T lymphocytes targeting CLD18A2 and preparation method and application thereof

Abstract

The present invention relates to T lymphocytes targeting CLD18A2 and a preparation method and application thereof. The inventors have successfully found a target gene CLD18A2 suitable for developing CAR T cells from a number of tumor-related genes for the first time, and successfully prepared CAR T cells targeting CLD18A2, thereby providing a new treatment method for pancreatic cancer, gastric cancer and other tumors.

Description

[0001] This application is a divisional application of application number 201410341504.X, filed on July 17, 2014, and the invention name is T lymphocytes targeting CLD18A2, preparation method and application thereof. Technical Field

[0002] The present invention belongs to the field of tumor cell therapy, and more specifically, relates to T lymphocytes targeting CLD18A2 and a preparation method and application thereof. Background Art

[0003] The role of T lymphocytes in tumor immune response has been increasingly valued. Adoptive immunotherapy based on T lymphocytes has achieved certain results in some tumors, and this immunotherapy method can overcome the above-mentioned defects of antibody therapy, but the efficacy in most tumors is still unsatisfactory [Grupp SA, et al. Adoptive cellular therapy. Curr Top Microbiol Immunol., 2011; 344: 149-72.]. In recent years, based on the discovery that the recognition specificity of CTL to target cells depends on the T cell receptor (T Cell Receptor, TCR), the scFv of antibodies against tumor cell-associated antigens is fused with intracellular signal activation motifs such as CD3δ or FcεRIγ of the T lymphocyte receptor to form a chimeric antigen receptor (Chimeric antigen receptor, CAR), and it is genetically modified on the surface of T lymphocytes by means such as lentiviral infection. This CAR T lymphocyte can selectively direct T lymphocytes to tumor cells and specifically kill tumors in a non-restrictive manner of the major histocompatibility complex (Major Histocompatibility Complex, MHC). CAR T lymphocytes are a new immunotherapy strategy in the field of tumor immunotherapy [Schmitz M, et al. Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer. J Biomed Biotechnol, 2010, doi: 10.1155 / 2010 / 956304.].

[0004] Chimeric antigen receptors include an extracellular binding region, a transmembrane region, and an intracellular signaling region. Usually, the extracellular region contains scFv that can recognize tumor-associated antigens, the transmembrane region uses the transmembrane region of molecules such as CD8 and CD28, and the intracellular signaling region uses the intracellular signaling region of the immunoreceptor tyrosine-based activation motif (ITAM) CD3δ or FcεRIγ and co-stimulatory signal molecules CD28, CD27, CD137, CD134, etc.

[0005] The first generation of CAR T lymphocytes contain only ITAM in the intracellular signaling region, in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-ITAM. This type of CAR T can stimulate anti-tumor cytotoxic effects, but the cytokine secretion is relatively small and cannot stimulate a lasting anti-tumor effect in vivo [Zhang T. et al. Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways, Can Res 2007, 67 (22): 11029-11036.].

[0006] The second generation of CAR T lymphocytes subsequently developed added the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-ITAM or scFv-TM- / CD137-ITAM. The B7 / CD28 or 4-1BBL / CD137 co-stimulation in the intracellular signaling region causes the continuous proliferation of T lymphocytes, and can increase the level of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, while improving the survival cycle and anti-tumor effect of CAR T in vivo [Dotti G. et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients. J Clin Invest, 2011, 121 (5): 1822-1826.].

[0007] The third generation of CAR T lymphocytes developed in recent years, in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improves the survival period of CAR T in vivo and its anti-tumor effect [Carpenito C., et al. Control of large established tumorxenografts with genetically retargeted human T cells containing CD28 andCD137 domains. PNAS, 2009, 106(9): 3360–3365.].

[0008] Although CAR T lymphocytes have attractive prospects in tumor immunotherapy, their higher risks also need to be considered. For example, due to the low expression of specific antigens that can be recognized by CAR in certain normal tissues, CAR T lymphocytes may cause damage to normal tissues expressing the corresponding antigens. For example, the first case of CAR T lymphocyte adoptive therapy for the antigen carbonic anhydrase IX (CAIX) expressed on tumor cells in patients with renal cell carcinoma was the first case of CAR T lymphocyte adoptive therapy used in clinical practice, and it was also the first case to report the off-target effect of CAR cells. Patients developed grade 2-4 liver toxicity after multiple infusions of CAR T lymphocytes. The reason for the analysis was that CAIX was lowly expressed in hepatobiliary epithelial cells, and the original clinical trial was forced to be interrupted, excluding any evaluation of the patient's treatment effect [Stoter G. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol, 2006, 24 (13): e20-e22.; Ngo MC., et al. Ex vivo gene transfer for improved adoptive immunotherapy of cancer. Human Molecular Genetics, 2011, R1-R7]. In addition, excessive co-stimulatory signals in CAR will reduce the threshold required for effector cell activation, so that genetically modified T lymphocytes may also be activated under conditions of low levels of antigen or no antigen triggering, resulting in the release of a large number of cytokines, which may trigger the so-called "cytokine storm". This signal leakage (singal leakage) will lead to off-target cytotoxicity, thereby producing nonspecific tissue damage. For example, during the clinical treatment of a patient with advanced colon cancer with liver and lung metastases using the third-generation CAR targeting Her2, the so-called "cytokine storm" was triggered due to the low expression of Her2 in normal lung tissue, leading to the patient's sudden death [Morgan RA., et al. Report of aserious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing Erbb2. Molecular Therapy, 2010, 18(4):843-851.].

[0009] When designing CAR T, the antigen gene targeted is a key choice. Given the complexity of gene expression in the body and various uncontrollable factors, it is very difficult to select a suitable gene for CAR T. In addition, it is difficult to find specific molecules for many tumor-specific antigens that are suitable for constructing CAR T cells. After establishing CAR T, it is often impossible to obtain active extracellular binding regions, which is also a difficulty in developing CAR T technology. Summary of the invention

[0010] The object of the present invention is to provide T lymphocytes targeting CLD18A2 and a preparation method and application thereof.

[0011] In a first aspect of the present invention, a chimeric antigen receptor (CAR) expressed on the surface of T lymphocytes is provided, wherein the chimeric antigen receptor comprises: an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises a protein that specifically recognizes CLD18A2 (claudin 18.2).

[0012] In a preferred embodiment, the protein that specifically recognizes CLD18A2 is an antibody or a ligand; preferably, the antibody is a single-chain antibody or a domain antibody.

[0013] In another preferred embodiment, the transmembrane region is a sequence comprising the transmembrane region and hinge region of CD8 or CD28.

[0014] In another preferred embodiment, the intracellular signal region is selected from the group consisting of: intracellular signal region sequences of CD3δ, FcεRIγ, CD27, CD28, CD137, CD134, or a combination thereof.

[0015] In another preferred embodiment, the chimeric antigen receptor comprises the following sequentially connected extracellular binding region, transmembrane region and intracellular signaling region:

[0016] A single-chain antibody that specifically recognizes CLD18A2, CD8, and CD3δ;

[0017] A single-chain antibody that specifically recognizes CLD18A2, CD8, CD137, and CD3δ;

[0018] A single chain antibody that specifically recognizes CLD18A2, the transmembrane region of the CD28 molecule (CD28a), the intracellular signaling region of the CD28 molecule (CD28b), and CD3δ; or

[0019] A single-chain antibody that specifically recognizes CLD18A2, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, CD137, and CD3δ.

[0020] In another preferred embodiment, the chimeric antigen receptor has an amino acid sequence as described in any one of SEQ ID NOs: 19 to 22.

[0021] In another aspect of the present invention, a nucleic acid encoding the chimeric antigen receptor is provided.

[0022] In a preferred example, the nucleic acid has a nucleotide sequence as described in any one of SEQ ID NOs: 15 to 18.

[0023] In another aspect of the present invention, an expression vector is provided, which comprises the nucleic acid.

[0024] In a preferred example, the expression vector is derived from the lentiviral plasmid pWPT (or pWPT-eGFP).

[0025] In another aspect of the present invention, a virus is provided, wherein the virus (such as a lentivirus) comprises the vector.

[0026] In another aspect of the present invention, there is provided a use of the chimeric antigen receptor, or the nucleic acid, or the expression vector, or the virus for preparing genetically modified T lymphocytes targeting CLD18A2.

[0027] In another aspect of the present invention, a genetically modified T lymphocyte is provided, which is transduced with the nucleic acid, the expression vector or the virus.

[0028] In another aspect of the present invention, a genetically modified T lymphocyte is provided, which expresses a chimeric antigen receptor on its surface, and the amino acid sequence of the chimeric antigen receptor is selected from any one of the amino acid sequences described in SEQ ID NO: 19-22.

[0029] In another aspect of the present invention, there is provided a use of the genetically modified T lymphocytes for preparing a drug for inhibiting tumors, wherein the tumor is a CLD18A2-positive (highly expressed) tumor.

[0030] In another preferred embodiment, the CLD18A2-positive tumors include: pancreatic cancer and gastric cancer.

[0031] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 , a schematic diagram of the structure of the lentiviral vector pWPT-eGFP-F2A-CAR containing a CAR encoding sequence of the present invention as an example.

[0033] Figure 2, schematic diagram of the connection order of the various parts of the chimeric antigen receptor.

[0034] Figure 3 , Electrophoresis diagram of purification of single-chain antibody against CLD18A2 in Example 1.

[0035] Figure 4 The results of Western Blot detection of stable expression cell lines of CLD18A1 and CLD18A2.

[0036] Figure 5 , flow cytometry was used to detect the binding specificity of CLD18A2 single-chain antibody and CLD18A1 and CLD18A2.

[0037] Figure 6 , electrophoresis identification of the spliced ​​chimeric antigen receptor. DETAILED DESCRIPTION

[0038] After extensive and in-depth research, the inventors disclosed for the first time a CAR T lymphocyte based on the CLD18A2 gene and a preparation method thereof.

[0039] CLD18A2 gene

[0040] The inventors have previously investigated many types of tumor-specific genes and found that a considerable portion of these genes are also expressed in normal cells of some tissues and cannot be applied to chimeric antigen receptor T cell technology; some tumor-specific genes have good tumor-specific expression characteristics, but the CAR T cells designed based on them have no tumor cell killing activity or very low activity, which may be due to the low antigenicity of the protein expressed by the corresponding gene, or the unreasonable expression position or insufficient expression level, or it may be due to the fact that the T lymphocytes obtained after the recombinant construction are affected and the tumor killing ability is weakened or lost.

[0041] After repeated investigations and screening, the inventors found the CLD18A2 gene as the target gene for designing CAR T cells.

[0042] Claudin 18 (CLD18) molecule (Genbank accession number: splice variant 1 (CLD18A1): NP.sub.-057453, NM.sub.-016369, and splice variant 2 (CLD18A2): NM.sub.-001002026, NP.sub.-001002026) is a transmembrane protein with a molecular weight of approximately 27.9 / 27.72. Claudins are membrane proteins located in the tight junctions between epithelium and endothelium.

[0043] Studies have shown that CLD18A1 is selectively expressed in normal lung and gastric epithelium, while CLD18A2 is only expressed in differentiated short-lived cells of gastric epithelium and not in gastric stem cells; and existing studies have shown that CLD18A2 is expressed in many tumor cells. In view of the above characteristics of CLD18A2, the inventors speculate that CLD18A2 may be an important therapeutic target for these tumors; and, after a large amount of subsequent work, this speculation has been implemented.

[0044] Chimeric antigen receptor and nucleic acid encoding same

[0045] The present invention provides a chimeric antigen receptor expressed on the surface of T lymphocytes, wherein the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region and an intracellular signal region connected in sequence, wherein the extracellular binding region comprises a protein that specifically recognizes CLD18A2 (claudin 18.2). The chimeric antigen receptor is expressed on the surface of T lymphocytes, so that the T lymphocytes have a highly specific cytotoxic effect on tumor cells that highly express CLD18A2.

[0046] As a preferred embodiment of the present invention, the extracellular binding region comprises a single-chain antibody scFv (CLD18A2) that specifically recognizes CLD18A2. The extracellular binding region of the chimeric antigen receptor protein is connected to the transmembrane region of CD8 or CD28 through the CD8 hinge region, and the intracellular signal region is immediately behind the transmembrane region.

[0047] The present invention also includes nucleic acids encoding the chimeric antigen receptor. The nucleic acid sequence 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 nucleic acid codons encoding the amino acid sequence of the chimeric antigen receptor protein of the present invention may be degenerate, that is, multiple degenerate nucleic acid sequences encoding the same amino acid sequence are included in the scope of the present invention. Degenerate nucleic acid codons encoding corresponding amino acids are well known in the art. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. The variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it.

[0048] Monoclonal antibodies that specifically recognize human CLD18A2 can be selected from antibodies disclosed in the prior art. A variety of monoclonal antibodies that recognize the C-terminal epitope of CLD18A2 can be used in the present invention in a suitable manner, as long as they can finally obtain CAR T lymphocytes with killing activity after recombinant construction. As a preferred embodiment of the present invention, single-chain antibodies are used, preferably, the single-chain antibodies are 163 and 175 antibodies; 163 and 175 antibodies can specifically recognize CLD18A2 but not CLD18A1. More preferably, they are connected to Fc (ScFv-163 and scFv-175).

[0049] The term "single-chain antibody (scFv) fragment" used in the present invention refers to an antibody fragment defined as follows, which is a recombinant protein comprising a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker, and the linker associates these two domains to ultimately form an antigen binding site. 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. The single-chain antibody used in the present invention can be further modified using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination and / or other modification methods, alone or in combination. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY. The modification referred to is preferably performed at the nucleic acid level. The above-mentioned single-chain antibody may also include its derivatives. In the present invention, "antibody derivatives" include, for example, when the derivatives of the antibody are obtained by phage display technology, the efficiency of phage antibodies binding to the CLD18A2 antigen epitope can be increased using surface plasmon resonance technology such as used in the BIAcore system (Schier, Human Antibody Hybridoma 7 (1996), 97-105; Malmborg, Journal of Immunological Methods 183 (1995), 7-13). It also includes, for example, the method for producing chimeric antibodies described in WO 89 / 09622, the method for producing humanized antibodies described in EP-A10239400 and WO90 / 07861, and the method for producing heterologous antibodies such as human antibodies in mice described in WO91 / 10741, WO94 / 02602 and WO96 / 33735.

[0050] The term "specific recognition" of the present invention means that the bispecific antibody of the present invention does not or substantially does not cross-react with any polypeptide other than the target antigen. The degree of its specificity can be determined by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometric analysis, etc. In the present invention, specific recognition is preferably determined by flow cytometry, and the standard of specific recognition in specific cases can be determined by a person skilled in the art based on the common knowledge in the art.

[0051] The transmembrane region of the chimeric antigen receptor can be selected from the transmembrane region of proteins such as CD8 or CD28. CD8 or CD28 is a natural marker on the surface of T lymphocytes. The human CD8 protein is a heterodimer composed of two chains of αβ or γδ. In one embodiment of the present invention, the transmembrane region is selected from the transmembrane region of CD8α or CD28. In addition, the CD8α hinge region is a flexible region, so CD8 or CD28 and the transmembrane region plus the hinge region are used to connect the target recognition domain scFv of the chimeric antigen receptor CAR to the intracellular signaling region.

[0052] The intracellular signal region can be selected from the intracellular signal region of CD3δ, FcεRIγ, CD28, CD137, CD134 proteins, and combinations thereof. The CD3 molecule consists of five subunits, of which the CD3δ subunit (also known as CD3 zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. CD3δZ is a truncated CD3δ sequence without an ITAM motif, and is generally used as a negative control in the practice of the present invention. FcεRIγ is mainly distributed on the surface of mast cells and basophils, and contains an ITAM motif, which is similar to CD3δ in structure, distribution and function. In addition, as mentioned above, CD28, CD137, and CD134 are co-stimulatory signal molecules. After binding to their respective ligands, the co-stimulatory effect produced by their intracellular signal segments causes the continuous proliferation of T lymphocytes and can increase the level of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, while also increasing the survival cycle and anti-tumor effect of CAR T lymphocytes in the body.

[0053] The anti-CLD18A2 chimeric antigen receptor protein encoded by the nucleic acid of the present invention can be selected from the following sequence:

[0054] scFv(CLD18A2)-CD8-CD3δ,

[0055] scFv(CLD18A2)-CD8-CD137-CD3δ,

[0056] scFv(CLD18A2)-CD28a-CD28b-CD3δ,

[0057] scFv(CLD18A2)-CD28a-CD28b-CD137-CD3δ,

[0058] And combinations thereof, wherein CD28a in the relevant chimeric antigen receptor protein represents the transmembrane region of the CD28 molecule, and CD28b represents the intracellular signaling region of the CD28 molecule. The above-mentioned various anti-CLD18A2 chimeric antigen receptors are collectively referred to as scFv (CLD18A2) -CAR.

[0059] In one embodiment of the present invention, the nucleic acid of the present invention has a sequence as shown in SEQ ID NOs: 15 to 18. In another embodiment of the present invention, the nucleic acid of the present invention is a nucleic acid encoding a chimeric antigen receptor protein having one of SEQ ID NOs: 19-22.

[0060] Expression vector and cells

[0061] The present invention also provides a vector comprising the above-mentioned nucleic acid encoding the chimeric antigen receptor protein expressed on the surface of T lymphocytes. In a specific embodiment, the vector used in the present invention is a lentiviral plasmid vector pWPT-eGFP. The plasmid belongs to the third-generation self-inactivating lentiviral vector system, which has three plasmids, namely, the packaging plasmid psPAX2 encoding the protein Gag / Pol and the Rev protein; the envelope plasmid PMD2.G encoding the VSV-G protein; and the empty vector pWPT-eGFP, which can be used to recombinantly introduce the target nucleic acid sequence, i.e., the nucleic acid sequence encoding CAR. The expression of enhanced green fluorescent protein (eGFP) is regulated by the elongation factor-1α (EF-1α) promoter in the empty vector pWPT-eGFP (which itself is a mock in subsequent experiments). The recombinant expression vector pWPT-eGFP-F2A-CAR containing the target nucleic acid sequence encoding CAR achieves co-expression of eGFP and CAR through the ribosomal skipping sequence 2A (abbreviated as F2A) from foot-and-mouth disease virus (FMDV).

[0062] The present invention also includes viruses comprising the above-mentioned vectors. The viruses of the present invention include viruses with infectivity after packaging, and also include viruses to be packaged that contain the necessary components for packaging into infectious viruses. Other viruses known in the art that can be used to transduce foreign genes into T lymphocytes and their corresponding plasmid vectors can also be used in the present invention.

[0063] In one embodiment of the present invention, the virus is a lentivirus comprising the above-mentioned pWPT-eGFP-F2A-CAR recombinant vector (ie, containing scFv (CLD18A2)-CAR).

[0064] The present invention also provides genetically modified T lymphocytes, which are transduced with the nucleic acid of the present invention or transduced with the above-mentioned recombinant plasmid containing the nucleic acid of the present invention, or a virus containing the plasmid. Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral transduction methods include electroporation and transposon methods. The Nucleofector nuclear transfection instrument recently developed by Amaxa can directly introduce exogenous genes into the cell nucleus to obtain efficient transduction of the target gene. In addition, the transduction efficiency of transposon systems such as Sleeping Beauty transposon or PiggyBac transposon is greatly improved compared with ordinary electroporation. The combined use of nucleofector transfection instrument and Sleeping Beauty transposon system has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70 (10): OF1-10.] This method has both high transduction efficiency and can achieve site-specific integration of the target gene. In one embodiment of the present invention, the transduction method for T lymphocytes modified with chimeric antigen receptor genes is a transduction method based on viruses such as retroviruses or lentiviruses. This method has the advantages of high transduction efficiency, stable expression of exogenous genes, and shortening the time for in vitro cultured T lymphocytes to reach clinical-grade quantities. On the surface of the transgenic T lymphocytes, the transduced nucleic acid is expressed on its surface through transcription and translation. In vitro cytotoxicity experiments on various cultured tumor cells have demonstrated that the anti-CLD18A2 chimeric antigen receptor gene-modified T lymphocytes of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid encoding the chimeric antigen receptor protein of the present invention, the plasmid containing the nucleic acid, the virus containing the plasmid, and the transgenic T lymphocytes transduced with the above nucleic acid, plasmid or virus can be effectively used for tumor immunotherapy.

[0065] In one embodiment, the genetically modified T lymphocytes of the present invention express a chimeric antigen receptor on their surface, and the chimeric antigen receptor is encoded and expressed by a nucleic acid of one of SEQ ID NOs: 15 to 18. In another embodiment, the transgenic T lymphocytes of the present invention express a chimeric antigen receptor on their surface, and the amino acid sequence of the chimeric antigen receptor is selected from one of SEQ ID NOs: 19 to 22.

[0066] Since there are no reports on CAR T targeting CLD18A2, the inventors have successfully found a target gene CLD18A2 suitable for CAR T cells from many tumor-related genes for the first time, and successfully prepared CAR T cells targeting CLD18A2, thus providing a new treatment for pancreatic cancer, gastric cancer and other tumors.

[0067] 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 according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0068] Example 1. Expression of single-chain antibody against CLD18A2

[0069] After repeated research and analysis, the inventors identified several scFv antibodies that recognize CLD18A2, referred to as 125, 163 and 175.

[0070] The single-chain antibody sequences of 125 (SEQ ID NO: 1 (nucleotide), 2 (amino acid)), 163 (SEQ ID NO: 3 (nucleotide), 4 (amino acid)), and 175 (SEQ ID NO: 5 (nucleotide), 6 (amino acid)) were synthesized by gene synthesis technology based on PCR bridging, and double-digested with Nhe1 / BamH1 (purchased from NEB), and ligated with T4 DNA ligase (purchased from NEB) to the vector plasmid pCMV-V5-Fc (the vector is fused to express the human antibody Fc fragment downstream of the multiple cloning site, hereinafter referred to as V5-Fc, purchased from Shanghai Ruijin Biotechnology) which was also double-digested with Nhe1 / BamH1, and transformed into the host bacteria TOP10, and clones were picked, positive clones were identified by PCR, and confirmed by sequencing, and V5-scFv-125-Fc, V5-scFv-163-Fc, and V5-scFv-175-Fc eukaryotic expression plasmids were obtained, respectively.

[0071] The above expression plasmids were transfected into HEK-293F cells with good growth, and cultured continuously for 7 days at 37°C, 5% CO2, and 125rpm shaking. The cells were centrifuged at 4000rpm for 10min, the precipitate was removed, the supernatant was collected, and filtered with a 0.45μm filter membrane. The treated samples were affinity purified with a protein A (purchased from GE) affinity column to finally obtain purified single-chain antibody-Fc fusion proteins scFv-125-Fc (abbreviated as scFv-125), scFv-163-Fc (abbreviated as scFv-163), and scFv-175-Fc (abbreviated as scFv-175). The identification results are as follows: Figure 3 .

[0072] Example 2: Construction of a stable expression cell line of CLD18A1 or CLD18A2

[0073] 1. Construction of expression vectors for CLD18A1 and CLD18A2 and preparation of lentivirus

[0074] The complete coding sequence of CLD18A1 (GenBank: NM_016369) and the complete coding sequence of CLD18A1 (GenBank: NM_001002026) were synthesized by PCR-based gene synthesis technology, a Flag tag (DYKDDDK) was inserted at the C-terminus of the coding, and MluI / SalI (purchased from NEB) restriction sites were added to both ends of the synthetic gene fragment. After MluI / SalI double digestion, the vector plasmid pWPT (purchased from Addgene) that was also double digested with MluI / SalI was connected with T4 DNA, transformed into the host bacteria TOP10, and clones were picked for PCR identification and sequencing to confirm that the correct lentiviral vector plasmids PWPT-CLD18A1 and PWPT-CLD18A2 were obtained. The above plasmids were co-transfected into 293T cells with packaging auxiliary plasmids (pGag-pol, pREV, pVsv-g (all purchased from Addgene) at a certain ratio. CLD18A1 and CLD18A2 virus solutions were collected 48h and 72h after transfection, respectively, packaged, and stored at -80°C.

[0075] 2. Establishment of stable exogenous expression lines of CLD18A1 and CLD18A2 and Western blot detection

[0076] The CLD18A1 or CLD18A2 virus solution collected above was added to 293T cells spread on a 6 cm plate, and the cells were collected after 72 hours and lysed using a cell lysis solution. In addition, the CLD18A2 virus solution was used to infect human gastric cancer BGC-823 (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, TCHu 11) and NCI-N87 (purchased from ATCC, CRL-5822) cell lines, and the cells were collected after they were full and lysed using a cell lysis solution. 40 μg of the cell protein collected by lysis was taken for SDS-PAGE gel electrophoresis, and then the gel was immunoblotted and stained with mouse anti-Flag antibody (purchased from Sigma Aldrich). After washing with PBS, it was incubated with a goat anti-mouse antibody labeled with horseradish peroxidase (purchased from Santa Cluz) and then developed using an ECL reagent, and finally developed.

[0077] Western blot results showed that a band with a molecular weight of about 28KD was detected in 293T cells transfected with CLD18A1 or CLD18A2 (i.e., 293T-CLD18A1, 293T-CLD18A2) and BGC-823 and NCI-N87 cells transfected with CLD18A2 (i.e., BGC-823-CLD18A2, NCI-N87-CLD18A2), but no corresponding band was detected in untransfected empty cells ( Figure 4 ), indicating that the cell line exogenously expressing CLD18A1 and CLD18A2 was successfully constructed.

[0078] 3. Flow cytometry analysis of the binding of anti-CLD18A2 antibodies to various cell lines

[0079] The binding ability of the single-chain antibodies scFv-125, scFv-163 and scFv-175 to the following cell lines was analyzed by fluorescence activated cell sorter (FACS) (BD, FACSCalibur).

[0080] The specific method is as follows:

[0081] 1). Take 293T, 293T-CLD18A1, 293T-CLD18A2, BGC-823, BGC-823-CLD18A2, NCI-N87, NCI-N87-CLD18A2 tumor cells in the logarithmic growth phase and inoculate them into 6 cm dishes. The inoculation cell density is about 90% and cultured in a 37°C incubator overnight.

[0082] 2). Digest the cells with 10mM EDTA and collect the cells by centrifugation at 200g for 5min. 6 ~1×10 7The cells were resuspended in 1% phosphate buffered saline (NBS PBS) at a concentration of 1 / mL and added to flow cytometry tubes at a volume of 100 ul / tube.

[0083] 3). Centrifuge at 200g×5min and discard the supernatant.

[0084] 4). Add the test antibodies scFv-125, scFv-163 and scFv-175 respectively, and use an irrelevant antibody as a negative control. The final concentration of the antibody is 20 μg / ml. Add 100ul to each tube. Incubate on ice for 45 minutes.

[0085] 5). Add 2 ml of 1% NBS PBS to each tube and centrifuge at 200g×5min for two times.

[0086] 6). Discard the supernatant and add 1:50 diluted FITC fluorescent labeled goat anti-human antibody (from Shanghai Kangcheng Bioengineering Co., Ltd.), add 100ul to each tube. Incubate on ice for 45 minutes.

[0087] 7). Add 2 ml of 1% NBS PBS to each tube and centrifuge at 200g×5min for two times.

[0088] 8). Discard the supernatant, resuspend in 300ul 1% NBS PBS, and detect by flow cytometry.

[0089] 9). Analyze the data using flow cytometry data analysis software WinMDI 2.9.

[0090] Flow cytometry analysis results showed that the single-chain antibody scFv-125 could bind not only to CLD18A1, but also to 293T cells stably expressing CLD18A2 ( Figure 5 ), indicating that the single-chain antibody lacks binding specificity for CLD18A2. Fortunately, the single-chain antibodies ScFv-163 and scFv-175 can specifically recognize 293T cells stably expressing CLD8A2, but do not bind to 293T cells stably expressing CLD18A1, indicating that these two single-chain antibodies can specifically recognize CLD18A2. In addition, these two single-chain antibodies can also specifically recognize BGC-823 or NCI-N87 cell lines stably transfected with CLD18A2, but do not bind to BGC-823 or NCI-N87 cells that are not transfected with CLD18A2.

[0091] Example 3. Construction of lentiviral plasmid expressing chimeric antigen receptor protein encoded by nucleic acid of the present invention and viral packaging

[0092] Table 1 explains the order of connection of the various parts of the chimeric antigen receptor exemplified by the present invention. Figure 2 as shown in .

[0093] Table 1

[0094]

[0095]

[0096] 1. Amplification of nucleic acid fragments

[0097] (1) Amplification of scFv (CLD18A2-163, CLD18A2-175) sequences

[0098] Using V5-scFv-163-Fc plasmid as a template, the primer pair used was a forward primer (SEQ ID NO: 7) containing a portion of the 2A sequence and a reverse primer (SEQ ID NO: 8) containing a portion of the CD8 hinge sequence, and PCR amplification was performed to obtain scFv (CLD18A2-163); similarly, using V5-scFv-175-Fc plasmid as a template, the primer pair used was a forward primer containing a portion of the 2A sequence and a reverse primer (SEQ ID NO: 9) and a reverse primer containing a portion of the CD8 hinge sequence (SEQ ID NO: 10) to obtain PCR amplification of scFv (CLD18A2-175).

[0099] (2) Nucleic acid sequences of other parts of the chimeric antigen receptor

[0100] The nucleic acid sequences of the other parts of the anti-CLD18A2 chimeric antigen receptor protein except scFv (CLD18A2-163, CLD18A2-175) were obtained by PCR using the sequences SEQ ID NO: 18 and 21 disclosed in patent application No. 201310164725.X as templates.

[0101] The eGFP-F2A sequence was obtained by PCR amplification using the plasmid SEQ ID NO: 18 contained in patent application No. 201310164725.X as a template and a primer pair (SEQ ID NO: 11, 12).

[0102] Obtaining CD8-CD3δ(Z) and CD28a-CD28b-CD137-CD3δ(28BBZ): Using scFv(GPC3)-CD8-CD3δ (SEQ ID NO: 18 in patent application 201310164725.X) and scFv(GPC3)-CD28a-CD28b-CD137-CD3δ (SEQ ID NO: 21 in patent application 201310164725.X) as templates, primer pairs (SEQ ID NO: 13, 14) were used for PCR amplification to obtain CD8-CD3δ(Z) and CD28a-CD28b-CD137-CD3δ(28BBZ) fragments, respectively.

[0103] SEQ ID NO: 18 in 201310164725.X corresponds to SEQ ID NO: 23 in the present invention.

[0104] SEQ ID NO:21 in 201310164725.X corresponds to SEQ ID NO:24 in the present invention.

[0105] 2. Splicing of nucleic acid fragments

[0106] The eGFP-F2A nucleic acid fragment obtained as described above was mixed with an equal mole of scFv (CLD18A2-163) or scFv (CLD18A2-175) nucleic acid fragment and an equal mole of CD8-CD3δ (Z) or CD28a-CD28b-CD137-CD3δ (BBZ) nucleic acid fragment, respectively. Figure 2 The three fragments were spliced ​​and PCR was performed as shown, and the splicing conditions were: pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s; annealing: 60°C, 40s; extension: 68°C, 140s, for 5 cycles, and then a total extension of 68°C, 10min. DNA polymerase and forward primer (SEQ ID NO: 11) and reverse primer (SEQ ID NO: 14) were supplemented and PCR amplification was performed for 30 cycles. The amplification conditions were pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s; annealing: 60°C, 40s; extension: 68°C, 140s, for 30 cycles, and then a total extension of 68°C, 10min. The fragments obtained by amplification are (Table 2):

[0107] eGFP-scFv(CLD18A2)-163-Z (SEQ ID NO: 15, 19),

[0108] eGFP-scFv(CLD18A2)-163-BBZ (SEQ ID NO: 16, 20),

[0109] eGFP-scFv(CLD18A2)-175-Z (SEQ ID NO: 17, 21),

[0110] eGFP-scFv(CLD18A2)-175-BBZ (SEQ ID NO: 18, 22).

[0111] The results were identified as Figure 6 .

[0112] Table 2. Sequences in the present invention

[0113]

[0114] 3. Construction of Lentiviral Plasmid Vector

[0115] As an example, the vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third-generation self-inactivating lentiviral vector system. The system has three plasmids, namely, the packaging plasmid psPAX2 (purchased from addgene) encoding the protein Gag / Pol and encoding the Rev protein; the envelope plasmid PMD2.G (purchased from addgene) encoding the VSV-G protein, and the recombinant expression vector encoding the target gene CAR based on the empty vector pWPT-eGFP (purchased from addgene).

[0116] In the empty vector pWPT-eGFP, the promoter of elongation factor-1α (EF-1α) regulates the expression of enhanced green fluorescent protein (eGFP), while in the recombinant expression vector encoding the target gene CAR, the ribosomal skipping sequence (F2A) from foot-and-mouth disease virus (FMDV) is used to achieve co-expression of eGFP and the target gene CAR. F2A is a core sequence of 2A (or "self-cleaving polypeptide 2A") from foot-and-mouth disease virus, which has the "self-cleavage" function of 2A and can achieve co-expression of upstream and downstream genes. 2A provides an effective and feasible strategy for constructing gene therapy multicistronic vectors due to its high cleavage efficiency, high balance of upstream and downstream gene expression, and short self-sequence. In particular, this sequence is often used to achieve co-expression of the target gene with GFP or eGFP in immunotherapy based on chimeric antigen receptor gene-modified T lymphocytes, and the expression of CAR can be indirectly detected by detecting GFP or eGFP.

[0117] In this example, a lentiviral expression vector co-expressing eGFP and specific CAR linked to F2A was constructed, collectively referred to as pWPT-eGFP-F2A-CAR ( Figure 1 ). The target gene eGFP-F2A-CAR obtained in the above step 2 (see 1 (2) in Example 3, the element after F2A is referred to as CAR) is double-digested by MluI and SalI restriction endonucleases and connected to the pWPT vector that is also double-digested, thereby constructing a lentiviral vector expressing each chimeric antigen receptor. After the successfully constructed vector is identified by MluI and SalI digestion and the sequence is determined to be correct, it can be prepared for lentiviral packaging. As mentioned above, eGFP-F2A-CAR is transcribed into an mRNA, but is ultimately translated into two peptide chains of eGFP and anti-CLD18A2 chimeric antigen receptor, in which the anti-CLD18A2 chimeric antigen receptor will be located on the cell membrane under the guidance of the CD8α signal peptide.

[0118] The obtained vectors containing the CARs of various purposes are as follows:

[0119] pWPT-eGFP-F2A-scFv(CLD18A2)-163-Z;

[0120] pWPT-eGFP-F2A-scFv(CLD18A2)-163-BBZ;

[0121] pWPT-eGFP-F2A-scFv(CLD18A2)-175-Z;

[0122] pWPT-eGFP-F2A-scFv(CLD18A2)-175-BBZ.

[0123] 4. Plasmid transfection 293T packaging lentivirus

[0124] 6×10 6 HEK-293T cells (ATCC: CRL-11268) cultured to the 6th to 10th passages were inoculated at a density of 100 μg / mL in a 10 cm culture dish and cultured overnight at 37°C, 5% CO2 for transfection. The culture medium was DMEM (purchased from PAA) containing 10% fetal bovine serum (purchased from PAA). The next day, the culture medium was replaced with serum-free DMEM about 2 hours before transfection.

[0125] The steps for transfection are as follows:

[0126] 4.1 Dissolve 20 μg of empty plasmid pWPT-eGFP (mock control) or 20 μg of each target gene plasmid pWPT-eGFP-F2A-CAR, 15 μg of packaging plasmid PAX2: and 6 μg of envelope plasmid pMD2.G in 500 μL MillQ water and mix well.

[0127] 4.2 Add 62 μL 2.5M CaCl2 (purchased from Sigma) dropwise and mix at 1200 rpm / min vortex.

[0128] 4.3 Finally, add 500 μL 2×HeBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4·2H2O, 12 mM glucose, 50 mM Hepes (purchased from Sigma), pH 7.05, 0.22 μM filter sterilized) dropwise, and mix at 1200 rpm / min vortex for 10 s.

[0129] 4.4 Immediately add dropwise to the culture dish, shake gently, culture at 37°C, 5% CO2 for 4 to 6 hours, and then replace with DMEM containing 10% fetal bovine serum.

[0130] The transfection efficiency (i.e., the proportion of cells showing green fluorescence) was observed the day after transfection, and a positive transfection efficiency of 80% was considered successful. After 48h or 72h of transfection, the virus was collected by filtration using a 0.45μm filter (purchased from Millipore), and then centrifuged at 28000rpm and 4°C for 2 hours using a Beckman Optima L-100XP ultracentrifuge. The supernatant was discarded, and the precipitate obtained by centrifugation was resuspended in 1 / 10 to 1 / 50 of the original volume of Quantum 007 culture medium (purchased from PAA), and aliquoted in 100μL / tube and frozen at -80°C for virus titration or infection of T lymphocytes.

[0131] 5. Determine the titer of lentivirus packed with mock or eGFP-F2A-CAR

[0132] On the first day, 1×10 5 / mL inoculated 293T cells in a 96-well culture plate, 100 μL / well, 37°C, 5% CO2, culture medium is DMEM containing 10% fetal bovine serum. On the second day, discard 50 μL / well culture supernatant, add 50 μL / well fresh culture medium, and add polybrene with a final concentration of 6 μg / mL, incubate at 37°C, 5% CO2 for 30 minutes. Add 10 μL / well of virus stock solution or 1 μL / well of virus concentrate, dilute 5 times, 4 gradients, two replicates, 37°C, 5% CO2 culture. After 48 hours of infection, eGFP was detected by flow cytometry, and the number of cells with a positive rate of 5-20% was appropriate. The titer (U / mL) was calculated as follows = positive rate × dilution factor × 100 × 10 4 The titers of the above viruses containing mock, i.e., empty vector control and each eGFP-F2A-CAR packaged by calcium phosphate transfection were about 0.5-2×10 6U / mL, and the virus titer after concentration was about 2×10 7 U / mL.

[0133] Example 4: Recombinant lentivirus infection of CTL cells

[0134] Human peripheral blood mononuclear cells (provided by Shanghai Blood Center) were obtained from healthy human peripheral blood by density gradient centrifugation. CTLs were obtained by negative sorting of peripheral blood mononuclear cells using CTL cell magnetic beads (purchased from Stem Cell Technologies). The purity of the sorted CTL cells was detected by flow cytometry. The next step was preferably performed when the positive rate of CTL cells was ≥95%. 6 / mL density was added with Quantum 007 lymphocyte culture medium (purchased from PAA) and stimulated with magnetic beads coated with anti-CD3 and CD28 antibodies (Invitrogen) at a cell:magnetic bead ratio of 1:1 and recombinant human IL-2 (purchased from Shanghai Huaxin Biotech Co., Ltd.) at a final concentration of 100U / mL for 24h. Then, CTL cells were infected with the above recombinant lentivirus at an MOI of ≈ 5. After infection, 5×10 cells were used every other day. 5 / mL for passage, and at the same time, recombinant human IL-2 at a final concentration of 100U / mL was added to the lymphocyte culture medium.

[0135] On the 8th day of culture, the infected CTL cells were tested for the expression of different chimeric antigen receptors by flow cytometry. Since eGFP and CAR were co-expressed, the positive cells for eGFP were the positive cells expressing chimeric antigen receptors. Using uninfected T lymphocytes as negative controls, the positive rates of CTL cells infected with viruses expressing different chimeric antigen receptors are shown in Table 3. The positive rate results show that a certain positive rate of CAR can be obtained by lentiviral infection. + CTL cells.

[0136] Table 3

[0137]

[0138]

[0139] After CTL cells were infected with viruses packaged with different chimeric antigen receptors, the cell density was 5×10 5 / ml were subcultured and counted every other day, and IL-2 (final concentration was 100U / ml) was added to the culture medium of the subcultured cells. On the 11th day of culture, there was about 20-40 times of expansion, indicating that CTL cells expressing different chimeric antigen receptors can be expanded to a certain amount in vitro, which provides a guarantee for subsequent in vitro toxicity tests and in vivo tests.

[0140] Example 5: In vitro toxicity experiment of cells expressing chimeric antigen receptors

[0141] The materials used in the in vitro toxicity experiments are as follows:

[0142] The 293T and gastric cancer cell lines shown in Table 4 were used as target cells, and the effector cells were the positive cells expressing chimeric antigen receptor detected by FACS detected by in vitro culture for 12 days as verified in Example 4, and were recorded as chimeric antigen receptor positive (CAR) + ) CTL, the effector-target ratio was 3:1, 1:1 and 1:3 depending on the situation, the number of target cells was 10000 / well, and the effector cells were corresponding to different effector-target ratios. Each group had 5 replicate wells, and the average value of the 5 replicate wells was taken. The detection time was 18 hours.

[0143] The experimental groups and control groups are as follows:

[0144] Each experimental group: each target cell + CTL expressing different chimeric antigen receptors,

[0145] Control group 1: target cells release LDH to the maximum,

[0146] Control group 2: Target cells spontaneously released LDH,

[0147] Control group 3: effector cells spontaneously released LDH.

[0148] Detection method: CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used. This method is based on colorimetry and can replace 51 Cr release method. CytoTox The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis in a manner similar to 51 Cr is released in essentially the same way in radioactive assays. The released LDH in the culture supernatant can be detected by a 30-minute coupled enzyme reaction, in which LDH converts a tetrazolium salt (INT) into red formazan. The amount of red product generated is proportional to the number of cells lysed. For details, refer to the instructions for the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit.

[0149] The cytotoxicity calculation formula is:

[0150]

[0151] As shown in Tables 4 and 5, the chimeric antigen receptor (fused with single-chain antibody 163 or 175) CLD18A2-Z CAR of the present invention+ CTL and CLD18A2-28BBZ CAR + The CTLs of the present invention have a significant killing effect on 293T cells that highly express CLD18A2, but have no killing effect on 293T cells that highly express CLD18A1, indicating that they can selectively kill cells that express CLD18A2. + CTL and CLD18A2-28BBZCAR + The CTLs can also significantly kill two gastric cancer cell lines BGC-823 and NCI-N87 that highly express CLD18A2 (see Tables 4 and 5), and show a gradient dependence of the effector-target ratio, that is, the higher the effector-target ratio, the stronger the cytotoxic effect, but have no specific cytotoxic effect on BGC-823 and NCI-N87 that do not express CLD18A2.

[0152] The data of effector-target ratio dependence further showed that CTL expressing the anti-CLD18A2 chimeric antigen receptor of the present invention had a specific cytotoxic effect on gastric cancer cells that highly expressed CLD18A2.

[0153] In comparison, the CTL transfected with a virus containing a mock plasmid (empty plasmid vector pWPT-eGFP without CLD18A2-CAR) as a blank control showed very low cytotoxicity to the above three cell lines that highly express CLD18A2. Its cytotoxicity to cell lines that highly express CLD18A2 is very significantly different from the cytotoxicity data of CTL expressing the anti-CLD18A2 chimeric antigen receptor of the present invention.

[0154] The above results show that the chimeric antigen receptor constructed by selecting a single-chain antibody against CLD18A2 can selectively kill target cells that highly express CLD18A2. In addition, from the cytotoxicity data, CLD18A2-28BBZ CAR T is more toxic to cells expressing CLD18A2 than CLD18A2-Z CAR T.

[0155] Table 4. Cytotoxicity of CAR T cells expressing single-chain antibody 163

[0156]

[0157] Table 5. Cytotoxicity of CAR T cells expressing single-chain antibody 175

[0158]

[0159]

[0160] discuss

[0161] At present, CAR T cells have become a potential treatment. However, there are no reports on CAR T cell therapy for many tumors, such as gastric cancer. Studies have shown that CLD18A2 may be a specific marker for gastric tissue, and therefore, it may also become a therapeutic target for tumors such as gastric cancer. However, the only candidate drug for CLD18A2 as a therapeutic target is monoclonal antibody, and it is still unknown whether it can be successfully used for the treatment of corresponding tumors. Therefore, it is necessary to find new treatments. Considering the tissue specificity of CLD18A2, the present invention envisions that if CAR T cells can be used for targeted therapy, it is expected that a new anticancer preparation can be obtained. However, we know that the CLD18A2 antigen is a tight junction protein, and it is still unknown whether it can be contacted by CART cells and trigger the killing of corresponding target cells. In addition, since the spatial conformation of the protein is a ripple effect, many monoclonal antibodies evolve into single-chain antibodies and often lose antigen binding activity or specificity. Fortunately, the inventors found that two single-chain antibodies (163 and 175) retain the antigen binding specificity of monoclonal antibodies. Further studies have shown that the CAR T cells composed of these two single-chain antibodies retain the selective killing effect on CLD18A2-positive cells. The results of the present invention show that CLD18A2 can indeed become a target for CAR T cell therapy; CAR T cells targeting CLD18A2 are a new candidate for tumor treatment.

[0162] 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. Sequence Listing <110> Kaixing Life Science (Shanghai) Co., Ltd. <120> T lymphocytes targeting CLD18A2 and preparation method and application thereof <130> PD01100D1 <160> twenty four <170> PatentIn version 3.3 <210> 1 <211> 727 <212> DNA <213> Artificial sequence <220> <221> misc_feature Nucleic acid sequence encoding single-chain antibody 125 of CLD18A2 <400> 1 gctagcacag gttcagctgc agcagtctgg agctgagctg gcgaggcccg gggcttcagt 60 gaagctgtcc tgcaaggctt ctggctacac cttcactgac tactatataa actgggtgaa 120 gcagaggact ggacagggcc ttgagtggat tggagagatt tatcctggaa gtggtaatac 180 ttactacaat gagaagttca agggcaaggc cacactgact gcagacaaat cctccagcac 240 agcctacatg cagctcagca gcctgacatc tgaggactct gcagtctatt tctgtgcaag 300 atcgtatggt gcctttgact actggggcca aggcaccact ctcacagtct cctcaggtgg 360 aggcggttca ggcggaggtg gctctggcgg tggcggatcg gacattgtga tgacccagtc 420 tcaaaaattc atgtccacat cagtaggaga cagggtcagc atcacctgca aggccagtca 480 gaatgttcgt actgctgtag cctggtatca acagaaacca gggcagtctc ctaaagcact 540 gatttacttg gcatccaacc ggcacactgg agtccctgat cgcttcacag gcagtggatc 600 tgggacagat ttcactctca ccattagcaa tgtgcaatct gaagacctgg cagattattt 660 ctgtctgcaa cattggaatt atcctctgac gttcggtgga ggcaccaagc tggaaatcaa 720 aggatcc 727 <210> 2 <211> 238 <212> PRT <213> Artificial Sequence <220> <221> MISC_FEATURE <223> CLD18A2 single-chain antibody 125 amino acid sequence <400> 2 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala 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 Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr 130 135 140 Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val 145 150 155 160 Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 165 170 175 Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg 180 185 190 Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn 195 200 205 Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn 210 215 220 Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 225 230 235 <210> 3 <211> 751 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Nucleic acid sequence encoding single-chain antibody 165 of CLD18A2 <400> 3 gctagcacag atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc tgcaaggctt ctgggtatac cttcacaaac tatggaatga actgggtgaa gcaggctcca ggaaagggtt taaagtggat gggctggata aacaccaaca ctggagagcc aacctatgct gaagagttca agggacggtt tgccttctct ttggaaacct ctgccagcac 240 300. tgcctatttg cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaag actgggtttt ggtaatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc 360 aggtggaggc ggttcaggcg gaggtggctc tggcggtggc ggatcggaca ttgtgatgac 420 acagtctcca tcctccctga ctgtgacagc aggagagag gtcactatga gctgcaagtc 480 cagtcagagt ctgttaaca gtggaaatca aaagaactac ttgacctggt accagcagaa accagggcag cctcctaaac tgttgatcta ctggggcatcc actagggaat ctggggtccc tgatcgcttc acaggcagtg gatctggac agatttcact ctcaccatca gcagtgtgca ggctgaagac ctggcagttt attactgtca gaatgattat agttatccgc tcacgttcgg 720. tgctgggacc aagctggagc tgaaagaatt c 751 <210> 4 <211> 246 <212> PRT <213> Artificial Sequence <220> <221> MISC_FEATURE <223> Single-chain antibody 163 amino acid sequence of CLD18A2 <400> 4 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys 245 <210> 5 <211> 751 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> Nucleic acid sequence encoding CLD18A2 single-chain antibody 175 <400> 5 gctagcacag gtccaactgc agcagcctgg ggctgagctg gtgaggcctg gggcttcagt gagctgtcc tgcaaggctt ctggctacac cttcaccagc tactggataa actgggtgaa gcagaggcct ggacaaggcc ttgagtggat cggaaatatt tatccttctg atagttatac 240. taactcaat caaaagttca aggacaaggc cacattgact gtagacaat cctccagcac agcctacatg cagctcagca gcccgacatc tgaggactct gcggtctatt actgtacaag atcgtggagg ggtaactcct ttgactactg gggccaaggc accactctca cagtctcctc 360 aggtggaggc ggttcaggcg gaggtggctc tggcggtggc ggatcggaca ttgtgatgac 420 acagtctcca tcctccctga ctgtgacagc aggagagag gtcactatga gctgcaagtc 480 cagtcagagt ctgttaaca gtggaaatca aaagaactac ttgacctggt accagcagaa accagggcag cctcctaaac tgttgatcta ctggggcatcc actagggaat ctggggtccc tgatcgcttc acaggcagtg gatctggac agatttcact ctcaccatca gcagtgtgca ggctgaagac ctggcagttt attactgtca gaatgattat agttatccat tcacgttcgg 720 ctcggggaca aagttggaaa taaaagaatt c 751 <210> 6 <211> 246 <212> PRT <213> Artificial Sequence <220> <221> MISC_FEATURE <223> CLD18A2 single-chain antibody 175 amino acid sequence <400> 6 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg 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 Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys 245 <210> 7 <211> 42 <212> DNA <213> primer <400> 7 tgctccacgc cgccaggccg cagatccagt tggtgcagtc tg 42 <210> 8 <211> 42 <212> DNA <213> Primer <400> 8 cgcggcgctg gcgtcgtggt tttcagctcc agcttggtcc ca 42 <210> 9 <211> 40 <212> DNA <213> Primer <400> 9 tgctccacgc cgccaggccg caggtccaac tgcagcagcc 40 <210> 10 <211> 43 <212> DNA <213> Primer <400> 10 cgcggcgctg gcgtcgtggt ttttatttcc aactttgtcc ccg 43 <210> 11 <211> 22 <212> DNA <213> Primer <400> 11 gcaggggaaa gaatagtaga ca 22 <210> 12 <211> 20 <212> DNA <213> Primer <400> 12 cggcctggcg gcgtggagca 20 <210> 13 <211> 20 <212> DNA <213> Primer <400> 13 accacgacgc cagcgccgcg 20 <210> 14 <211> 22 <212> DNA <213> Primer <400> 14 tagcgtaaaa ggagcaacat ag 22 <210> 15 <211> 2183 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Nucleic acid sequence encoding chimeric antigen receptor protein CLD18A2-163-Z <400> 15 aagcttacgc gtcctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt 60 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 120 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 180 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 240 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 300 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 360 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 420 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 480 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 540 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 600 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 660 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 720 gatcactctc ggcatggacg agctgtacaa gtccggagtg aaacagactt tgaattttga 780 ccttctgaag ttggcaggag acgttgagtc caaccctggg cccatggcct taccagtgac 840 cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccgcaga tccagttggt 900 gcagtctgga cctgagctga agaagcctgg agagacagtc aagatctcct gcaaggcttc 960 tgggtatacc ttcacaaact atggaatgaa ctgggtgaag caggctccag gaaagggttt 1020 aaagtggatg ggctggataa acaccaacac tggagagcca acctatgctg aagagttcaa 1080 gggacggttt gccttctctt tggaaacctc tgccagcact gcctatttgc agatcaacaa 1140 cctcaaaaat gaggacacgg ctacatattt ctgtgcaaga ctgggttttg gtaatgctat 1200 ggactactgg ggtcaaggaa cctcagtcac cgtctcctca ggtggaggcg gttcaggcgg 1260 aggtggctct ggcggtggcg gatcggacat tgtgatgaca cagtctccat cctccctgac 1320 1380 1440 gttgatctac tgggcatcca ctagggaatc tggggtccct gatcgcttca caggcagtgg 1500 atctggaaca gatttcactc tcaccatcag cagtgtgcag gctgaagacc tggcagttta 1560 ttactgtcag aatgattata gttatccgct cacgttcggt gctgggacca agctggagct 1620 gaaaaccacg acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc 1680 cctgtccctg cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg 1740 gctggacttc gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct 1800 tctcctgtca ctggttatca ccagagtgaa gttcagcagg agcgcagacg cccccgcgta 1860 ccagcagggc cagaaccagc tctataacga gctcaatcta ggacgaag aggagtacga 1920 tgttttggac aagacgtg gccgggaccc tgagatgggg ggaaagccgc agaaaggaa 1980 gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag 2040 tgagattggg atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg 2100 tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg 2160 ctaggtcgac ctcgagggaa ttc 2183 <210> 16 <211> 2450 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> Nucleic acid sequence encoding chimeric antigen receptor protein CLD18A2-163-28BBZ <400> 16 aagcttacgc gtcctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt 60 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 120 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 180 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 240 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 300 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 360 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 420 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 480 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 540 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 600 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 660 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 720 gatcactctc ggcatggacg agctgtacaa gtccggagtg aaacagactt tgaattttga 780 ccttctgaag ttggcaggag acgttgagtc caaccctggg cccatggcct taccagtgac 840 cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccgcaga tccagttggt 900 gcagtctgga cctgagctga agaagcctgg agagacagtc aagatctcct gcaaggcttc 960 tgggtatacc ttcacaaact atggaatgaa ctgggtgaag caggctccag gaaagggttt 1020 aaagtggatg ggctggataa acaccaacac tggagagcca acctatgctg aagagttcaa 1080 gggacggttt gccttctctt tggaaacctc tgccagcact gcctatttgc agatcaacaa 1140 cctcaaaaat gaggacacgg ctacatattt ctgtgcaaga ctgggttttg gtaatgctat 1200 ggactactgg ggtcaaggaa cctcagtcac cgtctcctca ggtggaggcg gttcaggcgg 1260 aggtggctct ggcggtggcg gatcggacat tgtgatgaca cagtctccat cctccctgac 1320 tgtgacagca ggagagaagg tcactatgag ctgcaagtcc agtcagagtc tgttaaacag 1380 tggaaatcaa aagaactact tgacctggta ccagcagaaa ccagggcagc ctcctaaact 1440 gttgatctac tgggcatcca ctagggaatc tggggtccct gatcgcttca caggcagtgg 1500 atctggaaca gatttcactc tcaccatcag cagtgtgcag gctgaagacc tggcagttta 1560 ttactgtcag aatgattata gttatccgct cacgttcggt gctgggacca agctggagct 1620 gaaaaccacg acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc 1680 cctgtccctg cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg 1740 gctggacttc gcctgtgatt tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta 1800 tagcttgcta gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct 1860 cctgcacagt gactacatga acatgactcc ccgccgcccc gggccaaccc gcaagcatta 1920 ccagccctat gccccaccac gcgacttcgc agcctatcgc tccaaacggg gcagaaagaa 1980 actcctgtat atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga 2040 tggctgtagc tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt 2100 cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct 2160 caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga 2220 gatgggggga aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca 2280 gaaagataag atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg 2340 caaggggcac gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc 2400 ccttcacatg caggccctgc cccctcgcta ggtcgacctc gagggaattc 2450 <210> 17 <211> 2183 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Nucleic acid sequence encoding chimeric antigen receptor protein CLD18A2-175-Z <400> 17 aagcttacgc gtcctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt 60 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 120 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 180 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 240 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 300 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 360 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 420 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 480 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 540 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 600 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 660 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 720 gatcactctc ggcatggacg agctgtacaa gtccggagtg aaacagactt tgaattttga 780 ccttctgaag ttggcaggag acgttgagtc caaccctggg cccatggcct taccagtgac 840 cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccgcagg tccaactgca 900 gcagcctggg gctgagctgg tgaggcctgg ggcttcagtg aagctgtcct gcaaggcttc 960 tggctacacc ttcaccagct actggataaa ctgggtgaag cagaggcctg gacaaggcct 1020 tgagtggatc ggaaatattt atccttctga tagttatact aactacaatc aaaagttcaa 1080 ggacaaggcc acattgactg tagacaaatc ctccagcaca gcctacatgc agctcagcag 1140 cccgacatct gaggactctg cggtctatta ctgtacaaga tcgtggaggg gtaactcctt 1200 tgactactgg ggccaaggca ccactctcac agtctcctca ggtggaggcg gttcaggcgg 1260 aggtggctct ggcggtggcg gatcggacat tgtgatgaca cagtctccat cctccctgac 1320 tgtgacagca ggagagaagg tcactatgag ctgcaagtcc agtcagagtc tgttaaacag 1380 1440 gttgatctac tgggcatcca ctagggaatc tggggtccct gatcgcttca caggcagtgg 1500 atctggaaca gatttcactc tcaccatcag cagtgtgcag gctgaagacc tggcagttta 1560 ttactgtcag aatgattata gttatccatt cacgttcggc tcggggcaa agttggagaat 1620 aaaaaccacg acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc 1680 cctgtccctg cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg 1740 gctggacttc gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct 1800 tctcctgtca ctggttatca ccagagtgaa gttcagcagg agcgcagacg cccccgcgta 1860 ccagcagggc cagaaccagc tctataacga gctcaatcta ggacgaag aggagtacga 1920 tgttttggac aagacgtg gccgggaccc tgagatgggg ggaaagccgc agaaaggaa 1980 gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag 2040 tgagattggg atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg 2100 tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg 2160 ctaggtcgac ctcgagggaa ttc 2183 <210> 18 <211> 2450 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> Nucleic acid sequence encoding chimeric antigen receptor protein CLD18A2-175-28BBZ <400> 18 aagcttacgc gtcctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt 60 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 120 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 180 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 240 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 300 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 360 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 420 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 480 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 540 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 600 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 660 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 720 gatcactctc ggcatggacg agctgtacaa gtccggagtg aaacagactt tgaattttga 780 ccttctgaag ttggcaggag acgttgagtc caaccctggg cccatggcct taccagtgac 840 cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccgcagg tccaactgca 900 gcagcctggg gctgagctgg tgaggcctgg ggcttcagtg aagctgtcct gcaaggcttc 960 tggctacacc ttcaccagct actggataaa ctgggtgaag cagaggcctg gacaaggcct 1020 tgagtggatc ggaaatattt atccttctga tagttatact aactacaatc aaaagttcaa 1080 ggacaaggcc acattgactg tagacaaatc ctccagcaca gcctacatgc agctcagcag 1140 cccgacatct gaggactctg cggtctatta ctgtacaaga tcgtggaggg gtaactcctt 1200 tgactactgg ggccaaggca ccactctcac agtctcctca ggtggaggcg gttcaggcgg 1260 aggtggctct ggcggtggcg gatcggacat tgtgatgaca cagtctccat cctccctgac 1320 tgtgacagca ggagagaagg tcactatgag ctgcaagtcc agtcagagtc tgttaaacag 1380 tggaaatcaa aagaactact tgacctggta ccagcagaaa ccagggcagc ctcctaaact 1440 gttgatctac tgggcatcca ctagggaatc tggggtccct gatcgcttca caggcagtgg 1500 atctggaaca gatttcactc tcaccatcag cagtgtgcag gctgaagacc tggcagttta 1560 ttactgtcag aatgattata gttatccatt cacgttcggc tcggggacaa agttggaaat 1620 aaaaaccacg acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc 1680 cctgtccctg cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg 1740 gctggacttc gcctgtgatt tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta 1800 tagcttgcta gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct 1860 cctgcacagt gactacatga acatgactcc ccgccgcccc gggccaaccc gcaagcatta 1920 ccagccctat gccccaccac gcgacttcgc agcctatcgc tccaaacggg gcagaaagaa 1980 actcctgtat atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga 2040 tggctgtagc tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt 2100 cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct 2160 caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga 2220 gatgggggga aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca 2280 gaaagataag atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg 2340 caaggggcac gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc 2400 ccttcacatg caggccctgc cccctcgcta ggtcgacctc gagggaattc 2450 <210> 19 <211> 709 <212> PRT <213> Artificial Sequence <220> <221> MISC_FEATURE <223> Amino acid sequence of chimeric antigen receptor protein CLD18A2 - 163 - Z <400> 19 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser 225 230 235 240 Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255 Val Glu Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265 270 Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gln Ile Gln Leu 275 280 285 Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile 290 295 300 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp 305 310 315 320 Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn 325 330 335 Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg Phe 340 345 350 Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn 355 360 365 Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu Gly 370 375 380 Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 385 390 395 400 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 405 410 415 Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala 420 425 430 Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn 435 440 445 Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly 450 455 460 Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 465 470 475 480 Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 485 490 495 Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln 500 505 510 Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu 515 520 525 Leu Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 530 535 540 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 545 550 555 560 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 565 570 575 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 580 585 590 Leu Val Ile Thr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 595 600 605 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 610 615 620 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 625 630 635 640 Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 645 650 655 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 660 665 670 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 675 680 685 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 690 695 700 Ala Leu Pro Pro Arg 705 <210> 20 <211> 798 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <223> Amino acid sequence of chimeric antigen receptor protein CLD18A2-163-28BBZ <400> 20 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser 225 230 235 240 Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255 Val Glu Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265 270 Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gln Ile Gln Leu 275 280 285 Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile 290 295 300 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp 305 310 315 320 Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn 325 330 335 Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg Phe 340 345 350 Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn 355 360 365 Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu Gly 370 375 380 Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 385 390 395 400 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 405 410 415 Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala 420 425 430 Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn 435 440 445 Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly 450 455 460 Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 465 470 475 480 Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 485 490 495 Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln 500 505 510 Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu 515 520 525 Leu Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 530 535 540 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 545 550 555 560 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe 565 570 575 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 580 585 590 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 595 600 605 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 610 615 620 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 625 630 635 640 Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 645 650 655 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 660 665 670 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 675 680 685 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 690 695 700 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 705 710 715 720 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg 725 730 735 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 740 745 750 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 755 760 765 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 770 775 780 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 785 790 795 <210> twenty one <211> 709 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <223> Amino acid sequence of chimeric antigen receptor protein CLD18A2-175-Z <400> twenty one Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser 225 230 235 240 Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255 Val Glu Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265 270 Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gln Val Gln Leu 275 280 285 Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu 290 295 300 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn Trp 305 310 315 320 Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn Ile Tyr 325 330 335 Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala 340 345 350 Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser 355 360 365 Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser Trp 370 375 380 Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 385 390 395 400 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 405 410 415 Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala 420 425 430 Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn 435 440 445 Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly 450 455 460 Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 465 470 475 480 Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 485 490 495 Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln 500 505 510 Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu 515 520 525 Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 530 535 540 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 545 550 555 560 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 565 570 575 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 580 585 590 Leu Val Ile Thr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 595 600 605 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 610 615 620 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 625 630 635 640 Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 645 650 655 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 660 665 670 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 675 680 685 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 690 695 700 Ala Leu Pro Pro Arg 705 <210> twenty two <211> 798 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <223> Amino acid sequence of chimeric antigen receptor protein CLD18A2-175-28BBZ <400> twenty two Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser 225 230 235 240 Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255 Val Glu Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265 270 Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gln Val Gln Leu 275 280 285 Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu 290 295 300 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn Trp 305 310 315 320 Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn Ile Tyr 325 330 335 Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala 340 345 350 Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser 355 360 365 Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser Trp 370 375 380 Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 385 390 395 400 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 405 410 415 Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala 420 425 430 Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn 435 440 445 Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly 450 455 460 Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 465 470 475 480 Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 485 490 495 Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln 500 505 510 Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu 515 520 525 Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 530 535 540 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 545 550 555 560 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe 565 570 575 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 580 585 590 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 595 600 605 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 610 615 620 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 625 630 635 640 Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 645 650 655 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 660 665 670 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 675 680 685 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 690 695 700 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 705 710 715 720 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg 725 730 735 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 740 745 750 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 755 760 765 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 770 775 780 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 785 790 795 <210> 23 <211> 1266 <212> DNA <213> Artificial sequence <220> <221> misc_feature <223> scFv(GPC3)-CD8-CD3ζ nucleotide sequence <400> 23 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca gatctagtca gagccttgta cacagtaatg ccaacaccta tttacattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc caaccgattt 180 tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgct ctcaaaatac acatgttcct 300 cctacgtttg gccaggggac caagctggag atcaaacgtg gtggaggcgg ttcaggcgga 360 ggtggctctg gcggtggcgg atcgcaggtg cagctggtgc agtctggagc tgaggtgaag 420 aagcctgggg cctcagtgaa ggtctcctgc aaggcttctg gatacacctt caccgactat 480 gaaatgcact gggtgcgaca ggcccctgga caagggcttg agtggatggg agctcttgat 540 cctaaaactg gtgatactgc ctacagtcag aagttcaagg gcagagtcac gctgaccgcg 600 gacgaatcca cgagcacagc ctacatggag ctgagcagcc tgagatctga ggacacggcc 660 gtgtattact gtacaagatt ctactcctat acttactggg gccagggaac cctggtcacc 720 gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 780 cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840 agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900 gtccttctcc tgtcactggt tatcaccaga gtgaagttca gcaggagcgc agacgccccc 960 gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1020 tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgcagaga 1080 aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1140 tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1200 cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1260 cctcgc 1266 <210> 24 <211> 1533 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Nucleotide sequence of scFv(GPC3)-CD28a-CD28b-CD137-CD3ζ <400> 24 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca gatctagtca gagccttgta cacagtaatg ccaacaccta tttacattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc caaccgattt 180 tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgct ctcaaaatac acatgttcct 300 cctacgtttg gccaggggac caagctggag atcaaacgtg gtggaggcgg ttcaggcgga 360 ggtggctctg gcggtggcgg atcgcaggtg cagctggtgc agtctggagc tgaggtgaag 420 aagcctgggg cctcagtgaa ggtctcctgc aaggcttctg gatacacctt caccgactat 480 gaaatgcact gggtgcgaca ggcccctgga caagggcttg agtggatggg agctcttgat 540 cctaaaactg gtgatactgc ctacagtcag aagttcaagg gcagagtcac gctgaccgcg 600 gacgaatcca cgagcacagc ctacatggag ctgagcagcc tgagatctga ggacacggcc 660 gtgtattact gtacaagatt ctactcctat acttactggg gccagggaac cctggtcacc 720 gtctcctcaa ccacgacgcc agcgccgcga ccaccacac cggcgcccac catcgcgtcg 780 cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840 agggggctgg acttcgcctg tgatttttgg gtgctggtgg tggttggtgg agtcctggct 900 tgctatagct tgctagtaac agtggccttt attattct gggtgaggag taagaggagc 960 aggctcctgc acagtgacta catgaacatg acccccgcc gccccggcc aacccgcaag 1020 cattaccagc cctagcccc accacgcgac ttcgcagcct atcgctcca acggggcaga 1080 aagaaactcc tgtatattat caacaacca tttatgagac cagtacaac tactcagag 1140 gaagatggct gtagctgccg atttccagaa gagagaag gaggatgtga actgagagtg 1200 aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1260 gagctcaatc taggacgaag agaggagtac gatgttttgg acagagacg tggccgggac 1320 cctgagatgg ggggaaagcc gcagagaagg aagaaccctc aggaggcct gtacaatgaa 1380 ctgcagaaag atagatggc ggaggcctac agtgagattg ggatgaagg cgagcgccgg 1440 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1500 gacgcccttc acatgcaggc cctgccccct cgc 1533

Claims

1. A chimeric antigen receptor expressed on the surface of a T lymphocyte, characterized in that: The chimeric antigen receptor comprises: an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises a protein that specifically recognizes CLD18A2, the protein that specifically recognizes CLD18A2 is a single-chain antibody, and the chimeric antigen receptor targets CLD18A2-positive tumors, wherein the single-chain antibody is encoded by the nucleic acid shown in SEQ ID NO:

5.

2. The chimeric antigen receptor according to claim 1, wherein The single-chain antibody specifically recognizes CLD18A2 but not CLD18A1.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that The transmembrane region is a sequence comprising the transmembrane region and hinge region of CD8 or CD28.

4. The chimeric antigen receptor according to claim 1 or 2, characterized in that The intracellular signal region is selected from the group consisting of: intracellular signal region sequences of CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, or a combination thereof.

5. The chimeric antigen receptor according to claim 1 or 2, characterized in that The chimeric antigen receptor comprises the following sequentially connected extracellular binding region, transmembrane region and intracellular signaling region: A single-chain antibody that specifically recognizes CLD18A2, CD8, and CD3ζ; A single-chain antibody that specifically recognizes CLD18A2, CD8, CD137, and CD3ζ; A single chain antibody that specifically recognizes CLD18A2, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, and CD3ζ; or A single-chain antibody that specifically recognizes CLD18A2, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, CD137 and CD3ζ.

6. An expression vector, characterized in that: It comprises a nucleic acid with a nucleotide sequence as described in any one of SEQ ID NOs: 17 to 18, the expression vector is derived from a lentiviral plasmid pWPT, and the expression vector is a lentiviral expression vector that co-expresses eGFP connected to F2A and a specific CAR.

7. A genetically modified T lymphocyte, characterized in that: The expression vector described in claim 6 is transduced therein.

8. A genetically modified T lymphocyte, characterized in that: A chimeric antigen receptor is expressed on its surface, and the chimeric antigen receptor comprises: an extracellular binding region, a transmembrane region and an intracellular signal region connected in sequence, wherein the extracellular binding region comprises a protein that specifically recognizes CLD18A2, and the chimeric antigen receptor targets CLD18A2-positive tumors, wherein the protein that specifically recognizes CLD18A2 is a single-chain antibody, the sequence of the single-chain antibody is shown in SEQ ID NO: 6 or the single-chain antibody is encoded by the nucleic acid shown in SEQ ID NO:

5.

9. The T lymphocyte according to claim 8, characterized in that The single-chain antibody specifically recognizes CLD18A2 but not CLD18A1.

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