A humanized chimeric antigen receptor targeting cells expressing CLDN 18.2

Abstract

The present invention discloses a humanized chimeric antigen receptor (CAR) targeting cells expressing CLDN 18.2. Specifically, a chimeric antigen receptor having an amino acid sequence as shown in SEQ ID NO: 14 is disclosed, wherein the chimeric antigen receptor comprises: a humanized single-chain antibody targeting CLDN 18.2, a hinge region, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor targeting CLDN 18.2 of the present invention can effectively and specifically target malignant cells (e.g., tumor cells) expressing CLDN 18.2 surface antigens, reduce the immunogenicity of heterologous antibodies, and can be better applied to the human body, thereby providing a more efficient method for treating some tumors expressing CLDN 18.2 surface antigens with fewer side effects and adverse reactions.

Description

Technical Field

[0001] The present invention relates to the field of cell immunotherapy, and in particular to a humanized chimeric antigen receptor targeting cells expressing CLDN 18.2. Background Art

[0002] Cellular immunotherapy is an emerging method of treating tumors. It uses molecular biology techniques to construct a chimeric antigen receptor (CAR) expression vector, which is then introduced into immune cells isolated from the human body. The cells express CAR on their surface, which is then cultured and amplified before being infused back into the human body. Immune cells expressing CAR can specifically recognize and bind to target cells, and kill them by releasing specific immune factors. Although the first-generation CAR-T can mediate the killing effect on tumor cells, it does not transduce proliferation signals or induce cytokine production, and its sustained action time in vivo is not long, and it can only cause short-term T cell proliferation. Therefore, the anti-tumor effect is minimal; the second-generation CAR-T prolongs its survival time in vivo and promotes its rapid expansion ability by increasing the intracellular domain of co-stimulatory molecules (such as 4-1BB or CD28). By constructing scFv / 4-1BB (or CD28) / CD3-z CAR-T, it can lyse target cells, transmit activation signals, and produce a large amount of IFN-γ, IL-2 and other cytokines; the second-generation CAR-T has better ability to enhance T cell activation, expansion, anti-tumor and promote transgene expression than the first generation.

[0003] The main advantages of CAR-T in killing tumors are: first, because its antigen recognition binding domain can recognize tumor antigens, it has high specificity for tumors; second, because its intracellular signal transduction domain can quickly and simultaneously turn on the downstream signals of CAR-T killing and amplification, the tumor killing efficiency is high, and CART cells can continue to proliferate in vivo; third, because CAR-T does not need to start tumor killing through the combination of TCR and MHC molecules, it is non-MHC restricted and can directly recognize tumor surface antigens, so the recognition efficiency of tumors is high and specific, and it can kill tumor cells with MHC downregulation. Based on these advantages of CAR-T, CAR-T has achieved significant therapeutic effects in the treatment of blood tumors such as B-cell acute leukemia and non-Hodgkin's lymphoma, has been approved by the FDA, and has shown preliminary efficacy in the treatment of solid tumors.

[0004] The scFv in the CART structure comes from mice. Mouse monoclonal antibodies have heterologous reactions to humans and can induce human anti-mouse antibodies (HAMA reactions), which delays the therapeutic effect of CART.

[0005] Gastrointestinal and pancreatic tumors are a great threat to human life. Although surgical treatment, radiotherapy, chemotherapy, interventional therapy and other treatment methods have certain effects on these tumors, the survival rate of patients has not been significantly improved. At present, the highly anticipated CAR-T cell therapy technology is expected to become a breakthrough in treatment. Therefore, it is urgent to develop a new CAR-T cell for gastrointestinal and pancreatic tumors. Summary of the invention

[0006] The object of the present invention is to provide a humanized chimeric antigen receptor targeting cells expressing CLDN 18.2.

[0007] In a first aspect of the present invention, a humanized chimeric antigen receptor (CAR) targeting cells expressing CLDN 18.2 is provided, wherein the chimeric antigen receptor comprises: a humanized single-chain antibody targeting CLDN 18.2, a hinge region, a transmembrane domain and an intracellular signaling domain, wherein the amino acid sequence of the humanized single-chain antibody targeting CLDN 18.2 is shown in SEQ ID NO: 1.

[0008] In another preferred embodiment, the CAR has a structure as shown in Formula I:

[0009] L-scFv-H-TM-C-CD3ζ (Formula I)

[0010] in,

[0011] L is none or a signal peptide sequence;

[0012] scFv is the variable region sequence of a humanized single-chain antibody targeting CLDN 18.2;

[0013] H is none or hinge region;

[0014] TM is the transmembrane domain;

[0015] C is a co-stimulatory signal molecule;

[0016] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0017] Each "-" independently represents a connecting peptide or a peptide bond connecting the above-mentioned elements.

[0018] In another preferred example, the single-chain antibody comprises an antibody heavy chain variable region as shown in SEQ ID NO:17 and an antibody light chain variable region as shown in SEQ ID NO:18.

[0019] In another preferred embodiment, the antibody heavy chain variable region includes the following three complementarity determining regions CDR:

[0020] CDR1 as shown in SEQ ID NO:19;

[0021] CDR2 as shown in SEQ ID NO:20; and

[0022] CDR3 as shown in SEQ ID NO:21;

[0023] Furthermore, the affinity of the heavy chain variable region is on the same order of magnitude as that of the murine heavy chain variable region shown in SEQ ID NO:25.

[0024] In another preferred embodiment, the heavy chain variable region further comprises a humanized framework region (FR).

[0025] In another preferred embodiment, the antibody light chain variable region includes the following three complementarity determining regions CDR:

[0026] CDR1' as shown in SEQ ID NO:22;

[0027] CDR2' as shown in SEQ ID NO:23; and

[0028] CDR3' as shown in SEQ ID NO:24;

[0029] Furthermore, the affinity of the light chain variable region is on the same order of magnitude as that of the mouse light chain variable region shown in SEQ ID NO: 26.

[0030] In another preferred embodiment, the light chain variable region further comprises a humanized framework region (FR).

[0031] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0032] In another preferred embodiment, the hinge region is CD8 Hinge, and its amino acid sequence is shown in SEQ ID NO:3.

[0033] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0034] In another preferred embodiment, the transmembrane domain is CD8 TM, and its amino acid sequence is shown in SEQ ID NO:5.

[0035] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0036] In another preferred embodiment, C includes a co-stimulatory signal molecule derived from 4-1BB and / or a co-stimulatory signal molecule derived from CD28.

[0037] In another preferred example, the intracellular signaling domain is composed of 4-1BB and CD3ζ, and its amino acid sequence is shown in SEQ ID NO:6.

[0038] In another preferred embodiment, the intracellular signaling domain consists of CD28 and CD3ζ.

[0039] In another preferred embodiment, the intracellular signaling domain consists of 4-1BB, CD28 and CD3ζ.

[0040] In another preferred example, the amino acid sequence of CD28 is shown in SEQ ID NO:8.

[0041] In another preferred embodiment, the amino acid sequence of 4-1BB is shown in SEQ ID NO:10.

[0042] In another preferred example, the amino acid sequence of CD3ζ is shown in SEQ ID NO:12.

[0043] In another preferred example, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:14.

[0044] The second aspect of the present invention provides a nucleic acid molecule, which encodes the humanized chimeric antigen receptor targeting cells expressing CLDN 18.2 as described in the first aspect of the present invention, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:13.

[0045] In another preferred embodiment, the nucleic acid molecule contains a nucleotide sequence encoding a CAR construct as shown in Formula I;

[0046] L-scFv-H-TM-C-CD3ζ (Formula I)

[0047] in,

[0048] L is none or a signal peptide sequence;

[0049] scFv is the variable region sequence of a humanized single-chain antibody targeting CLDN 18.2;

[0050] H is none or hinge region;

[0051] TM is the transmembrane domain;

[0052] C is a co-stimulatory signal molecule;

[0053] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0054] Each "-" independently represents a connecting peptide or a peptide bond connecting the above-mentioned elements.

[0055] In another preferred example, the nucleic acid molecule encodes the amino acid sequence of the chimeric antigen receptor as shown in SEQ ID NO:14.

[0056] In another preferred example, the nucleotide sequence of the nucleic acid molecule comprises a sequence encoding the CD8 Hinge hinge region, a sequence encoding the transmembrane domain CD8 TM, and a sequence encoding the intracellular signaling domains 4-1BB, CD28 and CD3 ζ.

[0057] In another preferred example, the sequence encoding the CD8 Hinge hinge region is shown as SEQ ID NO: 2.

[0058] In another preferred example, the sequence encoding the transmembrane domain CD8 TM is shown as SEQ ID NO:4.

[0059] In another preferred example, the sequence encoding CD28 is shown as SEQ ID NO:7.

[0060] In another preferred example, the sequence encoding 4-1BB is shown as SEQ ID NO:9.

[0061] In another preferred embodiment, the sequence encoding CD3ζ is shown in SEQ ID NO: 11

[0062] The third aspect of the present invention provides a recombinant expression vector expressing the humanized chimeric antigen receptor targeting cells expressing CLDN 18.2 as described in the first aspect of the present invention, wherein the promoter of the recombinant expression vector is connected to a leader peptide and a nucleic acid fragment comprising the nucleic acid molecule as described in the second aspect of the present invention.

[0063] In another preferred embodiment, the promoter is EF1alpha promoter.

[0064] In another preferred embodiment, the nucleic acid sequence encoding the leader peptide is shown as SEQ ID NO:15.

[0065] In another preferred example, the amino acid sequence of the leader peptide is shown in SEQ ID NO:16.

[0066] In another preferred embodiment, the expression vector is selected from DNA, RNA, or a combination thereof.

[0067] In another preferred embodiment, the expression vector is selected from the following group: plasmid, viral expression vector, transposon, or a combination thereof.

[0068] In another preferred embodiment, the viral expression vector is selected from the following group: a lentiviral vector, an adenoviral vector, a retroviral vector, or a combination thereof.

[0069] The fourth aspect of the present invention provides an engineered immune cell, which contains the recombinant expression vector as described in the third aspect of the present invention, or has the nucleic acid molecule as described in the second aspect of the present invention integrated into its chromosome, or expresses the chimeric antigen receptor as described in the first aspect of the present invention.

[0070] In another preferred embodiment, the immune cells are T cells, NK cells or a combination thereof.

[0071] In another preferred embodiment, the immune cells are T cells.

[0072] In another preferred embodiment, the immune cells are chimeric antigen receptor T cells (CAR-T cells).

[0073] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises the humanized chimeric antigen receptor as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention or the engineered immune cell as described in the fourth aspect of the present invention, and a pharmaceutically acceptable carrier, excipient or diluent.

[0074] In another preferred embodiment, the pharmaceutical composition is a liquid pharmaceutical composition.

[0075] In another preferred embodiment, the pharmaceutical composition is an injection.

[0076] In another preferred embodiment, the immune cells are chimeric antigen receptor T cells (CAR-T cells).

[0077] In another preferred embodiment, in the pharmaceutical composition, the concentration of the CAR-T cells is 1×10 6 -5× 10 6 cells / ml, preferably 1×10 6 -2×10 6 cells / ml.

[0078] In a sixth aspect, the present invention provides a use of the humanized chimeric antigen receptor as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention, the engineered immune cell as described in the fourth aspect of the present invention, or the pharmaceutical composition as described in the fifth aspect of the present invention in preventing and / or treating cancer containing tumor cells expressing CLDN 18.2.

[0079] In another preferred embodiment, the cancer is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, prostate cancer, small intestinal adenocarcinoma, oral cancer or nasopharyngeal cancer.

[0080] The seventh aspect of the present invention provides a method for preparing the engineered immune cells as described in the fourth aspect of the present invention, comprising the following steps: transducing the nucleic acid molecule as described in the second aspect of the present invention or the vector as described in the third aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.

[0081] In another preferred embodiment, the immune cells are T cells, NK cells or a combination thereof.

[0082] In another preferred embodiment, the immune cells are T cells.

[0083] In a seventh aspect, the present invention provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a humanized chimeric antigen receptor targeting cells expressing CLDN 18.2 as described in the first aspect of the present invention, an engineered immune cell as described in the fourth aspect of the present invention, or a pharmaceutical composition as described in the fifth aspect of the present invention.

[0084] In another preferred embodiment, the disease is cancer or tumor.

[0085] In another preferred embodiment, the cancer is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, prostate cancer, small intestinal adenocarcinoma, oral cancer or nasopharyngeal cancer.

[0086] In another preferred embodiment, the subject in need is a human or non-human mammal.

[0087] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 The diagrams show three exemplary structures of the chimeric antigen receptor of the present invention.

[0089] Figure 2 This is a schematic diagram of the structure of the lentiviral expression vector used in the embodiments of the present invention.

[0090] Figure 3 The figure is a restriction endonuclease XbaI electrophoresis identification diagram of a lentiviral expression vector inserted with a nucleic acid fragment encoding a chimeric antigen receptor targeting CLDN 18.2.

[0091] Figure 4 The figure shows the killing effect of CLDN 18.2-CART cells and control T cells on CLDN 18.2-positive K562 tumor cells.

[0092] Figure 5 The figure shows the killing effect of CLDN 18.2-CART cells and control T cells on CLDN 18.2-negative K562 tumor cells.

[0093] Figure 6 The figure shows the results of IL-2 secretion by T cells expressing chimeric antigen receptor targeting CLDN 18.2.

[0094] Figure 7 The figure shows the results of IFN-γ secretion by T cells expressing chimeric antigen receptor targeting CLDN 18.2. DETAILED DESCRIPTION

[0095] After extensive and in-depth research, the inventors unexpectedly developed a humanized chimeric antigen receptor for the first time that targets cells expressing Claudin 18.2 (CLDN 18.2). In vitro experiments have shown that the humanized chimeric antigen receptor can effectively and specifically target malignant cells (such as tumor cells) that express the CLDN 18.2 surface antigen, and can be expressed on the surface of immune cells such as T cells, specifically killing malignant cells that express the CLDN 18.2 surface antigen, and achieved stronger cell killing and cytokine release effects than the positive control CAR-T cells. On this basis, the present invention was completed.

[0096] the term

[0097] CLDN 18.2

[0098] CLDN 18.2 is highly specific in normal tissue expression and is activated in a variety of cancers. Therefore, CLDN 18.2 has become a very potential target for epithelial tumors. CLDN 18.2 is a broad-spectrum tumor marker that can be expressed in a variety of tumors.

[0099] As used herein, "CLDN 18.2 positive" refers to cells that highly express CLDN 18.2 on the surface of the cell, and "CLDN 18.2 negative" refers to cells that lowly express or do not express CLDN 18.2 on the surface of the cell.

[0100] The humanized chimeric antigen receptor or CAR-T cell of the present invention can specifically target CLDN 18.2 expressed on the surface of tumor cells, thereby killing tumor cells.

[0101] Antibody

[0102] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0103] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration and are connected by three CDRs (CDR1, CDR2, and CDR3) that form a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0104] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0105] The variable regions of the heavy and / or light chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having monoclonal antibody light and heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0106] As used herein, the term "heavy chain variable region" is synonymous with "V H ” can be used interchangeably.

[0107] As used herein, the terms "light chain variable region" and "V L ” can be used interchangeably.

[0108] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0109] scFv

[0110] As used herein, the terms "single-chain antibody" and "scFv" refer to antibodies formed by connecting the heavy chain variable region and the light chain variable region of an antibody through a short peptide (linker) of 15 to 20 amino acids. scFv can better retain its affinity activity for antigens and has the characteristics of small molecular weight, strong penetration and weak antigenicity.

[0111] The chimeric antigen receptor targeting cells expressing CLDN 18.2 of the present invention comprises a single-chain antibody targeting CLDN 18.2, and the amino acid sequence of the single-chain antibody targeting CLDN 18.2 is shown in SEQ ID NO.:1.

[0112] Furthermore, the single-chain antibody comprises an antibody heavy chain variable region as shown in SEQ ID NO.:17 and an antibody light chain variable region as shown in SEQ ID NO.:18.

[0113] Humanization

[0114] The present invention provides a humanized chimeric antigen receptor targeting cells expressing CLDN 18.2, comprising a humanized single-chain antibody targeting CLDN 18.2.

[0115] As used herein, the term "humanization" refers to the use of gene manipulation to replace or remove the mouse-derived protein fragment in the monoclonal antibody gene after the monoclonal antibody is produced, thereby weakening its antigenicity in the human body and achieving therapeutic effect.

[0116] The humanized single-chain antibody targeting CLDN 18.2 of the present invention is obtained by replacing the framework region (FR) of the mouse antibody, and the affinity of the heavy chain variable region and the light chain variable region of the prepared antibody is at the same order of magnitude as the affinity of the heavy chain variable region / light chain variable region shown in SEQ ID NO: 25 and 26 of the mouse.

[0117] Chimeric Antigen Receptor (CAR)

[0118] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0119] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0120] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting Claudin 18.2. When the CAR of the present invention is expressed in T cells, it can recognize antigens based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells not to grow, to be caused to die or to be affected in other ways, and causing the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with an intracellular domain from one or more of the costimulatory molecules and the ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0121] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the heavy chain and the variable region of the light chain of the antibody, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the antigen binding domain comprises an antibody that specifically recognizes CLDN 18.2, preferably a single-chain antibody.

[0122] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0123] The intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3ζ.

[0124] Polynucleotide molecules and vectors

[0125] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof and the above-mentioned chimeric antigen receptors. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 13, 2, 4, 7, 9 and 11 or may be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to the polypeptide of the present invention, but different from the coding region sequence shown in SEQ ID NO.: 13, 2, 4, 7, 9 and 11.

[0126] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0127] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0128] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NOs..14, 3, 5, 6, 8, 10 and 12.

[0129] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Generally, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0130] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0131] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0132] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, T cells, and NK cells.

[0133] Chimeric Antigen Receptor T Cells (CAR-T Cells)

[0134] As used herein, the terms "CAR-T cell", "CAR-T", and "CAR-T cell of the present invention" include the CAR-T cell comprised in the fourth aspect of the present invention.

[0135] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigens, once the CAR gene construction targeting a certain tumor antigen is completed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) The use of the patient's own cells reduces the risk of rejection; and (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0136] The present invention provides a CAR-T cell comprising a CAR targeting CLDN 18.2, as described in the fourth aspect of the present invention.

[0137] In a preferred embodiment of the present invention, the CAR-T cells constructed using humanized CLDN 18.2scFv can further improve their killing effect and tumor clearance ability.

[0138] Pharmaceutical composition

[0139] The present invention provides a pharmaceutical composition comprising a humanized chimeric antigen receptor as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, or an engineered immune cell as described in the fourth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the pharmaceutical composition is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 5 -1×10 8 cells / ml, preferably 1×10 5 -1×10 6 cells / ml.

[0140] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0141] Therapeutic applications

[0142] The present invention also provides a use of the humanized chimeric antigen receptor as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention, the engineered immune cell as described in the fourth aspect of the present invention, or the pharmaceutical composition as described in the fifth aspect of the present invention in preventing and / or treating cancer containing tumor cells expressing CLDN 18.2.

[0143] The cancer is a solid tumor, and the cancer is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, prostate cancer, small intestinal adenocarcinoma, oral cancer, or nasopharyngeal cancer.

[0144] For ex vivo immune cell preparation, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0145] Ex vivo cell processing procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing the CAR disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cells can be autologous, allogeneic, or syngeneic relative to the recipient.

[0146] The present invention provides a method for treating tumors, comprising administering an effective amount of the universal CAR-T cells of the present invention to a subject in need thereof.

[0147] The universal CAR-T cells of the present invention may be administered alone or as a pharmaceutical composition with a diluent and / Or in combination with other components such as IL-2, IL-15, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include target cells as described herein, combined with one or more pharmaceutically or clinically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0148] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the characteristics of the patient's condition, the type and severity of the disease - although the appropriate dosage can be determined by clinical trials.

[0149] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 7 The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered using well-known infusion techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be determined by monitoring the patient's disease signs, and the treatment regimen is determined by a medical technician.

[0150] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The composition described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, intravenously (iv) or intraperitoneally, intrapleurally. In another embodiment, the T cell composition of the present invention is preferably administered by iv intravenous injection. The composition of T cells can be directly injected into tumors, lymph nodes or infection sites. (CART cell products are mainly intravenous infusions, and can be directly injected into tumors, lymph nodes or infection sites)

[0151] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to patients in combination with any number of related treatment modalities (e.g., before, simultaneously or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir, interleukin-2, IFN-γ, cytarabine (also known as ARA-C) and other cytotoxic chemotherapy drugs, checkpoint inhibitors such as PD-1 antibodies, anti-CTLA-4 antibodies, and drugs that inhibit cytokine storms, such as tocilizumab for anti-IL-6 receptors, and other treatments. In further embodiments, the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to patients in combination with bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0152] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The universal CAR-DNT cells of the present invention are administered to patients, for example, by intravenous infusion.

[0153] The main advantages of the present invention are:

[0154] (1) The CLDN 18.2 mouse-derived antibody of the present invention has undergone humanized antibody modification, which reduces the immunogenicity of the heterologous antibody while retaining the high affinity and specificity for binding to the antigen, and is advantageous for application in the human body.

[0155] (2) The Claudin18.2 CAR-T cells of the present invention have stronger killing and cytokine release functions than the positive control CAR-T cells, indicating that CAR-T cells have more effective effects in vivo.

[0156] (3) The Claudin 18.2 CAR-T cells of the present invention continuously maintain a high proportion of CAR positive expression throughout the in vitro culture process, indicating that CAR-T cells can maintain a high growth rate in in vitro culture, which also indicates that CAR-T cells can have a good therapeutic effect in vivo.

[0157] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0158] Unless otherwise specified, the materials, reagents, instruments, etc. used in the examples can be obtained from commercial sources.

[0159] Example 1

[0160] Construction of chimeric antigen receptor expression plasmid containing scFv targeting CLDN 18.2

[0161] A 1461 bp DNA fragment as shown in SEQ ID NO: 13 was artificially synthesized, wherein the nucleotides at positions 1-63 encode the leader, the nucleotides at positions 64-789 encode the CLDN 18.2scFv (scFv targeting carcinoembryonic antigen), the nucleotides at positions 790-924 encode the CD8 hinge region, the nucleotides at positions 925-996 encode the CD8 transmembrane region, the nucleotides at positions 997-1122 encode 4-1BB, and the nucleotides at positions 1123-1458 encode CD3ζ.

[0162] The above DNA fragment was inserted into the downstream of the EF1alpha promoter of the lentiviral expression vector to obtain a chimeric antigen receptor expression plasmid targeting CLDN18.2 (pLV-CLDN 18.2-scFv-BBz). Figure 3 The figure shows the electrophoresis identification of restriction endonuclease XbaI digestion of the lentiviral expression vector into which the chimeric antigen receptor targeting CLDN 18.2 is inserted.

[0163] Example 2

[0164] T cells were transfected with humanized chimeric antigen receptor expression plasmid targeting CLDN 18.2

[0165] (1) Lentivirus packaging preparation

[0166] The humanized chimeric antigen receptor expression plasmid, structural plasmid, and envelope plasmid were transfected into 293T / 17 cells at a ratio of 3:2:1 using calcium phosphate transfection. After 12 hours of transfection, fresh DMEM medium containing 10% FBS was replaced, and sodium butyrate with a final concentration of 5mM was added. After 48 hours of transfection, the cell culture supernatant containing the virus was aspirated into a centrifuge tube, centrifuged at 1500rpm for 10 minutes at 4℃, and the supernatant was transferred to a new centrifuge tube, filtered with a 0.45μm filter, and stored at -80℃.

[0167] (2) Preparation of T cells

[0168] Take 10 ml of fresh blood from a healthy person and separate peripheral blood mononuclear cells using lymphocyte separation medium (Yunfei Biotechnology). For specific methods, refer to the instructions. Use T551 medium containing 4% autologous serum to adjust the cell density to 2×10 6 / ml and added IL-2 with a final concentration of 300U / ml and CD3 monoclonal antibody with 100ng / ml to induce culture for 24h to obtain T cells.

[0169] (3) Lentivirus infection of T cells and proliferation and culture of infected T cells

[0170] The lentiviral solution was added to 2×10 6 The cells were co-cultured in a 37°C, 5% CO2 incubator. Three days later, the cells were washed by centrifugation, and fresh T551 medium containing 300 IU / ml IL-2, 100 ng / ml CD3 monoclonal antibody, and 4% human autologous serum was added, and the cell density was adjusted to 2x10 6 / ml and continue to culture. Check the cell density every 2-3 days, centrifuge and adjust the cell density with fresh culture medium, and continue to expand the culture. Repeat this process until the cells have expanded to a sufficient amount.

[0171] After verifying that the humanized chimeric antigen receptor is effectively expressed in T cells, CAR-T cells targeting CLDN18.2 are obtained.

[0172] Example 3

[0173] Specific killing activity of CAR-T cells against CLDN 18.2-positive malignant cells

[0174] Take 5×10 4K562 cells that highly express CLDN 18.2 (CLDN18.2 positive, represented by CLDN 18.2-K562) and control K562 cells that highly express CLDN 18.1 (CLDN18.2 negative, represented by CLDN18.1-K562) were inoculated in 96-well plates, and CAR-T cells (CLDN 18.2-CART), CAR-T positive control cells (CLDN 18.2-CART positive control) and unmodified T cells (T) were added to the two cell culture wells at three ratios of effector cells (Effector): target cells (Target) = 10:1, 3:1, and 1:1. After incubation for 18 hours, the LDH cytotoxicity assay kit (Biyuntian) was used to detect the specific killing activity of CAR-T cells against K562 cells that highly express CLDN18.2. The results showed that the CAR-T cell group had a stronger killing effect on K562 cells that highly expressed CLDN 18.2 compared with the CAR-T positive control cell group and the unmodified T cell group, and this difference was significant (p<0.05) ( Figure 4 ); There was no significant difference in the killing effect on K562 cells expressing CLDN 18.1 (p>0.05) ( Figure 5 ).

[0175] Example 4

[0176] ELISA to detect the expression of IFN-gamma and IL-2

[0177] Take 5×10 4 K562 target cells with high expression of CLDN18.2 (CLDN18.2 positive) and control CLDN18.1-K562 target cells (CLDN18.2 negative) were inoculated in 96-well plates, and CAR-T cells (CLDN18.2-CART), CLDN18.2-CART positive control cells (CLDN18.2-CART positive control) and unmodified T cells (T) were added to the two cell culture wells at a ratio of effector cells (Effector): target cells (Target) = 3:1. After incubation for 18 hours, the Human IL-2ELISA Kit (Dakoway) was used to operate and the supernatant was taken to detect the content of IL-2 and IFN-. Figure 6 and Figure 7As shown, the results showed that the CAR-T cell group produced a large amount of IL-2 and IFN- to K562 cells that highly expressed CLDN18.2 when the effector-target ratio was 3:1 compared with the CAR-T positive control cell group and the T cell group, and this difference was significant (p<0.05); K562 cells that highly expressed CLDN18.1 had no induction effect on cytokines, and there was no significant difference (p>0.05). Therefore, CAR-T cells have a specific cytokine induction effect on tumor cells that highly express CLDN18.2.

[0178] In summary, the humanized chimeric antigen receptor targeting CLDN18.2 of the present invention can effectively and specifically target malignant cells expressing the CLDN 18.2 surface antigen, thereby providing a more efficient method with fewer side effects and adverse reactions for treating some tumors expressing the CLDN 18.2 surface antigen.

[0179] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0180] The sequence information involved in the present invention is shown in the following table:

[0181] Sequence information

[0182]

[0183]

[0184] Sequence Listing <110> Shanghai Life Medical Technology Co., Ltd. <120> A humanized chimeric antigen receptor targeting cells expressing CLDN 18.2 <130> P2020-1999 <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> 247 <212> PRT <213> Artificial sequence <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ser 180 185 190 Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Three Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys With Glu Ile Lys 245 <210> 2 <211> 135 <212> DNA <213> Homo sapiens <400> 2 accaccacac cagctcctag acctcctaca ccagccccta caatcgccctc tcagccctg 60 agcctgagac ccgaagcttg tagaccagcc gcaggaggag ccgtgcatac cagaggcctg 120 gatttcgctt gcgac 135 <210> 3 <211> 45 <212> PRT <213> Homo sapiens <400> 3 Thr Thr Thr Pro Ala Pro Arg Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 4 <211> 72 <212> DNA <213> Homo sapiens <400> 4 atctacattt gggcccccct gggcaggaact tgcggagtgc tgctgctgtc tctggtcatc 60 accctgtatt gc 72 <210> 5 <211> 24 <212> PRT <213> Homo sapiens <400> 5 Tyr Ile Trp Ile Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leo Val Ile Thr Lion Tyr Cys 20 <210> 6 <211> 154 <212> PRT <213> Homo sapiens <400> 6 Lys Arg Gly Arg Lys Leu Leu Tyr Ile Phe Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 35 40 45 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 50 55 60 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 65 70 75 80 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 85 90 95 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 100 105 110 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 115 120 125 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 130 135 140 Ala Leu His Met Gln Ala Leu Pro Pro Arg 145 150 <210> 7 <211> 123 <212> DNA <213> Homo sapiens <400> 7 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 8 <211> 41 <212> PRT <213> Homo sapiens <400> 8 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 9 <211> 126 <212> DNA <213> Homo sapiens <400> 9 aagcggggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgag gcccgtgcag 60 acaacacagg aggaggacgg ctgttcttgc aggttcccag aggaagaaga gggcggttgc 120 gagctg 126 <210> 10 <211> 42 <212> PRT <213> Homo sapiens <400> 10 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 11 <211> 336 <212> DNA <213> Homo sapiens <400> 11 agagtgaagt tcagcagaag cgccgacgct ccagcttatc agcagggcca gaaccagctg 60 tacaacgagc tgaacctggg caggagggag gaatacgacg tgctggacaa gcggagaggc 120 agagatccag agatgggcgg caagcccaga agaaagaacc cccaggaggg cctgtacaac 180 gagctgcaga aggacaagat ggccgaggcc tacagcgaga tcggaatgaa gggcgagagg 240 aggagaggca agggacacga cggactgtac cagggactga gcaccgccac caaggatacc 300 tacgatgccc tgcatatgca ggctctgcct ccaaga 336 <210> 12 <211> 112 <212> PRT <213> Homo sapiens <400> 12 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 13 <211> 1476 <212> DNA <213> Artificial sequence <400> 13 atggctctgc cagtgacagc tctgctgctg cctctggctc tgctgctgca cgcagctaga 60 ccacaggttc agctggttca gtctggcgcc gaagtgaaga aacctggcgc ctctgtgaag 120 gtgtcctgca aggcttccgg ctacaccttt accagctact ggatgcactg ggtccgacag 180 gctccaggac aaggcctgga atggatcggc atgatccatc ctaactccgg ctccaccaac 240 tacaacggca agttcaagtc ccgcgtgacc atgaccagag acacctctac ctccaccgcc 300 tacatggaac tgtccagcct gagatctgag gacaccgccg tgtactactg tgccagaggc 360 ggctactacg gcaactccct ggatttttgg ggccagggca ccaccgtgac agtgtcatct 420 ggaggcggag gctctggagg aggaggatct ggcggaggag gcagcgacat ccagatgacc 480 cagtctccat cctctctgtc cgcctctgtg ggcgacagag tgaccatgac ctgtcgggct 540 tctcagtccc tgctgaactc cggcaaccag aagaactacc tgacctggta tcagcagaag 600 cccggcaagg ctcccaagct gctgatctac tgggcctcca ccagagaatc tggcgtgccc 660 tctagattct ccggctctgg ctctggcacc gactttaccc tgaccatctc ctccgtgcag 720 cctgaggatt tcgccaccta ctactgccag aacgcctaca gctacccctt cacctttggc 780 cagggcacca agctggaaat caagaccacc acaccagctc ctagacctcc tacaccagcc 840 cctacaatcg cctctcagcc tctgagcctg agcccgag cttgtagacc agccgcag ggagccgtgc ataccagagg cctggatttc gcttgcgaca tctacatttg ggcccccctg 960 gcaggaactt gcggagtgct gctgctgtct ctggtcatca ccctgtattg caagcggggc 1020 cggaagaagc tgctgtacat cttcaagcag cccttcatga ggcccgtgca gacaacacag gaggaggacg gctgttcttg caggttccca gaggagaag agggcggttg cgagctgaga gtgaagttca gcagaagcgc cgacgctcca gcttatcagc agggccagaa ccagctgtac aacgagctga acctgggcag gagggagga tacgacgtgc tggacaagcg gaggcaga gatccagaga tgggcggcaa gcccagaga aagaaccccc aggagggcct gtacaacgag ctgcagaagg acaagatggc cgaggcctac agcgagatcg gaatgaaggg cgagaggagg agaggcaagg gacacgacgg actgtaccag ggactgagca ccgccacca ggatacctac gatgccctgc atatgcaggc tctgcctcca agataa 1476 <210> 14 <211> 491 <212> PRT <213> Artificial sequence (Artificial sequence) <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn 65 70 75 80 Tyr Asn Gly Lys Phe Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser 85 90 95 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp 115 120 125 Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 145 150 155 160 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met 165 170 175 Thr Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn 180 185 190 Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 195 200 205 Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln 225 230 235 240 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro 245 250 255 Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro 260 265 270 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 305 310 315 320 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375 380 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 385 390 395 400 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 15 <211> 63 <212> DNA <213> Homo sapiens <400> 15 atggctctgc cagtgacagc tctgctgctg cctctggctc tgctgctgca cgcagctaga 60 cca 63 <210> 16 <211> 21 <212> PRT <213> Homo sapiens <400> 16 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 17 <211> 119 <212> PRT <213> Mus musculus <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 18 <211> 113 <212> PRT <213> Mouse (Mus musculus) <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 19 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 19 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 20 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 20 Met Ile His Pro Asn Ser Gly Ser Thr Asn 1 5 10 <210> 21 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 21 Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe 1 5 10 <210> 22 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 22 Arg Ala Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 23 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 23 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 24 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 24 Gln Asn Ala Tyr Ser Tyr Pro Phe Thr 1 5 <210> 25 <211> 119 <212> PRT <213> Mouse (Mus musculus) <400> 25 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Ser Leu Thr Val Ser Ser 115 <210> 26 <211> 113 <212> PRT <213> Mouse (Mus musculus) <400> 26 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Arg 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Light

Claims

1. A humanized chimeric antigen receptor (CAR) targeting cells expressing CLDN 18.2, characterized in that The chimeric antigen receptor comprises: a humanized single-chain antibody targeting CLDN 18.2, a hinge region, a transmembrane domain and an intracellular signaling domain, wherein the amino acid sequence of the humanized single-chain antibody targeting CLDN 18.2 is shown in SEQ ID NO:

1.

2. The chimeric antigen receptor according to claim 1, wherein The CAR has a structure as shown in Formula I: L-scFv-H-TM-C-CD3ζ (Formula I) in, L is none or a signal peptide sequence; scFv is the variable region sequence of a humanized single-chain antibody targeting CLDN 18.2; H is the hinge region; TM is the transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; Each "-" independently represents a connecting peptide or a peptide bond connecting the above elements.

3. The chimeric antigen receptor according to claim 1, wherein The single-chain antibody comprises an antibody heavy chain variable region as shown in SEQ ID NO:17 and an antibody light chain variable region as shown in SEQ ID NO:

18.

4. The chimeric antigen receptor according to claim 3, wherein The antibody heavy chain variable region includes the following three complementarity determining regions CDR: CDR1 as shown in SEQ ID NO:19; CDR2 as shown in SEQ ID NO:20; and CDR3 as shown in SEQ ID NO:21; Furthermore, the affinity of the heavy chain variable region is on the same order of magnitude as that of the murine heavy chain variable region shown in SEQ ID NO:

25.

5. The chimeric antigen receptor according to claim 3, wherein The antibody light chain variable region includes the following three complementarity determining regions CDR: CDR1' as shown in SEQ ID NO:22; CDR2' as shown in SEQ ID NO:23; and CDR3' as shown in SEQ ID NO:24; Furthermore, the affinity of the light chain variable region is on the same order of magnitude as that of the mouse light chain variable region shown in SEQ ID NO:

26.

6. The chimeric antigen receptor according to claim 2, wherein The H is a hinge region selected from the following histones: CD8, CD28, CD137, or a combination thereof.

7. The chimeric antigen receptor according to claim 2, wherein The TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

8. The chimeric antigen receptor according to claim 2, wherein The C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

9. The chimeric antigen receptor according to claim 1, wherein The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:

14.

10. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the humanized chimeric antigen receptor targeting cells expressing CLDN18.2 as claimed in claim 1, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

13.

11. A recombinant expression vector expressing the humanized chimeric antigen receptor targeting cells expressing CLDN 18.2 as claimed in claim 1, characterized in that: The promoter of the recombinant expression vector is connected with a leading peptide and a nucleic acid fragment encoding the chimeric antigen receptor.

12. An engineered immune cell, characterized in that: The immune cell contains the recombinant expression vector according to claim 11, or has the nucleic acid molecule according to claim 10 integrated into its chromosome, or expresses the chimeric antigen receptor according to claim 1.

13. The engineered immune cell according to claim 12, characterized in that The immune cells are T cells, NK cells or a combination thereof.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the humanized chimeric antigen receptor as described in claim 1, the nucleic acid molecule as described in claim 10, the recombinant expression vector as described in claim 11 or the engineered immune cell as described in claim 13, and a pharmaceutically acceptable carrier, excipient or diluent.

15. Use of the humanized chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 10, the recombinant expression vector according to claim 11, the engineered immune cell according to claim 13 or the pharmaceutical composition according to claim 14 in the preparation of a medicament for treating cancer containing tumor cells expressing CLDN 18.2, wherein the cancer is selected from: gastric cancer, pancreatic cancer.

16. A method for preparing the engineered immune cell according to claim 11, characterized in that: The following steps are involved: The nucleic acid molecule according to claim 10 or the vector according to claim 11 is transduced into immune cells to obtain the engineered immune cells.

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