Claudin 18.2-targeting chimeric antigen receptor fused with chimeric switch receptor, immune cell modified thereby, and use thereof

Abstract

Disclosed are a Claudin 18.2-targeting chimeric antigen receptor fused with a chimeric switch receptor, an immune cell modified thereby, and the use thereof. Specifically disclosed are a chimeric antigen receptor having an amino acid sequence of SEQ ID NO: 1, and a T cell modified thereby. According to the CAR of the present application, a dual-epitope design is used, and fusion with an interferon and a chimeric switch receptor is achieved, so as to enhance the affinity of a CAR-T cell and the ability of the CAR-T cell to resist a tumor microenvironment. The CAR-T cell of the present application can secrete the specific effector molecule IFN-γ, and exhibits the ability to kill Claudin 18.2-positive tumor cells in vitro and tumor-killing activity in vivo.

Description

Chimeric antigen and chimeric switch receptor targeting Claudin 18.2, modified immune cells thereof and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of cellular immunotherapy, and particularly relates to chimeric antigen and chimeric switch receptor targeting Claudin 18.2, modified immune cells thereof and uses thereof. BACKGROUND

[0002] Claudins (CLDNs) are a family of proteins that are important in normal tissue tight junctions, with four transmembrane domains, and function in the regulation of cell paracellular permeability and conductance. The Claudin family contains at least 27 members, of which Claudin 18.2 (CLDN18.2) is a member of the Claudin protein family, which is a transmembrane protein with high tissue expression specificity. It has very low expression level in normal tissues, but is highly selectively expressed in non-malignant gastric mucosa. It forms a tight junction complex with Claudin 18.2-molecules expressed on the surface of adjacent cells, forms a selectively permeable barrier, and realizes tissue-specific permeability. This tight junction protein is exposed on the surface of tumor cells during malignant transformation, making it a targetable target for cancer treatment. Studies have found that under normal physiological conditions, Claudin 18.2 is only expressed in differentiated epithelial cells in the gastric mucosa, and is not expressed in other healthy tissues. However, Claudin 18.2 is highly expressed in gastrointestinal tumors such as gastric cancer, colorectal adenocarcinoma, esophageal cancer, pancreatic cancer, as well as lung cancer, lung adenocarcinoma, liver cancer, ovarian cancer, and kidney cancer. The disorder of tumor cell polarity can lead to the exposure of Claudin 18.2 protein epitopes on the cell surface and abnormal activation. This normal tissue-limited expression and specific high expression in tumors make Claudin 18.2 an ideal target for solid tumor treatment.

[0003] Existing CAR-T cell therapy shows therapeutic potential in clinical trials of CLDN18.2-positive advanced gastrointestinal tumors, with good overall tolerability and controllable safety. However, CAR-T therapy still faces many challenges in solid tumors such as gastrointestinal tumors. The low pH, low oxygen, high permeability, and other environments in the tumor microenvironment have immunosuppressive effects, which are not conducive to the survival and anti-tumor effects of CAR-T cells. At the same time, there are many immunosuppressive cells in the tumor microenvironment, which secrete transforming growth factor beta (TGF-beta) to promote tumor cell growth and metastasis and inhibit cytotoxic T cells. On the other hand, there is an endogenous regulatory mechanism for the immune activity of T cells. Activated T cells up-regulate the expression of various immunosuppressive molecules such as PD-1, causing T cells to enter an exhausted state and undergo apoptosis.

[0004] Claudin 18.2 is an important target for treating solid tumors, and research on CAR-T cells targeting Claudin 18.2 is still in its infancy. There is no drug targeting Claudin 18.2 on the market in China. Therefore, it is of great significance and wide application value to improve and perfect the structure of CAR, innovate and develop new CAR-T cells and their therapies, change the immunosuppressive microenvironment, and improve the specificity and anti-tumor activity of CAR-T cells, which can more accurately locate tumor targets and more efficiently kill tumors.

[0005] SUMMARY

[0006] One of the purposes of the present application is to provide a chimeric antigen receptor targeting Claudin 18.2, immune effector cells containing the chimeric antigen receptor, and applications thereof. The technical problems to be solved are not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0007] To achieve the above-mentioned purpose, the present application first provides a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain and an intracellular signaling domain, the antigen binding domain comprises a first single domain antibody and a second single domain antibody, the first single domain antibody comprises complementarity determining regions CDR1 of 47-54 of SEQ ID NO: 1, complementarity determining regions CDR2 of 72-78 of SEQ ID NO: 1 and complementarity determining regions CDR3 of 116-130 of SEQ ID NO: 1.

[0008] The second single domain antibody comprises complementarity determining regions CDR1 of 182-188 of SEQ ID NO: 1, complementarity determining regions CDR2 of 206-215 of SEQ ID NO: 1 and complementarity determining regions CDR3 of 254-267 of SEQ ID NO: 1.

[0009] The chimeric antigen receptor, the first single domain antibody and the second single domain antibody all target Claudin 18.2.

[0010] The name of the first single domain antibody can be Claudin 18.2-VHH#223. The name of the second single domain antibody can be Claudin 18.2-VHH#357.

[0011] The sequence of the complementarity determining region (CDR) is defined according to the Kabat numbering system.

[0012] The single-domain antibody only has a variable region (VHH) of a heavy chain antibody, which is composed of a framework region FR1, a complementarity determining region CDR1, a framework region FR2, a complementarity determining region CDR2, a framework region FR3, a complementarity determining region CDR3, and a framework region FR4 in sequence.

[0013] The amino acid sequence of FR1 of the first single-domain antibody is shown in SEQ ID NO: 1 at positions 22-46; the amino acid sequence of FR2 is shown in SEQ ID NO: 1 at positions 55-71; the amino acid sequence of FR3 is shown in SEQ ID NO: 1 at positions 79-115; and the amino acid sequence of FR4 is shown in SEQ ID NO: 1 at positions 131-141.

[0014] The amino acid sequence of FR1 of the second single-domain antibody is shown in SEQ ID NO: 1 at positions 157-181; the amino acid sequence of FR2 is shown in SEQ ID NO: 1 at positions 189-205; the amino acid sequence of FR3 is shown in SEQ ID NO: 1 at positions 216-253; and the amino acid sequence of FR4 is shown in SEQ ID NO: 1 at positions 268-278.

[0015] In the above-mentioned chimeric antigen receptor, the first single-domain antibody can be any one of the following:

[0016] A1) a protein with an amino acid sequence shown in SEQ ID NO: 1 at positions 22-141;

[0017] A2) a protein with 80% or more identity to the protein shown in A1) and having the same function, obtained by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 1 at positions 22-141;

[0018] A3) a fusion protein with the same function, obtained by connecting a tag or a signal peptide to the N-terminus and / or the C-terminus of A1) or A2);

[0019] The second single-domain antibody can be any one of the following:

[0020] B1) a protein with an amino acid sequence shown in SEQ ID NO: 1 at positions 157-278;

[0021] B2) a protein with 80% or more identity to the protein shown in B1) and having the same function, obtained by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 1 at positions 157-278;

[0022] B3) connecting a tag or a signal peptide at the N-terminus and / or C-terminus of the fusion protein of B1) or B2) to obtain a fusion protein with the same function.

[0023] Further, the amino acid sequence can be inconsistent at the framework region (FR), for example, having one or several amino acid substitutions, deletions or additions in the framework region.

[0024] The framework region (FR) is known in the art, which refers to the region in the variable region (VHH) other than the CDR, including FR1, FR2, FR3 and FR4.

[0025] The several can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0026] Further, the second single-domain antibody can be located at the N-terminus or C-terminus of the first single-domain antibody. Specifically, the second single-domain antibody is located at the C-terminus of the first single-domain antibody.

[0027] Further, the first single-domain antibody and the second single-domain antibody can be connected by a linker.

[0028] The linker can be a flexible peptide linker, for example, a peptide linker including glycine, serine, proline and / or lysine residues. The peptide linker can consist of 1-40 amino acids.

[0029] Further, the linker includes but is not limited to: (G)n, (S)n, (GxS)n, (SxG)n, (GSSGG)n (SEQ ID NO: 11), (GGSGG)n (SEQ ID NO: 12), (GSGGSG)n (SEQ ID NO: 13), (GSGSGS)n (SEQ ID NO: 14), (GGQGG)n (SEQ ID NO: 15), (EAAAK)n (SEQ ID NO: 16) and IEGRMD (SEQ ID NO: 17), and various combinations thereof. Wherein: n can be any integer between 1-10; x can be any integer between 1-6. In an embodiment of the present application, the linker is (GGGGS)3 (SEQ ID NO: 18).

[0030] The first single-domain antibody (including antigen-binding fragments thereof) and the second single-domain antibody (including antigen-binding fragments thereof) can be connected by a linker to form a biparatopic single-domain antibody targeting two different epitopes of Claudin 18.2. The biparatopic single-domain antibody constitutes the antigen-binding domain of the chimeric antigen receptor of the present application.

[0031] Further, the antigen binding domain of the chimeric antigen receptor can be the first single-domain antibody, the linker, and the second single-domain antibody in sequence from N-terminus to C-terminus. The amino acid sequence of the antigen binding domain can be represented by SEQ ID NO: 1 at positions 22-278.

[0032] In the above chimeric antigen receptor, the intracellular signaling domain comprises a costimulatory domain, an activating domain, an interferon, and a chimeric switch receptor comprising an extracellular region of PD-1, an extracellular region of TGFβ receptor type II, and a transmembrane region and a cytoplasmic region of CD27.

[0033] The chimeric switch receptor can be named PD-1&TRII&CD27, and can be used to convert the immunosuppressive signals conducted by PD-1 and TGFβ into the T cell activation signals mediated by CD27, so as to achieve efficient and persistent killing of tumor cells and improve the ability of CAR cells to resist immunosuppressive tumor microenvironment.

[0034] Further, the chimeric switch receptor can be located at the C-terminus of the chimeric antigen receptor.

[0035] Further, the chimeric switch receptor can be the extracellular region of PD-1, the extracellular region of TGFβ receptor type II, the transmembrane region and the cytoplasmic region of CD27 in sequence from N-terminus to C-terminus.

[0036] Further, the amino acid sequence of the chimeric switch receptor can be represented by SEQ ID NO: 1 at positions 739-1144.

[0037] The interferon can be a type I interferon (such as IFNα (IFN-α), IFNβ (IFN-β), or IFNω (IFN-ω)).

[0038] Further, the interferon can be IFNα.

[0039] Further, the amino acid sequence of the IFNα can be represented by SEQ ID NO: 1 at positions 529-716.

[0040] The intracellular signaling domain can comprise one, two or more costimulatory domains. The costimulatory domain can comprise the entire intracellular portion of a costimulatory molecule, or a functional fragment thereof. A “costimulatory molecule” can refer to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell.

[0041] The co-stimulatory domain can be an intracellular signaling domain from a co-stimulatory molecule, for example, the co-stimulatory domain can be derived from an intracellular signaling domain of CARD11, CD2, CD7, CD27, CD28, CD30, ICAM-1 (CD54), LFA-1 (CD11a), CD134 (OX40), CD137 (4-1BB), 2B4, CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), or DAP10.

[0042] The activation domain can be derived from an intracellular signaling domain of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d.

[0043] In the above chimeric antigen receptor, the co-stimulatory domain can be derived from 4-1BB, and / or the activation domain can be derived from CD3 zeta.

[0044] Further, the co-stimulatory domain can be an intracellular signaling domain of 4-1BB.

[0045] Further, the amino acid sequence of the co-stimulatory domain can be as shown in positions 348-394 of SEQ ID NO: 1.

[0046] Further, the activation domain can be an intracellular signaling domain of CD3 zeta.

[0047] Further, the amino acid sequence of the activation domain can be as shown in positions 395-506 of SEQ ID NO: 1.

[0048] The intracellular signaling domain can further comprise a linker for connecting the CD3 zeta, IFN alpha, and chimeric switch receptor.

[0049] Further, the linker can be a cleavable linker.

[0050] Further, the cleavable linker comprises P2A, F2A, T2A, and E2A, or any combination thereof, but is not limited thereto. The amino acid sequence of the P2A can be as shown in SEQ ID NO: 7. The amino acid sequence of the F2A can be as shown in SEQ ID NO: 8. The amino acid sequence of the T2A can be as shown in SEQ ID NO: 9. The amino acid sequence of the E2A can be as shown in SEQ ID NO: 10.

[0051] In one or more embodiments of the present application, the cleavable linker is P2A.

[0052] Although the P2A is used as the connecting linker in the chimeric antigen receptor designed in the present application, the present application is not limited to this specific linker, and the P2A in the chimeric antigen receptor of the present application can be replaced by those skilled in the art, for example, F2A, T2A and E2A which have the same "self-cleavage" function as P2A. As long as the chimeric antigen receptor constructed is functionally equivalent to the chimeric antigen receptor of the present application, it is considered as a chimeric antigen receptor equivalent to the chimeric antigen receptor of the present application.

[0053] The intracellular signaling domain can be, from N-terminal to C-terminal, co-stimulatory domain 4-1BB, activation domain CD3 zeta, P2A, IFN alpha, P2A, chimeric switch receptor PD-1 & TRII & CD27.

[0054] Further, the amino acid sequence of the intracellular signaling domain can be as shown in SEQ ID NO: 1 at positions 348-1144.

[0055] In the chimeric antigen receptor described herein, the transmembrane domain can be derived from the transmembrane domain of the alpha, beta or zeta chain of the T cell receptor, CD3 zeta, CD8a (CD8 alpha), CD4, CD9, CD45, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152 or CD154.

[0056] Further, the transmembrane domain is located between the antigen binding domain and the intracellular signaling domain, responsible for maintaining the stability of the CAR molecule on the T cell membrane.

[0057] Further, the transmembrane domain can be the CD8a transmembrane region (TM).

[0058] The chimeric antigen receptor described herein can also include a hinge domain (Hinge) located at the N-terminal of the transmembrane domain.

[0059] Further, the hinge domain can be derived from the hinge region of CD8a (CD8 alpha), CD4, CD5, CD7, CD28, CD134, CD137, ICOS, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM.

[0060] Further, the hinge domain can be the CD8a hinge region (Hinge).

[0061] Further, the amino acid sequence of the CD8a hinge region (Hinge) and transmembrane region (TM) can be as shown in SEQ ID NO: 1 at positions 279-347.

[0062] The chimeric antigen receptor described herein can further comprise a signal peptide (Signal) at the N-terminus of the antigen binding domain.

[0063] Further, the signal peptide can be derived from the signal peptide of CD4, CD8, CD8a, CD28, CD137, and GM-CSF.

[0064] Further, the signal peptide can be a human CD8 signal peptide. The amino acid sequence of the human CD8 signal peptide can be as shown in SEQ ID NO: 1 at positions 1-21.

[0065] The chimeric antigen receptor described herein can be, from N-terminus to C-terminus, a signal peptide (Signal), a first single-domain antibody, a linker, a second single-domain antibody, a CD8a hinge region, a CD8a transmembrane region, a costimulatory domain 4-1BB, an activation domain CD3 zeta, a cleavable linker P2A, IFN a, a cleavable linker P2A, a chimeric switch receptor PD-1 & TRII & CD27.

[0066] Further, the chimeric antigen receptor described herein can be any one of the following:

[0067] C1) a protein having an amino acid sequence as shown in SEQ ID NO: 1 at positions 22-1144;

[0068] C2) a protein having 80% or more identity to the protein shown in C1) and having the same function, obtained by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 1 at positions 22-1144;

[0069] C3) a fusion protein having the same function, obtained by connecting a tag or a signal peptide at the N-terminus and / or the C-terminus of C1) or C2).

[0070] Further, the amino acid sequence of the fusion protein in C3) can be as shown in SEQ ID NO: 1.

[0071] The protein having an amino acid sequence of SEQ ID NO: 1 at positions 22-1144 is a chimeric antigen receptor without a signal peptide, and the amino acid sequence of the chimeric antigen receptor with a signal peptide can be as shown in SEQ ID NO: 1.

[0072] The substitution described herein can be a conservative substitution.

[0073] The connection in A3), B3), and C3) can be directly connected by a peptide bond or connected through a linker.

[0074] To facilitate the isolation, purification, detection and / or localization of the chimeric antigen receptors described herein, a tag protein can be attached to the amino-terminal or carboxy-terminal end of the chimeric antigen receptors. The tags include, but are not limited to, a GST (glutathione S-transferase) tag protein, a Trx (thioredoxin) tag protein, a NusA tag protein, a His tag protein (His-tag), a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA (influenza hemagglutinin) tag protein, a Myc tag protein, a LacZ tag protein, a CBD (cellulose binding domain) tag protein, a bacteriophage T7 protein kinase (T7PK) tag protein, a GFP (green fluorescent protein), a CFP (cyan fluorescent protein), a YFP (yellow-green fluorescent protein), a mCherry (monomeric red fluorescent protein), or an AviTag tag protein. Those skilled in the art know how to select a suitable tag protein according to the desired purpose. The use of a tag does not change the function of the protein of interest, and its purpose is to isolate, purify, detect or track, so the tag proteins suitable for the present application are not limited to a particular kind. The tag can be separated from the protein of interest by chemical cleavage or enzymatic cleavage known in the art, such as introducing a protease cleavage site to remove the tag using TEV protease cleavage.

[0075] The present application also provides a biological material, which can be any one of the following:

[0076] D1) a nucleic acid molecule encoding any of the chimeric antigen receptors described herein;

[0077] D2) an expression cassette containing the nucleic acid molecule of D1);

[0078] D3) a recombinant vector containing the nucleic acid molecule of D1);

[0079] D4) a recombinant microorganism containing the nucleic acid molecule of D1);

[0080] D5) a recombinant host cell containing the nucleic acid molecule of D1).

[0081] The biological material can express any of the chimeric antigen receptors described herein.

[0082] The recombinant vector can be a recombinant vector obtained by cloning a nucleic acid molecule encoding any of the chimeric antigen receptors described herein (such as the DNA molecule shown in SEQ ID NO: 2) into a backbone vector. Although the examples provided by the present application utilize a pUC57 vector and a retroviral vector as the backbone vector, the present application can not be limited to this particular vector. Those skilled in the art can use other suitable vectors, such as a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a non-viral vector transposition system, a CRISPR-Cas system, or other backbone vectors for cloning or expression, etc. As long as the vector can clone or express a nucleic acid molecule encoding any of the chimeric antigen receptors described herein.

[0083] Further, the recombinant vector can be specifically a recombinant retroviral vector MP71-Claudin 18.2 CAR-IC. The MP71-Claudin 18.2 CAR-IC is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment whose nucleotide sequence is SEQ ID NO: 2 in the sequence listing, while keeping other nucleotide sequences of the retroviral vector MP71 unchanged.

[0084] In the above-mentioned biological material, the nucleic acid molecule can be any of the following:

[0085] E1) a coding sequence or a nucleotide sequence such as positions 64-3432 of SEQ ID NO: 2 or a DNA molecule shown in SEQ ID NO: 2;

[0086] E2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in positions 64-3432 of SEQ ID NO: 2, and encoding a chimeric antigen receptor whose amino acid sequence is shown in positions 22-1144 of SEQ ID NO: 1;

[0087] E3) a DNA molecule having 75% or more identity to the nucleotide sequence defined in SEQ ID NO: 2, and encoding a chimeric antigen receptor whose amino acid sequence is shown in SEQ ID NO: 1.

[0088] The DNA molecule shown in SEQ ID NO: 2 encodes a chimeric antigen receptor whose amino acid sequence is SEQ ID NO: 1 (including the signal peptide).

[0089] The DNA molecule shown in positions 64-3432 of SEQ ID NO: 2 encodes a chimeric antigen receptor whose amino acid sequence is shown in positions 22-1144 of SEQ ID NO: 1 (not including the signal peptide).

[0090] One of ordinary skill in the art can readily employ known methods, such as site-directed mutagenesis (including oligonucleotide primer-directed site- directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis, etc.) or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random priming recombination, etc.) to mutate the nucleotide sequence encoding any of the chimeric antigen receptors described herein. Those nucleotide sequences that are artificially constructed, which are more than 75% identical to the nucleotide sequence encoding any of the chimeric antigen receptors described herein (e.g., SEQ ID NO: 2), and which encode any of the chimeric antigen receptors described herein and have the same function as any of the chimeric antigen receptors described herein, are derived from the nucleotide sequences of the present application and are equivalent to the sequences of the present application.

[0091] The nucleic acid molecules described herein can also include nucleic acid molecules that are codon-preference engineered based on the nucleotide sequence set forth in SEQ ID NO: 2. One of skill in the art can use codons appropriate for expression in a particular species, taking into account the degeneracy of the code and the preference of codons in different species, as needed.

[0092] Variants having improved affinity to the sequence of any of the chimeric antigen receptors described herein can be obtained by employing methods known in the art and are included within the scope of the present application. For example, amino acid substitutions can be used to obtain chimeric antigen receptors having further improved affinity. Alternatively, codon optimization of the nucleotide sequence can also be used to improve the efficiency of translation in an expression system used to produce the chimeric antigen receptor.

[0093] In certain embodiments, substitutions, insertions, or deletions can occur within one or more complementarity determining regions or framework regions of the single domain antibody in any of the chimeric antigen receptors of the present application, so long as such changes do not substantially reduce the ability of the chimeric antigen receptor to bind to an antigen. For example, conservative changes (e.g., conservative substitutions of amino acids, well known to those skilled in the art, which do not change the properties and functions of the protein) can be made to the complementarity determining regions and / or framework regions that do not substantially reduce binding affinity. Such changes can be outside of antigen contact residues in the complementarity determining regions, for example.

[0094] The present application also provides modified immune effector cells comprising any of the chimeric antigen receptors described herein or the nucleic acid molecules.

[0095] The modified immune effector cells can be immune effector cells modified with any of the chimeric antigen receptors described herein (i.e., CAR-modified immune effector cells, CAR cells) that express any of the chimeric antigen receptors described herein.

[0096] Further, the immune effector cells can include T cells, NK cells, B cells, macrophages, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), and monocytes (MCs).

[0097] The immune effector cells can also include (or be derived from) stem cells capable of being induced to differentiate into immune effector cells in vivo or in vitro, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells (HSCs).

[0098] The immune effector cells can be autologous or non-autologous (allogeneic, syngeneic, or xenogeneic). Further, the immune effector cells are autologous or allogeneic.

[0099] Further, the modified immune effector cells can include CAR-T cells, CAR-NK cells, CAR-NKT cells, CAR-γδT cells, CAR-αβT cells, CAR-regulatory T cells (CAR-Treg cells), CAR-MAIT cells, CAR-B cells, CAR-macrophages (CAR-M cells), CAR-dendritic cells (CAR-DC cells), CAR-peripheral blood mononuclear cells (CAR-PBMC cells), CAR-monocytes (CAR-MC cells), CAR-iPSC cells, and CAR-PSC cells, but are not limited thereto.

[0100] Further, the immune effector cells can be T cells.

[0101] The T cells can include CD8 + T cells, CD4 + T cells, regulatory T cells (Tregs), naive T cells, effector T cells, memory T cells (including stem memory T cells, central memory T cells, and effector memory T cells), αβT cells, γδT cells, Natural killer T cells (NKT cells), and Mucosal-associated invariant T cells (MAIT cells), etc., but are not limited thereto.

[0102] Further, the T cells can be derived from tumor patients or healthy volunteers.

[0103] Further, the T cells can be derived from peripheral blood, monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, infected site tissue, ascites, pleural effusion, spleen tissue, and tumors, but are not limited thereto.

[0104] The T cells can be isolated using various techniques known to those skilled in the art (e.g. density gradient separation techniques, adherence separation methods, magnetic bead separation techniques, flow cytometry sorting techniques, etc.).

[0105] Further, the modified immune effector cell can be a CAR-T cell.

[0106] Further, the CAR-T cell can be a Claudin 18.2 CAR-IC T cell.

[0107] The Claudin 18.2 CAR-IC T cell is a CAR-T cell obtained by stably expressing a gene of a chimeric antigen receptor constructed by the present application into a T cell through a method of genetic engineering in vitro recombination.

[0108] Further, the Claudin 18.2 CAR-IC T cell contains and / or expresses a DNA molecule shown in SEQ ID NO: 2 or positions 64-3432 of SEQ ID NO: 2.

[0109] The Claudin 18.2 CAR-IC T cell can have at least any one of the following properties:

[0110] (1) secreting a T cell specific effector molecule IFN-γ;

[0111] (2) specifically killing Claudin 18.2 positive tumor cells;

[0112] (3) inhibiting Claudin 18.2 positive tumor cell proliferation;

[0113] (4) inhibiting Claudin 18.2 positive tumor growth;

[0114] (5) anti-tumor microenvironment immunosuppression and / or T cell exhaustion;

[0115] (6) converting PD-1 and TGFβ mediated immunosuppressive signals into CD27 mediated T cell activation signals.

[0116] The present application also provides the use of any of the chimeric antigen receptors described herein, the biomaterials or the modified immune effector cells described herein in the preparation of a product having any of the following functions:

[0117] F1) preventing or treating tumors;

[0118] F2) preventing or treating Claudin 18.2 target related diseases;

[0119] F3) killing tumor cells positive for Claudin 18.2;

[0120] F4) inhibiting proliferation of tumor cells positive for Claudin 18.2;

[0121] F5) inhibiting growth of tumors positive for Claudin 18.2;

[0122] F6) anti-tumor microenvironment immune suppression and / or T cell exhaustion;

[0123] F7) converting the immunosuppressive signals of PD-1 and TGFβ into CD27- mediated T cell activation signals.

[0124] The tumor or Claudin 18.2 target-related disease described herein can be a Claudin 18.2-positive tumor.

[0125] Further, the Claudin 18.2-positive tumor can be a Claudin 18.2-positive solid tumor.

[0126] Further, the Claudin 18.2-positive solid tumor can be a Claudin 18.2-positive digestive system tumor.

[0127] Further, the Claudin 18.2-positive tumor can be a Claudin 18.2-positive cancer, and can also be a Claudin 18.2-positive mesothelioma.

[0128] Further, the cancer can be selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, lung cancer, lung adenocarcinoma, liver cancer, kidney cancer, ovarian cancer, bronchial cancer, breast cancer, bladder cancer, head and neck cancer, gallbladder cancer, cholangiocarcinoma, and gastroesophageal junction (GEJ) adenocarcinoma.

[0129] Further, the cancer includes early-stage cancer, mid-stage cancer, mid-advanced stage cancer, advanced stage cancer, recurrent cancer, metastatic cancer, etc.

[0130] The present application also provides the use of any of the chimeric antigen receptors or the biological material described herein in the preparation of modified immune effector cells (such as CAR-T cells).

[0131] The present application also provides a pharmaceutical composition for preventing or treating a Claudin 18.2 target-related disease, which includes any of the chimeric antigen receptors or the biological material described herein, or any of the modified immune effector cells described herein, and one or more pharmaceutically acceptable carriers.

[0132] The pharmaceutically acceptable carrier is selected from the group consisting of diluents, excipients, fillers, binders, humectants, disintegrants, preservatives, stabilizers, absorption promoters, adsorptive carriers, surfactants, lubricants, atomizing agents, suspending agents, plasticizers, and dispersants.

[0133] The dosage form of the pharmaceutical composition can include, but is not limited to, inhalation preparations, oral preparations, and injection preparations. The inhalation preparations can be inhalation aerosols, inhalation powder sprays, inhalation spray, inhalation liquid preparations, inhalation atomization preparations, inhalation lyophilized powder injections, or nasal sprays. The oral preparations can be tablets, powders, capsules, granules, pills, powders, pastes, solid beverages, or oral liquids. The injection preparations can be injection liquids or injection powders.

[0134] To prepare the pharmaceutical composition into injection preparations such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents (carriers) commonly used in the art can be used, for example, water, saline, phosphate buffered saline, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like. In addition, to prepare isotonic injection liquids, an appropriate amount of sodium chloride, glucose, glycerol, or the like can be added to the injection preparations, and in addition, conventional cosolvents, buffers, pH adjustors, and the like can be added.

[0135] The pharmaceutical composition can further include a cell cryopreservation solution. The cell cryopreservation solution is used to freeze-preserve cells, prevent ice crystals from damaging cells during freeze-thawing, and the main components can include dimethyl sulfoxide, sodium chloride, human blood albumin, dextran glucose, dextrose, and / or plasma electrolyte-A, and the like.

[0136] The pharmaceutical composition can have at least one of the following uses: (1) for preventing or treating Claudin 18.2 target-related diseases; (2) killing Claudin 18.2-positive tumor cells; (3) inhibiting the proliferation of Claudin 18.2-positive tumor cells; (4) inhibiting the growth of Claudin 18.2-positive tumors; (5) resisting the immunosuppressive effect and / or T cell exhaustion effect of the tumor microenvironment; (6) converting the immunosuppressive signal of PD-1 and TGFβ into a CD27-mediated T cell activation signal.

[0137] The active ingredient of the pharmaceutical composition can be any of the modified immune effector cells described herein (such as Claudin 18.2 CAR-IC T cells).

[0138] The pharmaceutical compositions can be prepared according to methods that are well known and generally practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 22ndEd., Loyd V. Allen, et al., eds., Pharmaceutical Press (2012). The pharmaceutical compositions are preferably manufactured under GMP or cGMP conditions.

[0139] The present application also provides a kit for preparing a CAR-T cell, which can comprise the chimeric antigen receptor or the biological material described herein.

[0140] Further, the kit can further comprise one or more of the following reagents: lymphocyte separation medium, CD3 / CD28 monoclonal antibody coated magnetic beads (CD3 / CD28 magnetic beads), phosphate buffer (such as Dulbecco's phosphate buffer), cell culture medium, IL-2, IL-7, IL-15, IL-21, antibiotics, cell cryopreservation solution, and viral vector.

[0141] The various reagent components of the kit can be present in separate containers, or can be pre-combined, in whole or in part, into a reagent mixture.

[0142] The components of the kit can be provided in solution, for example, in the form of an aqueous solution. In the case of being present in the form of an aqueous solution, the concentration or content of these components can be readily determined by one skilled in the art according to different needs. For example, for the purpose of storage, the components can be present in a higher concentration, and when in working condition or for use, the concentration can be reduced to the working concentration by diluting the above-mentioned solution of higher concentration.

[0143] The present application also provides a method for preparing the modified immune effector cell described herein, which comprises introducing a nucleic acid molecule encoding the chimeric antigen receptor described herein into an immune effector cell for expression, to obtain the modified immune effector cell.

[0144] Further, the method of introduction can include, but is not limited to, calcium phosphate coprecipitation, cationic polymer method (such as DEAE-dextran transfection), cationic liposome method, electroporation method (i.e., electroporation), microinjection, gene gun method, or viral mediated method (such as retroviral infection, adenoviral infection, lentiviral infection), etc.

[0145] Further, the immune effector cell can be a T cell, and the modified immune effector cell can be a CAR-T cell.

[0146] Further, the method for preparing the CAR-T cell can comprise the following steps:

[0147] (1) cloning a nucleic acid molecule encoding any of the chimeric antigen receptors described herein into a viral vector to obtain a recombinant viral vector;

[0148] (2) introducing the recombinant viral vector into a packaging cell for packaging to obtain a recombinant virus;

[0149] (3) infecting (transfecting) human T cells with the recombinant virus to obtain the CAR-T cells.

[0150] Further, the nucleic acid molecule can be a nucleotide sequence as shown in SEQ ID NO: 2 or the Claudin 18.2 CAR-IC gene shown in positions 64-3432 of SEQ ID NO: 2.

[0151] Further, the viral vector can include baculovirus, retrovirus, lentivirus, adenovirus, adeno-associated virus, poxvirus, papillomavirus, papovavirus (such as SV40), and herpesvirus (such as herpes simplex virus), but is not limited thereto. Preferably, the viral vector is a retrovirus, adenovirus, adeno-associated virus, or lentivirus.

[0152] Further, the viral vector can be a retroviral vector (such as retroviral vector MP71).

[0153] Further, the packaging cell is used for packaging a viral vector plasmid lacking at least one gene encoding a protein required for viral packaging, which can provide comprehensive viral proteins for viral packaging into a recombinant virus, and which itself cannot produce any form of viral particles. Any cell that provides a protein or polypeptide lacking in viral packaging can be considered for use as a packaging cell. Suitable packaging cells are well known to those skilled in the art, such as ECO cells, PG13 cells, HEK293 cells, CHO cells, 293T cells, 293 cells, MDCK cells, NIH3T3 cells, PA317 cells, COS cells, HeLa cells, Vero cells, PsiCRIP cells, etc.

[0154] In one or more embodiments of the present application, the packaging cell is a PG13 cell and a Phoenix-ECO cell.

[0155] In the above preparation method, before the step (1), there can be further included a step of isolating and extracting peripheral blood mononuclear cells (PBMCs), and a step of isolating and activating T cells.

[0156] The isolation of the mononuclear cells (PBMCs) can employ a density gradient centrifugation method known in the art for isolating PBMCs, which utilizes the difference in specific gravity of cells to separate cells by using a Ficoll reagent.

[0157] The isolation and activation of the T cells can achieve effective enrichment of T cells and their subgroups by CD3 / CD28 monoclonal antibodies coupled with magnetic beads. Meanwhile, CD3 is a surface marker of T cells, and CD28 is a second signal for T cell activation, and CD3 / CD28 magnetic beads can also effectively activate T cells. The technology of separating and activating T cells by CD3 / CD28 magnetic beads is known in the art.

[0158] In the above preparation method, after the step (3), the method can further comprise the steps of expanding culture and detection of the CAR-T cells.

[0159] The present application also provides a method for preventing or treating Claudin 18.2 target-related diseases, which comprises administering the modified immune effector cell or the pharmaceutical composition of any one of the present application to a subject suffering from Claudin 18.2 target-related diseases.

[0160] In the above method, the modified immune effector cell can be any one of the chimeric antigen receptor modified T cells described herein.

[0161] Further, the modified immune effector cell can be the CAR-T cell (such as Claudin 18.2 CAR-IC T cell) described in the present application.

[0162] Further, the CAR-T cell can be autologous or allogeneic.

[0163] Further, the method can comprise the following steps:

[0164] (1) isolating and extracting peripheral blood mononuclear cells (PBMCs) from a sample (such as peripheral blood) of a subject (including a patient or a healthy volunteer);

[0165] (2) isolating and activating T cells from the PBMCs;

[0166] (3) genetically modifying the T cells with the gene of any one of the chimeric antigen receptors described in the present application to obtain CAR-T cells;

[0167] (4) expanding culture and quality detection of the CAR-T cells to obtain qualified CAR-T cells;

[0168] (5) administering the qualified CAR-T cells to the subject suffering from Claudin 18.2 target-related diseases.

[0169] Further, the sample can be derived from peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, infected site tissue, ascites, pleural effusion, spleen tissue and tumor, but is not limited thereto.

[0170] In the above method, the Claudin 18.2 target related disease can be a Claudin 18.2 positive tumor.

[0171] In the above method, the Claudin 18.2 positive tumor can be a Claudin 18.2 positive solid tumor.

[0172] Further, the solid tumor can be a digestive system tumor (such as gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma).

[0173] In the above method, the Claudin 18.2 positive tumor can be a Claudin 18.2 positive cancer or a Claudin 18.2 positive mesothelioma.

[0174] In the above method, the cancer can be selected from gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, lung cancer, lung adenocarcinoma, liver cancer, kidney cancer, ovarian cancer, bronchial cancer, breast cancer, bladder cancer, head and neck cancer, gallbladder cancer, bile duct cancer, and gastroesophageal junction adenocarcinoma.

[0175] In the above method, the cancer can include early stage cancer, intermediate stage cancer, middle-late stage cancer, late stage cancer, recurrent cancer, and metastatic cancer.

[0176] In the above method, the amount administered can be a therapeutically effective amount. The therapeutically effective amount can refer to the amount of the drug that (i) treats or prevents a particular disease, condition, or disorder; (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder; or (iii) prevents or delays onset of one or more symptoms of a particular disease, condition, or disorder described herein. A therapeutically effective amount can be determined by testing in known in vitro or in vivo (e.g., animal model) systems.

[0177] Further, the therapeutically effective amount described herein can be about (0.3-35) x 10 6 , (0.3-30) x 10 6 , (0.3-25) x 10 6 , (0.3-20) x 10 6 , (0.3-15) x 10 6 , (0.3-10) x 10 6 , (0.3-9) x 10 6 , (0.3-8) x 10 6 , (0.3-7) x 10 6 , (0.3-6) x 10 6 , (0.3-5) x 10 6 , (0.3-4) x 10 6one, (0.3-3) x 10 6 one, (0.3-2) x 10 6 one, (0.3-1) x 10 6 one, (1-35) x 10 6 one, (1-30) x 10 6 one, (1-25) x 10 6 one, (1-20) x 10 6 one, (1-15) x 10 6 one, (1-10) x 10 6 one, (1-9) x 10 6 one, (1-8) x 10 6 one, (1-7) x 10 6 one, (1-6) x 10 6 one, (1-5) x 10 6 one, (1-4) x 10 6 one, (1-3) x 10 6 one, (1-2) x 10 6 one, (2-35) x 10 6 one, (2-30) x 10 6 one, (2-25) x 10 6 one, (2-20) x 10 6 one, (2-15) x 10 6 one, (2-10) x 10 6 one, (2-9) x 10 6 one, (2-8) x 10 6 one, (2-7) x 10 6 one, (2-6) x 10 6 one, (2-5) x 10 6 one, (2-4) x 10 6 one, (2-3) x 10 6 one, (4-35) x 10 6 one, (4-30) x 10 6 one, (4-25) x 10 6 one, (4-20) x 10 6 one, (4-15) x 10 6 one, (4-10) x 10 6 one, (4-9) x 10 6 one, (4-8) x 10 6 one, (4-7) x 10 6 one, (4-6) x 10 6 one, or (4-5) x 10 6(1-10)xlO 6 (1-9)xlO 6 (2-9)xlO 6 (1-5)xlO 6 (2-5)xlO 6 (1-10)xlO

[0178] Further, the administration includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumoral injection, mucosal administration, oral administration, oral and nasal cavity spray, aerosol inhalation, in vivo implantation and in vitro carrying device administration.

[0179] The preferred mode of administration (route of administration) is parenteral (e.g., intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular). The skilled artisan will appreciate that the route of administration will vary depending on the intended purpose. In certain embodiments, the modified immune effector cells (e.g., CAR-T cells) or pharmaceutical compositions of the present application can be administered by injection or continuous infusion (including, but not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and intraportal).

[0180] In some embodiments, the prophylactic or therapeutic methods provided by the present application can also be used in combination with other therapies, including surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, etc. For example, a second therapeutic agent can be used for treatment, which can be an antitumor drug, an anti-inflammatory drug, an immunosuppressant, or other therapeutically active drug, including but not limited to gefitinib, imatinib, icotinib, sorafenib, infliximab, adalimumab, rituximab, etanercept, filgrastim, pegfilgrastim, epirubicin, capecitabine, cisplatin, carboplatin, gemcitabine, pemetrexed, paclitaxel, oxaliplatin, and fluorouracil.

[0181] The modified immune effector cells (e.g., CAR-T cells) or pharmaceutical compositions of the present application can be administered to the subject separately (by different routes of administration at the same time or substantially at the same time), sequentially (at different times, the routes of administration can be the same or different), or simultaneously (by the same route of administration at the same time or substantially at the same time) with the second therapeutic agent.

[0182] The present application also provides a method for inhibiting the growth of Claudin 18.2 positive tumor cells or killing Claudin 18.2 positive tumor cells, comprising contacting the Claudin 18.2 positive tumor cells with any of the modified immune effector cells or the pharmaceutical composition described herein.

[0183] Further, the method can be an in vitro method or an in vivo method.

[0184] The present application also provides any of the modified immune effector cells or the pharmaceutical composition described herein for use as a medicament.

[0185] The present application also provides any of the modified immune effector cells or the pharmaceutical composition described herein for use in preventing or treating Claudin 18.2 target related diseases.

[0186] According to the use of any of the modified immune effector cells or the pharmaceutical composition described herein, the Claudin 18.2 target related disease can be a Claudin 18.2 positive tumor.

[0187] Further, the Claudin 18.2 positive tumor can be a Claudin 18.2 positive solid tumor.

[0188] Further, the solid tumor can be a digestive system tumor (such as gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma).

[0189] Further, the Claudin 18.2 positive tumor can be a Claudin 18.2 positive cancer or a Claudin 18.2 positive mesothelioma.

[0190] Further, the cancer can be selected from gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, lung cancer, lung adenocarcinoma, liver cancer, kidney cancer, ovarian cancer, bronchial cancer, breast cancer, bladder cancer, head and neck cancer, gallbladder cancer, bile duct cancer and gastroesophageal junction adenocarcinoma.

[0191] Further, the cancer can include early stage cancer, intermediate stage cancer, advanced stage cancer, late stage cancer, recurrent cancer and metastatic cancer.

[0192] The purposes of the uses and methods of the present application can be disease diagnosis purposes, disease prognosis purposes and / or disease treatment purposes, and their purposes can also be non-disease diagnosis purposes, non-disease prognosis purposes and non-disease treatment purposes; their direct purposes can be to obtain information of intermediate results of disease diagnosis results, disease prognosis results and / or disease treatment results, and their direct purposes can be non-disease diagnosis purposes, non-disease prognosis purposes and / or non-disease treatment purposes.

[0193] The products described herein can include reagents, kits, formulations, drugs, and pharmaceutical compositions, among others.

[0194] The subject described herein can be a human or a non-human animal. In certain embodiments, the subject has a Claudin 18.2 target-related disease.

[0195] The non-human animal described herein can be a non-human mammal.

[0196] The non-human mammal described herein can be any one of, but not limited to, a mouse, a rat, a guinea pig, a hamster, a pig, a dog, a sheep, a monkey, a rabbit, a cat, a cow, a horse.

[0197] The inventors of the present application have conducted extensive and in-depth research and designed a chimeric antigen receptor (CAR) targeting Claudin 18.2. The CAR adopts a design of a double epitope and is fused with interferon and a chimeric switch receptor (CR), and the structure is shown in Figure 1. The antigen binding domain of the CAR is composed of a double epitope single domain antibody targeting two different epitopes of Claudin 18.2, which is composed of two single domain antibodies connected by a linker, which is mainly responsible for recognizing and specifically binding to the tumor surface antigen Claudin 18.2. The design of the double epitope optimizes the affinity of CAR-T cells and can enhance the anti-tumor effect. A signal peptide is provided at the N-terminus of the CAR for guiding the peptide chain of the antigen binding domain to transfer to the extracellular, so as to recognize the tumor cell surface antigen Claudin 18.2. The hinge region (Hinge) connects the extracellular antigen binding domain and the transmembrane region, which can promote the stable expression of the CAR molecule on the membrane surface of the T cell, provide a certain flexibility for the antigen binding domain, and avoid the influence of steric hindrance. The transmembrane region (TM) is used to anchor the antigen binding domain on the cell membrane. The intracellular signal domain includes, from N-terminus to C-terminus, the costimulatory domain 4-1BB, the activation domain CD3ζ, IFNα, and the chimeric switch receptor PD-1&TRII&CD27. CD3ζ and 4-1BB provide the first signal and the second signal for the activation of CAR-T cells, respectively, so that the CAR-T cells can be fully activated after being stimulated by the antigen to kill tumor cells. IFNα is used for broad-spectrum anti-tumor and remodeling of the tumor immune microenvironment to activate immune response. The chimeric switch receptor PD-1&TRII&CD27 is used to convert the immune suppression signal in the tumor microenvironment into the CD27 activation signal in the CAR-T cells, thereby enhancing the tumor killing effect. PD-1 can bind to PD-L1 on tumor cells to block the PD-1 signaling pathway on T cells and inhibit T cell exhaustion, and the extracellular region of TRII can bind to TGFβ to block the TGFβ inhibition pathway, thereby inhibiting the tumor microenvironment and enhancing immune cell infiltration. The designed CAR of the present application endows the CAR-T cells with stronger tumor killing ability, the ability to resist the immunosuppressive tumor microenvironment, and stronger in vivo persistence.

[0198] The present application successfully constructs CAR-T cells targeting Claudin 18.2. Experiments prove that the CAR-T cells of the present application can secrete T cell-specific effector molecules IFN-γ well, effectively specifically kill Claudin 18.2 positive cells, and have no killing effect on Claudin 18.2 negative cells, especially Claudin 18.1 (which is extremely similar in structure to Claudin 18.2, with a homology of up to 92%) positive cells. The CAR-T cells of the present application have good killing activity on tumor cells in vitro, can effectively specifically kill Claudin 18.2 positive tumor cells, and also have good in vivo killing activity, can significantly clear tumor cells in mice, inhibit tumor growth, and the tumor completely disappears in about 3 weeks, showing significant potential in resisting Claudin 18.2 positive cancer and having broad clinical application prospects.

[0199] Definitions of terms

[0200] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Meanwhile, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0201] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a gene of interest. A typical expression cassette comprises a promoter, a MCS (multiple cloning site) and a terminator. The expression cassette can also include a gene of interest, a marker gene (such as a TK gene, a DHFR gene, a CAT gene and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) addition signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly connected or indirectly connected through a linker.

[0202] The term "vector" generally refers to a vehicle that is capable of carrying foreign DNA or a gene of interest into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are amplified and / or expressed in the host cell. A person skilled in the art can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vector includes but is not limited to: plasmid, phage (such as lambda phage or M13 phage), cosmid (i.e. cos plasmid), phagemid, shuttle vector (such as yeast expression vector), Ti plasmid, artificial chromosome (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC) or Ti plasmid artificial chromosome (TAC)), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papovavirus (such as SV40), herpesvirus (such as herpes simplex virus)). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0203] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, and the like. For example, the bacteria can be from Escherichia sp. (e.g., E. coli), Erwinia sp., Agrobacterium sp. (e.g., A. tumefaciens), Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., and Bacillus sp. (e.g., B. subtilis), and the like. The viruses can include rotavirus, baculovirus, retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papovavirus (e.g., SV40), and herpesvirus (e.g., herpes simplex virus), and the like. The fungi can be from Saccharomyces sp. (e.g., S. cerevisiae, S. pombe, S. pichia), Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., and Cephalosporium sp., and the like. The actinomycetes can be from Streptomyces sp. (e.g., S. coelicolor). The algae can be from Cyanophyta (e.g., cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., and Boekelovia sp., and the like.

[0204] The term "host cell" is also referred to as recipient cell, and generally refers to any type of cell that can be used for introducing a vector, such as plant cells and animal cells. The host cell can be understood to refer not only to the particular recipient cell, but also to the progeny of such a cell, which progeny will in most instances, but not always, contain the same genetic makeup as the original parent cell and which may, but not always, be capable of reproducing. Suitable host cells are known in the art, wherein: the plant cell can be, but is not limited to, Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc.; the animal cell can be, but is not limited to, mammalian cells (e.g. Chinese hamster ovary cells (CHO cells), Chinese hamster ovary subline (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse mammary tumor cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g. chicken or duck cells), amphibian cells (e.g. Xenopus laevis cells or Andrias davidianus cells), fish cells (e.g. grass carp, common carp, rainbow trout or catfish cells), insect cells (e.g. Sf21 cells, Sf-9 cells or Hi-5), etc.

[0205] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed in vitro by ligating an exogenous gene of interest with a vector, which can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous gene of interest into a recipient cell, and provide the exogenous gene of interest with the ability of replication, integration, amplification and / or expression in the recipient cell.

[0206] The term "recombinant microorganism" generally refers to a recombinant microorganism obtained by manipulating and modifying the genes of a microorganism of interest, so that the function of the recombinant microorganism is changed. For example, an exogenous gene of interest or a recombinant vector is introduced into the microorganism of interest, or the endogenous genes of the microorganism of interest are directly genetically edited.

[0207] The term "recombinant host cell" generally refers to a recombinant host cell obtained by manipulating and modifying the genes of a host cell, so that the function of the recombinant host cell is changed. For example, an exogenous gene of interest or a recombinant vector is introduced into the host cell, or the endogenous genes of the host cell are directly genetically edited.

[0208] The term "linkage" generally refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage described herein can be directly linked by a peptide bond, or linked by a linker.

[0209] The term "linker" can also be referred to as a connecting peptide, a peptide linker, or a linker, which is used to fuse, couple, link, or join two proteins or polypeptides, which can prevent steric hindrance. The linker is an amino acid chain that connects two fusion proteins, which has a certain flexibility to allow the proteins on both sides to complete their respective independent functions. It should be understood that the presence of the linker is optional, and the length of the flexible linker can also be adjusted to allow the correct folding of the fusion protein or to achieve optimal biological activity. The characteristics of the linker and its suitability for a particular purpose are known in the art, and a person skilled in the art can independently select and optimize each peptide linker.

[0210] The term "signal peptide" generally refers to a short peptide chain (usually 5-30 amino acids in length) that directs the newly synthesized protein to the secretory pathway. The antigen binding region of the chimeric antigen receptor described in the present application can be preceded (N-terminally) by a signal peptide (Signal). The signal peptide of the CAR structure can guide the antigen binding region peptide chain to transfer to the outside of the cell, thereby recognizing tumor cell surface antigens. The signal peptide can be a signal peptide derived from CD4, CD8, CD8a, CD28, CD137, and GM-CSF.

[0211] The term "identity" generally refers to the extent to which two (nucleotide or amino acid) sequences have the same residues at a given position in an alignment, and is usually expressed as a percentage. Identity as described herein can refer to identity of an amino acid sequence or a nucleotide sequence. Two copies having exactly the same sequence have 100% identity. One skilled in the art is aware that identity of an amino acid sequence or a nucleotide sequence can be determined using identity search sites on the internet, such as the BLAST page of the NCBI home page website. For example, the value of identity (%) can be obtained by performing a search in Advanced BLAST 2.1 using blastp as the program, setting Expect value to 10, setting all Filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and then using a computer program BLAST, especially BLASTP or TBLASTN with default parameters. Identity as described herein of more than 75% can be at least 75%, 80%, 85%, 90% or 95% or more. Identity as described herein of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.

[0212] The term "conservative substitution" generally refers to the replacement of one amino acid residue by another possessing a similar physico-chemical property. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and He), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gin), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). Conservative substitutions generally do not produce a significant change in the conformational structure of a protein and do not have a qualitative effect on the function of the protein, and thus the biological activity of the protein can be retained.

[0213] The term "single domain antibody (sdAb)" also known as nanobody, generally refers to an antibody that consists of only one H chain V region, and can also be referred to as a VHH antibody. Single domain antibodies are extremely stable in their ability to bind to antigens and their stability is essentially identical to that of a full antibody. In the present context, unless the context clearly indicates otherwise, when the term "single domain antibody" is referred to, it includes not only intact single domain antibodies, but also antigen binding fragments of single domain antibodies. An antigen binding fragment refers to a polypeptide comprising a fragment of a single domain antibody that retains the ability to specifically bind to the same antigen to which the single domain antibody binds, and / or competes with the single domain antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". In some embodiments, the "antigen binding fragment" of the single domain antibody can be truncated at the N- or C-terminus compared to the full length single domain antibody to comprise only part of FR1 and / or FR4, or lack one or both of those framework regions, as long as it essentially retains antigen binding and specificity.

[0214] The term "complementarity determining region" or "CDR" generally refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. CDRs can be defined according to various numbering systems known in the art, e.g., according to the definition in the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. For a given antibody, it is easy for one skilled in the art to identify the CDRs defined by each numbering system. And the correspondence between different numbering systems is well known to one skilled in the art.

[0215] The term "framework region (FR)" generally refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0216] The term "epitope" generally refers to any antigenic determinant with which an antibody specifically binds. Both linear and conformational epitopes are included. Epitopes can be identified by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural determination methods (e.g., nuclear magnetic resonance spectroscopy).

[0217] The term "introducing" generally refers to the transfer of an exogenous gene into a recipient cell, such as a eukaryotic recipient cell or a prokaryotic recipient cell. The method of introduction is not particularly limited, and any known transformation method can be used as long as it can transfer the gene of interest (e.g., the DNA molecule of the present application) into the recipient cell. The method of introduction can include any one of the following: (1) introducing the gene of interest or a recombinant vector containing the gene of interest into a host bacterium by a chemical transformation method (e.g., Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation) or a physical transformation method (e.g., electroporation transformation); (2) transducing the gene of interest into a host bacterium by a bacteriophage transduction method; (3) directly transferring the gene of interest into a plant recipient cell by a physical or chemical method, such as a chemical stimulation method, an electric shock method, a liposome-mediated method, a microinjection method, a gene gun method, a laser microbeam method, a pollen tube channel method, an ultrasonic wave method, an air gun method, and a vortex method; (4) transferring the gene of interest into a plant recipient cell using a vector as a medium, such as an Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems) mediated method; (5) introducing the gene of interest into an isolated animal cell (transfection) by a calcium phosphate coprecipitation method, a cationic polymer method (e.g., a DEAE-dextran transfection method), a cationic liposome method, an electroporation method (i.e., an electroporation transfection method), microinjection, a gene gun method, or a virus-mediated method (e.g., a retrovirus infection method, an adenovirus infection method, a lentivirus infection method); and (6) introducing the gene of interest into an in vivo animal cell by a microinjection method, a retroviral vector method, a somatic cell nuclear transfer method, a sperm vector method, or an embryonic stem cell method.

[0218] The term "Claudin18.2-positive tumor" generally refers to a tumor that expresses Claudin18.2 protein. Methods for detecting Claudin18.2 protein expression in a tumor are well known to those skilled in the art, for example, the expression of Claudin18.2 protein can be determined by immunological detection methods based on the specific reaction of antigen-antibody (e.g., immunohistochemistry, FACS, etc.), and the expression of Claudin18.2 mRNA can be determined by real-time fluorescent quantitative PCR, Northern blotting, or RNA in situ hybridization.

[0219] The term "preventing" generally refers to a method performed to stop or delay the occurrence of a disease or disorder or a symptom in a subject.

[0220] The term "treatment" generally refers to a method implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, narrowing of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief of disease status, and remission (whether partial or complete), whether detectable or undetectable. Furthermore, treatment can also refer to an extension of survival compared to the expected survival of a subject without treatment.

[0221] The term "cleavable linker (also known as a shearable linker)" generally refers to a linker that contains a cleavage site so that it can be selectively cleaved upon expression to produce two or more products. Such cleavable linkers include, but are not limited to, P2A, F2A, T2A, and E2A, or any combination thereof.

[0222] The term "chimeric antigen receptor (CAR)" generally refers to an artificially constructed transmembrane molecule encoded by a fusion gene that guides immune cells to specifically track, recognize, and eliminate tumor cells expressing relevant target ligands, exhibiting high targeting specificity. The basic structure of a CAR can be divided into four parts, from extracellular to intracellular: an extracellular tumor antigen-binding domain (antigen-binding domain), a hinge domain, a transmembrane domain, and an intracellular signaling domain that provides activation signals to T cells. Methods for preparing chimeric antigen receptors and immune effector cells (e.g., T cells) containing such receptors are known in the art and may include transfecting cells with at least one nucleic acid molecule encoding a CAR (CAR gene) and expressing said nucleic acid molecule in the cells. For example, the nucleic acid molecule encoding the CAR of the present invention can be constructed into an expression vector (such as a retroviral vector) that can be expressed in host cells, such as T cells, to prepare the CAR.

[0223] The term "Chimeric Switch Receptor (CSR)," also known as a chimeric switching receptor, generally refers to a genetically engineered T cell that, upon receiving inhibitory signals from suppressor molecules, transforms them into activating signals, allowing the T cell to maintain its offensive capabilities even in a suppressive environment. CSRs work by recognizing ligands of inhibitory receptors, thereby converting the original immunosuppressive signal into an immune-activating signal, activating immune cells, and enhancing the anti-tumor activity of CAR-T cells.

[0224] The term "CAR cell" is also referred to as modified immune effector cell herein, and generally refers to a kind of genetically modified immune effector cell which is obtained by introducing the gene of chimeric antigen receptor (CAR) (CAR gene) into immune effector cell through molecular biology and genetic engineering technology, and expressing CAR on the surface of immune effector cell, so as to specifically recognize and kill target cells. In relation to CAR-T cell, CAR cell is not limited to a specific receptor cell type, and CAR cell can include CAR-T cell, CAR-NK cell, CAR-NKT cell, CAR-γδT cell, CAR-αβT cell, CAR-regulatory T cell (CAR-Treg cell), CAR-MAIT cell, CAR-B cell, CAR-macrophage (CAR-M cell), CAR-dendritic cell (CAR-DC cell), CAR-peripheral blood mononuclear cell (CAR-PBMC cell), CAR-monocyte (CAR-MC cell), CAR-iPSC cell and CAR-PSC cell, etc. but not limited thereto.

[0225] The term "CAR-T cell (chimeric antigen receptor T cell)" generally refers to a T cell expressing a chimeric antigen receptor (CAR), which can specifically recognize and kill cells expressing a target antigen recognized by the chimeric antigen receptor. CAR-T cell can be prepared by introducing CAR gene into T cell in vitro through various vectors. CAR-T cell is activated after specific recognition of tumor antigen by CAR molecule, and realizes specific killing effect on tumor cells by releasing perforin, granzyme B, cytokine IFN-γ, etc. The definition of CAR-T cell covers all categories and subclasses of T lymphocytes, including CD8 + T cell, CD4 + T cell, regulatory T cell (Treg), naive T cell, effector T cell, memory T cell (including stem memory T cell, central memory T cell and effector memory T cell), αβT cell, γδT cell, natural killer T cell (NKT cell) and mucosal-associated invariant T cell (MAIT cell), etc.

[0226] The term "antigen binding region" is also referred to as antigen binding domain, and generally refers to the part of chimeric antigen receptor which specifically binds to target antigen, responsible for the recognition of tumor antigen, and can include antibody light chain variable region (VL), antibody heavy chain variable region (VH), antibody Fab fragment, antibody Fab' fragment, single chain antibody variable region (scFv), single domain antibody (sdAb), etc. The antigen binding region can be preceded by a signal peptide.

[0227] The term "Hinge" also known as hinge domain, generally refers to any oligo- or polypeptide used to connect the extracellular antigen binding region and the transmembrane region, which is a segment of peptide chain located outside the cell of CAR. Selecting a suitable hinge region can improve the flexibility of the antigen recognition region, reduce the spatial constraints between the antigen and CAR, thereby promoting the synapse formation of CAR-T cells and target cells. The hinge region is located at the N-terminus of the transmembrane region and the C-terminus of the antigen binding region, which can be derived from the hinge region of CD8a (CD8a), CD4, CD5, CD7, CD28, CD134, CD137, ICOS, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM.

[0228] The term "transmembrane domain" (TD) also known as transmembrane domain, is used to connect the antigen binding region and the intracellular signal region of the chimeric antigen receptor, which is mainly responsible for maintaining the stability of the CAR molecule on the T cell membrane. It can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein, and is usually composed of homodimeric or heterodimeric membrane proteins. The transmembrane region of the chimeric antigen receptor of the present application can be any protein structure known in the art, as long as it can be thermodynamically stable in the cell membrane (especially the eukaryotic cell membrane). The transmembrane region can be derived from the transmembrane domain of the a, b or z chain of the T cell receptor, CD3 zeta, CD8a (CD8a), CD4, CD9, CD45, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152 or CD154.

[0229] The term“intracellular signaling region (also referred to as an intracellular signaling domain)” generally refers to the portion of a chimeric antigen receptor that converts an antigen stimulus into a T cell activation signal. The intracellular signaling region can comprise an activating domain and / or a costimulatory domain. The activating domain can be derived from the intracellular signaling domain of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. The costimulatory domain can be from the intracellular signaling domain of a costimulatory molecule, for example, the costimulatory domain can be derived from the intracellular signaling domain of CARD11, CD2, CD7, CD27, CD28, CD30, ICAM-1 (CD54), LFA-1 (CD1 la), CD134 (OX40), CD137 (4-1BB), 2B4, CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), or DAP10. The intracellular signaling region can comprise one, two, or more costimulatory domains. The costimulatory domain can comprise the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. A“costimulatory molecule” refers to a cognate binding partner that specifically binds with a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell.

[0230] The term“immune effector cell” generally refers to an immune cell that participates in an immune response, performing an immune effector function (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). The immune effector cell includes T cells (such as CD8 + T cells, CD4 +T cells, regulatory T cells (Tregs), naive T cells, effector T cells, memory T cells (including stem memory T cells, central memory T cells, and effector memory T cells), alpha beta T cells, gamma delta T cells, Natural killer T cells (NKT cells), and Mucosal-associated invariant T cells (MAIT cells), NK cells, B cells, macrophages, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), and monocytes (MCs). The immune effector cells described herein can also include stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells (HSCs), that can be induced to differentiate into immune effector cells in vivo or in vitro. The immune effector cells described herein can be differentiated from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells (HSCs). The immune effector cells can be autologous or non-autologous (allogeneic, syngeneic, or xenogeneic), preferably autologous or allogeneic. In one embodiment of the application, the immune effector cells are T cells. T cells are found in a variety of sources, including, but not limited to, peripheral blood, mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, site of infection tissue, ascites, pleural effusion, spleen tissue, and tumors. T cells can be isolated using a variety of techniques known to those of skill in the art, such as density gradient separation techniques, adherence separation methods, magnetic bead separation techniques, flow cytometric sorting techniques, and the like.

[0231] The term "autologous" generally refers to a cell from the same subject.

[0232] The term "allogeneic" generally refers to a cell of the same species that is genetically different from the cell under comparison.

[0233] The term "syngeneic" generally refers to a cell of a different subject that is genetically the same as the cell under comparison.

[0234] The term "xenogeneic" generally refers to a cell of a different species than the cell under comparison. BRIEF DESCRIPTION OF DRAWINGS

[0235] FIG. 1 is a schematic diagram of a Claudin 18.2 CAR-IC (#131 CAR-IC) structure.

[0236] FIG. 2 is a result of Claudin 18.2 CAR-IC T cell CAR expression detection.

[0237] Figure 3 is a detection of Claudin 18.2 expression in different cell lines and a detection of Claudin 18.2 CAR-IC T cell antigen-specific effector molecule secretion.

[0238] Figure 4 is a detection of Claudin 18.2 CAR-IC T cell antigen-specific cytotoxic function.

[0239] Figure 5 is a detection of Claudin 18.2 CAR-IC T cell antigen-specific in vivo tumor inhibition function. Embodiments of the present application

[0240] The present application is further described in detail by the specific embodiments below, which are only intended to illustrate the present application, and not to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0241] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0242] The retroviral vector MP71 in the following examples is described in the following literature: Engels B, Cam H, et al. Retroviral Vectors for High-Level Transgene Expression in T Lymphocytes [J]. Human Gene Therapy, 2003, 14(12): 1155-1168. The biological material can be obtained from the applicant by the public, and the biological material is only used for repeating the experiments of the present application, and cannot be used for other purposes.

[0243] The Phoenix-ECO cells in the following examples are purchased from the American Type Culture Collection (ATCC) with the accession number CRL-3214.

[0244] The PG13 cells in the following examples are purchased from the American Type Culture Collection (ATCC) with the accession number CRL-3597.

[0245] The ASPC1 cells in the following examples were purchased from the American Type Culture Collection (ATCC) with the accession number CRL-1682.

[0246] The HepG2 cells in the following examples were derived from the Concordia Cell Bank with the resource number 1101HUM-PUMC000035.

[0247] The Jurkat cells in the following examples were derived from the Concordia Cell Bank with the resource number 1101HUM-PUMC000075.

[0248] The BGC823 cells in the following examples were derived from the Biyun Tian Company with the product number C6123.

[0249] The SNU601 cells in the following examples were derived from the Nanjing Kebai Biological Technology Company with the product number CBP60507.

[0250] The peripheral blood mononuclear cells (PBMC) in the following examples were derived from the peripheral blood of healthy volunteers.

[0251] The amino acid sequence of the CLDN18.2 protein in the following examples is shown in SEQ ID NO: 3, and the nucleotide sequence of the gene encoding the CLDN18.2 protein is shown in SEQ ID NO: 4; the amino acid sequence of the CLDN18.1 protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the CLDN18.1 protein is shown in SEQ ID NO: 6.

[0252] The data in the following examples were processed using Graphpad statistical software, and the experimental results were expressed as mean ± standard deviation. The log rank test method was used, P<0.05 (*) indicated statistical difference, P<0.01 (**) indicated significant statistical difference, and P<0.001 (***) indicated extremely significant statistical difference. In the quantitative experiments in the following examples, three replicates were set unless otherwise specified, and the results were averaged.

[0253] Example 1, Structure and sequence of Claudin 18.2 CAR-T chimeric antigen receptor and chimeric switch receptor

[0254] The present embodiment designs and constructs a chimeric antigen receptor (CAR) for modifying T cells, named Claudin 18.2 CAR-IC (also known as #131 CAR-IC). The antigen binding region in the CAR molecule includes 2 single domain antibodies, which can target and bind to two different epitopes of a single tumor antigen Claudin 18.2. In addition, the C-terminus of the CAR molecule is further fused with IFNα and a chimeric switch receptor (CSR) PD-1&TRII&CD27, and the structural schematic diagram is shown in Figure 1. The specific structure and sequence are as follows.

[0255] The Claudin 18.2 CAR-IC molecule is sequentially composed of a signal peptide (Signal), a double epitope single domain antibody #131VHHs (i.e. Claudin 18.2-VHH#223-VHH#357), a CD8a hinge region (Hinge) and a transmembrane region, a costimulatory domain 4-1BB, an activation domain CD3ζ, a cleavable linker P2A, an interferon IFNα, a cleavable linker P2A, and a chimeric switch receptor PD-1&TRII&CD27 (including the extracellular region of PD-1, the extracellular region of TGFβ receptor type II, and the transmembrane region and cytoplasmic region of CD27) from N-terminus to C-terminus.

[0256] The amino acid sequence of Claudin 18.2 CAR-IC is shown as SEQ ID NO: 1. Among them: the amino acid sequence of SEQ ID NO: 1 at positions 1-21 is the amino acid sequence of signal peptide (Signal); the amino acid sequence of SEQ ID NO: 1 at positions 22-141 is the amino acid sequence of single domain antibody (Claudin 18.2-VHH#223); the amino acid sequence of SEQ ID NO: 1 at positions 142-156 is the amino acid sequence of G4S linker; the amino acid sequence of SEQ ID NO: 1 at positions 157-278 is the amino acid sequence of single domain antibody (Claudin 18.2-VHH#357); the amino acid sequence of SEQ ID NO: 1 at positions 279-347 is the amino acid sequence of CD8a hinge region (Hinge) and transmembrane region (TM); the amino acid sequence of SEQ ID NO: 1 at positions 348-394 is the amino acid sequence of co-stimulatory domain 4-1BB; the amino acid sequence of SEQ ID NO: 1 at positions 395-506 is the amino acid sequence of activation domain CD3 zeta; the amino acid sequence of SEQ ID NO: 1 at positions 507-528 is the amino acid sequence of P2A; the amino acid sequence of SEQ ID NO: 1 at positions 529-716 is the amino acid sequence of interferon IFN alpha; the amino acid sequence of SEQ ID NO: 1 at positions 717-738 is the amino acid sequence of P2A; the amino acid sequence of SEQ ID NO: 1 at positions 739-1144 is the amino acid sequence of chimeric switch receptor PD-1 & TRII & CD27.

[0257] In the single domain antibody (Claudin 18.2-VHH#223) (SEQ ID NO: 1 at positions 22-141), the amino acid sequence of SEQ ID NO: 1 at positions 47-54 is the complementarity determining region CDR1; the amino acid sequence of SEQ ID NO: 1 at positions 72-78 is the complementarity determining region CDR2; the amino acid sequence of SEQ ID NO: 1 at positions 116-130 is the complementarity determining region CDR3.

[0258] In the single domain antibody (Claudin 18.2-VHH#357) (SEQ ID NO: 1 at positions 157-278), the amino acid sequence of SEQ ID NO: 1 at positions 182-188 is the complementarity determining region CDR1; the amino acid sequence of SEQ ID NO: 1 at positions 206-215 is the complementarity determining region CDR2; the amino acid sequence of SEQ ID NO: 1 at positions 254-267 is the complementarity determining region CDR3.

[0259] The single-domain antibody (Claudin 18.2-VHH#223) and the single-domain antibody (Claudin 18.2-VHH#357) are connected by a linker to form a biparatopic single-domain antibody (Claudin 18.2-VHH#223-VHH#357) targeting two different epitopes of Claudin 18.2, named #131VHHs.

[0260] The amino acid sequence of the biparatopic single-domain antibody #131VHHs is shown in SEQ ID NO: 1, positions 22-278.

[0261] The nucleotide sequence of the Claudin 18.2 CAR-IC is shown in SEQ ID NO: 2. The DNA molecule shown in SEQ ID NO: 2 (also referred to as the Claudin 18.2 CAR-IC gene) encodes the chimeric antigen receptor Claudin 18.2 CAR-IC with the amino acid sequence shown in SEQ ID NO: 1.

[0262] Example 2, Preparation of Claudin 18.2 CAR-IC T cells and detection of CAR expression

[0263] The chimeric antigen receptor T cells specifically targeting Claudin 18.2 prepared in this example (i.e., Claudin 18.2 CAR-IC modified T cells) are recombinant cells stably expressing the Claudin 18.2 CAR-IC gene (SEQ ID NO: 2) introduced into T cells, named Claudin 18.2 CAR-IC T cells (also referred to as #131 CAR-IC T cells), and the specific steps are as follows:

[0264] 1. Construction of recombinant retroviral vector

[0265] (1) The recombinant vector pUC57-Claudin 18.2 CAR-IC was double digested with NotI (NEB) and EcoRI (NEB), and the Claudin 18.2 CAR-IC gene fragment of interest was recovered by gel recovery. The recombinant vector pUC57-Claudin 18.2 CAR-IC is a recombinant expression vector obtained by cloning the Claudin 18.2 CAR-IC gene (SEQ ID NO: 2) into the pUC57 vector, which was synthesized and provided by GenScript Biotech Co., Ltd.

[0266] (2) The retroviral vector MP71 was double digested with NotI and EcoRI, and the large fragment of the vector was recovered by gel recovery.

[0267] (3) The above-mentioned target gene fragment Claudin 18.2 CAR-IC gene is connected with the vector large fragment by using T4 ligase (NEB), and a recombinant retrovirus vector MP71-Claudin 18.2 CAR-IC carrying the Claudin 18.2 CAR-IC gene is obtained.

[0268] (4) The recombinant retrovirus vector MP71-Claudin 18.2 CAR-IC is transformed into competent E. coli DH5α, and a plasmid is extracted and purified by using a plasmid purification kit of Qiagen Company, and the MP71-Claudin 18.2 CAR-IC plasmid is obtained. The recombinant retrovirus vector MP71-Claudin 18.2 CAR-IC (i.e. the MP71-Claudin 18.2 CAR-IC plasmid) is obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retrovirus vector MP71 with a DNA fragment with the nucleotide sequence of SEQ ID NO: 2 in the sequence listing, while keeping other nucleotide sequences of the retrovirus vector MP71 unchanged.

[0269] 2. Retrovirus packaging

[0270] The MP71-Claudin 18.2 CAR-IC plasmid prepared in step 1 is introduced into a packaging cell for packaging, and virus assembly is completed to obtain a retrovirus. The specific steps of virus packaging are as follows:

[0271] a) Day 1: Phoenix-ECO cells should be less than 20 passages and not too full. Plate at a cell density of 0.5 x 10 6 / mL, add 10 mL of DMEM medium (10% Fetal Bovine Serum, 2 mM L-glutamine, 1% Penicillin / Streptomycin) to a 10 cm cell culture dish, mix the cells thoroughly, and culture at 37°C overnight;

[0272] b) Day 2: Phoenix-ECO cells reach about 90% confluence for transfection (usually 14-18 h after plating), and chloroquine is added half an hour before transfection; prepare 15 μg of plasmid, 1.5 M CaCl2 250 μL, add H2O 1 mL to a total volume of 1.25 mL; add an equal volume of 2x HBS to the plasmid complex, mix well, and stand at room temperature for 15 minutes; then add the mixture to the ECO cell dish. Culture at 37°C for 6 h, remove the culture medium, wash with PBS once, and add 10 mL of preheated fresh culture medium;

[0273] c) Day 4: Collect the supernatant 48h after transfection, filter through a 0.45 μm filter to obtain the retrovirus solution, and store in aliquots at -80°C.

[0274] d) Add 1.2 mL of a 15 μg / mL Retronectin coating solution per well in the NTC 6-well plate, and incubate overnight at 4°C.

[0275] e) Carefully remove the blocking solution, wash with 2 mL / well PBS, add 5 mL of the virus solution per well, centrifuge at 2000 x g for 2 h at 32°C, and discard the unbound virus supernatant.

[0276] f) Wash the logarithmic phase PG13 cells once with 10 mL of PBS, add 1 mL of 0.25% recombinant trypsin, and incubate at room temperature for 2-3 min.

[0277] g) Add 5 mL of complete medium containing 10% FBS to stop the digestion, and centrifuge at 1500 rpm for 5 min.

[0278] h) Discard the supernatant, adjust the cell density to 0.5 x 10 5 cells / mL with complete medium, and add 3 mL / well to the virus-coated NTC 6-well plate, so that the final cell number is 1.5 x 10 5 cells / well.

[0279] i) Incubate at 37°C in 5% CO2for 48 h.

[0280] j) After 1-2 passages, transfer to a T175 flask and incubate for 2 d in DEME medium containing 12% FBS.

[0281] k) Replace the medium with fresh DEME medium containing 12% FBS, and continue incubation for 48 h. Collect the supernatant, filter through a 0.45 μm filter to obtain the retrovirus solution, and store in aliquots at -80°C.

[0282] 3. Retroviral infection of human T cells

[0283] a) Thaw the frozen healthy human peripheral blood PBMC, and adjust the cell density to 1 x 10 6 cells / mL with RPMI-1640 medium containing 10% fetal bovine serum (FBS).

[0284] b) PBMC was separated by Ficoll solution (Tianjin Haoyang, LTS1077-1) to obtain lymphocytes, and then CD3+T cells were obtained by magnetic bead method (Miltenyi, item number 130-097-043). The T cells were activated by adding clinical grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen, item number: 11141D) at a ratio of magnetic beads: CD3+cells = 3: 1, and the T cells were cultured in a 24-well cell culture plate to obtain an activated T cell suspension.

[0285] c) The non-tissue treated culture plate was coated with RetroNectin (TAKARA) diluted with PBS to a final concentration of 15 μg / mL, 1.2 mL per well of a 6-well plate. Avoid light, 4°C overnight for standby.

[0286] d) After two days of T cell activation culture, the coated 6-well plate was taken out, and the coating solution was aspirated and washed once with PBS.

[0287] e) The reverse transcriptase solution prepared in step 2 was added to the well, 2 mL per well, centrifuged at 2000 x g for 2 h at 32°C, and the supernatant was discarded. 3 mL of activated T cell suspension containing hIL-2 (500 U / mL) was added to each well, and the culture was continued for 1 day.

[0288] f) After cell infection, the cell density was observed every day, and T cell culture solution containing 500 U / mL IL-2 was added in time to maintain the T cell density at about 5 x 10 5 / mL, which was convenient for cell expansion.

[0289] g) Thus, CAR-T cells infected with the reverse transcriptase prepared in step 2 (i.e. Claudin 18.2 CAR-IC T cells) were obtained. Claudin 18.2 CAR-IC T cells are T cells expressing the Claudin 18.2 CAR-IC gene with the nucleotide sequence of SEQ ID NO: 2. The constructed Claudin 18.2 CAR-IC T cells and CTR T cells (i.e. T cells without virus transfection, as negative control) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C, and recorded as D0 days, and cultured to D10 days for functional detection.

[0290] 4. CAR expression detection in Claudin 18.2 CAR-IC T cells

[0291] a) After centrifugation (1500 rpm x 5 min), the supernatant was discarded, and 200 μL of FACS buffer (1x PBS containing 2% FBS) was added to each well of a 96-well round-bottom plate to resuspend the cells, which were then centrifuged at 1500 rpm for 5 min;

[0292] b) 60 μL of prepared fluorescently labeled anti-human Claudin 18.2 (rp)-APC (APC anti-human IgG Fc Antibody, Biolegend, 410712) and TRII-APC (APC anti-human TGF-β Receptor II Antibody, Biolegend, 399706) were added to each well, mixed well, and incubated at 4°C for 30 min;

[0293] c) 200 μL of FACS buffer was added to each well, which was then centrifuged at 1500 rpm for 5 min;

[0294] d) The supernatant was discarded, and the cells were resuspended with 400 μL of FACS buffer and transferred to a flow reading tube. The cells were read by flow cytometry (BD Canto-II), and the percentage of the two antibodies in Claudin 18.2 CAR-IC T cells was analyzed.

[0295] The detection results are shown in Figure 2. Claudin 18.2 CAR-IC T cells (i.e., #131 CAR-IC T cells, labeled as #131 in Figure 2) can well express Claudin 18.2 antibodies and TGF-β Receptor II proteins, indicating that the expression of CAR molecules in Claudin 18.2 CAR-IC T cells meets the expected design.

[0296] Example 3, Detection of Claudin 18.2 expression in different cell lines and detection of IFN-γ secretion in Claudin 18.2 CAR-IC T cells

[0297] 1. Detection of Claudin 18.2 expression in different cell lines

[0298] 1-1. Construction of recombinant cells

[0299] (1) Construction of CLDN18.2 overexpressing HEK293T cells (HEK293T-CLDN18.2 cells): The gene encoding human CLDN18.2 protein (SEQ ID NO: 4) was cloned into the eukaryotic expression vector pSecTag2A-BN (Invitrogen, Cat No: V90020) after removing the stop codon, and the recombinant expression vector was transfected into HEK293T cells (Co-Cell, Cat No: 4201 PAT-CCTCC01347). The recombinant cells stably expressing human CLDN18.2 were obtained by screening, and named HEK293T-CLDN18.2 cells.

[0300] (2) Construction of CLDN18.2 overexpressing ASPC1 cells (ASPC1-CLDN18.2 cells): The construction steps were the same as step (1) except that the transfected cells were replaced by ASPC1 cells.

[0301] (3) Construction of CLDN18.1 overexpressing HEK293T cells (HEK293T-CLDN18.1 cells): The gene encoding human CLDN18.1 protein (SEQ ID NO: 6) was cloned into the eukaryotic expression vector pSecTag2A-BN (Invitrogen, Cat No: V90020) after removing the stop codon, and the recombinant expression vector was transfected into HEK293T cells (Co-Cell, Cat No: 4201 PAT-CCTCC01347). The recombinant cells stably expressing human CLDN18.1 were obtained by screening, and named HEK293T-CLDN18.1 cells.

[0302] 1-2, Detection of Claudin 18.2 expression

[0303] The test cells were: HEK293T-CLDN18.1, ASPC1, HepG2, Jurkat, BGC823, HEK293T-CLDN18.2, ASPC1-CLDN18.2 and SNU601 cells. The detection steps were as follows:

[0304] a) After centrifugation (1500 rpm x 5 min), the supernatant was discarded, and 200 μL FACS buffer (1x PBS containing 2% FBS) was added to each well of a 96-well round-bottom plate for resuspension, and centrifuged at 1500 rpm for 5 min;

[0305] b) Add 60 μL of prepared fluorescently labeled antibody VHH131 (Claudin 18.2-VHH#223 antibody) to each well, incubate at 4°C for 30 min; add secondary antibody (Biolegend, 410720), incubate at 4°C for 30 min.

[0306] c) Add 200 μL of FACS buffer to each well, centrifuge at 1500 rpm for 5 min;

[0307] d) Discard the supernatant, resuspend the cells with 400 μL of FACS buffer, transfer them to a flow reading tube, and read the cells by flow cytometry (BD Canto-II), and analyze the expression level of Claudin 18.2 protein.

[0308] The results are shown in Figure 3A. HEK293T-CLDN18.1, ASPC1, HepG2, Jurkat and BGC823 cell lines do not express Claudin 18.2 protein, and HEK293T-CLDN18.2, ASPC1-CLDN18.2 and SNU601 have different levels of Claudin 18.2 protein expression.

[0309] 2. Cell function detection of Claudin 18.2 CAR-IC T cell secretion of IFN-γ

[0310] Determining the release level of cytokine IFN-γ is an effective method for evaluating the therapeutic effect of CAR-T cells. Cytokine IFN-γ is essential for CAR-T cell toxicity and can induce apoptosis of target cells. High levels of IFN-γ are an important indicator of well-functioning CAR-T, and its expression is consistent with T cell cytotoxicity, which can indicate the activation level of CAR-T cells. It is detected by intracellular staining and flow cytometry, and the steps are as follows:

[0311] a) Adjust the cell density of the tested cells Claudin 18.2 CAR-IC T cells (#131 CAR-IC T cells) and CTR T cells (i.e. T cells without viral transfection, as negative control) to 2×10 6Incubate for 6 hours at 37°C. 100 μL of the sample is added to a 96-well U-bottom plate, and target cells (human embryonic kidney cells overexpressing CLDN18.1 (HEK293T-CLDN18.1), human pancreatic cancer cells (ASPC1), human hepatocarcinoma cells (HepG2), human gastric cancer cells (BGC823), human T lymphocyte leukemia cells (Jurkat), human embryonic kidney cells overexpressing CLDN18.2 (HEK293T-CLDN18.2), pancreatic cancer cells overexpressing CLDN18.2 (ASPC1-CLDN18.2), and human gastric cancer cells (SNU601)) are added at a ratio of 1:1, and 5 μg / mL of Brefeldin A (Med Chem Express, HY-16592) is added to each well, and incubation is performed in a 37°C incubator for 6 hours.

[0312] b) After incubation, flow cytometry staining is performed, and the steps are as follows:

[0313] (1) After centrifugation (1500 rpm x 5 min), the supernatant is discarded, 200 μL of FACS buffer (1x PBS containing 2% FBS) is added to each well, and centrifugation is performed at 1500 rpm for 5 min. This step is repeated twice.

[0314] (2) 60 μL of prepared fluorescently labeled G4S-FITC (Cell Signaling Technology, #50515) antibody is added to each well, and the mixture is mixed well, incubated at room temperature for 10 min, and protected from light.

[0315] (3) 200 μL of FACS buffer is added to each well, centrifuged at 1500 rpm for 5 min, and the supernatant is discarded.

[0316] (4) 150 μL of Cytofix / Cytoperm (BD, #55472) is added to each well, mixed well, incubated at room temperature for 15 min, and protected from light.

[0317] (5) After centrifugation at 1500 rpm for 5 min and discarding the supernatant, 200 μL of Perm / Wash buffer (BD, #554723) is added to each well, mixed well, centrifuged at 1500 rpm for 5 min, and washed twice by centrifugation.

[0318] (6) 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, #506510) is added to each well, mixed well, incubated at room temperature for 20 min, and protected from light.

[0319] (7) Add 200 μL Perm buffer to each well, centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells with 400 μL FACS buffer and transfer to flow reading tubes, and read the cells with a flow cytometer (BD Canto-II) to analyze the percentage of functional effector molecule IFN-γ.

[0320] The results are shown in Figures 3B and C. After co-incubation of Claudin 18.2 CAR-IC T cells (i.e. #131 CAR-IC T cells) with CLDN18.2 positive tumor cells HEK293T-CLDN18.2, ASPCl-CLDN18.2 and SNU601, the percentage of IFN-γ+ cells in #131 CAR-IC T cells was significantly increased, being 28.8%, 24.1% and 22.6%, respectively. However, after co-incubation with CLDN18.2 negative tumor cells HEK293T-CLDN18.1, HepG2, BGC823, ASPCl and Jurkat, the percentage of IFN-γ+ cells was similar to that without target cells (+Medium). The level of specific secretion of IFN-γ by Claudin 18.2 CAR-IC T cells was consistent with the results of CLDN18.2 antigen expression level of the above tumor cells (Figure 3A). This result shows that Claudin 18.2 CAR-IC T cells can specifically secrete effector molecule IFN-γ after co-incubation with CLDN18.2 positive cells, but not with CLDN18.2 negative cells.

[0321] Example 4, Detection of antigen-specific cytotoxic function of Claudin 18.2 CAR-IC T cells

[0322] 1. Adjust the density of effector cells (Claudin 18.2 CAR-IC T cells) and tumor cell SNU601 (CLDN18.2 positive tumor cell) suspensions to 2 x 105cells / well and 1 x 105cells / well, respectively. 6Cells were added at a ratio of 1:1 (100 μL / well) to 96-well U-plates. Claudin 18.2 CAR-IC T cells without tumor cells served as a negative control. 1 μL of APC anti-human CD107a antibody (BioLegend, 328620) was added to each well and incubated for 1 h. Then, 1 μL of Monensin solution (1 mg / mL) was added to each well and incubated for another 3 h. After incubation, the cells were centrifuged, the supernatant was discarded, and the cells were washed once with FACS buffer. Then, 60 μL / well of G4S-FITC antibody was added. Cells were incubated at room temperature in the dark for 10 min. Afterward, the cells were washed once with 200 μL of FACS buffer, resuspended in 400 μL of FACS buffer, and transferred to flow cytometry tubes. Cell analysis data were read using a flow cytometer (BD Canto-II). As shown in Figure 4A, after co-incubation of Claudin 18.2 CAR-IC T cells (#131 CAR-IC T cells) with SNU601 tumor cells, CD107a expression was significantly upregulated (50.8%), while no expression was observed in the negative control group. The degranulation molecule CD107a was used to determine the activation level of CAR-T cells. This result indicates that after co-incubation with SNU601 tumor cells, Claudin 18.2 CAR-IC T cells were in a state of T cell activation with a high activation level, exhibiting excellent killing activity against tumor cells.

[0323] 2. Adjust the density of the effector cell (Claudin 18.2 CAR-IC T cells) and SNU601 tumor cell suspension to 2×10⁻⁶. 6 CTR T cells (i.e., virus-free T cells) were used as a negative control. Target cell ratios of 9:1, 3:1, 1:1, 0.3:1, and 100 μL / well were added to 96-well U-plates. Cells were incubated at 37°C for 4 hours. Fluorescein sodium dilution buffer (1 μL / mL) was prepared and added to each well in the dark. Fluorescence values ​​were measured using a TECAN Spark microplate reader, and the average value of three replicates was taken. The specific killing activity (Cytotoxicity) of the effector cells was calculated. Cytotoxicity% = 100 - 100 × (Eexp - Emin) / (Tmax - Tmin)

[0324] Eexp: RLU value when effector cells and target cells are co-cultured;

[0325] Emin: The RLU value of spontaneous death of effector cells in the absence of target cells;

[0326] Tmax: RLU value of spontaneous death of target cells without effect cells;

[0327] Tmin: RLU value under the condition of maximum killing rate.

[0328] The detection results are shown in FIG. 4B. The negative control CTR T cells had no killing effect on the Claudin 18.2 positive target cells SNU601, and the Claudin 18.2 CAR-IC T cells (#131 CAR-IC T cells) could effectively and specifically kill the target cells SNU601 at different effector-to-target ratios (P value < 0.01).

[0329] Example 5: Evaluation of the in vivo tumor killing effect of Claudin 18.2 CAR-IC T cells in SNU601-NSG model

[0330] The in vivo tumor killing effect and animal survival of Claudin 18.2 CAR-IC T cells were evaluated using a SNU601 subcutaneously tumor-bearing NSG mouse model (SNU601-NSG model). The specific steps are as follows:

[0331] NSG mice (6-7 weeks old) (B-NDG, Bio-Oriented) were subcutaneously inoculated with SNU601 cells on the back of the right lower limb, 1x10 7 After 7 days of inoculation, the mice were randomly divided into 2 groups according to the tumor size, 5 mice in each group. The Claudin 18.2 CAR-IC T cells cultured for 10 days were prepared into a cell suspension, and the T cells without virus transfection were used as a control. A single injection of the drug was given through the tail vein, 3.0x10 6 The Claudin 18.2 CAR-IC T cells or the same number of control T cells were measured for tumor size by vernier caliper on D7, D14, D21, D28, and D35 days after administration, with D0 being the administration day.

[0332] The results are shown in FIG. 5. After administration, the tumors of the control group of mice continued to grow rapidly, and the tumors of the mice injected with Claudin 18.2 CAR-IC T cells (#131 CAR-IC T cells) gradually regressed after D7, and completely disappeared around 3 weeks. The difference between the two groups was significant (P value < 0.01), indicating that the Claudin 18.2 CAR-IC T cells could significantly inhibit tumor growth, kill tumor cells, and effectively exert an anti-tumor effect.

[0333] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the application, and without unnecessary experiments. Although specific examples are given in the application, it should be understood that further improvements can be made to the application. In summary, according to the principles of the application, the application is intended to include any changes, uses or improvements of the application, including changes made outside the scope disclosed in the application, using conventional techniques known in the art. Industrial applicability

[0334] The CAR-T cells of the application can secrete T cell-specific effector molecules IFN-γ well, effectively specifically kill Claudin 18.2 positive cells, and have no killing effect on Claudin 18.2 negative cells, especially Claudin 18.1 (extremely similar in structure to Claudin 18.2, with a homology of up to 92%) positive cells. The CAR-T cells of the application have good killing activity on tumor cells in vitro, can effectively specifically kill Claudin 18.2 positive tumor cells, and also have good in vivo killing activity, can significantly clear tumor cells in mice, inhibit tumor growth, and the tumor completely disappears in about 3 weeks, showing significant potential in combating Claudin 18.2 positive cancer, and having a broad clinical application prospect.

[0335] Cross-reference to related applications

[0336] The present application claims priority to the Chinese patent application No. 202410868595.6, filed on July 01, 2024, and entitled “Targeting Claudin 18.2 Chimeric Antigen and Chimeric Switch Receptor, Modified Immune Cells Thereof and Uses Thereof”, the entire content of which is incorporated herein by reference.

Claims

1. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and an intracellular signaling domain, the antigen binding domain comprises a first single domain antibody and a second single domain antibody, the first single domain antibody comprises complementarity determining region CDR1 of 47-54 of SEQ ID NO: 1, complementarity determining region CDR2 of 72-78 of SEQ ID NO: 1 and complementarity determining region CDR3 of 116-130 of SEQ ID NO: 1; The second single domain antibody comprises complementarity determining region CDR1 of 182-188 of SEQ ID NO: 1, complementarity determining region CDR2 of 206-215 of SEQ ID NO: 1 and complementarity determining region CDR3 of 254-267 of SEQ ID NO:

1.

2. The chimeric antigen receptor of claim 1, wherein, The first single domain antibody is any one of the following: A1) a protein with an amino acid sequence shown as 22-141 of SEQ ID NO: 1; A2) a protein with more than 80% identity to the protein shown as A1) and with the same function, obtained by substitution, deletion and / or addition of amino acid residues of the amino acid sequence shown as 22-141 of SEQ ID NO: 1; A3) a fusion protein with the same function, obtained by connecting a tag or a signal peptide to the N terminal and / or C terminal of A1) or A2); The second single domain antibody is any one of the following: B1) a protein with an amino acid sequence shown as 157-278 of SEQ ID NO: 1; B2) a protein with more than 80% identity to the protein shown as B1) and with the same function, obtained by substitution, deletion and / or addition of amino acid residues of the amino acid sequence shown as 157-278 of SEQ ID NO: 1; B3) a fusion protein with the same function, obtained by connecting a tag or a signal peptide to the N terminal and / or C terminal of B1) or B2).

3. The chimeric antigen receptor of claim 1 or 2, wherein, The intracellular signaling domain comprises a costimulatory domain, an activation domain, an interferon and a chimeric switch receptor, the chimeric switch receptor comprises an extracellular region of PD-1, an extracellular region of TGFβ receptor type II, and a transmembrane region and a cytoplasmic region of CD27.

4. The chimeric antigen receptor of any one of claims 1-3, wherein, The costimulatory domain is derived from 4-1BB, and / or the activation domain is derived from CD3ζ.

5. The chimeric antigen receptor of any one of claims 1-4, wherein, The chimeric antigen receptor is any one of the following: C1) a protein with an amino acid sequence shown as 22-1144 of SEQ ID NO: 1; C2) a protein with more than 80% identity to the protein shown as C1) and with the same function, obtained by substitution, deletion and / or addition of amino acid residues of the amino acid sequence shown as 22-1144 of SEQ ID NO: 1; C3) a fusion protein with the same function, obtained by connecting a tag or a signal peptide to the N terminal and / or C terminal of C1) or C2).

6. Biomaterials characterized in that, The biological material is any one of the following: D1) a nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-5; D2) an expression cassette comprising the nucleic acid molecule of D1); D3) a recombinant vector comprising the nucleic acid molecule of D1); D4) a recombinant microorganism comprising the nucleic acid molecule of D1); D5) a recombinant host cell comprising the nucleic acid molecule of D1).

7. The biomaterial of claim 6, wherein, The nucleic acid molecule is any one of the following: E1) a DNA molecule encoding a sequence or a nucleotide sequence as set forth in positions 64-3432 of SEQ ID NO: 2 or SEQ ID NO: 2; E2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in positions 64-3432 of SEQ ID NO: 2, and encoding a chimeric antigen receptor having an amino acid sequence as set forth in positions 22-1144 of SEQ ID NO: 1; E3) a DNA molecule having 75% or more identity to the nucleotide sequence defined in SEQ ID NO: 2, and encoding a chimeric antigen receptor having an amino acid sequence as set forth in SEQ ID NO:

1.

8. A modified immune effector cell, characterized in that, The modified immune effector cell comprises the chimeric antigen receptor of any one of claims 1-5 or the nucleic acid molecule of claim 6 or 7.

9. The modified immune effector cell of claim 8, wherein, The immune effector cell comprises T cells, NK cells, B cells, macrophages, dendritic cells, peripheral blood mononuclear cells, and monocytes.

10. The modified immune effector cell of claim 8 or 9, wherein, The immune effector cell is a T cell.

11. Use of the chimeric antigen receptor of any one of claims 1-5, the biological material of claim 6 or 7, or the modified immune effector cell of any one of claims 8-10 in the manufacture of a product having any one of the following functions: F1) preventing or treating a tumor; F2) preventing or treating a Claudin 18.2 target-related disease; F3) killing tumor cells positive for Claudin 18.2; F4) inhibiting proliferation of tumor cells positive for Claudin 18.2; F5) inhibiting growth of tumors positive for Claudin 18.2; F6) resisting immunosuppression and / or T cell exhaustion of a tumor microenvironment; F7) converting immunosuppressive signals of PD-1 and TGFβ into T cell activation signals mediated by CD27.

12. A pharmaceutical composition for preventing or treating a disease associated with Claudin 18.2 target, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor of any one of claims 1-5, the biological material of claim 6 or 7, or the modified immune effector cell of any one of claims 8-10, and one or more pharmaceutically acceptable carriers.

13. A method of making a modified immune effector cell of any one of claims 8-10, characterized in that, The method comprises introducing a nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-5 into an immune effector cell for expression, to obtain the modified immune effector cell.

14. A method for preventing or treating a Claudin 18.2 target-related disease, characterized by, The method comprises administering the modified immune effector cell of any one of claims 8-10 or the pharmaceutical composition of claim 12 to a subject having a Claudin 18.2 target-related disease.

15. The method of claim 14, wherein, The modified immune effector cell is a T cell modified with the chimeric antigen receptor of any one of claims 1-5.

16. The method according to claim 14 or 15, characterized in that The Claudin 18.2 target-related disease is a Claudin 18.2-positive tumor.

17. The method of claim 16, wherein, The Claudin 18.2-positive tumor is a Claudin 18.2-positive solid tumor.

18. The method of claim 16, wherein, The Claudin 18.2-positive tumor is a Claudin 18.2-positive cancer or a Claudin 18.2-positive mesothelioma.

19. The method of claim 18, wherein, The cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, lung cancer, lung adenocarcinoma, liver cancer, kidney cancer, ovarian cancer, bronchial cancer, breast cancer, bladder cancer, head and neck cancer, gallbladder cancer, cholangiocarcinoma, and gastroesophageal junction adenocarcinoma.

20. The method of claim 18 or 19, wherein, The cancer includes early-stage cancer, mid-stage cancer, mid-advanced stage cancer, advanced stage cancer, recurrent cancer, and metastatic cancer.

21. A method of inhibiting the growth of or killing a Claudin 18.2-positive tumor cell, characterized by, The method comprises contacting the Claudin 18.2-positive tumor cell with the modified immune effector cell of any one of claims 8-10 or the pharmaceutical composition of claim 12.

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