cancer gastric metastasis, breast cancer gastric metastasis, and / or gastric cancer breast metastasis containing EpCAM gene mutations N120S, G222S, and / or S228Y.

18. A method for preparing a cell expressing the chimeric antigen receptor of claim 7, comprising introducing a vector into the cell, wherein the vector comprises a polynucleotide encoding the chimeric antigen receptor.

19. The method of claim 18, wherein the cells comprise any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages.

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