EpCAM-targeted immune cell and medical application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an EpCAM (Epithelial Cell Amplifier)-targeted immune cell and medical application thereof. The invention provides application of immune cells in preparation of drugs for preventing or treating tumors. The immune cells are cells for adoptive immune cell therapy, and can target and kill EpCAM expression cells; the tumor expresses EpCAM and is a tumor with metastatic focus.

Description

Immune cells targeting EpCAM and their medical uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from Chinese patent application No. 202210656644.0 filed on June 10, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine technology, and in particular to immune cells targeting EpCAM and their medical uses. Background Art

[0004] The development of immunotherapy in recent years has profoundly transformed the field of cancer treatment, particularly immune checkpoint therapy, represented by PD1 / PDL1 pathway inhibitors, and adoptive cell therapy, represented by CAR-T. Adoptive cell therapies include TIL, NK, TCR-T, and CAR-T. Among them, CAR-T therapy targeting CD19 has achieved excellent clinical efficacy in B-cell tumors. To date, five CAR-T products have been approved by the FDA for the treatment of B-cell leukemia or lymphoma, and two CAR-T products have also been approved for marketing in China.

[0005] The concept of CAR (chimeric antigen receptors) was originally proposed by scientists such as Zelig Eshhar. CAR-T cells are capable of highly specific targeting of tumor antigens without relying on MHC (major histocompatibility complex) antigens. The CAR structure consists of an extracellular single-chain variable fragment (scFv) recognition region, a transmembrane region, a costimulatory domain, and an intracellular signaling domain. First-generation CARs contain only a single signaling unit, primarily derived from the CD3ζ or FcRγ subunit. Second-generation CARs build on the first-generation CARs by incorporating a costimulatory factor, possessing both a costimulatory signal and a signaling domain. Third-generation CAR-Ts incorporate two costimulatory factors, such as CD28 and 4-1BB, to enhance the anti-tumor effects of T cells. The fourth-generation CAR (TRUCK T cells) adds one or more constitutive or inducible expression components to the original antibody recognition region and signal transduction region, allowing CAR-T cells to express specific proteins, thereby enhancing the survival ability of CAR-T cells themselves, promoting T cell infiltration into tumor tissues, and resisting the tumor suppressive microenvironment.

[0006] CAR-T therapy has achieved remarkable efficacy in hematologic malignancies, exemplified by CD19CAR-T targeting the CD19 antigen. CD19CAR-T has been approved for the treatment of adult B-cell acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL), among other diseases. Most products have achieved complete remission rates exceeding 70% in clinical trials.

[0007] Solid tumor treatments have yet to achieve effective results. With advancements in tumor treatment, more and more studies have found that cancer recurrence and metastasis are the main causes of death in cancer patients, and controlling cancer recurrence and metastasis is key to prolonging the lives of cancer patients.

[0008] Epithelial cell adhesion molecule (EpCAM) is the main surface antigen associated with human cancer cells such as colon cancer, gastric cancer, breast cancer, ovarian cancer and pancreatic cancer. It can be highly expressed in various human epithelial tissues, cancers, progenitor cells and stem cells, and therefore has the potential to become an important target for anti-tumor immunotherapy.

[0009] Summary of the Invention

[0010] The present invention relates to the use of immune cells in preparing a drug for preventing or treating tumors;

[0011] The immune cells are cells used for adoptive immune cell therapy, which can target and kill cells expressing EpCAM;

[0012] The tumors expressed EpCAM and were metastatic.

[0013] The present invention also relates to a method for preventing or treating tumors in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the immune cells or pharmaceutical composition described above.

[0014] The present invention discovered for the first time that immune cells that can target and kill EpCAM expression can effectively treat tumors with metastases, and verified the application of specific CAR-T cells therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 shows the fluorescence intensity of metastatic lesions in mice after treatment with EpCAM-CAR-T or control in the CTC metastasis model;

[0017] Figure 2 shows the fluorescence intensity of metastatic foci in the lungs and livers of mice treated with EpCAM-CAR-T or a control in a CTC metastasis model;

[0018] Figure 3 shows the fluorescence intensity of metastatic lesions in the gastric cancer peritoneal metastasis model after treatment with EpCAM-CAR-T or control. DETAILED DESCRIPTION

[0019] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0020] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0021] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0022] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0023] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0024] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for a 2% concentration, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are also permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted.

[0025] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0026] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0027] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0028] The first aspect of the present invention relates to the use of immune cells in the preparation of drugs for preventing or treating tumors;

[0029] The immune cells are cells used for adoptive immune cell therapy, which can target and kill cells expressing EpCAM;

[0030] The tumors expressed EpCAM and were metastatic.

[0031] In some embodiments, the tumor is a digestive tract tumor, pancreatic cancer, ovarian cancer, or bladder cancer.

[0032] In some embodiments, the digestive tract tumor is gastric cancer or colorectal cancer.

[0033] In some embodiments, the tumor is gastric cancer with peritoneal metastasis.

[0034] In some embodiments, the tumor is colorectal cancer with liver metastases.

[0035] In some embodiments, the tumor is an advanced tumor.

[0036] In some embodiments, the tumor metastasizes via circulating tumor cells.

[0037] In the present invention, adoptive immune cell therapy includes NK therapy, LAK therapy, DC therapy, CIK therapy, TIL therapy, DC-CIK therapy, CAR-T therapy, TCR-T therapy, CAR-NK therapy or TCR-NK therapy.

[0038] In a more preferred embodiment, the immune cells are T cells, NK cells or DC cells.

[0039] In some embodiments, the T cell is any one of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a MAIT cell, or a γδ T cell.

[0040] In some embodiments, the immune cells are TCR-T, CAR-T, or CAR-NK cells.

[0041] In some embodiments, the immune cells are autologous immune cells.

[0042] In other embodiments, the immune cells are allogeneic immune cells.

[0043] In some preferred embodiments, the immune cell expresses a chimeric antibody receptor having an extracellular antigen recognition domain for recognizing the EpCAM antigen.

[0044] As used herein, "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor" or "chimeric immune receptor (CIR)". An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that can bind to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits a signal to cause activation or inhibition of a biological process.

[0045] In some embodiments, the chimeric antigen receptor comprises a hinge region, a transmembrane region, and an intracellular signaling region.

[0046] As used herein, a "region" or "domain" within a chimeric antigen receptor refers to a region within a polypeptide that can fold into a specific structure independently of other regions. These "regions" or "domains" can be sequences of murine or other animal origin, preferably human. Furthermore, unless otherwise specified or emphasized, "region" or "domain" should be understood to refer to a known sequence, which can be a full-length or partial active segment.

[0047] In some embodiments, the hinge region is selected from the hinge region of CD8, CD28, IgG1, IgG4, 4-1BB, ICOS, OX40, CD40, CD80, CD7, or CH3, CH2-CH3 constant region.

[0048] In some embodiments, the transmembrane region is selected from the transmembrane region of CD8a, CD28, CD4, ICOS, CD7, CD2, CD80, CD40, OX40, CD27, LFA-1, 4-1BB, ICOS, CD3ζ or CD3ε.

[0049] In some embodiments, the intracellular signaling region includes a CD3ζ signaling domain. In some embodiments, the intracellular signaling region further comprises a protein selected from the following table or its intracellular signaling region (or costimulatory region); in some embodiments, the intracellular signaling region further comprises one or more selected from the following proteins or their intracellular signaling regions: CD28, 4-1BB, OX40, ICOS, CD27, MYD88, KIR2DS2, DAP10, DAP12, CD3ζ, TLRs, CD2, LFA-1, CD8α, CD40, CD80, and CD3ε.

[0050] In some embodiments, the antigen recognition domain is a single-chain antibody (preferably scFv).

[0051] The single-chain antibody may be a chimeric, humanized or human antibody fragment that is capable of recognizing the EpCAM antigen binding domain.

[0052] In the present invention, the term "humanization" or "humanization treatment" refers to replacing an animal-derived (such as mouse-derived) antibody sequence with a human-derived antibody sequence, thereby reducing or eliminating the human anti-mouse antibody (HAMA) response. This replacement can be a framework replacement, for example, replacing the FR sequence in the variable region with a human one, and / or replacing the constant region of the antibody (if any) with a human one. This replacement can also be converting a mouse monoclonal antibody into a fully human antibody (ie, the CDR is also replaced) by chain replacement, using methods such as phage antibody library technology. It should be noted that during the humanization process, the replaced human sequence may include partial amino acid substitutions or additions and subtractions, so that the replaced sequence may not be an exact copy of the expressed human immunoglobulin sequence or germline gene sequence. The antibody thus produced may be referred to as a human-mouse chimeric antibody, a humanized antibody, or a fully human antibody.

[0053] In some embodiments, the sc-Fv comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NOs: 1 to 3, and light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NOs: 4 to 6, respectively.

[0054] As used herein, the term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to a region comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope. In another specific embodiment, the CDR region or CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by IMGT.

[0055] In some embodiments, the sc-Fv comprises a heavy chain variable region shown in any one of SEQ ID NOs: 7 to 11 (preferably SEQ ID NO: 8) and a light chain variable region shown in any one of SEQ ID NOs: 12 to 15 (preferably SEQ ID NO: 13).

[0056] In the present invention, the term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0057] In the present invention, the term "connector peptide" can be a flexible or rigid peptide, for example, consisting of a repeated GGGGS amino acid sequence or a variant thereof, for example, a variant with 1 to 4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other connecting peptides that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0058] In some embodiments, the number of amino acids in the connecting peptide is 1 to 30; can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5 to 20.

[0059] In some embodiments, the amino acids of the connecting peptide are nonsense polypeptides that do not have additional functions other than connection (eg, protein localization, enzyme cleavage sites, etc.).

[0060] In some embodiments, the connecting peptide is a flexible connecting peptide;

[0061] In some embodiments, the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.

[0062] In some embodiments, the amino acid sequence of the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.

[0063] Modified forms of single-chain antibodies are also within the scope of protection of the present invention, for example, those modified by covalent attachment of polyethylene glycol or other suitable polymers. Variants of single-chain antibodies are also within the scope of the present invention, wherein the variants of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 may each comprise up to three amino acid mutations (e.g., substitutions, deletions, or additions of one, two, or three amino acids, or any combination thereof) compared to any of the complementary determining region combinations shown in SEQ ID NOs: 1 to 6; preferably, the mutations are conservative mutations. "Conservative substitutions" refer to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity, etc.), allowing frequent changes without altering the biological activity of the protein.

[0064] The substitutions generally considered as conservative substitutions are the substitutions of aliphatic amino acids Ala, Val, Leu and Ile, the interchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the substitution between amide residues Asn and Gln, the exchange of basic residues Lys and Arg, and the substitution between aromatic residues Phe and Tyr. It is known to those skilled in the art that, in general, single amino acid substitutions in the non-essential regions of a polypeptide do not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)). In addition, substitutions of amino acids with similar structure or function are unlikely to destroy the biological activity.

[0065] Variants of SEQ ID NOs: 7-15 are also within the scope of the present invention. Variants may have, for example, 90% to 99.9% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth in SEQ ID NOs: 7-15. Variants may also have modifications or conservative substitutions as described above.

[0066] In a specific embodiment, the chimeric antigen receptor targeting EpCAM (abbreviated as EpCAM-CAR) comprises an antigen binding region, a CD28 hinge region, a CD28 transmembrane region, and an intracellular signaling domain.

[0067] The antigen binding region is a humanized scFv antibody that binds to the EpCAM antigen.

[0068] The VH sequence of the humanized antibody is:

[0069] The sequence of the G4S x3 linker region is: GGGGSGGGGSGGGGS;

[0070] The VL sequence of the humanized antibody is:

[0071] The sequence of the CD28 hinge region is:

[0072] The sequence of the CD28 transmembrane region is: FWVLVVVGGVLACYSLLVTVAFIIFWV;

[0073] The sequence of the CD28 costimulatory signal region is:

[0074] The sequence of the CD3ζ costimulatory signal region is:

[0075] Specifically, the amino acid sequence of EpCAM-CAR is shown in SEQ ID NO:16.

[0076] The second aspect of the present invention relates to a method for preventing or treating tumors in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the immune cells or pharmaceutical composition described above.

[0077] The tumor defined in the first aspect of the present invention is also applicable to the second aspect.

[0078] It should be understood that the envisioned treatment methods will also include the administration of other immunotherapeutic entities, particularly preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic E. coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), chemotherapy drugs, antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell transplants (e.g., allogeneic or autologous), and tumor-targeted cytokines (e.g., NHS-IL12, IL-12 coupled to a tumor-targeted antibody or fragment thereof). In some embodiments, the envisioned treatment methods further include radiotherapy of the patient. In some embodiments, the envisioned treatment methods further include surgery of the patient, such as tumor resection surgery.

[0079] The method of administration may be intravenous administration and / or intracavitary administration (preferably intraperitoneal administration or intravesical administration).

[0080] A "patient" is a mammal, including but not limited to humans, monkeys, pigs and other farm animals, sports animals, pets, primates, horses, dogs, cats, giant pandas, rodents (including mice, rats, guinea pigs), etc.

[0081] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0082] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0083] For the preparation and characterization of the effects of humanized antibodies against EpCAM, as well as the construction of CAR-T cells targeting EpCAM, please refer to the Chinese patent application with publication number CN113527491A and publication date 2021-10-22. All documents mentioned in the present invention are cited in this application as references, just as if each document was cited as a reference individually. Unless it conflicts with the invention objectives and / or technical solutions of the present application, the cited documents involved in the present invention are cited in their entirety and for all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but only to the extent that the present invention can be implemented. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0084] Example 1 Clinical study of autologous T cells modified with chimeric antigen receptor targeting EpCAM for advanced gastric cancer

[0085] Study Drug

[0086] Autologous T cells modified with a chimeric antigen receptor targeting EpCAM, codenamed EpCAM-CAR-T, contain an antigen recognition domain, including a heavy chain variable region and a light chain variable region. The sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and the sequence of the light chain variable region is shown in SEQ ID NO: 13. The amino acid sequence of the chimeric antigen receptor of EpCAM-CAR-T is shown in SEQ ID NO: 16.

[0087] 1.2 Administration

[0088] Administer intravenously.

[0089] 1.3 Experimental design

[0090] This study is a multicenter, single-arm, open-label, dose-escalation clinical study. This study is an open-label clinical study to evaluate the safety and efficacy of EpCAM-CAR-T in the treatment of advanced gastric cancer. The recommended starting dose is 3×10 5 cells / kg, and the dose was escalated according to the 3+3 dose escalation principle.

[0091] 1.4 Test results

[0092] This study has enrolled 6 patients with advanced gastric cancer, and histopathological staining showed that the expression rate of EpCAM was 10%-90%. 5 cells / kg or 1×10 6 The EpCAM-CAR-T therapy with 100 cells / kg was safe for patients without dose-limiting toxicity. Efficacy evaluation showed that 5 patients had disease control, with a disease control rate (DCR) of 83.3%, of which 2 patients had partial remission (PR).

[0093] Among them, 3×10 5 The second patient in the 50 cells / kg dose group was a 40-year-old male with antral cancer involving the duodenum and pancreatic head. The pathological diagnosis was poorly differentiated adenocarcinoma (antral), with some cases being signet ring cell carcinoma. He underwent laparoscopic exploration and gastrojejunostomy in January 2021. After surgery, he received multiple lines of treatment, including single-agent S-1a, 7 cycles of FTP, FOLFIRI chemotherapy, and apatinib mesylate tablets, but none of them were effective. On November 16, 2021, he received an infusion of 3×10 5 / kg CAR-T cell therapy, no CRS or ICANS occurred after infusion. The efficacy evaluation results of the patients are shown in the following table. The patients were still alive 60 weeks after administration:

[0094] 1×10 6 The first patient in the cells / kg dose group was a 50-year-old male with gastric body adenocarcinoma. He underwent left gastric artery angiography and intra-arterial chemoembolization (raltitrexed regimen) in May 2021. He was not suitable for radical surgery. On February 15, 2022, MRI enhancement showed gastric cancer with metastasis to the gastroepiploic sac and para-antral lymph nodes and multiple intrahepatic nodules. The patient was considered to have disease progression after second-line treatment. On June 8, 2021, 1×10 6The patient's efficacy evaluation results are shown in the following table. The investigator determined that the patient had recovered to the point of being eligible for radical surgery 24 weeks after dosing and underwent radical gastrectomy on January 3, 2023. The patient was still alive 52 weeks after dosing:

[0095] Example 2 Clinical study of autologous T cells modified with chimeric antigen receptor targeting EpCAM for advanced digestive system malignancies

[0096] 2.1 Study Drug

[0097] Autologous T cells modified with a chimeric antigen receptor targeting EpCAM, codenamed EpCAM-CAR-T, contain an antigen recognition domain, including a heavy chain variable region and a light chain variable region. The heavy chain variable region sequence is shown in SEQ ID 8, and the light chain variable region sequence is shown in SEQ ID 13. The amino acid sequence of the chimeric antigen receptor of EpCAM-CAR-T is shown in SEQ ID NO: 16.

[0098] 2.2 Administration

[0099] Administer intravenously.

[0100] 2.3 Experimental design

[0101] This study is a multicenter, single-arm, open-label, dose-escalation clinical study. This study is an open-label clinical study to evaluate the safety and efficacy of EpCAM-CAR-T in the treatment of advanced gastrointestinal tumors. The recommended starting dose is 3×10 5 cells / kg, and the dose was escalated according to the 3+3 dose escalation principle.

[0102] 2.4 Test results

[0103] Three patients with advanced gastrointestinal malignancies were enrolled, all with colorectal cancer and multiple metastases. Histopathological staining showed that the EpCAM expression rate was 80%-90%. 5 28 days after administration of EpCAM-CAR-T cells / kg, the three patients had good safety and no dose-limiting toxic reactions occurred. Efficacy evaluation showed that two of the three patients had disease control, with a disease control rate (DCR) of 66.7%.

[0104] Example 3 Blocking effect of autologous T cells modified with chimeric antigen receptor targeting EpCAM on metastatic tumors in vivo

[0105] In order to demonstrate the blocking effect of EpCAM-CAR-T on CTC metastasis (the amino acid sequence of the chimeric antigen receptor of EpCAM-CAR-T is shown in SEQ ID NO: 16), we constructed a pharmacodynamic model simulating CAR-T treatment of CTC metastasis. First, HCT116 cells without fluorescent labeling were subcutaneously inoculated into M-NSG mice. When the tumor grew to 100-150 mm 3 After that, EpCAM-CAR-T cells, unT, or DPBS were infused back through the tail vein. Two days after CAR-T infusion, mice were inoculated with CTC cells (simulated with HCT116-Luc) through the tail vein to verify whether EpCAM-CAR-T cells can eliminate CTCs in vivo and block their formation of metastases. The results are shown in Figures 1A and 1B. The results show that there is no metastatic fluorescence in the CAR-T treatment group, while multiple metastases were formed in the bodies of mice in the DPBS and unT groups, and the fluorescence intensity is significantly higher than that in the treatment group. In order to determine whether CTCs have formed metastases in key organs and the therapeutic effect of CAR-T, at the end of the study, we performed ex vivo imaging analysis of the lungs and liver. The results showed that the unT group and the DPBS group had strong fluorescence signals in the liver and lungs, while the EpCAM-CAR-T treatment group had no obvious fluorescence signals in the lungs and liver, as shown in Figure 2. The above results show that EpCAM-CAR-T cells can eliminate CTCs and prevent them from forming metastatic lesions in key organs.

[0106] Example 4 Study on the Treatment of Gastric Cancer Peritoneal Metastasis with Autologous T Cells Modified with Chimeric Antigen Receptor Targeting EpCAM

[0107] To investigate whether EpCAM-CAR-T (the amino acid sequence of the chimeric antigen receptor of EpCAM-CAR-T is shown in SEQ ID NO: 16) can eliminate gastric cancer peritoneal metastases and whether the chemotherapy drug docetaxel can enhance the efficacy of CAR-T, we established a gastric cancer peritoneal metastasis model based on the gastric cancer cell line MKN-45-luc. Model construction: Human gastric cancer cells MKN-45-luc were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Cells were cultured for ten generations before being inoculated into the peritoneal cavity of mice. Approximately 1.5×10 6 MKN-45-luc cells were resuspended in PBS and inoculated into NCG mice via intraperitoneal injection in a volume of 100 μL. Grouping and administration: 7 days after inoculation, the growth of peritoneal tumor cells was detected using a small animal in vivo imaging fluorescence instrument. Six groups were randomly divided according to body weight and tumor size (fluorescence intensity), with 5 mice per group. The day of grouping was defined as day 0. The grouping and dosing schedule are shown in the following table:

[0108] The results of the study, as shown in Figure 3, show that compared with control T cells (unT), the 0.5M and 2M EpCAM-CAR-T cell treatment groups effectively inhibited tumor growth, with significantly reduced tumor fluorescence values. The efficacy was positively correlated with dose, particularly in the 2M dose group, where the tumor inhibition effect was particularly significant. A single treatment with 1.5mg / kg docetaxel did not produce significant efficacy. Furthermore, we observed that the combination of 1.5mg / kg docetaxel with 0.5M EpCAM-CAR-T cells enhanced the tumor inhibition effect of CAR-T, with tumor fluorescence values ​​lower than those in the 0.5M EpCAM-CAR-T treatment group alone. These results demonstrate that EpCAM-CAR-T can effectively inhibit the growth of peritoneal metastases of gastric cancer cells MKN45-Luc, and that the combination with docetaxel can enhance the efficacy of EpCAM-CAR-T.

[0109] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. The use of immune cells in the preparation of drugs for preventing or treating tumors; The immune cells are cells used for adoptive immune cell therapy, which can target and kill cells expressing EpCAM; The tumors expressed EpCAM and were metastatic.

2. The use according to claim 1, wherein the tumor is a digestive tract tumor, pancreatic cancer, ovarian cancer or bladder cancer.

3. The use according to claim 2, wherein the digestive tract tumor is gastric cancer or colorectal cancer. The method according to claim 3 , wherein the tumor is gastric cancer with peritoneal metastasis. The use according to claim 3 , wherein the tumor is colorectal cancer with liver metastasis. The use according to claim 1 , wherein the immune cells are T cells, NK cells or DC cells. 7 . The use according to claim 1 , wherein the cell expresses a chimeric antigen receptor having an extracellular antigen recognition domain for recognizing EpCAM antigen.

8. The use according to claim 7, wherein the antigen recognition domain comprises sc-Fv, preferably comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NOs: 1 to 3, and light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NOs: 4 to 6, respectively. 9 . The use according to claim 8 , wherein the sc-Fv comprises a heavy chain variable region shown in any one of SEQ ID NOs: 7 to 11 and a light chain variable region shown in any one of SEQ ID NOs: 12 to 15. 10 . The use according to claim 9 , wherein the sc-Fv comprises the heavy chain variable region shown in SEQ ID NO: 8 and the light chain variable region shown in SEQ ID NO:

13.

11. The use according to claim 9, wherein the chimeric antigen receptor amino acid sequence is shown as SEQ ID NO:

16.

12. A method for preventing or treating a tumor in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the immune cell or pharmaceutical composition described above; The immune cell and the tumor are as defined in any one of claims 1 to 11.

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