Chimeric antigen receptor immune cell specifically binding to claudin 18.2 and use thereof

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

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202411302196.X (22) Application Date 2024.09.18 (66) Domestic Priority Data 202410556865.X 2024.05.07 CN (71) Applicant Genxi Biotechnology (Shanghai) Co., Ltd. Address 12th Floor, Building 1, No. 926 Yishan Road, Xuhui District, Shanghai 200233 (72) Inventors Dong Qi, Shen Lianjun, Yin Wenjie, Cao Wei (74) Patent Agency China Patent Agency (Hong Kong) Limited 72001 Patent Attorney Li Tang, Peng Chang (51) Int.Cl. C12N 5 / 10 (2006.01) C07K 19 / 00 (2006.01) A61K 39 / 00(2006.01) A61P 35 / 00(2006.01) (54) Invention Title: Chimeric Antigen Receptor Immune Cells Specifically Binding to Claudin18.2 and Their Applications (57) Abstract: This invention relates to chimeric antigen receptor immune cells specifically binding to Claudin18.2 and their applications. Specifically, this invention provides a CAR-T cell with a Claudin18.2-targeting nanobody as the antigen-binding domain, which can highly specifically target Claudin18.2-positive tumor cells; the CAR-T cell co-expresses the extracellular domain of the TGF-β receptor and, optionally, the intracellular signaling domain of the IL-7 receptor, which can effectively block or reduce the TGF-β-induced inhibitory signal, promote the expansion, survival, and persistence of immune cells, thereby enhancing their specific killing effect on tumor cells. The chimeric antigen receptor immune cells of this invention can be used to treat Claudin18.2-positive tumors. Claims (2 pages), Description (20 pages), Sequence Listing (electronic publication), Drawings (9 pages), CN 120944823 A, 2025.11.14, CN 1 20 94 48 23 A. 1. An engineered immune cell, characterized in that the engineered immune cell expresses 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) a signaling protein, wherein the signaling protein comprises: an extracellular domain of an immunosuppressive receptor, a transmembrane domain, and optionally an intracellular domain of an immunostimulatory receptor. 2. The engineered immune cell of claim 1, characterized in that the CAR has a structure represented by 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) Where, L1 is a no-signal peptide sequence; VHH is a nanobody targeting Claudin18.2; H is a no-hinge region; TM1 is a transmembrane domain; C is a no-co-stimulatory signaling domain; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild type or its mutant / modified form); A is a self-cleaved 2A peptide; E1 and E2 are each independently signal proteins (including wild type or its mutant / modified form); each "-" is independently a linking peptide or peptide bond. 3. The engineered immune cell according to claim 1, characterized in that the signal protein has the structure shown in formula V: L2-Z1-TM2-Z2(V) where L2 is a no-signal peptide sequence; Z1 is the extracellular domain of an immunosuppressive receptor; TM2 is a transmembrane region; Z2 is the intracellular domain of a no-immunostimulatory receptor; and each of the "-" symbols independently represents a linking peptide or a peptide bond. 4. A chimeric antigen receptor (CAR) construct, characterized in that the CAR has the following structural formula Ia or Ib: L1-VHH-H-TM1-C-CD3ζ-A-E1 (Ib) L1-VHH-H-TM1-C-CD3ζ-A-E1-A-E2 (Ic) Wherein, L1 is a no-signal peptide sequence; VHH is a nanobody targeting Claudin18.2; H is a no-hinge region; TM1 is a transmembrane domain; C is a no-co-stimulatory signal domain; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild-type or its mutant / modified form); AA is a self-cleaved 2A peptide; E1 and E2 are each independently signal proteins (including wild-type or its mutant / modified form); each "-" is independently a linking peptide or peptide bond. 5. The CAR construct of claim 4, wherein the sequence of the CAR is as shown in any one of SEQ ID NO: 27-29, 31-36. 6. A polynucleotide, wherein the polynucleotide encodes the CAR fusion protein of claim 4. 7. A vector, wherein the vector contains the polynucleotide of claim 5. 8. A formulation, wherein the formulation contains the engineered immune cells of claim 1, and a pharmaceutically acceptable vector, diluent, or excipient. 9. A kit for preparing the engineered immune cells of claim 1, the kit comprising a container, and within the container: (1) a first nucleic acid sequence containing a first expression cassette for expressing the CAR; and(2) A second nucleic acid sequence, wherein the second nucleic acid sequence contains a second expression cassette for co-expressing a signaling protein. 10. Use of the engineered immune cells of claim 1, characterized in that they are used to prepare drugs or preparations for the prevention and / or treatment of cancer or tumors. Claims 2 / 2 Page 3 CN 120944823 A Chimeric antigen receptor immune cells that specifically bind to Claudin18.2 and their application Technical Field

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

[0002] Claudin family proteins are widely distributed in the tight junction structures of epithelial cells. Claudin18.2 is a membrane protein with four transmembrane regions, the N-terminus and C-terminus are in the cytoplasm, and there are two extracellular loop regions. These two extracellular regions can serve as specific targets for targeted therapy. Claudin18.2 is specifically expressed in highly differentiated epithelial cells of the stomach in normal tissues. Besides gastric cancer, it is also expressed in a high proportion of tumor cells such as cholangiocarcinoma, ovarian cancer, lung cancer, esophageal cancer, and pancreatic cancer.

[0003] Chimeric antigen receptor T cells (CAR T cells) are a novel immunotherapy method targeting specific antigens on the surface of tumor cells. Currently, they are used to develop cell therapies for hematologic malignancies and solid tumors. While immunotherapy for hematologic malignancies has shown remarkable efficacy, its efficacy in solid tumors is not ideal. CAR-T therapy for solid tumors faces several major challenges: lack of safe tumor-specific antigen targets (TSA), 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 the field to develop more effective and sustained immunotherapies targeting tumors expressing Claudin18.2.

[0005] The object of the present 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, the engineered immune cell expressing the following exogenous proteins:

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

[0008] (b) a signaling protein 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 immune agonist 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 without an immune agonist 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: Specification 1 / 20 page 4 CN 120944823 A

[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 combinations 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 an IL-7 receptor.

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

[0024] In another preferred embodiment, the CAR has a structure represented by 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] Wherein,

[0029] L1 is a no-signal peptide sequence;

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

[0031] H is a no-hinge region;

[0032] TM1 is a transmembrane domain;

[0033] C is a no-co-stimulatory signal domain;

[0034] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild type or its mutant / modified form);

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

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

[0037] The "-" is each independently a linking peptide or peptide bond.

[0038] In another preferred embodiment, the VHH includes a complementarity-determining 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, the 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 specifically binding 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. Specification 2 / 20 pages 5 CN 120944823 A

[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 one 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 that 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 human or non-human mammalian Claudin18.2.

[0048] In another preferred embodiment, the Claudin18.2 is 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 their mutants / modified forms, or combinations thereof.

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

[0051] In another preferred embodiment, 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, H also carries a Flag tag sequence.

[0054] In another preferred embodiment, TM1 is selected from the transmembrane region 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 a combination thereof.

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

[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 the co-stimulatory domain of the CD28 protein.

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

[0059] In another preferred embodiment, A is T2A, P2A, or combinations 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 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] Wherein,

[0065] L2 is a no-signal peptide sequence;

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

[0067] TM2 is a transmembrane region;

[0068] Z2 is the intracellular domain of a no-immunostimulatory receptor;

[0069] Each "-" is independently a linking peptide or peptide bond.

[0070] In another preferred embodiment, the engineered immune cell contains 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. Specification 3 / 20 pages 6 CN 120944823 A

[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: TGFBR2 receptor isoform A or TGFBR2 receptor isoform B.

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

[0079] In another preferred embodiment, 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 combinations 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 mutants / modified forms thereof).

[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 further comprises a transmembrane segment and a juxtamembrane 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 juxtamembrane segment is 10-50 aa.

[0087] In another preferred embodiment, the transmembrane segment and the juxtamembrane 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, TM2 has the amino acid sequence shown in SEQ ID NO:22.

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

[0091] In another preferred embodiment, 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, 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, Z2 has the amino acid sequence shown in SEQ ID NO:19.

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

[0099] (a) the cell expresses a chimeric antigen receptor CAR that targets Claudin18.2; and

[0100] (b) the engineered immune cell induces the expression of a signaling protein upon contact with an inducer.

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

[0102] In another preferred embodiment, in the CAR cell, 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. Instructions for Use, Page 4 / 20, CN 120944823 A

[0106] L1-VHH-H-TM1-C-CD3ζ-A-E1(Ib)

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

[0108] Wherein,

[0109] L1 is a no-signal peptide sequence;

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

[0111] H is a no-hinge region;

[0112] TM1 is a transmembrane domain;

[0113] C is a no-co-stimulatory signal domain;

[0114] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild type or its mutant / modified form);

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

[0116] E1 and E2 are each independently signal proteins (including wild-type or mutants / modified forms thereof);

[0117] The "-" is independently a linking peptide or 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, the polynucleotide encoding 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 vector is provided, the vector 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 a fifth method of the invention, a method for preparing engineered immune cells according to the first aspect of the invention is provided, comprising the steps of:

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

[0125] (b) modifying the immune cell to express a CAR molecule and an exogenous signaling protein, thereby obtaining the engineered immune cells according to the first aspect of the invention.

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

[0127] (b1) introducing a first expression vector expressing the CAR into the immune cell; and

[0128] (b2) introducing a second expression vector expressing a signaling protein into the immune cell;

[0129] wherein 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 or a plasmid.

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

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

[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 on the same vector.

[0137] In another preferred embodiment, when the first and second expression vectors are located on the same vector, a third expression vector for expressing the self-cleaving 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 the viral vector contains the 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 comprises the CAR-T cells, and 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 formulation includes an injectable formulation.

[0146] In another preferred embodiment, the concentration of the engineered immune cells (such as CAR-T cells) in the formulation is 1 × 10³–1 × 10⁸ cells / mL, preferably 1 × 10⁴–1 × 10⁷ cells / mL.

[0147] In a seventh aspect of the invention, a kit for preparing engineered immune cells according to the first aspect of the invention is provided, the kit comprising a container, and within the container:

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

[0149] (2) a second nucleic acid sequence, the second nucleic acid sequence comprising a second expression cassette for co-expressing a signaling protein.

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

[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 on the same vector.

[0154] In another preferred embodiment, when the first and second nucleic acid sequences are located on the same vector, a third nucleic acid sequence is further included between the first and second nucleic acid sequences, the third nucleic acid sequence comprising a third expression cassette for expressing a 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 preparations 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, hematologic malignancies, 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 the present invention, the above-mentioned technical features of the present 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. Specification 6 / 20 pages 9 CN 120944823 A Brief Description of the Drawings

[0163] Figure 1 shows a schematic diagram of CAR-T of the present invention.

[0164] Figures 2, 3, and 4 show the expression and phenotypic detection results of CAR in different donor cells, respectively.

[0165] Figure 5 shows the results of reporter gene detection of the TGFβ signaling pathway.

[0166] Figure 6 shows the transient killing ability of each group of CAR-T cells.

[0167] Figure 7 shows the cytokine secretion levels of each group of CAR-T cells.

[0168] Figures 8, 9, and 10 show the sustained killing ability of each group of CAR-T cells after multiple antigen stimulations.

[0169] Figure 11 shows the cell expansion ability of CAR-T cells after multiple rounds of antigen stimulation.

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

[0171] Figure 13 shows the in vivo tumor killing experiment results of each group of CAR-T cells.

[0172] Figure 14 shows the results of pSTAT5 expression detected by FACS method.

[0173] Figure 15 shows the results of pSTAT5 and pSMAD / 3 expression detected by Western Blot method. Detailed Embodiments

[0174] Through extensive and in-depth research, the inventors have developed engineered immune cells that co-express CAR structures and signaling proteins (including the TGFBR2 extracellular domain and the optional IL-7Rα intracellular signaling domain) after extensive screening. In vitro and in vivo experiments showed that the extracellular structure of TGFBR2 can effectively block or reduce TGF-β signaling, protect CAR-T cells, and counteract the effects of the immunosuppressive environment; the intracellular domain of the IL-7 receptor can further enhance downstream cytokine signals, improve the proliferation capacity, exhaustion level, and persistence of CAR-T cells, and increase the therapeutic effect.

[0175] The engineered immune cells of the present invention can not only recognize Claudin18.2 target antigens with high specificity through CAR, targeting solid tumors with high expression of Claudin18.2; 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 signals of TGF-β immunosuppressive factors that are unfavorable to the survival of immune cells in the tumor microenvironment, and further enhance downstream cytokine signals through the intracellular domain of the IL-7 receptor, thereby enhancing the survival of engineered immune cells andSustained tumor killing effect, reducing tumor recurrence. Based on this, the present invention was completed.

[0176] This article takes CAR-T cells as an example to describe in detail 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. The engineered immune cells of the present invention have 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 contains 261 amino acids in total length. It is a four-transmembrane protein with four transmembrane hydrophobic regions. The extracellular structure consists of two ring structures. Ring 1 is formed by transmembrane region 1 and transmembrane region 2. Ring 2 is formed by transmembrane region 3 and transmembrane region 4. The N-terminus, transmembrane region 1, and extracellular loop 1 of human CLDN18.1 and CLDN18.2 differ in 8 amino acids, while the remaining parts are completely 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 CAR is expressed in T cells, the extracellular domain recognizes a specific antigen, which is then transduced through an intracellular domain, inducing cell activation and proliferation, cytotoxicity, and secretion of cytokines such as IL-2 and IFN-γ, affecting tumor cells, causing tumor cells to stop growing, die, or be otherwise affected, and leading to a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with one or more intracellular domains from co-stimulatory molecules and the CD3ζ chain.

[0181] The CAR of the present invention uses VHH targeting CLND18.2 as the antigen-binding domain. In one embodiment of the present 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 first method of the present invention.The CAR-T cells described above. The CAR-T cells of the present invention can be used to treat tumors with high expression of CLND18.2, such as gastric cancer, cholangiocarcinoma, 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 limited by MHC; (2) Given that many tumor cells express the same tumor antigens, once the CAR gene for 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 wild-type and mutant TGF-β. In this invention, TGF-β includes human and non-human mammalian TGF-β.

[0190] As used herein, 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. This factor, as an immunosuppressive factor, can significantly inhibit the activation and proliferation of T cells, promote their differentiation into Tregs, and reduce their effector function.

[0192] Signaling protein

[0193] In this invention, a signaling protein is provided, which comprises an extracellular domain of an immunosuppressive receptor, a transmembrane domain, and optionally an intracellular domain of an immunostimulatory receptor. In one embodiment of the invention, the signaling protein of the invention 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 the present invention can convert the inhibitory signal of TGF-β immunosuppressive factor, which is detrimental to the survival of immune cells in the tumor microenvironment, into an activation signal, thereby enhancing the sustained effect of immune cell survival and tumor killing, improving the efficacy of immunotherapy, and reducing tumor recurrence.

[0194] Expression cassette specification 8 / 20 pages 11 CN 120944823 A

[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 the present invention, the signaling protein may 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 cassette and the second expression cassette respectively further include a promoter and / or a terminator. The promoter of the second expression cassette can be a constitutive or inducible promoter.

[0199] Formulation

[0200] The present invention provides an engineered immune cell (such as CAR-T cells) containing the first aspect of the present 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 1×10³-1×10⁸ cells / mL, more preferably 1×10⁴-1×10⁷ cells / mL.

[0201] In one embodiment, the formulation may include buffers 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] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a vector (e.g., a lentiviral vector) containing the expression cassette of the present invention. The transduced T cells can target surface markers of tumor cells and express signaling proteins, synergistically and significantly enhancing their killing efficiency against tumor cells.

[0204] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response targeting a mammalian tumor cell population or tissue, comprising the steps of: 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 T cells (or allogeneic donor cells) are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This approach results in a very 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-term persistence that leads 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, CAR-mediated immune responses can be part of adoptive immunotherapy steps, wherein CAR-T cells can induce specific immune responses against tumor cells that highly express antigens recognized by the CAR antigen-binding domain. For example, the present inventionThe CAR-T cells of the 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 cancers that can be treated with the CAR of the present invention include, but are not limited to, gastric cancer, bile duct cancer, ovarian cancer, lung cancer, esophageal cancer, and pancreatic cancer.

[0208] Generally, activated and expanded cells as described herein can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method of treating cancer comprising administering a therapeutically effective amount of the CAR-T cells of the present 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. Briefly, 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 disease to be treated (or prevented). 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 amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," 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 condition. Pharmaceutical compositions including T cells described herein can be administered at a dose of 10⁴ to 10⁹ cells / kg body weight, preferably 10⁵ to 10⁷ cells / kg body weight (inclusive of all integer values ​​within the range). T cell compositions can also be administered multiple times at these doses. 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 dosage and treatment regimen for a specific patient can be easily determined by a medical professional by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0212] The subject 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 the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by 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 disease being treated and the recipient of the treatment. The dosage ratio administered to a person can be implemented according to practices accepted in the art. Typically, 1 × 10⁵ to 1 × 10¹⁰ of the modified T cells of the present invention can be administered to the patient per treatment or per course of treatment, for example, by intravenous infusion.

[0214] The main advantages of the present invention include:

[0215] 1. The engineered immune cells of the present invention contain both CAR and signaling proteins, thereby activating immune cells more efficiently.

[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 durable 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 a 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 signaling protein co-expressed by CAR-T cells 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 activation 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 only for illustrating the present invention and are not intended to limit the scope of the present invention. Experimental methods not specifically described in the following examples 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 Structures

[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, CAR9-CAR11 adopt the following as SEQ IDThe sequence shown in NO:1 is used 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 TGFβR2 extracellular region and the IL-7 intracellular region; CAR2 contains only the TGFβR2 extracellular region and does not contain the IL-7 intracellular region; CAR3, CAR4, CAR6, CAR7, CAR8 and CAR9 contain the TGFβR2 extracellular region and the IL-7R intracellular region; CAR10 and CAR11 contain the TGFβR2 extracellular region, the IL-7R intracellular region, the TGFβR1 extracellular region and the IL-2R γ intracellular region. The specific CAR structures are shown in Table 1.

[0225] Table 1 CAR molecular structure of the embodiment

[0226]

[0227]

[0228] The sequences of each structural element for constructing the CAR molecule are shown in Table 2. Specification 11 / 20 pages 14 CN 120944823 A

[0229] Table 2 Sequence of each structural element for constructing the CAR molecule

[0230] Specification 12 / 20 pages 15 CN 120944823 A

[0231]

[0232] *The underlined part is the near-membrane 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 density gradient centrifugation was performed using Ficoll. T cells were enriched (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 a 37 ℃, 5% CO2 incubator.

[0235] Example 2: Target Cell Culture and Construction

[0236] A cell line expressing CLDN18.2 was constructed by 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] A cell line expressing PD-L1 was constructed using the following method: the PD-L1 gene was transduced into target cells AsPC-1-CLDN18.2 via lentivirus, and after cloning and selection, a stable cell line AsPC-1-CLDN18.2-PD-L1 was obtained. The culture medium was prepared according to 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, and then concentrated lentivirus was added. The cells were cultured in a 37 ℃, 5% CO2 incubator. The CAR positivity rate was detected by flow cytometry 72 hours after cell infection.

[0240] After 8-10 days of in vitro culture, CAR-T cells were harvested. The 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 detected, and the cell memory phenotype was analyzed. The proliferation rate of CAR-positive cells was statistically analyzed. There was no significant difference in cell proliferation among the CAR-T groups.

[0241] The experimental results are shown in Figure 2. As shown in the first row of Figure 2, the CAR expression of CAR-T cells was above 20%. As shown in the fourth and fifth rows of Figure 2, CAR2-CAR4 and CAR6-8 cells co-expressed CAR and TGFβR2 on their surface.

[0242] Flow cytometry was performed on CAR+ cells to analyze the cell memory phenotype (CD45RO-CCR7+, / Tscm). The proportion of CAR2-CAR4 was significantly higher than that of CAR5-6. The proportion of CAR2 and CAR3 was 18%-20%, which was higher than that of CAR1 (8.68%). Flow cytometry was also performed on CAR+CD45RO-CCR7+ cells. CAR1-CAR4 cells were all CD27+CD95+, indicating that CAR1-CAR4 cells were all Tscm cells, while only 31.2%-56.3% of CAR5-CAR6 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. The CAR expression rate and the proportions of CD4+ and CD8+ are shown in Figures 3 and 4, respectively. The results showed that the CAR structure of the present invention could be effectively expressed in cells from different donor sources.

[0244] Example 4: Reporter Gene Detection of TGFβ / Smad Signaling Pathway

[0245] CAR expression plasmid, two reporter gene vectors pNL (NLucP / SBE-RE / Hygro, with TGFβ signaling pathway regulatory elements) and pGL4.51 (luc2 / CMV / Neo, with 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 incubated for another 20 hours. The Reporter Assay System was used for detection, and the light signal values ​​were read to compare the inhibitory effects of each molecular structure on downstream TGFβ signals.

[0246] The experimental results are shown in Figure 5. Under different concentrations of TGFβ1 treatment, extracellular TGFBR2 receptors and / orIntracellular tIL-7R protein (CAR2-4) has a significant inhibitory effect on the TGFβ / Smad signaling pathway.

[0247] Example 5: Detection of CAR-T cell killing effect on target cells by 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 screening.

[0249] Target cells and CAR-T cells of various effector cells were mixed and cultured at different effector-target ratios. After culturing for 6 or 24 hours, luciferase substrate was added. The activity of luciferase could be determined by detecting the fluorescence intensity. The cell survival rate could be detected to obtain the killing effect of each CAR-T cell.

[0250] The results are shown in Figure 6a. After co-culturing for 24 hours, 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 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 shown in Figure 6b. After co-culturing for 6 hours, under the culture conditions of E:T = 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% at E:T = 3:1; 50-60% vs 30% at 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 IFN-γ in the co-culture supernatant were measured using the CBA method.

[0254] The results are shown in Figure 7a. The levels of cytokines (IL-2, IFN-γ) produced by CAR cells and target cells AsPC-1-CLDN18.2-PD-L1 after co-culture were significantly lower than those of target cells AsPC-1-CLDN18.2.

[0255] CAR4 cells produced more IL-2 than CAR1-3 after co-culturing with target cells AsPC-1-CLDN18.2-Luc; CAR2-3 cells produced more IL-2 than CAR1 after co-culturing with target cells; and CAR1-4 cells produced more IL-2 than CAR5 and CAR6 after co-culturing with target cells.

[0256] There was no significant difference in the production of IFN-γ by CAR cells in each group against target cells AsPC-1-CLDN18.2-Luc.

[0257] The results are shown in Figure 7b. After co-culturing CAR cells with target cells AGS-CLDN18.1, the production of cytokines (IL-2, IFN-γ) was significantly different among the groups.The levels of CAR-T cells were significantly lower than those of the target cells AGS-CLDN18.2, indicating that CAR-T cells specifically kill CLDN18.2 target cells.

[0258] Example 7: In vitro efficacy study of multiple rounds of killing of cancer cells

[0259] The T cells harvested in Example 3 were subjected to multiple rounds of killing experiments in vitro. The schematic diagram of the experimental method is shown in 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 analysis of T cell phenotype, and Luciferase method to detect the killing rate of CAR-T cells against target cells. 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 above method was used to kill cells for several rounds.

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

[0261] As shown in Figure 8c, under the co-culture conditions of CAR-T and AsPC1-CLDN18.2 (E:T = 1:2) without the addition of TGFβ1 (5 ng / mL), CAR1 showed significantly weaker killing ability than CAR3 and CAR4 in the third round; under the addition of TGFβ1 (5 ng / mL), CAR1 had no killing ability in the third round, while CAR3 and CAR4 still had significant killing ability in the fourth round, showing that the sustained killing ability of CAR3 and CAR4 (which have an extracellular TGFBR2 receptor and an intracellular tIL-7R) was significantly better than that of CAR1.

[0262] As shown in Figure 9, under the 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 showed significantly weaker killing ability in the 5th 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-2R γ).(protein). The results showed that the sustained killing ability of CAR3 and CAR11 was significantly better than that of CAR1, CAR2 and CAR9.

[0263] As shown in Figure 10, using CAR-T cells from different donor sources than those in Figure 9, the sustained killing ability of CAR2, CAR3 and CAR11 after multiple rounds of in vitro antigen stimulation was compared. Under the co-culture conditions of CAR-T cells and AGS-CLDN18.2 (E:T = 1:2), without the addition of TGFβ1 (10 ng / mL), CAR2 showed no significant ability to lyse target cells in the 7th round, while CAR3 and CAR11 showed significant killing ability. The killing ability of CAR2 was significantly weaker than that of CAR3 and CAR11; under the conditions of adding TGFβ1 (10 ng / mL), CAR2, CAR3 and CAR11 still had significant killing ability in the 7th round.

[0264] The cell expansion capacity of CAR-T cells after multiple rounds of antigen stimulation was analyzed. The results are shown in Figure 11. Under the culture condition without the addition of TGFβ1 (10 ng / mL), the cell expansion rate after the 6th round of antigen stimulation was analyzed. CAR2 was significantly slower than CAR3 and CAR11. After the 7th round of antigen stimulation, no significant target cell lysis ability was observed. The cell expansion rates of CAR3 and CAR11 also decreased significantly after the 7th round of antigen stimulation. Under the culture condition with the addition of TGFβ1 (10 ng / mL), the cell expansion rate after the 7th round of antigen stimulation was analyzed. CAR11 had 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. The results are shown in Figure 12. The trend of CAR expression rate change was consistent with the CAR-T cell expansion rate.

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

[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 (NOD.Cg-B2mem1Tac Prkdcscid H2-Ab1tm1Doi Il2rgtm1Sug / JicCrl) model.

[0269] 2×106 AsPC-1-CLDN18.2-Luc cells were subcutaneously injected. When the tumor burden was about 200 mm3, the mice were divided into groups of 3-4. One day after grouping, 200 μL of CLDN18.2 CAR-T cells and NT cells were injected via the tail vein, 2×106 CAR-T cells / 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 vivo. Blood was taken once a week thereafter.Similarly, various phenotypes of CAR-T cells were detected, and the size of subcutaneous tumors and the weight of mice were detected twice a week.

[0270] The results are shown in Figure 13. The tumors of mice that were infused with CAR1 and CAR3 cells were significantly reduced to clear. On Day 50 after the infusion of CAR-T cells, the mice were still alive and there was no tumor recurrence.

[0271] 2×106 AsPC-1-CLDN18.2-Luc cells were inoculated on the left side of the mouse tumor. On Day 100 after the infusion of CAR-T cells, the size of the tumors on both sides was detected. No tumors were detected in mice that were infused with CAR3 (extracellular TGFBR2 receptor and intracellular tIL-7R protein). In mice that were infused with CAR1, there was a tumor burden on the left side in 3 / 3 of the mice, and one mouse on the right side had a recurrence of tumor.

[0272] On Day 106 after CAR-T cell infusion, 2 × 10⁶ AsPC-1-CLDN18.2-Luc cells were inoculated on the right side of the mouse tumor. On Day 160 after CAR-T cell infusion, the tumor size on both sides of the mice after CAR3 infusion was measured. It was found that the left tumor (first re-challenge) did not recur. Observation of the right tumor (second re-challenge) showed that one of the three mice (1 / 3) had no detectable tumor burden, while the tumors of the other two mice grew, but the tumor burden was significantly smaller than that of the control group (400 mm³ vs 1000 mm³).

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

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

[0275] (I) FACS method detection:

[0276] FACS detection of TGFβ / Smad signaling pathway in CAR-T cells of each group was performed. The specific method is as follows: After culturing CAR-T cells of each group in a 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 are shown in Figure 14a, and the statistical data are shown in Figure 14b. CAR-T cells treated with IL-2 as the positive control group showed a significant increase in pSTAT5, and the pSTAT5 positive cells were all above 80%. Comparing the intracellular pSTAT5 levels of the TGFβ-treated 1 hour group and the 3 hour group, the 3 hour group was significantly lower than the 1 hour group. The analysis of the TGFβ-treated 1 hour and 3 hour groups showed that the levels of pSTAT5 in the 3 hour group were significantly lower than those in the 1 hour group.pSTAT5 levels were highest in CAR11 cells, followed by CAR9 and CAR3. Figure 14c further analyzes the pSTAT5 expression levels in CAR3, CAR9, and CAR11 cells for CAR-negative (red) and CAR-positive (blue) cells. Flow cytometry data shows that CAR-positive cells have higher MFI than CAR-negative cells, indicating that pSTAT5 expression is higher in CAR-positive cells than in CAR-negative cells, with CAR11-positive cells showing the highest pSTAT5 expression.

[0278] (II) Western Detection Method:

[0279] CAR-T cells expressing CAR were screened. After culturing each group of CAR-T cells in a medium without IL-2 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. The positive control group was treated with IL-2 for 0.5 hours. Cells were collected, cells were 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 then developed with chemiluminescent reagents 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 shown in Figure 15. In 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 CAR11 cells showed significantly increased pSTAT5 expression after TGFβ1 treatment, 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 Blot detection is lower than that of FACS detection. In CAR1 and CAR2 cells, significant pSMAD2 / 3 expression was detected, and this was determined by a TGFβ concentration gradient, meaning that pSMAD2 / 3 expression in CAR-T cells in the high-concentration TGFβ1 group was higher than in the low-concentration group. Sequence

[0282] VHH1 (SEQ ID NO: 1)

[0283] QVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFVAGITWSGGIKDYADSVKGRFT IARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSS

[0284] VHH2 (SEQ ID NO:2)

[0285] EVQLVESGGGLVQPGGSLRLSCAASGNILSINYMGWYRQAPGKERELVATITRGGSTNYQGNLKGRFIISGDNTKDTVYLQMNNLQPEDTAVYYCYFRINFGILGGLRDYWGQGTQVTVSS Page 16 / 20 of the specification 19 CN 120944823 A

[0286] VHH3(SEQ ID NO:3)

[0287] QVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWVRQAPGKGLEWVAGITWSGGIKDYADSVKGRFT IARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSS

[0288] VHH4(SEQ ID NO:4)

[0289] QVQLVESGGGLVQPGGSLRLSCAASDSTFSSYIMGWFRQAPGKGLEFVAGITWSGGIKDYADSVKGRFT IARDNSKNTLYLQMNSLRAEDAAVYYCAANAIQLTRRSGDYAYWGQGTLVTVSS

[0290] CAR1(SEQ ID NO:26)

[0291] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0292] CAR2(SEQ ID NO:27)

[0293] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTTTDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR EGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINN DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYC YRVNRQQKLSS

[0294] CAR3(SEQ ID NO:28)

[0295] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR EGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILAC VLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQ RLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0296] CAR4(SEQ ID NO:29)

[0297] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DESCRIPTION PAGE 17 / 20 20 CN 120944823 A DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR EGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINN DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCCSSDECNDNIIFSEEKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMD PILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQA RDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRES GKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0298] CAR5(SEQ ID NO:30)

[0299] MALPVTALLLPLALLHAARPEVQLVESGGGLVQPGGSLRLSCAASGNILSINYMGWYRQAPGKERELV ATITRGGSTNYQGNLKGRFIISGDNTKDTVYLQMNNLQPEDAVYYCYFRINFGILGGLRDYWGQGTQVTVSSTTTD YKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAAFIIFWVR SKRSRLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0300] CAR6(SEQ ID NO:31)

[0301] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGNILSINYMGWYRQAPGKERELV ATITRGGSTNYQGNLKGRFIISGDNTKDTVYLQMNNLQPEDTAVYCYFRINFGILGGLRDYWGQGTQVTVSSTTTD YKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVVVGGVLACYSLLTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREG RGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFIL EDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILACVL WKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRL GGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPP PFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0302] CAR7(SEQ ID NO:32)

[0303] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWVRQAPGKGLEWV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR EGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINN DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILAC VLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQ RLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0304] CAR8(SEQ ID NO:33)

[0305] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASDSTFSSYIMGWFRQAPGKGLEFV DESCRIPTION 18 / 20 pages 21 CN 120944823 A AGITWSGGIKDYADSVKGRFTIARDNSKNTLYLQMNSLRAEDAAVYYCAANAIQLTRRSGDYAYWGQGTLVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINN DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILAC VLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQ RLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTL PPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0306] CAR9(SEQ ID NO:34)

[0307] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR EGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTCPTISILSFFSVALLVI LACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEES EKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTN STLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0308] CAR10(SEQ ID NO:35)

[0309] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ

[0310] GLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGP

[0311] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMI

[0312] VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE

[0313] TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLL

[0314] LVIFQHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIK

[0315] PIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQ

[0316] LEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESG

[0317] KNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQ

[0318] NQEGRGSLLTCGDVEENPGPMEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHL

[0319] CTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQ

[0320] DHCNKIELPTTVKSSPGLGPVELPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISV Page 19 / 20 of the specification 22 CN 120944823 A

[0321] GSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERL

[0322] CLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0323] CAR11(SEQ ID NO:36)

[0324] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQTGGSLRLSCAASDSTFSSYIMGWFRQAPGKEREFV AGITWSGGIKDYADSVKGRFTIARDNAKNTVYLQMNSLKPEDAAIYYCAANAIQLTRRSGDYAYWGQGTQVTVSSTT TDYKDDDDKPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GSGEGRGSLLTCGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDV RFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTL EHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPES FGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPI LTSLGSNQEEAYVTMSSFYQNQGSGEGRGSLLTCGDVEENPGPMEAAVAAPRPRLLLLVLAAAAAAAAALLPGATAL QCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIEL PTTVKSSPGLGPVELVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPD YSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[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 the invention, and these equivalent forms also fall within the scope defined by the appended claims. Specification 20 / 20 pages 23 CN 120944823 A Figure 1 Description DrawingsPage 1 / 9 24 CN 120944823 A Figure 2 Appendix to the Specification Page 2 / 9 25 CN 120944823 A Figure 3 Figure 4 Figure 5 Appendix to the Specification Page 3 / 9 26 CN 120944823 A Figure 6 Figure 7 Appendix to the Specification Page 4 / 9 27 CN 120944823 A Figure 8 Figure 9 Appendix to the Specification Page 5 / 9 28 CN 120944823 A Figure 10 Figure 11 Figure 12 Appendix to the Specification Page 6 / 9 29 CN 120944823 A Figure 13 Appendix to the Specification Page 7 / 9 30 CN 120944823 A Appendix to the Specification Page 8 / 9 31 CN 120944823 A Figure 14 Figure 15 Appendix to the Specification Page 9 / 9 32 CN 120944823 A Abstract The present invention relates to a chimeric antigen receptor immune cell specifically binding to Claudin 18.2, and a use thereof. Specifically, provided is a CAR-T cell using a Claudin 18.2-targeting nanobody as an antigen-binding domain, capable of highly specifically targeting Claudin 18.2-positive tumor cells; the CAR-T cell co-expresses the extracellular domain of a TGF-β receptor, and optionally the intracellular signaling domain of an IL-7 receptor, which can effectively block or reduce TGF-β-induced inhibitory signals, and promote immune cell expansion, survival and persistence, thereby enhancing specific killing of tumor cells. Thechimeric antigen receptor immune cell can be used for treating Claudin 18.2-positive tumors.

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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