Chimeric antigen receptor immune cell specifically combined with Claudin18.2 and application of chimeric antigen receptor immune cell

Abstract

The invention relates to a chimeric antigen receptor immune cell specifically combined with Claudin18.2 and application of the chimeric antigen receptor immune cell. Specifically, the invention provides a CAR-T cell taking a Claudin18.2 targeting nano antibody as an antigen binding domain, and the CAR-T cell can be used for targeting Claudin18.2 positive tumor cells in a high-specificity manner; the CAR-T cell co-expresses an extracellular domain of a TGF-beta receptor and an optional intracellular signal domain of an IL-7 receptor, TGF-beta induced inhibition signals can be effectively blocked or reduced, and amplification, survival and durability of immune cells are promoted, so that specific killing of the CAR-T cell on tumor cells is enhanced. The chimeric antigen receptor immune cell disclosed by the invention can be used for treating Claudin18.2 positive tumors.

Description

Technical Field

[0001] This invention relates to the field of cell therapy, and more specifically to chimeric antigen receptor immune cells that specifically bind to Claudin18.2 and their applications. Background Technology

[0002] Claudin family proteins are widely distributed in the tight junction structures of epithelial cells. Claudin 18.2 is a membrane protein with four transmembrane regions: the N-terminus and C-terminus are located in the cytoplasm, while two extracellular loop regions can serve as specific targets for targeted therapy. In normal tissues, Claudin 18.2 is specifically expressed in highly differentiated epithelial cells of the stomach. Besides gastric cancer, Claudin 18.2 is also expressed in a high proportion of tumor cells in cholangiocarcinoma, ovarian cancer, lung cancer, esophageal cancer, and pancreatic cancer.

[0003] Chimeric antigen receptor T cell (CAR T) therapy is a novel immunotherapy method targeting specific antigens on the surface of tumor cells. Currently, it is being used to develop cell therapies for hematologic malignancies and solid tumors. While immunotherapy has shown remarkable efficacy in hematologic malignancies, its efficacy in solid tumors is less than ideal. CAR-T therapy for solid tumors faces several major challenges: the lack of safe tumor-specific antigen (TSA) targets, tumor heterogeneity, CAR-T cell infiltration, immunosuppression in the tumor microenvironment, and endogenous T cell inhibitory signals, all of which are detrimental to immune cell survival and thus inhibit the effectiveness of immunotherapy.

[0004] Therefore, there is an urgent need in this field to develop more effective and sustained immunotherapies targeting tumors that express Claudin18.2. Summary of the Invention

[0005] The purpose of this invention is to provide a more effective and sustained immunotherapy for tumors that target the expression of Claudin18.2.

[0006] In a first aspect of the invention, an engineered immune cell is provided, said engineered immune cell expressing the following exogenous proteins:

[0007] (a) A chimeric antigen receptor (CAR) wherein the antigen-binding domain of the CAR comprises a nanobody (VHH) targeting Claudin18.2; and

[0008] (b) Signaling proteins comprising: an extracellular domain of an immunosuppressive receptor, a transmembrane domain, and optionally an intracellular domain of an immunostimulatory receptor.

[0009] In another preferred embodiment, the signaling protein comprises: an extracellular domain of an immunosuppressive receptor, a transmembrane domain, and an intracellular domain of an immunostimulatory receptor.

[0010] In another preferred embodiment, the signaling protein comprises an extracellular domain of an immunosuppressive receptor and a transmembrane domain (i.e., an intracellular domain that does not contain an immunostimulatory receptor).

[0011] In another preferred embodiment, the immune cells are selected from the group consisting of T cells, NK cells, NKT cells, or combinations thereof.

[0012] In another preferred embodiment, the immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or combinations thereof.

[0013] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0014] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0015] (ii) Chimeric antigen receptor γδ T cells (CAR-T cells);

[0016] (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);

[0017] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0018] In another preferred embodiment, the immunosuppressive receptor is a TGF-β receptor.

[0019] In another preferred embodiment, the TGF-β receptor is a TGFBR2 and / or TGFBR1 receptor.

[0020] In another preferred embodiment, the immune agonist receptor is selected from the group consisting of IL-7 receptor, IL-15 receptor, IL-12 receptor, IL-2 receptor, IL-18 receptor, IL-21 receptor, or a combination thereof.

[0021] In another preferred embodiment, the immune agonist receptor is an IL-7 and / or IL-2 receptor.

[0022] In another preferred embodiment, the immune agonist receptor is the IL-7 receptor.

[0023] In another preferred embodiment, the transmembrane region is derived from an immunosuppressive receptor, an immunostimulatory receptor, or a combination thereof.

[0024] In another preferred embodiment, the CAR has the structure shown in formula Ia, Ib or Ic:

[0025] L1-VHH-H-TM1-C-CD3ζ (Ia)

[0026] L1-VHH-H-TM1-C-CD3ζ-A-E1 (Ib)

[0027] L1-VHH-H-TM1-C-CD3ζ-A-E1-A-E2(Ic)

[0028] In the formula,

[0029] L1 is either absent or a signal peptide sequence;

[0030] VHH is a nanobody targeting Claudin18.2;

[0031] H represents the area with no hinge or no connection.

[0032] TM1 is a transmembrane domain;

[0033] C represents the domain with no or no co-stimulatory signal;

[0034] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0035] A is a self-cleaving 2A peptide;

[0036] E1 and E2 are each independently signaling proteins (including wild type or its mutant / modified forms);

[0037] Each of the "-" symbols independently represents a linking peptide or a peptide bond.

[0038] In another preferred embodiment, the VHH includes a complementary determination region (CDR) selected from the group consisting of:

[0039] (1) CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7;

[0040] (2) CDR1 shown in SEQ ID NO:8, CDR2 shown in SEQ ID NO:9, and CDR3 shown in SEQ ID NO:10.

[0041] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0042] In another preferred embodiment, the nanobody that specifically binds to Claudin18.2 includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0043] In another preferred embodiment, the amino acid sequence of the VHH is as shown in any one of SEQ ID NO:1-4.

[0044] In another preferred embodiment, the CDR region of the nanobody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to any one of SEQ ID NO:1-4.

[0045] In another preferred embodiment, the amino acid sequence of the CDR region of the nanobody VHH chain contains one or more amino acid substitutions compared to any of SEQ ID NO:1-4, preferably conserved amino acid substitutions.

[0046] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to specifically bind to Claudin18.2.

[0047] In another preferred embodiment, the Claudin18.2 is Claudin18.2 of a human or non-human mammal.

[0048] In another preferred embodiment, the Claudin18.2 is a human Claudin18.2.

[0049] In another preferred embodiment, L1 is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or combinations thereof.

[0050] In another preferred embodiment, L1 is the signal peptide of the CD8 protein.

[0051] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG4, or a combination thereof.

[0052] In another preferred embodiment, H is the hinge region of the CD8 protein.

[0053] In another preferred embodiment, the H also carries a Flag tag sequence.

[0054] In another preferred embodiment, the TM1 is selected from the transmembrane regions of the following histones: CD28, CD137, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0055] In another preferred embodiment, TM1 is the transmembrane region of the CD28 and CD137 proteins.

[0056] In another preferred embodiment, C is selected from the co-stimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0057] In another preferred embodiment, C is a co-stimulatory domain of the CD28 protein.

[0058] In another preferred embodiment, C is a co-stimulatory domain of the 4-1BB protein.

[0059] In another preferred embodiment, A is T2A, P2A, or a combination thereof.

[0060] In another preferred embodiment, E can be constitutive expression or inducible expression.

[0061] In another preferred embodiment, the sequence of the CAR is as shown in any one of SEQ ID NO:27-29, 31-36.

[0062] In another preferred embodiment, the signaling protein has the structure shown in Formula V:

[0063] L2-Z1-TM2-Z2(V)

[0064] In the formula,

[0065] L2 is either absent or a signal peptide sequence;

[0066] Z1 is the extracellular domain of an immunosuppressive receptor;

[0067] TM2 is the transmembrane region;

[0068] Z2 is an intracellular domain of receptors that are absent or do not have immune agonist receptors.

[0069] Each of the "-" symbols independently represents a linking peptide or a peptide bond.

[0070] In another preferred embodiment, the engineered immune cells contain two or more different signaling proteins.

[0071] In another preferred embodiment, the different signaling proteins have different Z1, TM2, and / or Z2.

[0072] In another preferred embodiment, the different signaling proteins are expressed independently or in tandem.

[0073] In another preferred embodiment, the different signaling proteins are expressed in tandem via self-cleaving of the 2A peptide.

[0074] In another preferred embodiment, the immunosuppressive receptor is selected from the group consisting of TGF-β receptors and their subtypes or combinations thereof (including wild type, or mutants / modifiers thereof).

[0075] In another preferred embodiment, the TGF-β receptor is a TGFBR2 and a TGFBR1 receptor.

[0076] In another preferred embodiment, the TGF-β receptor is a TGFBR2 receptor.

[0077] In another preferred embodiment, the TGFBR2 receptor includes either TGFBR2 receptor isoform A or TGFBR2 receptor isoform B.

[0078] In another preferred embodiment, L2 is the signal peptide sequence of the TGFBR2 or TGFBR1 receptor.

[0079] In another preferred embodiment, the Z1 has the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:18.

[0080] In another preferred embodiment, TM2 is selected from the transmembrane regions of the following histones: IL-7 receptor, IL-15 receptor, IL-12 receptor, IL-2 receptor, IL-18 receptor, IL-21 receptor, or a combination thereof.

[0081] In another preferred embodiment, TM2 is the transmembrane region of the IL-7 receptor and its subtypes or combinations thereof (including wild type, or its mutants / modifiers).

[0082] In another preferred embodiment, TM2 is selected from the transmembrane region of the IL-7 receptor.

[0083] In another preferred embodiment, TM2 has the amino acid sequence shown in SEQ ID NO:21.

[0084] In another preferred embodiment, TM2 also includes a transmembrane segment and a pilomembrane segment connected to the transmembrane segment.

[0085] In another preferred embodiment, the juxtamembrane segment is located extracellularly.

[0086] In another preferred embodiment, the length of the near-membrane segment is 10-50 aa.

[0087] In another preferred embodiment, the transmembrane segment and the near-membrane segment originate from the same protein.

[0088] In another preferred embodiment, TM2 is selected from the transmembrane region of an IL-7 receptor mutant.

[0089] In another preferred embodiment, the TM2 has the amino acid sequence shown in SEQ ID NO:22.

[0090] In another preferred embodiment, TM2 comprises a transmembrane segment and a juxtamembrane segment of the IL-7 receptor.

[0091] In another preferred embodiment, the TM2 has the amino acid sequence shown in SEQ ID NO:23.

[0092] In another preferred embodiment, TM2 is the transmembrane region of the IL-2 receptor γ chain.

[0093] In another preferred embodiment, the TM2 has the amino acid sequence shown in SEQ ID NO:24 or 37.

[0094] In another preferred embodiment, the immune agonist receptor is selected from the group consisting of IL-7 receptors and their subtypes or combinations thereof (including wild type, or mutants / modifiers thereof).

[0095] In another preferred embodiment, the IL-7 receptor is IL-7Rα.

[0096] In another preferred embodiment, Z2 is an intracellular domain of IL-7Rα.

[0097] In another preferred embodiment, the Z2 has the amino acid sequence shown in SEQ ID NO:19.

[0098] In another preferred embodiment, the engineered immune cells have one or more of the following characteristics:

[0099] (a) The cells express a chimeric antigen receptor CAR that targets Claudin18.2; and

[0100] (b) When exposed to an inducer, the engineered immune cells induce the expression of signaling proteins.

[0101] In another preferred embodiment, in the CAR cells, the CAR and the signaling protein are expressed in tandem.

[0102] In another preferred embodiment, in the CAR cells, the CAR and the signaling protein are expressed independently.

[0103] In another preferred embodiment, the chimeric antigen receptor (CAR) is located on the cell membrane of the engineered immune cell.

[0104] In another preferred embodiment, the signaling protein is located on the cell membrane of the engineered immune cell.

[0105] In a second aspect of the invention, a chimeric antigen receptor (CAR) construct is provided, the CAR having the structural formula shown in formula Ia or Ib below.

[0106] L1-VHH-H-TM1-C-CD3ζ-A-E1(Ib)

[0107] L1-VHH-H-TM1-C-CD3ζ-A-E1-A-E2(Ic)

[0108] In the formula,

[0109] L1 is either absent or a signal peptide sequence;

[0110] VHH is a nanobody targeting Claudin18.2;

[0111] H represents the area with no hinge or no connection.

[0112] TM1 is a transmembrane domain;

[0113] C represents the domain with no or no co-stimulatory signal;

[0114] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0115] A is a self-cleaving 2A peptide;

[0116] E1 and E2 are each independently signaling proteins (including wild type or its mutant / modified forms);

[0117] Each of the "-" symbols independently represents a linking peptide or a peptide bond.

[0118] In another preferred embodiment, the sequence of the CAR is as shown in any one of SEQ ID NO:27-29, 31-36.

[0119] In a third aspect of the invention, a polynucleotide is provided that encodes a CAR construct as described in the second aspect of the invention.

[0120] In another preferred embodiment, the polynucleotide is DNA, RNA, or a combination thereof.

[0121] In a fourth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the third aspect of the invention.

[0122] In another preferred embodiment, the vector is selected from the group consisting of plasmids, viral vectors, transposons, or combinations thereof.

[0123] In the fifth method of the present invention, a method for preparing the engineered immune cells described in the first aspect of the present invention is provided, comprising the following steps:

[0124] (a) Provide an immune cell to be modified; and

[0125] (b) The immune cells are modified to express CAR molecules and exogenous signaling proteins, thereby obtaining the engineered immune cells described in the first aspect of the present invention.

[0126] In another preferred embodiment, step (b) includes:

[0127] (b1) Introducing the first expression vector expressing the CAR into the immune cells; and

[0128] (b2) The second expression vector expressing the signaling protein is introduced into the immune cells;

[0129] The step (b1) may be performed before, after, simultaneously with, or alternately with step (b2).

[0130] In another preferred embodiment, the first expression vector and the second expression vector are the same or different expression vectors.

[0131] In another preferred embodiment, the expression vector includes a viral vector and a plasmid.

[0132] In another preferred embodiment, the CAR has the structure shown in formula Ia, Ib or Ic.

[0133] In another preferred embodiment, when the immune cells to be modified in step (a) have already expressed CAR, step (b) includes (b2) introducing a second expression vector expressing a signaling protein into the immune cells.

[0134] In another preferred embodiment, the transcription directions of the first expression vector and the second expression vector are in the same direction (→→), opposite direction (→←), or opposite direction (←→).

[0135] In another preferred embodiment, the first expression vector and the second expression vector are located on the same or different vectors.

[0136] In another preferred embodiment, the first expression vector and the second expression vector are located in the same vector.

[0137] In another preferred embodiment, when the first and second expression vectors are located in the same vector, a third expression vector for expressing the self-cleaved 2A peptide is further included between the first and second expression vectors.

[0138] In another preferred embodiment, the self-cleaving 2A peptide is T2A, P2A, or a combination thereof.

[0139] In another preferred embodiment, the vector is a viral vector, preferably containing first and second expression vectors in tandem.

[0140] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

[0141] In another preferred embodiment, the vector is a lentiviral vector.

[0142] In a sixth aspect of the invention, a formulation is provided comprising the engineered immune cells described in the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0143] In another preferred embodiment, the formulation contains the CAR-T cells, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0144] In another preferred embodiment, the formulation is a liquid formulation.

[0145] In another preferred embodiment, the dosage form of the preparation includes an injection.

[0146] In another preferred embodiment, the concentration of the engineered immune cells (such as CAR-T cells) in the formulation is 1 × 10⁻⁶. 3 -1×10 8 cells / mL, preferably 1×10⁻⁶. 4 -1×10 7 Cells / mL.

[0147] In a seventh aspect of the invention, a kit is provided for preparing engineered immune cells as described in the first aspect of the invention, the kit comprising a container, and the following:

[0148] (1) A first nucleic acid sequence, the first nucleic acid sequence containing a first expression cassette for expressing the CAR; and

[0149] (2) A second nucleic acid sequence containing a second expression cassette for co-expression of a signaling protein.

[0150] In another preferred embodiment, the first and second nucleic acid sequences are independent or linked.

[0151] In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

[0152] In another preferred embodiment, the first and second nucleic acid sequences are located on the same or different vectors.

[0153] In another preferred embodiment, the first and second nucleic acid sequences are located in the same vector.

[0154] In another preferred embodiment, when the first and second nucleic acid sequences are located in the same vector, a third nucleic acid sequence is further included between the first and second nucleic acid sequences, the third nucleic acid sequence containing a third expression cassette for expressing the self-cleaved 2A peptide.

[0155] In another preferred embodiment, the self-cleaving 2A peptide is P2A, T2A, or a combination thereof.

[0156] In another preferred embodiment, the vector is a viral vector, preferably containing first and second nucleic acid sequences in tandem.

[0157] In an eighth aspect of the invention, the use of the engineered immune cells described in the first aspect of the invention is provided for the preparation of medicaments or formulations for the prevention and / or treatment of cancer or tumors.

[0158] In another preferred embodiment, the tumor expresses Claudin18.2.

[0159] In another preferred embodiment, the tumor is selected from the group consisting of solid tumors, hematomas, or combinations thereof.

[0160] In another preferred embodiment, the tumor is a solid tumor.

[0161] In another preferred embodiment, the tumor is selected from the group consisting of: gastric cancer, bile duct cancer, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, or combinations thereof.

[0162] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0163] Figure 1 A schematic diagram of the CAR-T of the present invention is shown.

[0164] Figure 2 , Figure 3 , Figure 4 The results of CAR expression and phenotypic detection in different donor cells are shown.

[0165] Figure 5 The results of reporter gene detection of the TGFβ signaling pathway are shown.

[0166] Figure 6The transient killing ability of each group of CAR-T cells was demonstrated.

[0167] Figure 7 The cytokine secretion levels of CAR-T cells in each group were shown.

[0168] Figure 8 , Figure 9 , Figure 10 The study demonstrated the sustained killing ability of each group of CAR-T cells after repeated antigen stimulation.

[0169] Figure 11 This demonstrates the cell expansion capacity of CAR-T cells after multiple rounds of antigen stimulation.

[0170] Figure 12 The expression rate of CAR on the surface of CAR-T cells was shown after multiple rounds of antigen stimulation.

[0171] Figure 13 The results of in vivo tumor-killing experiments of each group of CAR-T cells are shown.

[0172] Figure 14 The results of the FACS method for detecting pSTAT5 expression are shown.

[0173] Figure 15 The results of Western blotting detection of pSTAT5 and pSMAD / 3 expression are shown. Detailed Implementation

[0174] Through extensive and in-depth research and screening, the inventors have developed engineered immune cells that co-express CAR structures and signaling proteins (including the TGFBR2 extracellular domain and, optionally, the IL-7Rα intracellular domain). In vitro and in vivo experiments show that the extracellular structure of TGFBR2 can effectively block or reduce TGF-β signaling, protecting CAR-T cells and counteracting the effects of an immunosuppressive environment; the IL-7 receptor intracellular domain can further enhance downstream cytokine signaling, improve CAR-T cell proliferation, exhaustion, and persistence, thereby increasing therapeutic efficacy.

[0175] The engineered immune cells of this invention can not only highly specifically recognize the Claudin18.2 target antigen via CAR and target solid tumors with high Claudin18.2 expression, but also, through the chimeric protein containing the extracellular domain of the TGF-β receptor and the intracellular signaling domain of IL-7, reduce the inhibitory signal of the TGF-β immunosuppressive factor, which is unfavorable to the survival of immune cells in the tumor microenvironment, and further enhance downstream cytokine signaling through the intracellular domain of the IL-7 receptor, thereby enhancing the survival of engineered immune cells and the sustained tumor-killing effect, and reducing tumor recurrence. This invention was completed based on these principles.

[0176] This article uses CAR-T cells as an example to provide a detailed description of the engineered immune cells of the present invention. The engineered immune cells of the present invention are not limited to the CAR-T cells described in the context; they possess the same or similar technical features and beneficial effects as the CAR-T cells described in the context. Specifically, when immune cells express chimeric antigen receptor CAR, NK cells are equivalent to T cells (or T cells can be replaced by NK cells).

[0177] CLDN18.2

[0178] As used herein, the terms "CLDN18.2" and "Claudin18.2" are used interchangeably. Human CLDN18 is a 261-amino acid protein, a four-transmembrane protein with four transmembrane hydrophobic regions and two extracellular loop structures. Loop 1 is formed by transmembrane regions 1 and 2; loop 2 is formed by transmembrane regions 3 and 4. Human CLDN18.1 and CLDN18.2 differ in 8 amino acids at the N-terminus, transmembrane region 1, and extracellular loop 1; the remaining parts are identical, with a sequence similarity of 92%.

[0179] Chimeric antigen receptor (CAR)

[0180] As used herein, a chimeric antigen receptor (CAR) comprises an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes an optional signal peptide and a target-specific binding domain (also known as an antigen-binding domain). The intracellular domain includes a co-stimulatory domain and a portion of the CD3ζ chain. When expressed in T cells, the extracellular domain recognizes a specific antigen, which is then transduced through the intracellular domain, leading to cell activation and proliferation, cytotoxicity, and the secretion of cytokines such as IL-2 and IFN-γ. This affects tumor cells, causing them to stop growing, die, or otherwise be affected, resulting in a reduction or elimination of the tumor burden in the patient. The antigen-binding domain is preferably fused with one or more intracellular domains derived from the co-stimulatory molecule and the CD3ζ chain.

[0181] The CAR of this invention uses VHH targeting CLND18.2 as the antigen-binding domain. In one embodiment of this invention, the amino acid sequence of the VHH is as shown in any of SEQ ID NO: 1-4. The CDR sequence of the VHH is shown in Table A.

[0182] Table A: VHH and its CDR sequence of the present invention.

[0183]

[0184]

[0185] Chimeric antigen receptor T cells (CAR-T cells)

[0186] As used herein, the terms "CAR-T cell," "CAR-T," and "CAR-T cell of the present invention" all refer to the CAR-T cell described in the first aspect of the present invention. The CAR-T cell of the present invention can be used to treat tumors with high CLND18.2 expression, such as gastric cancer, bile duct cancer, ovarian cancer, lung cancer, esophageal cancer, and pancreatic cancer.

[0187] CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action of CAR-T cells is not restricted by MHC; (2) Given that many tumor cells express the same tumor antigens, once the CAR gene targeting a certain tumor antigen is constructed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, thus expanding the target range of tumor antigens; (4) Using the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0188] TGF-β and its receptor

[0189] As used herein, the term "TGF-β" refers to Transforming growth factor beta. It should be understood that the term includes both wild-type and mutant TGF-β. In this invention, TGF-β includes TGF-β from humans and non-human mammals.

[0190] As used in this article, the term "TGF-β receptor" refers to the Transforming growth factor beta receptor.

[0191] Existing literature has confirmed that tumor cells such as ovarian cancer, breast cancer, and prostate cancer secrete large amounts of TGF-β cytokine. As an immunosuppressive factor, this factor can significantly inhibit the activation and proliferation of T cells, promote their differentiation into Tregs, and reduce their effector function.

[0192] Signal proteins

[0193] This invention provides a signaling protein comprising an extracellular domain of an immunosuppressive receptor, a transmembrane domain, and optionally an intracellular domain of an immune agonist receptor. In one embodiment, the signaling protein is a fusion protein comprising the extracellular domain of the dominant inactivating receptor TGFBR2 and an intracellular element of the IL-7Rα protein. Unexpectedly, the signaling protein of this invention can convert the inhibitory signal of the TGF-β immunosuppressive factor, which is detrimental to immune cell survival in the tumor microenvironment, into an activating signal, thereby enhancing the sustained effects of immune cell survival and tumor killing, improving the efficacy of immunotherapy, and reducing tumor recurrence.

[0194] Expression Box

[0195] As used herein, "expression cassette" or "expression cassette of the present invention" includes a first expression cassette and a second expression cassette. As described in the fifth aspect of the present invention, the first expression cassette contains a nucleic acid sequence encoding the CAR. The second expression cassette expresses an exogenous signaling protein.

[0196] In this invention, the signaling protein can be constitutively expressed or inducibly expressed.

[0197] Under induced expression conditions, when the CAR-T cells are activated by the corresponding inducer, the second expression cassette expresses the signaling protein; thus, when the CAR-T cells of the present invention are not exposed to the corresponding inducer, the second expression cassette does not express the signaling protein.

[0198] In one embodiment, the first expression box and the second expression box each further include a promoter and / or a terminator. The promoter of the second expression box can be a constitutive or inductive promoter.

[0199] preparation

[0200] This invention provides a formulation containing engineered immune cells (such as CAR-T cells) as described in the first aspect of this invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / mL, more optimal 1×10 4 -1×10 7 Cells / mL.

[0201] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0202] Therapeutic applications

[0203] This invention includes therapeutic applications of cells (e.g., T cells) transduced with a vector (such as a lentiviral vector) containing the expression cassette of this invention. The transduced T cells can target surface markers of tumor cells and express signaling proteins, synergistically and significantly enhancing their tumor-killing efficiency.

[0204] Therefore, the present invention also provides a method for stimulating T-cell-mediated immune responses targeting mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to a mammal.

[0205] In one embodiment, the present invention includes a type of cell therapy in which patient-associated (or allogeneic) T cells are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This approach results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0206] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and persist for months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-T cells can induce a specific immune response against tumor cells that highly express antigens recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention elicit a specific immune response against tumor cells that highly express the NKG2D ligand.

[0207] The treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Types of cancer treated with the CAR of this invention include, but are not limited to: gastric cancer, bile duct cancer, ovarian cancer, lung cancer, esophageal cancer, and pancreatic cancer.

[0208] Typically, activated and expanded cells, as described herein, can be used to treat and prevent diseases such as tumors. Therefore, this invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the CAR-T cells of this invention to a subject in need of them.

[0209] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may include target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0210] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the treatment (or prevention) of a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.

[0211] When referring to "immunologically effective dose," "antitumor effective dose," "tumor-inhibitory effective dose," or "therapeutic dose," the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. Pharmaceutical compositions including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 7 The T-cell composition can be administered at a dose of cells per kg of body weight (inclusive of all integer values ​​within the range). These doses can also be administered multiple times. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a medical professional by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0212] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient via intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered via intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0213] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 5 One to 1×10 10 The modified T cells of this invention are administered to a patient, for example, via intravenous infusion.

[0214] The main advantages of this invention include:

[0215] 1. The engineered immune cells of this invention contain both CAR and signaling proteins, thereby enabling more efficient activation of immune cells.

[0216] 2. The CAR of the present invention uses a nanobody targeting CLND18.2 as the antigen-binding domain, which can target CLND18.2 positive tumor cells with high specificity and has high cytotoxicity and long-lasting anti-tumor activity.

[0217] 3. The signaling protein of the present invention can not only effectively block or reduce the inhibitory signal of TGF-β immunosuppressive factor on T cells, but also further convert the TGF-β immunosuppressive signal into cytokine activation signal, thereby prolonging the survival time of CAR-T cells in vivo and effectively and continuously killing tumor cells, reducing tumor recurrence.

[0218] 4. The intracellular molecule of the CAR-T cell co-expressed signaling protein of the present invention can also be the intracellular domain of other exogenous recombinant proteins (such as IL-15R, IL-12Rα, IL-2Rα, IL-18R, IL-21R, etc.), which can convert the TGFβ immunosuppressive signal into an activating signal and perform the corresponding function.

[0219] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0220] Materials and Methods

[0221] CAR molecules and their structure

[0222] In the examples, 11 CAR molecules targeting CLDN18.2 were constructed and named CAR1-CAR11, and their structures are shown in Table 1. CAR1-CAR11 all contain the following identical structures: CD8 signal peptide [SP], CD8 hinge region [CD8H] and CD28 transmembrane domain [28TMD], CD8 intracellular region [CD8C] and CD3ζ cytoplasmic signal transduction sequence [CD3ζ].

[0223] CAR1-CAR4 and CAR9-CAR11 use the sequence shown in SEQ ID NO:1 as the antigen-binding domain [abbreviated as VHH1]; CAR5 and CAR6 use the sequence shown in SEQ ID NO:2 as the antigen-binding domain [abbreviated as VHH2]; CAR7 uses the sequence shown in SEQ ID NO:3 as the antigen-binding domain [abbreviated as VHH3]; CAR8 uses the sequence shown in SEQ ID NO:4 as the antigen-binding domain [abbreviated as VHH4].

[0224] CAR1 and CAR5 do not contain the extracellular region of TGFβR2 and the intracellular region of IL-7; CAR2 contains only the extracellular region of TGFβR2 and does not contain the intracellular region of IL-7; CAR3, CAR4, CAR6, CAR7, CAR8 and CAR9 contain the extracellular region of TGFβR2 and the intracellular region of IL-7R, and CAR10 and CAR11 contain the extracellular region of TGFβR2, the intracellular region of IL-7R, the extracellular region of TGFβR1 and the intracellular region of IL-2Rγ. The specific CAR structures are shown in Table 1.

[0225] Table 1. CAR molecular structure of the examples

[0226]

[0227]

[0228] The sequences of the structural elements used to construct the CAR molecule are shown in Table 2.

[0229] Table 2. Sequences of the structural elements used to construct the CAR molecule

[0230]

[0231]

[0232] *The underlined section represents the juxtamembranous region of IL-7R.

[0233] Example 1: Isolation of PBMCs and Expansion of T Cells from Donor Blood

[0234] Monocytes were isolated from donor peripheral blood, and T cells were enriched by density gradient centrifugation using Ficoll (EasySep Human T Cell Enrichment Kit, Stemcell Technologies Inc.). T cells were activated and expanded using magnetic beads coupled with anti-CD3 / anti-CD28. The cell culture system used was x-vivo 15 (3% SR, 300 IU / mL rhIL-2), and the cells were continuously cultured in an incubator at 37°C and 5% CO2.

[0235] Example 2: Target Cell Culture and Construction

[0236] The cell line expressing CLDN18.2 was constructed using the following method: the luciferase gene was transferred into target cells, and after cloning and screening, a stable cell line AsPC-1-CLDN18.2 was obtained. The AGS-CLDN18.2 medium was prepared according to the ATCC guidelines.

[0237] The cell line expressing PD-L1 was constructed using the following method: the PD-L1 gene was transferred into the target cell AsPC-1-CLDN18.2 via lentivirus, and after cloning and screening, a stable cell line AsPC-1-CLDN18.2-PD-L1 was obtained. The culture medium was prepared according to the ATCC guidelines.

[0238] Example 3: CAR-T cell preparation, CAR positivity rate and memory phenotype detection

[0239] Primary T cells were activated with anti-CD3 / anti-CD28 magnetic beads for 48 hours, then concentrated lentivirus was added and cultured in a 37°C, 5% CO2 incubator. The CAR positivity rate was detected by flow cytometry 72 hours after cell infection.

[0240] CAR-T cells were cultured in vitro for 8-10 days, and then harvested. CAR expression on the surface of CAR-T cells was detected using anti-Flag antibody and anti-TGFβTRII. The CAR-T cell phenotype at harvest was also assessed, and cell memory phenotype was analyzed. The proliferation rate of CAR-positive cells was statistically analyzed, and there was no significant difference in cell proliferation among the CAR-T groups.

[0241] Experimental results are as follows Figure 2 As shown. Figure 2 As shown in the first row, CAR expression in CAR-T cells is all above 20%. Figure 2 As shown in rows 4 and 5, CAR2-CAR4 and CAR6-8 cells co-express CAR and TGFβR2 on their surface.

[0242] Flow cytometry gating was performed on CAR+ cells to analyze cellular memory phenotypes (CD45RO-CCR7+). In Tscm, the proportion of CAR2-CAR4 was significantly higher than that of CAR5-6, and the proportion of CAR2 and CAR3 was 18%-20%, higher than that of CAR1 (8.68%). Flow cytometry gating of CAR+CD45RO-CCR7+ cells showed that CAR1-CAR4 cells were all CD27+CD95+, indicating that CAR1-CAR4... The cells were all Tscm, while CAR5-CAR6 cells were... Only 31.2-56.3% of the cells were Tscm cells, i.e. stem cell-like memory T cells.

[0243] Two batches of CAR-T cells from different donors were further prepared, and their CAR expression rates and CD4+ and CD8+ ratios were as follows: Figure 3 and Figure 4 As shown in the figure. The results show that the CAR structure of the present invention can be effectively expressed in cells from different donor sources.

[0244] Example 4: Reporter gene detection of the TGFβ / Smad signaling pathway

[0245] CAR expression plasmid, two reporter gene vectors pNL (NLucP / SBE-RE / Hygro, containing TGFβ signaling pathway regulatory elements) and pGL4.51 (luc2 / CMV / Neo, containing internal control reporter gene) were co-transfected into 293T cells. After 24 hours, TGFβ1 at concentrations of 10 μg / ml and 50 μg / ml were added, respectively, and the cells were incubated for another 20 hours. The Reporter Assay System detects and reads optical signal values ​​to compare the inhibitory effects of various molecular structures on downstream TGFβ signals.

[0246] Experimental results are as follows Figure 5 As shown, treatment with different concentrations of TGFβ1 significantly inhibited the TGFβ / Smad signaling pathway by the extracellular TGFBR2 receptor and / or the intracellular tIL-7R protein (CAR2-4).

[0247] Example 5: Detection of CAR-T cell killing of target cells using the Luciferase method

[0248] The killing ability of tumor target cells labeled with luciferase was detected. By transferring the luciferase gene into target cells, stable cell lines AsPC-1-CLDN18.2, AsPC-1-CLDN18.2-PD-L1, and AGS-CLDN18.2 were obtained after clone selection.

[0249] Target cells and various effector cells (CAR-T cells) are mixed and cultured at different effector-to-target ratios. After 6 or 24 hours of culture, a luciferin substrate is added. The activity of luciferase can be determined by detecting the fluorescence intensity, and the cell survival rate can be detected to obtain the killing effect of each CAR-T cell.

[0250] The results are as follows Figure 6 As shown in a, after 24 hours of co-culture, there was no significant difference in the killing ability of CAR-T cells against target cells AsPC-1-CLDN18.2 and AsPC-1-CLDN18.2-PD-L1 among the different groups at E:T=1:1 and E:T=1:3. The killing rate of CAR5-CAR6 was higher than that of other CAR-T cells.

[0251] The results are as follows Figure 6 As shown in b, after 6 hours of co-culture, under culture conditions with E:T ratios of 3:1, 1:1, and 1:3, the killing rate of humanized CAR7 and CAR8 was significantly higher than that of non-humanized CAR3 (90% vs 50% when E:T = 3:1; 50-60% vs 30% when E:T = 1:1).

[0252] Example 6: Release of cytokines during CAR-T cell killing of target cells

[0253] After the target cell killing experiment in Example 5 (co-incubation for 24 hours), the levels of cytokines IL-2 and IFNγ in the co-culture supernatant were measured using the CBA method.

[0254] The results are as follows Figure 7 As shown in Figure a, the levels of cytokine (IL-2, IFNγ) produced by CAR cells and target cells AsPC-1-CLDN18.2-PD-L1 after co-culturing were significantly lower than those of target cells AsPC-1-CLDN18.2.

[0255] Co-culturing CAR4 cells with target cells AsPC-1-CLDN18.2-Luc resulted in higher IL-2 levels compared to CAR1-3. Co-culturing CAR2-3 cells with target cells resulted in higher IL-2 levels compared to CAR1. Co-culturing CAR1-4 cells with target cells resulted in higher IL-2 levels compared to CAR5 and CAR6.

[0256] There was no significant difference in the production of IFNγ by CAR cells in target cells AsPC-1-CLDN18.2-Luc among the different groups.

[0257] The results are as follows Figure 7 As shown in b, the production of cytokines (IL-2, IFNγ) in CAR cells co-cultured with target cells AGS-CLDN18.1 was significantly lower than that in target cells AGS-CLDN18.2, indicating that CAR-T cells specifically kill CLDN18.2 target cells.

[0258] Example 7: In vitro efficacy study of continuous multi-round killing of cancer cells

[0259] The T cells harvested in Example 3 were subjected to multiple rounds of in vitro killing experiments. A schematic diagram of the experimental method is shown below. Figure 8a. The specific method is as follows: CAR-T cells and target cells were co-incubated at an E:T ratio of 1:2 and divided into two groups. One group was cultured in RPMI 1640 + 10% FBS basal medium, and the other group was cultured in basal medium with 5 ng / ml TGFβ1 added. The cells were cultured continuously for 3 days. The growth of target cells AsPC-1-CLDN18.2-Luc or AGS-CLDN18.2-Luc was observed under a microscope. On the third day of co-incubation, after the cells were mixed, a small number of cells were taken for staining, flow cytometry to detect and analyze the phenotype of T cells, and the killing rate of CAR-T cells against target cells was detected by Luciferase method. The killing efficiency of CAR-T cells = (control group - experimental group) / control group * 100%. The remaining cells were co-cultured with new target cells as shown in the figure, and the killing was carried out continuously for several rounds using the above method.

[0260] like Figure 8 As shown in b, when CAR-T cells were co-cultured with AsPC1-CLDN18.2 (E:T = 1:2), CAR2 showed significantly weaker killing ability than CAR3 (which has an extracellular TGFBR2 receptor and an intracellular tIL-7R protein) in the third round of culture without the addition of TGFβ1 (5 ng / mL).

[0261] like Figure 8 As shown in c, when CAR-T cells were co-cultured with AsPC1-CLDN18.2 (E:T = 1:2), CAR1 showed significantly weaker killing ability than CAR3 and CAR4 in the third round of culture without the addition of TGFβ1 (5 ng / mL). When TGFβ1 (5 ng / mL) was added, CAR1 had no killing ability in the third round, while CAR3 and CAR4 still had significant killing ability in the fourth round. This shows that the sustained killing ability of CAR3 and CAR4 (with extracellular TGFBR2 receptor and intracellular tIL-7R) is significantly better than that of CAR1.

[0262] like Figure 9 As shown, under co-culture conditions of CAR-T cells and AGS-CLDN18.2 (E:T = 1:2), with or without the addition of TGFβ1 (10 ng / mL), CAR1, CAR2, and CAR9 exhibited significantly weaker cytotoxic activity in the fifth round compared to CAR3 (extracellular TGFBR2 receptor + intracellular IL-7R protein) and CAR11 (extracellular TGFBR2 receptor + intracellular IL-7R protein and TGFBR1 receptor + intracellular IL-2Rγ protein). The results indicate that CAR3 and CAR11 demonstrated significantly superior sustained cytotoxic activity compared to CAR1, CAR2, and CAR9.

[0263] like Figure 10 As shown, the method adopted is the same as Figure 9The sustained killing ability of CAR-T cells from different donor sources after multiple rounds of in vitro antigen stimulation was compared among CAR2, CAR3, and CAR11. In co-culture of CAR-T cells with AGS-CLDN18.2 (E:T = 1:2), without the addition of TGFβ1 (10 ng / mL), CAR2 showed no significant target cell lysis ability in round 7, while CAR3 and CAR11 exhibited significant killing ability. The killing ability of CAR2 was significantly weaker than that of CAR3 and CAR11; however, with the addition of TGFβ1 (10 ng / mL), CAR2, CAR3, and CAR11 still showed significant killing ability in round 7.

[0264] The cell proliferation capacity of CAR-T cells after multiple rounds of antigen stimulation was analyzed, and the results are as follows: Figure 11 As shown, under culture conditions without the addition of TGFβ1 (10 ng / mL), the cell expansion rate after the 6th round of antigen stimulation was analyzed. CAR2 showed a significantly slower expansion rate than CAR3 and CAR11, and after the 7th round of antigen stimulation, it showed no significant ability to lyse target cells. The cell expansion rates of CAR3 and CAR11 also decreased significantly after the 7th round of antigen stimulation. Under culture conditions with the addition of TGFβ1 (10 ng / mL), the cell expansion rate after the 7th round of antigen stimulation was analyzed. CAR11 showed the highest cell growth rate, followed by CAR3 and CAR2.

[0265] After multiple rounds of antigen stimulation, the CAR expression rate on the surface of CAR-T cells was detected, and the results are as follows: Figure 12 As shown, the trend of CAR expression rate changes is consistent with the CAR-T cell expansion rate.

[0266] The results above show that CAR3 and CAR11 cells carrying the signaling protein of the present invention have significantly enhanced sustained cell killing ability, and CAR11 cells exhibit significantly higher cell proliferation and killing ability under the condition of TGFβ addition.

[0267] Example 8: In vivo efficacy study

[0268] The in vivo antitumor efficacy of Claudin18.2 CAR-T cells was evaluated in an Aspc-1 18.2-Luc pancreatic cancer NOG-DKO mouse model (NOD.Cg-B2mem1Tac Prkdcscid H2-Ab1tm1Doi Il2rgtm1Sug / JicCrl).

[0269] 2×10 subcutaneous injection 6 AsPC-1-CLDN18.2-Luc cells were collected until the tumor burden reached 200 mmHg. 3At approximately 10:00 AM, mice were divided into groups of 3-4. One day after grouping, 200 μL of CLDN18.2 CAR-T cells and NT cells (2 × 10⁻⁶ cells) were injected via the tail vein. 6 One CAR-T cell per mouse; On the first day after CAR-T injection, a small amount of mouse blood was taken to detect the number of CAR-T cells surviving in the body. Blood samples were taken once a week thereafter to detect various CAR-T cell phenotypes. The size of the subcutaneous tumor and the weight of the mouse were detected twice a week.

[0270] The results are as follows Figure 13 As shown, the tumors in mice that were infused with CAR1 and CAR3 cells were significantly reduced to the point of being cleared. Even 50 days after the infusion of CAR-T cells, the mice were still alive and there was no tumor recurrence.

[0271] 2×10⁻⁶ cells were injected into the left side of the mouse tumor. 6 One AsPC-1-CLDN18.2-Luc cell was infused into CAR-T cells. Day 100 after the infusion, the tumor size on both sides was measured. No tumors were detected in mice infused with CAR3 (which have extracellular TGFBR2 receptor and intracellular tIL-7R protein). In mice infused with CAR1 (3 / 3), there was a tumor burden on the left side, and one mouse on the right side had a recurrent tumor.

[0272] Day 106 after CAR-T cell infusion, 2 × 10⁶ cells were injected into the right side of the mouse tumor. 6 AsPC-1-CLDN18.2-Luc cells were infused into CAR-T cells. Day 160 after infusion, the tumor size on both sides of the CAR3-infused mice was examined. The left tumor (first re-challenge) showed no recurrence. Observation of the right tumor (second re-challenge) revealed that one in three mice (1 / 3) had no detectable tumor burden, while the other two mice showed tumor growth, but the tumor burden was significantly smaller than the control group (400 mm). 3 vs 1000mm 3 ).

[0273] In vivo experiments showed that cells carrying CAR3 (with extracellular TGFBR2 receptor and intracellular IL-7R protein) exhibited significantly enhanced antitumor activity and persistence.

[0274] Example 9: Detection of the TGFβ / Smad signaling pathway

[0275] (I) FACS method detection:

[0276] FACS was used to detect the TGFβ / Smad signaling pathway in CAR-T cells of each group. The specific method is as follows: CAR-T cells of each group were cultured in medium without IL-2 for 24 hours. The TGFβ1 group was treated with 10 ng / ml TGFβ1 added to the basal medium for 1 hour or 3 hours. The positive control group was treated with IL-2 for 0.5 hours. Then the extracellular CAR expression and intracellular pSTAT5 expression levels were detected.

[0277] The results show that... Figure 14 As shown in a, the statistical data can be found in [the table]. Figure 14 b. IL-2-treated CAR-T cells served as a positive control group, and pSTAT5 levels were significantly increased in all groups, with pSTAT5-positive cells exceeding 80%. Comparing the intracellular pSTAT5 levels in the 1-hour and 3-hour TGFβ-treated groups, the 3-hour treatment group showed significantly lower levels than the 1-hour treatment group. Analysis of pSTAT5 levels in the 1-hour and 3-hour TGFβ-treated groups revealed that CAR11 cells expressed the highest levels of pSTAT5, followed by CAR9 and CAR3. Figure 14 In step c, the pSTAT5 expression levels of CAR-negative cells (red markers) and CAR-positive cells (blue markers) in CAR3, CAR9, and CAR11 were further analyzed. Flow cytometry data showed that the MFI of CAR-positive cells was higher than that of CAR-negative cells, indicating that the pSTAT5 expression of CAR-positive cells was higher than that of CAR-negative cells, with the highest pSTAT5 expression in CAR11-positive cells.

[0278] (II) Western spectroscopy method detection:

[0279] CAR-T cells expressing CAR were screened. After culturing each group of CAR-T cells in IL-2-free medium for 24 hours, the TGFβ1 group was treated with 10 ng / ml or 30 ng / ml TGFβ1 added to the basal medium for 1 hour, and the positive control group was treated with IL-2 for 0.5 hours. Cells were collected, lysed and proteins were extracted. After SDS-PAGE electrophoresis, the cells were transferred to a membrane and incubated with antibodies. The cells were then treated with pSTAT5, actin and pSMAD2 / 3 antibodies, respectively. The secondary antibody was HRP-labeled. The cells were developed with chemiluminescent reagent and the chemiluminescent signals were recorded using an imaging system. The protein expression levels of pSTAT5, pSMAD2 / 3 and Actin (internal control) were analyzed.

[0280] The results are as follows Figure 15In CAR11 cells treated with different concentrations of TGFβ1, pSTAT5 expression was significantly higher than in other CAR-T groups, and pSMAD2 / 3 expression was the lowest. This indicates that TGFβ1 treatment significantly increased pSTAT5 expression in CAR11 cells and blocked pSMAD2 / 3 expression. CAR3 cells showed a significant decrease in pSMAD2 / 3 expression, but no significant change in pSTAT5 expression was detected, possibly because the sensitivity of Western blotting is lower than that of FACS. In contrast, significant pSMAD2 / 3 expression was detectable in CAR1 and CAR2 cells, showing a TGFβ concentration gradient dependence, meaning that pSMAD2 / 3 expression in CAR-T cells with high TGFβ1 concentrations was higher than in those with low concentrations.

[0281] sequence

[0282] VHH1 (SEQ ID NO:1)

[0283] QVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSS

[0284] VHH2 (SEQ ID NO:2)

[0285] EVQLVESGGGLVQPGGSLRLSCAASGNILSINYMGWYRQAPGKERELVATITRGGSTNYQGNLKGRFIISGDNTKDTVYLQMNNLQPEDTAVYYCYFRINFGILGGLRDYWGQGTQVTVSS

[0286] VHH3 (SEQ ID NO:3)

[0287] QVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWVRQAPGKGLEWVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSS

[0288] VHH4 (SEQ ID NO:4)

[0289] QVQLVESGGGLVQPGGSLRLSCAASDSTFSSYIMGWFRQAPGKGLEFVAGITWSGGIKDYADSVKGRFTIARDNSKNTLYLQMNSLRAEDAAVYYCAANAIQLTRRSGDYAYWGQGTLVTVSS

[0290] CAR1(SEQ ID NO:26)

[0291] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0292] CAR2(SEQ ID NO:27)

[0293] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS

[0294] CAR3(SEQ ID NO:28)

[0295] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0296] CAR4(SEQ ID NO:29)

[0297] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0298] CAR5(SEQ ID NO:30)

[0299] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGNILSINYMGWYRQAPGKERELVATITRGGSTNYQGNLKGRFIISGDNTKDTVYLQMNNLQPEDTAVYYCYFRINFGILGGLRDYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0300] CAR6(SEQ ID NO:31)

[0301] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGNILSINYMGWYRQAPGKERELVATITRGGSTNYQGNLKGRFIISGDNTKDTVYLQMNNLQPEDTAVYYCYFRINFGILGGLRDYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0302] CAR7(SEQ ID NO:32)

[0303] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWVRQAPGKGLEWVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0304] CAR8(SEQ ID NO:33)

[0305] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASDSTFSSYIMGWFRQAPGKGLEFVAGITWSGGIKDYADSVKGRFTIARDNSKNTLYLQMNSLRAEDAAVYYCAANAIQLTRRSGDYAYWGQGTLVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0306] CAR9(SEQ ID NO:34)

[0307] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTCPTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0308] CAR10(SEQ ID NO:35)

[0309] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ

[0310] GLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGP

[0311] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMI

[0312] VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE

[0313] TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLL

[0314] LVIFQHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIK

[0315] PIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQ

[0316] LEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESG

[0317] KNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQ

[0318] NQEGRGSLLTCGDVEENPGPMEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHL

[0319] CTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQ

[0320] DHCNKIELPTTVKSSPGLGPVELPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISV

[0321] GSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERL

[0322] CLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0323] CAR11(SEQ ID NO:36)

[0324]

[0325] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An engineered immune cell, characterized in that, The engineered immune cells express the following exogenous proteins: (a) A chimeric antigen receptor (CAR) wherein the antigen-binding domain of the CAR comprises a nanobody (VHH) targeting Claudin18.2; and (b) Signaling proteins comprising: an extracellular domain of an immunosuppressive receptor, a transmembrane domain, and optionally an intracellular domain of an immunostimulatory receptor.

2. The engineered immune cells as described in claim 1, characterized in that, The CAR described herein has the structure shown in formula Ia, Ib or Ic: L1-VHH-H-TM1-C-CD3ζ(Ia) L1-VHH-H-TM1-C-CD3ζ-A-E1(Ib) L1-VHH-H-TM1-C-CD3ζ-A-E1-A-E2(Ic) In the formula, L1 is either absent or a signal peptide sequence; VHH is a nanobody targeting Claudin18.2; H represents the area with no hinge or no connection. TM1 is a transmembrane domain; C represents the domain with no or no co-stimulatory signal; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers); A is a self-cleaving 2A peptide; E1 and E2 are each independently signaling proteins (including wild type or its mutant / modified forms); Each of the "-" symbols independently represents a linking peptide or a peptide bond.

3. The engineered immune cells as described in claim 1, characterized in that, The signaling protein described has the structure shown in Formula V: L2-Z1-TM2-Z2(V) In the formula, L2 is either absent or a signal peptide sequence; Z1 is the extracellular domain of an immunosuppressive receptor; TM2 is the transmembrane region; Z2 is an intracellular domain of receptors that are absent or do not have immune agonist receptors. Each of the "-" symbols independently represents a linking peptide or a peptide bond.

4. A chimeric antigen receptor (CAR) construct, characterized in that, The structural formula of the CAR is shown in formula Ia or Ib below: L1-VHH-H-TM1-C-CD3ζ-A-E1(Ib) L1-VHH-H-TM1-C-CD3ζ-A-E1-A-E2(Ic) In the formula, L1 is either absent or a signal peptide sequence; VHH is a nanobody targeting Claudin18.2; H represents the area with no hinge or no connection. TM1 is a transmembrane domain; C represents the domain with no or no co-stimulatory signal; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers); A is a self-cleaving 2A peptide; E1 and E2 are each independently signaling proteins (including wild type or its mutant / modified forms); Each of the "-" symbols independently represents a linking peptide or a peptide bond.

5. The CAR construct as described in claim 4, characterized in that, The sequence of the CAR is shown in any one of SEQ ID NO:27-29 or 31-36.

6. A polynucleotide, characterized in that, The polynucleotide encodes the CAR fusion protein as described in claim 4.

7. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5.

8. A formulation, characterized in that, The formulation contains the engineered immune cells as described in claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. A kit for preparing engineered immune cells according to claim 1, the kit comprising a container, and the following within the container: (1) A first nucleic acid sequence, the first nucleic acid sequence containing a first expression cassette for expressing the CAR; and (2) A second nucleic acid sequence containing a second expression cassette for co-expression of a signal protein.

10. The use of the engineered immune cells according to claim 1, characterized in that, Used to prepare drugs or preparations for the prevention and / or treatment of cancer or tumors.

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