T lymphocyte targeting CLD18A2 as well as preparation method and application of T lymphocyte

Abstract

The invention relates to a CLD18A2-targeted T lymphocyte as well as a preparation method and application thereof. The inventor successfully finds a target gene CLD18A2 suitable for developing CAR T cells from a plurality of tumor related genes for the first time, and successfully prepares CLD18A2-targeted CAR T cells, so that a brand new treatment means is provided for pancreatic cancer, gastric cancer and other tumors.

Description

[0001] This application is a further divisional application of the divisional application with the filing date of July 17, 2014, the divisional filing date of April 12, 2021, the application number of 202110389401.0, and the invention title of "T lymphocytes targeting CLD18A2, their preparation method and application"; among them, the divisional application with the application number of 202110389401.0 is a divisional application of the parent application with the application number of 201410341504.X, the filing date of July 17, 2014, and the invention title of "T lymphocytes targeting CLD18A2, their preparation method and application". Technical Field

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

[0003] The role of T lymphocytes in tumor immune response has been increasingly emphasized. Adoptive immunotherapy based on T lymphocytes has achieved certain effects in some tumors, and this immunotherapy method can overcome the above-mentioned defects of antibody therapy, but the efficacy in most tumors is still not satisfactory [Grupp SA, et al. Adoptive cellular therapy. Curr Top Microbiol Immunol., 2011; 344: 149-72.]. In recent years, according to the discovery that the recognition specificity of CTL for target cells depends on the T cell receptor (TCR), the scFv of the antibody against tumor cell-related antigens is fused with the intracellular signal activation motifs such as CD3ζ or FcεRIγ of the T cell receptor to form a chimeric antigen receptor (CAR), and it is gene-modified on the surface of T lymphocytes by means such as lentiviral infection. Such CAR T lymphocytes can selectively direct T lymphocytes to tumor cells and specifically kill tumors in a major histocompatibility complex (MHC)-unrestricted manner. 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. Generally, the extracellular region contains a scFv that can recognize tumor-associated antigens, the transmembrane region uses the transmembrane regions of molecules such as CD8 and CD28, and the intracellular signaling region uses the intracellular signaling regions of immunoreceptor tyrosine-based activation motifs (ITAM) CD3ζ or FcεRIγ and costimulatory signaling molecules such as CD28, CD27, CD137, and CD134.

[0005] The first-generation CAR T lymphocytes with only ITAM in the intracellular signaling region have the chimeric antigen receptor parts connected in the following form: scFv-TM-ITAM. This type of CAR T can stimulate anti-tumor cytotoxic effects, but has relatively low cytokine secretion and cannot stimulate a persistent 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] Subsequently developed second-generation CAR T lymphocytes added the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), and the chimeric antigen receptor parts are connected in the following form: scFv-TM-CD28-ITAM or scFv-TM- / CD137-ITAM. The B7 / CD28 or 4-1BBL / CD137 costimulation occurring in the intracellular signaling region causes the continuous proliferation of T lymphocytes, can increase the levels of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, and at the same time increase the survival period 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 CAR T lymphocytes developed in recent years have the chimeric antigen receptor parts connected in the following forms: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, which further improves the survival period of CAR T in vivo and its anti-tumor effect [Carpenito C., et al. Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. PNAS, 2009, 106(9): 3360–3365.].

[0008] Although CAR T lymphocytes have promising prospects in cancer immunotherapy, their relatively high risks also need to be considered. For example, the low expression of specific antigens recognized by CAR in certain normal tissues may cause damage to normal tissues expressing the corresponding antigens by CAR T lymphocytes. For instance, the antigen carbonic anhydrase IX (CAIX) expressed on the tumor cells of renal cell carcinoma patients was the first case used for the adoptive therapy of CAR T lymphocytes in clinical practice and also the first case reporting the off-target effect of CAR-containing cells. After multiple infusions of CAR T lymphocytes, the patient developed grade 2-4 hepatotoxicity. The analysis showed that cholangiocytes in the liver had low expression of CAIX, and the original clinical trial was forced to be interrupted and any evaluation of the patient's treatment effect was excluded [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 lower the threshold required for effector cell activation, enabling gene-modified T lymphocytes to be activated under conditions of low-level antigens or without antigen triggering, leading to the release of a large amount of cytokines and potentially triggering the so-called "cytokine storm". This signal leakage will cause off-target cytotoxicity, resulting in non-specific 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 tissues, resulting in the sudden death of the patient [Morgan RA., et al. Report of a serious 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 to be targeted is a crucial choice. Given the complexity of in vivo gene expression and various uncontrollable factors, it is very difficult to select a suitable gene for CAR-T. Moreover, for many tumor-specific antigens, it is difficult to find specific molecules that target them and are suitable for constructing CAR-T cells. After establishing CAR-T, it is often impossible to obtain an active extracellular binding region, which is also a difficulty in the development of CAR-T technology. Summary of the Invention

[0010] The object of the present invention is to provide T lymphocytes targeting CLD18A2, their preparation methods and applications.

[0011] In the first aspect of the present invention, a chimeric antigen receptor (CAR) expressed on the surface of T lymphocytes is provided. The chimeric antigen receptor comprises, in sequential connection: an extracellular binding region, a transmembrane region and an intracellular signaling region, 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 signaling region is selected from: the intracellular signaling region sequences of CD3ζ, 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 CD3ζ;

[0017] A single-chain antibody that specifically recognizes CLD18A2, CD8, CD137 and CD3ζ;

[0018] A single-chain antibody that specifically recognizes CLD18A2, the transmembrane region of CD28 molecule (CD28a), the intracellular signaling region of CD28 molecule (CD28b) and CD3ζ; or

[0019] A single-chain antibody that specifically recognizes CLD18A2, the transmembrane region of CD28 molecule, the intracellular signaling region of CD28 molecule, CD137 and CD3ζ.

[0020] In another preferred example, the chimeric antigen receptor has any one of the amino acid sequences set forth in SEQ ID NOs: 19-22.

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

[0022] In a preferred example, the nucleic acid has any one of the nucleotide sequences set forth in SEQ ID NOs: 15-18.

[0023] In another aspect of the present invention, there is provided an expression vector comprising 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, there is provided a virus, such as a lentivirus, comprising the vector.

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

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

[0028] In another aspect of the present invention, there is provided a gene-modified T lymphocyte that expresses on its surface a chimeric antigen receptor, the amino acid sequence of which is selected from any one of the amino acid sequences set forth in SEQ ID NOs: 19-22.

[0029] In another aspect of the present invention, there is provided the use of the gene-modified T lymphocyte for the preparation of a medicament for inhibiting a tumor, which is a CLD18A2-positive (high-expression) tumor.

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

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

[0032] Figure 1 Structural schematic diagram of the lentiviral vector pWPT-eGFP-F2A-CAR of the present invention containing the CAR-encoding sequence by way of example.

[0033] Figure 2, Schematic diagram of the connection order of each part of the chimeric antigen receptor.

[0034] Figure 3 , SDS-PAGE of purified single-chain antibody against CLD18A2 in Example 1.

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

[0036] Figure 5 , Results of detection of binding specificity of CLD18A2 single-chain antibody with CLD18A1 and CLD18A2 by flow cytometry.

[0037] Figure 6 , SDS-PAGE identification of the assembled chimeric antigen receptor. Detailed implementation manners

[0038] The present inventors have, through extensive and in-depth research, for the first time revealed a CAR T lymphocyte based on the CLD18A2 gene and its preparation method.

[0039] CLD18A2 gene

[0040] The present inventors previously investigated many tumor-specific genes and found that a quite large proportion of such 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, however, the CAR T cells designed based on them have no tumor cell killing activity or very low activity, which may be due to reasons such as low protein antigenicity of the corresponding gene expression, unreasonable expression position, insufficient expression level, etc., or may be due to the fact that the T lymphocytes obtained after recombinant construction are affected and the tumor killing ability is weakened or lost.

[0041] After repeated investigation and screening, the present 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 of epithelia and endothelia.

[0043] Studies have shown that CLD18A1 is selectively expressed in normal lung and gastric epithelium, while the expression of CLD18A2 is limited to the short-lived differentiated cells of gastric epithelium and is not expressed in gastric stem cells; moreover, existing studies have shown that CLD18A2 is expressed in many tumor cells. In view of the above characteristics of CLD18A2, the inventors of the present invention speculated that CLD18A2 might be an important therapeutic target for these tumors; and through a large amount of subsequent work verification, this speculation was confirmed.

[0044] Chimeric Antigen Receptor and Its Encoding Nucleic Acid

[0045] The present invention provides a chimeric antigen receptor expressed on the surface of T lymphocytes, which comprises, sequentially connected: an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes CLD18A2 (claudin 18.2). Expressing this chimeric antigen receptor on the surface of T lymphocytes can enable T lymphocytes to have a highly specific cytotoxic effect on tumor cells highly expressing 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 above chimeric antigen receptor protein is connected to the transmembrane region of CD8 or CD28 through the CD8 hinge region, and the intracellular signaling region is immediately adjacent to the transmembrane region.

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

[0048] Monoclonal antibodies that specifically recognize human CLD18A2 can be selected from the antibodies disclosed in the prior art. A variety of monoclonal antibodies that recognize the C-terminal epitope of CLD18A2 can be applied to 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 applied. Preferably, the single-chain antibodies are antibody 163 and antibody 175; the two antibodies, 163 and 175, can specifically recognize CLD18A2 but not CLD18A1. More preferably, they are linked 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 containing a heavy-chain variable region (VH) and a light-chain variable region (VL) linked by a linker, and the linker associates these two domains to finally form an antigen-binding site. The size of scFv is generally 1 / 6 of a complete antibody. The single-chain antibody is preferably an amino acid sequence of a single amino acid chain encoded by a single nucleotide chain. The single-chain antibodies used in the present invention can be further modified alone or in combination with conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination and / or other modification methods. The method of introducing such modifications into the DNA sequence according to the amino acid sequence of an antibody is well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.. The modifications referred to are preferably carried out at the nucleic acid level. The above single-chain antibodies may also include their derivatives. "Derivatives of antibodies" in the present invention include, for example, when obtaining derivatives of the antibodies by phage display technology, surface plasmon resonance technology such as that used in the BIAcore system can be used to increase the efficiency of phage antibodies binding to the CLD18A2 antigen epitope (Schier, Human Antibody Hybridomas 7 (1996), 97-105; Malmborg, Journal of Immunological Methods 183 (1995), 7-13). It also includes, for example, the method for generating chimeric antibodies described in WO 89 / 09622, the method for generating humanized antibodies described in EP-A10239400 and WO90 / 07861, and the derivatives of antibodies generated by the methods for generating xenogeneic antibodies such as human antibodies in mice described in WO91 / 10741, WO94 / 02602 and WO96 / 33735.

[0050] The term "specifically recognize" in the present invention means that the bispecific antibody of the present invention does not cross-react with or substantially does not cross-react with any polypeptide other than the target antigen. The degree of its specificity can be judged by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry, etc. In the present invention, specific recognition is preferably determined by flow cytometry, and the specific recognition criteria in specific cases can be judged by those of ordinary skill in the art according to the common knowledge in the art they have mastered.

[0051] The transmembrane region of the chimeric antigen receptor can be selected from the transmembrane regions of proteins such as CD8 or CD28. CD8 or CD28 are natural markers on the surface of T lymphocytes. Human CD8 protein is a heterodimer composed of two chains, 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. Therefore, 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 and the intracellular signaling region.

[0052] The intracellular signaling region can be selected from the intracellular signaling regions of CD3ζ, FcεRIγ, CD28, CD137, CD134 proteins, and combinations thereof. The CD3 molecule is composed of five subunits, and the CD3ζ subunit (also known as CD3 zeta, abbreviated as Z) contains 3 ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. CD3δZ is a truncated CD3ζ sequence without ITAM motifs and is generally used as a negative control construct in the practice of the present invention. FcεRIγ is mainly distributed on the surface of mast cells and basophils and contains one ITAM motif, which is similar to CD3ζ in structure, distribution, and function. In addition, as mentioned above, CD28, CD137, and CD134 are co-stimulatory signal molecules, and the co-stimulatory effects generated by their intracellular signal segments after binding to their respective ligands cause continuous proliferation of T lymphocytes and can increase the levels of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, while increasing the survival period and anti-tumor effect of CAR T lymphocytes in vivo.

[0053] The anti-CLD18A2 chimeric antigen receptor protein encoded by the nucleic acid of the present invention can be selected and sequentially connected in the following manner:

[0054] scFv(CLD18A2)-CD8-CD3ζ,

[0055] scFv(CLD18A2)-CD8-CD137-CD3ζ,

[0056] scFv(CLD18A2)-CD28a-CD28b-CD3ζ,

[0057] scFv(CLD18A2)-CD28a-CD28b-CD137-CD3ζ,

[0058] and combinations thereof, wherein in the relevant chimeric antigen receptor protein, CD28a represents the transmembrane region of the CD28 molecule, and CD28b represents the intracellular signaling region of the CD28 molecule. The above 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 the sequences described 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 the sequences as set forth in SEQ ID NOs: 19-22.

[0060] Expression vectors and cells

[0061] The present invention also provides a vector comprising the above nucleic acid encoding a 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. This plasmid belongs to the third-generation self-inactivating lentiviral vector system, which has a total of 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 for recombinantly introducing a target nucleic acid sequence, that is, the nucleic acid sequence encoding CAR. In the empty vector pWPT-eGFP (which itself is the mock in subsequent experiments), the expression of enhanced green fluorescent protein (eGFP) is regulated by the elongation factor-1α (EF-1α) promoter. The recombinant expression vector pWPT-eGFP-F2A-CAR containing the target nucleic acid sequence encoding CAR achieves the co-expression of eGFP and CAR through the ribosomal skipping sequence 2A (abbreviated as F2A) from the foot-and-mouth disease virus (FMDV).

[0062] The present invention also includes a virus comprising the above vector. The virus of the present invention includes the packaged infectious virus, and also includes the virus to be packaged containing the necessary components for packaging into an infectious virus. Other viruses known in the art that can be used to transfer 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 containing the above-mentioned pWPT-eGFP-F2A-CAR recombinant vector (i.e., containing scFv(CLD18A2)-CAR).

[0064] The present invention also provides gene-modified T lymphocytes that are transduced with the nucleic acid of the present invention or are 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-based transduction methods include electroporation and transposon methods. The recently developed Nucleofector by Amaxa can directly introduce foreign genes into the cell nucleus to obtain efficient transduction of the target gene. In addition, the transduction efficiency of transposon systems such as the Sleeping Beauty system or PiggyBac transposon is much higher than that of ordinary electroporation. The combined application of the nucleofector and the 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 not only has a high transduction efficiency but also can achieve site-specific integration of the target gene. In one embodiment of the present invention, the transduction method for achieving chimeric antigen receptor gene-modified T lymphocytes is based on viral transduction methods such as retroviruses or lentiviruses. This method has the advantages of high transduction efficiency, stable expression of foreign genes, and can shorten the time for in vitro culturing of T lymphocytes to reach clinical-grade numbers. On the surface of the transgenic T lymphocytes, the transduced nucleic acid is expressed on the surface through transcription and translation. Through in vitro cytotoxicity experiments on various cultured tumor cells, it is 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 the immunotherapy of tumors.

[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: 15-18. 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: 19-22.

[0066] Since there is currently no report on CAR T targeting CLD18A2, the inventors of the present invention have successfully found a target gene CLD18A2 suitable for CAR T cells from many tumor-related genes for the first time, and have successfully prepared CAR T cells targeting CLD18A2, thus providing a new treatment method for tumors such as pancreatic cancer and gastric cancer.

[0067] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, Third Edition, J. Sambrook et al., 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 of the present invention identified several scfv antibodies that recognize CLD18A2, simply referred to as 125, 163, and 175.

[0070] By gene synthesis technology based on PCR bridging, 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. After double digestion with Nhe1 / BamH1 (purchased from NEB), they were ligated with T4 DNA ligase (purchased from NEB) to the vector plasmid pCMV-V5-Fc (which fuses and expresses the human antibody Fc fragment downstream of the multiple cloning site, hereinafter referred to as V5-Fc, purchased from Shanghai Ruijin Biology) that was also double digested with Nhe1 / BamH1, and then transformed into the host bacterium TOP10. Positive clones were picked and identified by PCR and confirmed by sequencing to obtain the eukaryotic expression plasmids of V5-scFv-125-Fc, V5-scFv-163-Fc, and V5-scFv-175-Fc respectively.

[0071] The above expression plasmids were transfected into well-growing HEK-293F cells respectively and continuously cultured in a shaker at 37°C, 5% CO2, and 125 rpm for 7 days. Then, they were centrifuged at 4000 rpm for 10 min to remove the precipitate, and the supernatant was collected and filtered through a 0.45-μm filter membrane. The processed samples were affinity-purified using a protein A (purchased from GE) affinity column, and finally, the purified single-chain antibody-Fc fusion proteins scFv-125-Fc (abbreviation: scFv-125), scFv-163-Fc (abbreviation: scFv-163), and scFv-175-Fc (abbreviation: scFv-175) were obtained. The identification results are as Figure 3 .

[0072] Example 2. Construction of stable expression cell lines of CLD18A1 or CLD18A2

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

[0074] The complete coding sequences of CLD18A1 (GenBank: NM_016369) and CLD18A1 (GenBank: NM_001002026) were synthesized in full series by gene synthesis technology based on PCR bridging. A Flag tag (DYKDDDK) was inserted at the C-terminus of the coding sequence, and MluI / SalI (purchased from NEB) restriction enzyme sites were added to both ends of the synthesized gene fragment. After double digestion with MluI / SalI, the vector plasmid pWPT (purchased from addgene) that was also digested with MluI / SalI was ligated with T4 DNA and transformed into the host bacterium TOP10. Clones were picked for PCR identification and sequencing confirmation to obtain the correct lentiviral vector plasmids PWPT-CLD18A1 and PWPT-CLD18A2. The above plasmids were co-transfected into 293T cells with packaging helper plasmids (pGag-pol, pREV, pVsv-g, all purchased from addgene) in a certain proportion. The virus solutions of CLD18A1 and CLD18A2 were collected at 48 h and 72 h after transfection respectively, aliquoted, and stored at -80°C.

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

[0076] The above - collected CLD18A1 or CLD18A2 virus solutions were respectively added to 293T cells plated in 6 - cm dishes. After 72 h, the cells were collected and lysed using cell lysis buffer. In addition, the human gastric cancer cell lines BGC - 823 (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, TCHu 11) and NCI - N87 (purchased from ATCC, CRL - 5822) were infected with the CLD18A2 virus solution respectively. After the cells grew confluently, the cells were collected and lysed using cell lysis buffer. 40 μg of the cell proteins collected by lysis were subjected to SDS - PAGE gel electrophoresis, followed by immunoblotting of the gel, and stained with mouse anti - Flag antibody (purchased from Sigma Aldrich). After washing with PBS, it was incubated with horseradish peroxidase - labeled goat anti - mouse antibody (purchased from Santa Cluz) and developed using ECL reagent, and finally developed.

[0077] The results of Western blot showed that bands with a molecular weight of approximately 28 KD could be detected in 293T cells transfected with CLD18A1 or CLD18A2 (i.e., 293T - CLD18A1, 293T - CLD18A2) and in BGC - 823 and NCI - N87 cells transfected with CLD18A2 (i.e., BGC - 823 - CLD18A2, NCI - N87 - CLD18A2), while the corresponding bands were not detected in untransfected empty cells ( Figure 4 ), indicating that the cell lines expressing exogenous CLD18A1 and CLD18A2 were successfully constructed.

[0078] 3. Experimental procedures for analyzing the binding of each cell line to anti - CLD18A2 antibody by flow cytometry

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

[0080] The specific method is as follows:

[0081] 1). Tumor cells of 293T, 293T - CLD18A1, 293T - CLD18A2, BGC - 823, BGC - 823 - CLD18A2, NCI - N87, and NCI - N87 - CLD18A2 in the logarithmic growth phase were seeded into 6 - cm petri dishes, and the seeding cell density was approximately 90%, and cultured overnight in an incubator at 37 °C.

[0082] 2). The cells were digested with 10 mM EDTA, and the cells were collected by centrifugation at 200 g for 5 min. At a density of 1×10 6 ~1×10 7Resuspend at a concentration of / mL in 1% phosphate buffer saline containing calf serum (NBS PBS), and add 100 μl per tube to flow cytometry tubes.

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

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

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

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

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

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

[0089] 9). Analyze the data using the 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 293T cells stably expressing CLD18A1 but also to 293T cells stably expressing CLD18A2 ( Figure 5 ), indicating that this single-chain antibody lacked binding specificity for CLD18A2. Fortunately, the single-chain antibodies ScFv-163 and scFv-175 could specifically recognize 293T stably expressing CLD8A2 but not bind to 293T cells stably expressing CLD18A1, indicating that these two single-chain antibodies could specifically recognize CLD18A2. In addition, these two single-chain antibodies could also specifically recognize the BGC-823 or NCI-N87 cell lines stably transfected with CLD18A2 but not bind to BGC-823 or NCI-N87 cells not transfected with CLD18A2.

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

[0092] Table 1 explains the connection order of each part of the chimeric antigen receptor in the examples of the present invention. The connection can also be seen in Figure 2 as shown.

[0093] Table 1

[0094] Chimeric antigen receptor Extracellular binding domain - transmembrane domain - intracellular signaling domain 1 - intracellular signaling domain 2 etc CLD18A2-δZ scFv(CLD18A2)-CD8-CD3δzeta (negative control) CLD18A2-163-Z scFv(CLD18A2-163)-CD8-CD3 zeta CLD18A2-175-Z scFv(CLD18A2-175)-CD8-CD3 zeta CLD18A2-163-28BBZ scFv(CLD18A2-163)-CD28a-CD28b-CD137 (i.e., 4-1BB)-CD3 zeta CLD18A2-175-28BBZ scFv(CLD18A2-163)-CD28a-CD28b-CD137-CD3 zeta

[0095] 1. Amplification of nucleic acid fragments

[0096] (1) Amplification of scFv(CLD18A2-163, CLD18A2-175) sequences

[0097] Using the V5-scFv-163-Fc plasmid as a template, the forward primer of the primer pair (SEQ ID NO:7) contains a partial 2A sequence and the reverse primer (SEQ ID NO:8) contains a partial CD8 hinge sequence, and scFv(CLD18A2-163) is obtained by PCR amplification; similarly, using the V5-scFv-175-Fc plasmid as a template, the forward primer of the primer pair contains a partial 2A sequence and the reverse primer (SEQ ID NO:9) and the reverse primer contains a partial CD8 hinge sequence (SEQ ID NO:10), and scFv(CLD18A2-175) is obtained by PCR amplification.

[0098] (2) Nucleic acid sequences of other parts of the chimeric antigen receptor

[0099] The nucleic acid sequences of the parts other than scFv(CLD18A2-163, CLD18A2-175) of the anti-CLD18A2 chimeric antigen receptor protein are obtained by PCR using the sequences SEQ ID NO:18 and 21 disclosed in the patent application No. 201310164725.X as templates respectively.

[0100] Among them, the eGFP-F2A sequence is obtained by PCR amplification using the SEQ ID NO:18 plasmid described in the patent application No. 201310164725.X as a template and the primer pair (SEQ ID NO:11, 12).

[0101] Obtaining CD8-CD3ζ(Z) and CD28a-CD28b-CD137-CD3ζ(28BBZ): Using scFv(GPC3)-CD8-CD3ζ (SEQ ID NO:18 in the patent application 201310164725.X) and scFv(GPC3)-CD28a-CD28b-CD137-CD3ζ (SEQ ID NO:21 in the patent application 201310164725.X) as templates respectively, and performing PCR amplification using the primer pair (SEQ ID NO:13, 14) to obtain the CD8-CD3ζ(Z) and CD28a-CD28b-CD137-CD3ζ(28BBZ) fragments respectively.

[0102] The sequence of SEQ ID NO:18 in 201310164725.X corresponds to the sequence of SEQ ID NO:23 in the present invention.

[0103] The sequence of SEQ ID NO:21 in 201310164725.X corresponds to the sequence of SEQ ID NO:24 in the present invention.

[0104] 2. Ligation of nucleic acid fragments

[0105] Respectively, the eGFP-F2A nucleic acid fragment obtained as described above, the equimolar scFv(CLD18A2-163) or scFv(CLD18A2-175) nucleic acid fragment, and the equimolar CD8-CD3ζ(Z) or CD28a-CD28b-CD137-CD3ζ(BBZ) nucleic acid fragment were ligated in three fragments and subjected to PCR as Figure 2 shown. The ligation conditions were: pre-denaturation: 94°C, 4 min; denaturation: 94°C, 40 s; annealing: 60°C, 40 s; extension: 68°C, 140 s, for 5 cycles, and then a total extension at 68°C for 10 min. After adding DNA polymerase, forward primer (SEQ ID NO:11) and reverse primer (SEQ ID NO:14), PCR amplification was carried out for 30 cycles. The amplification conditions were pre-denaturation: 94°C, 4 min; denaturation: 94°C, 40 s; annealing: 60°C, 40 s; extension: 68°C, 140 s, for 30 cycles, and then a total extension at 68°C for 10 min. The amplified fragments were respectively (Table 2):

[0106] eGFP-scFv(CLD18A2)-163-Z (SEQ ID NO:15, 19),

[0107] eGFP-scFv(CLD18A2)-163-BBZ (SEQ ID NO:16, 20),

[0108] eGFP-scFv(CLD18A2)-175-Z (SEQ ID NO:17, 21),

[0109] eGFP-scFv(CLD18A2)-175-BBZ (SEQ ID NO:18, 22).

[0110] The results were identified as Figure 6 .

[0111] Table 2. Sequences in the present invention

[0112]

[0113]

[0114] 3. Construction of lentiviral plasmid vector

[0115] The vector system used for constructing the lentiviral plasmid vector of the present invention as an example belongs to the third-generation self-inactivating lentiviral vector system. This system has three plasmids, namely, the packaging plasmid psPAX2 encoding the Gag / Pol protein and the Rev protein (purchased from addgene); the envelope plasmid PMD2.G encoding the VSV-G protein (purchased from addgene); 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). In the recombinant expression vector encoding the target gene CAR, the co-expression of eGFP and the target gene CAR is achieved through the ribosomal skipping sequence (F2A) from the foot-and-mouth disease virus (FMDV). F2A is a core sequence of 2A (or called "self-cleaving polypeptide 2A") from the foot-and-mouth disease virus, which has the "self-cleaving" function of 2A and can achieve the co-expression of upstream and downstream genes. Due to its advantages of high cleavage efficiency, high expression balance of upstream and downstream genes, and short self-sequence, 2A provides an effective and feasible strategy for constructing gene therapy polycistronic vectors. Especially in immunotherapy based on chimeric antigen receptor gene-modified T lymphocytes, this sequence is often used to achieve the co-expression of the target gene and GFP or eGFP, 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 by F2A was constructed, collectively referred to as pWPT-eGFP-F2A-CAR( Figure 1) The target gene eGFP-F2A-CAR obtained in step 2 above (see 1(2) in Example 3, and the element after F2A is simply referred to as CAR) was digested with restriction endonucleases MluI and SalI, and ligated into the pWPT vector digested with the same enzymes, thereby constructing a lentiviral vector expressing each chimeric antigen receptor. After the successfully constructed vector was identified by digestion with MluI and SalI and the sequence was determined correctly, it could be prepared for lentiviral packaging. As described above, eGFP-F2A-CAR is transcribed into one mRNA, but finally translated into two peptide chains, eGFP and the anti-CLD18A2 chimeric antigen receptor. Under the guidance of the CD8α signal peptide, the anti-CLD18A2 chimeric antigen receptor will be localized on the cell membrane.

[0118] The vectors containing each target CAR obtained 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 of 293T for lentivirus packaging

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

[0125] The steps of transfection are as follows:

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

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

[0128] 4.3 Finally, add 500 μL of 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 filtered and sterilized by filtration) dropwise, vortex mix at 1200 rpm / min for 10 s,

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

[0130] On the day after transfection, observe the transfection efficiency (i.e., the proportion of cells showing green fluorescence), and a positive transfection efficiency of ~80% indicates a successful transfection experiment. At 48 h or 72 h after transfection, filter and collect the virus using a 0.45 μm filter (purchased from Millipore), then centrifuge at 28000 rpm, 4 °C for 2 h using a Beckman Optima L-100XP ultracentrifuge, discard the centrifugal supernatant, resuspend the centrifuged pellet with Quantum 007 culture medium (purchased from PAA) at 1 / 10 - 1 / 50 of the original volume, aliquot at 100 μL / tube and store frozen at -80 °C for virus titration or infection of T lymphocytes.

[0131] 5. Determine the titers of lentiviruses packaged with mock or eGFP-F2A-CAR

[0132] On the first day, seed 293T cells at 1×10 5 / mL into a 96-well culture plate, 100 μL / well, culture at 37 °C, 5% CO2, and the culture medium is DMEM containing 10% fetal bovine serum. On the second day, discard 50 μL / well of the culture supernatant, supplement with 50 μL / well of fresh above-mentioned culture medium and containing polybrene at a final concentration of 6 μg / mL, incubate at 37 °C, 5% CO2 for 30 min. Add 10 μL / well of the virus stock solution or 1 μL / well of the virus concentrate, dilute 5-fold, 4 gradients, two replicates, culture at 37 °C, 5% CO2. After 48 h of infection, detect eGFP by flow cytometry, and it is appropriate to use the number of cells with a positive rate of 5 - 20%, calculate the titer (U / mL) = positive rate × dilution factor × 100 × 10 4 . The titers of the above-mentioned viruses packaged by the calcium phosphate transfection method containing mock (i.e., empty vector control) and each eGFP-F2A-CAR are all about 0.5 - 2×10 6The level of U / mL, and the measured virus titer after concentration is about 2×10 7 U / mL.

[0133] Example 4. Recombinant lentivirus-infected CTL cells

[0134] Human peripheral blood mononuclear cells were obtained from healthy human peripheral blood by density gradient centrifugation (provided by Shanghai Blood Center). CTL cells were obtained from peripheral blood mononuclear cells by negative selection 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 carried out when the positive rate of CTL cells was ≥95%. They were cultured at a density of about 1×10 6 / mL in Quantum 007 lymphocyte culture medium (purchased from PAA), and magnetic beads coated with anti-CD3 and anti-CD28 antibodies (Invitrogen) and recombinant human IL-2 at a final concentration of 100 U / mL (purchased from Shanghai Huaxin Biotechnology Co., Ltd.) were added at a cell:magnetic bead ratio of 1:1 and stimulated for 24 h. Then, the above recombinant lentivirus was used to infect CTL cells at an MOI≈5. The infected cells were passaged every other day at a density of 5×10 5 / mL, and recombinant human IL-2 at a final concentration of 100 U / mL was supplemented in the lymphocyte culture medium.

[0135] On the 8th day of culture, the expression of different chimeric antigen receptors in the infected CTL cells was detected by flow cytometry. Since eGFP was co-expressed with CAR, the positive cells detected for eGFP were the positive cells expressing the chimeric antigen receptor. Uninfected T lymphocytes were used as a negative control. The positive rates of CTL cells infected with viruses expressing different chimeric antigen receptors are shown in Table 3. The positive rate results indicate that a certain positive rate of CAR + CTL cells can be obtained by the method of lentivirus infection.

[0136] Table 3

[0137] CTL cells transfected with the following CARs Positive rate of CTL cells for eGFP Mock (empty vector control) 56％ CLD18A2-Z fused with 163 single-chain antibody 51％ CLD18A2-28BBZ fused with 163 single-chain antibody 54％ CLD18A2-Z fused with 175 single-chain antibody 52％ CLD18A2-28BBZ fused with 175 single-chain antibody 55％

[0138] After CTL cells were respectively infected with viruses packaging different chimeric antigen receptors, they were passaged every other day at a cell density of 5×10 5 / ml, counted, and IL-2 (final concentration 100 U / ml) was supplemented in the culture medium of the passaged cells. On the 11th day of culture, there was an amplification of about 20-40 times, indicating that CTL cells expressing different chimeric antigen receptors can be amplified to a certain extent in vitro, providing a guarantee for subsequent in vitro toxicity tests and in vivo tests.

[0139] Example 5. In vitro Toxicity Effect Experiment of Chimeric Antigen Receptor-expressing Cells

[0140] The materials used in the in vitro toxicity experiment are as follows:

[0141] 293T and gastric cancer cell lines shown in Table 4 were used as target cells, and the effector cells were positive cells with chimeric antigen receptor expression detected by FACS after 12 days of in vitro culture as verified in Example 4, denoted as chimeric antigen receptor-positive (CAR + ) CTL. The effector-to-target ratios were 3:1, 1:1, and 1:3 respectively according to the situation, the number of target cells was 10,000 / well, and the effector cells were corresponding according to different effector-to-target ratios. Each group had 5 replicate wells, and the average value of the 5 replicate wells was taken. The detection time was the 18th hour.

[0142] Among them, each experimental group and each control group are as follows:

[0143] Each experimental group: each target cell + CTL expressing different chimeric antigen receptors,

[0144] Control group 1: maximum release of LDH by target cells,

[0145] Control group 2: spontaneous release of LDH by target cells,

[0146] Control group 3: spontaneous release of LDH by effector cells.

[0147] Detection method: It was carried out using the CytoTox 96 Non-Radioactive Cytotoxicity Detection Kit (Promega). This method is a colorimetric-based detection method and can replace 51 the Cr release method. CytoTox detects and quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released during cell lysis, and its release mode is basically the same as that of 51 Cr in radioactive analysis. The released LDH in the culture medium supernatant can be detected by a 30-minute coupled enzyme reaction. In the enzyme reaction, LDH can convert a tetrazolium salt (INT) into red formazan. The amount of the generated red product is proportional to the number of lysed cells. Specifically refer to the instruction manual of the CytoTox 96 Non-Radioactive Cytotoxicity Detection Kit.

[0148] The cytotoxicity calculation formula is:

[0149]

[0150] Specifically as shown in Table 4 and Table 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 CTL of + has obvious killing effect on 293T cells with high expression of CLD18A2, but has no killing effect on 293T cells with high expression of CLD18A1, indicating that they can selectively kill cells with CLD18A2. In addition, the CTL expressing the chimeric antigen receptor CLD18A2-Z CAR of the present invention + CTL and CLD18A2-28BBZ CAR + The CTL of + can also significantly kill two gastric cancer cell lines BGC-823 and NCI-N87 with high expression of CLD18A2 (see Tables 4 and 5), and shows effector-target ratio gradient dependence, that is, the higher the effector-target ratio, the stronger the cytotoxic effect, while there is no specific cytotoxic effect on BGC-823 and NCI-N87 that do not express CLD18A2.

[0151] The data of effector-target ratio dependence further shows the specific cytotoxic effect of CTL expressing the anti-CLD18A2 chimeric antigen receptor of the present invention on gastric cancer cells with high expression of CLD18A2.

[0152] In comparison, CTL as a blank control transfected with virus containing mock plasmid (empty plasmid vector pWPT-eGFP without carrying CLD18A2-CAR) shows very low cytotoxic effect on the above three cell lines with high expression of CLD18A2. Its cytotoxicity on cell lines with high expression of CLD18A2 shows a very significant difference from the cytotoxicity data of CTL expressing the anti-CLD18A2 chimeric antigen receptor of the present invention.

[0153] The above results show that the chimeric antigen receptor constructed by selecting a single-chain antibody against CLD18A2 can selectively kill target cells with high expression of CLD18A2. In addition, from the perspective of cytotoxicity data, CAR T of CLD18A2-28BBZ has stronger cytotoxicity on cells expressing CLD18A2 than CAR T of CLD18A2-Z.

[0154] Table 4. Cytotoxicity of CAR T cells expressing single-chain antibody 163

[0155]

[0156]

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

[0158]

[0159] Discussion

[0160] Currently, CAR T cells have become a potential treatment option. However, for many tumors, such as gastric cancer, there are no reports on CAR T cell therapy. Existing studies have shown that CLD18A2 may be a specific marker of gastric tissue, and thus may also become a therapeutic target for tumors such as gastric cancer. However, currently, the only candidate drug for CLD18A2 as a therapeutic target is a monoclonal antibody, and whether it can be successfully used for the treatment of corresponding tumors is still unknown. Therefore, it is necessary to search for new treatment methods. Considering the tissue specificity of CLD18A2, the present invention envisions that if CAR T cells can be used for targeted therapy, it is expected to obtain a new anti-cancer preparation. However, we know that the CLD18A2 antigen is a tight junction protein, and it is still unknown whether it can be contacted by CAR T cells and trigger the killing of corresponding target cells. In addition, since the protein conformation affects the whole when one part is touched, many monoclonal antibodies evolved into single-chain antibodies often lose their antigen-binding activity or specificity. Fortunately, the inventors of the present invention found that two single-chain antibodies (163 and 175) retained the antigen-binding specificity of the monoclonal antibody. Further research showed that the CAR T cells composed of these two single-chain antibodies retained the selective killing effect on CLD18A2-positive cells. The results of the present invention show that CLD18A2 can indeed become a therapeutic target for CAR T cell therapy; CAR T cells targeting CLD18A2 are a new candidate means for tumor treatment.

[0161] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. 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 appended claims of the present application.

Claims

1. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises, in sequential connection: an extracellular binding region, a transmembrane region, and an intracellular signaling region, and wherein the extracellular binding region comprises a protein that specifically recognizes CLD18A2.

2. The chimeric antigen receptor according to claim 1, wherein the extracellular binding region specifically recognizes CLD18A2, but does not recognize CLD18A1.

3. The chimeric antigen receptor according to claim 1, wherein the protein that specifically recognizes CLD18A2 is a fragment of an antibody that specifically recognizes CLD18A2 or a ligand; preferably, the antibody is a monoclonal antibody, a single-chain antibody, or a domain antibody; preferably, the extracellular binding region comprises a variable region; preferably, the extracellular binding region comprises a heavy-chain variable region and a light-chain variable region; preferably, the extracellular binding region comprises a polypeptide encoded by the nucleic acid shown in SEQ ID NO:3 or SEQ ID NO:

5.

4. The chimeric antigen receptor according to claim 3, wherein the single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6 or the single-chain antibody is encoded by the nucleic acid shown in SEQ ID NO:3 or SEQ ID NO:

5.

5. The chimeric antigen receptor according to any one of claims 1-4, wherein the intracellular signaling region comprises an intracellular signaling activation motif; preferably, the intracellular signaling region comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs); preferably, the intracellular signaling region comprises the intracellular signaling region of CD3ζ or FcεRIγ; more preferably, the intracellular signaling region further comprises the intracellular signaling region of CD28, CD137, or CD134, or a combination thereof.

6. The chimeric antigen receptor according to any one of claims 1-5, wherein the transmembrane region comprises the transmembrane region of a natural marker on the surface of T lymphocytes; preferably, the transmembrane region comprises the transmembrane region of CD8 or CD28; preferably, the transmembrane region comprises the transmembrane region of CD8α; More preferably, the transmembrane region further comprises a hinge region; preferably, the hinge region comprises the hinge region of CD8 or CD28.

7. The chimeric antigen receptor according to any one of claims 1-6, characterized in that Expressed on the surface of T lymphocytes.

8. The chimeric antigen receptor according to any one of claims 1-7, characterized in that The chimeric antigen receptor is capable of inducing cytotoxic effects of the T lymphocytes against CLD18A2-positive cells.

9. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, and an intracellular signaling region, and wherein the extracellular binding region specifically recognizes CLD18A2.

10. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes CLD18A2, and wherein the intracellular signaling region comprises an intracellular signaling activation motif.

11. A nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region specifically recognizes CLD18A2, and wherein the intracellular signaling region comprises an intracellular signaling activation motif.

12. A nucleic acid encoding a chimeric antigen receptor as claimed in any one of claims 1-10.

13. An immune effector cell expressing a chimeric antigen receptor as claimed in any one of claims 1-10.

14. The immune effector cell according to claim 13, which comprises a T cell, an NK cell or an NKT cell, preferably, comprises cytotoxic T lymphocytes (CTLs), and preferably, the T lymphocytes are stimulated and cultured with anti-CD3 antibody, anti-CD28 antibody and / or IL-2.

15. A method for preparing the immune effector cell as claimed in claim 13 or 14, comprising transducing the nucleic acid as claimed in claim 12 into a cell by a non-viral transduction method or a viral transduction method to obtain the immune effector cell.

16. Use of the immune effector cell as claimed in claim 13 or 14 in the preparation of a medicament for treating CLD18A2-positive tumors, preferably, wherein the CLD18A2-positive tumors include pancreatic cancer or gastric cancer.

17. A method for inducing cytotoxicity against CLD18A2-positive cells, wherein the method comprises contacting the CLD18A2-positive cells with an immune effector cell expressing a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular binding region, a transmembrane region and an intracellular signaling region, wherein the extracellular binding region specifically recognizes CLD18A2.

18. A method for inducing cytotoxicity against CLD18A2-positive cells, wherein the method comprises contacting the CLD18A2-positive cells with an immune effector cell expressing a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular binding region, a transmembrane region and an intracellular signaling region, wherein the extracellular binding region specifically recognizes CLD18A2, the intracellular signaling region comprises an intracellular signaling activation motif, and the nucleic acid encoding the chimeric antigen receptor is transduced into the immune effector cell, preferably, the nucleic acid is transduced by a non-viral transduction method or a viral vector.

19. A method for inducing cytotoxicity against CLD18A2-positive cells, wherein the method comprises transducing a nucleic acid encoding a chimeric antigen receptor into T lymphocytes, the chimeric antigen receptor comprising an extracellular binding region, a transmembrane region and an intracellular signaling region, wherein the extracellular binding region specifically recognizes CLD18A2, the intracellular signaling region comprises an intracellular signaling activation motif; the T lymphocytes transduced with the nucleic acid encoding the chimeric antigen receptor induce cytotoxicity against the CLD18A2-positive cells after contacting with the CLD18A2-positive cells.

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