CN106146666B - Immune effector cells targeting CLDN6, preparation method and application thereof - Google Patents

Abstract

The invention relates to an immune effector cell targeting CLDN6 (CLDN6), a preparation method and application thereof. For the first time, the inventors successfully found a target gene CLDN6 suitable for the development of chimeric antigen receptor (CAR) modified immune effector cells from many tumor-related genes, and successfully prepared a CAR-modified immune effector cell targeting CLDN6. cells, thus providing a new treatment method for ovarian cancer, gastric cancer, lung adenocarcinoma and other tumors.

Description

Immune effector cells targeting CLDN6, preparation method and application thereof

technical field

The invention belongs to the field of tumor cell therapy, and more specifically, the invention relates to immune effector cells targeting CLDN6 and its preparation method and application.

Background technique

The role of immune effector cells in the tumor immune response has been increasingly recognized. Adoptive immunotherapy based on immune effector cells has achieved certain effects in some tumors, and this immunotherapy method can overcome the above-mentioned defects of antibody therapy, but the curative effect in most tumors is still unsatisfactory [Grupp SA, et al. al.Adoptive cellular therapy.Curr Top Microbiol Immunol., 2011;344:149-72.]. In recent years, based on the discovery that cytotoxic T lymphocytes (cytotoxic lymphocytes, CTLs) specifically rely on T cell receptors (TCRs) for target cell recognition, the scFv of antibodies against tumor cell-associated antigens was combined with T Intracellular signal activation motifs such as CD3ζ or FcεRIγ of lymphocyte receptors are fused into a chimeric antigen receptor (CAR), which is genetically modified on the surface of T lymphocytes by means such as lentivirus infection. This CAR T lymphocyte can selectively direct T lymphocytes to tumor cells in a major histocompatibility complex (Major Histocompatibility Complex, MHC) non-restricted manner and specifically kill tumors. 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.] . In addition, CAR-modified NK cells (Klingemann H. Challenges of cancer therapy with natural killer cells.

Cytotherapy.2014Dec 18.pii:S1465-3249(14)00791-9) or NKT cells also showed good anti-tumor activity in preclinical studies (Heczey A1, Liu D1, Tian G2, Courtney AN1, Wei J1, Marinova E1, Gao X1, Guo L1, Yvon E3, Hicks J2, Liu H4, Dotti G5, Metelitsa LS6. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. Blood.2014;124(18):2824- 33).

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 adopts the transmembrane region of CD8, CD28 and other molecules, and the intracellular signal region adopts immunoreceptor tyrosine activation motif (ITAM) CD3ζ or FcεRIγ and co-stimulatory Intracellular signaling regions of signaling molecules CD28, CD27, CD137, CD134, etc.

The first-generation CAR T lymphocytes containing only ITAM in the intracellular signal region, in which each part of the chimeric antigen receptor is connected in the following form: scFv-TM-ITAM. This kind of CAR T can stimulate anti-tumor cytotoxic effect, but the secretion of cytokines is relatively small, and it cannot stimulate long-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.].

The subsequent development of the second-generation CAR T lymphocytes added the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the parts of the chimeric antigen receptor were 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 signal region causes the continuous proliferation of T lymphocytes, and can increase the levels of cytokines secreted by T lymphocytes such as IL-2 and IFN-γ, and at the same time increase the CAR T In vivo survival cycle and anti-tumor effect [Dotti G. et al. CD28costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphomapatients. J Clin Invest, 2011, 121(5): 1822-1826.].

In the third-generation CAR T lymphocytes developed in recent years, the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, which further improves the CAR T lymphocytes. The survival cycle of 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 and CD137 domains. PNAS, 2009, 106(9):3360–3365.].

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 recognized by CAR in certain normal tissues, CAR T lymphocytes may damage normal tissues expressing corresponding antigens. For example, targeting the antigen carbonic anhydrase IX (CAIX) expressed on the tumor cells of patients with renal cell carcinoma is the first case of adoptive therapy of CAR T lymphocytes used in clinical practice, and it is also the first case to report the off-target effect of CAR cells . The patient developed grade 2-4 hepatotoxicity after multiple infusions of CAR T lymphocytes. The reason for the analysis was the low expression of CAIX in hepatic bile duct epithelial cells. The original clinical trial was forced to be interrupted and any evaluation of the treatment effect of the patients was excluded [Stoter G. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydraseIX:first Clinical experience. J clin oncol, 2006, 24(13):e20-e22.; Ngo MC., et al. Ex vivo gene transfer for improved adopted 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 low-level or no antigen-triggered conditions, resulting in the release of a large number of cytokines As a result, the so-called "cytokine storm" may be triggered. This signal leakage can lead to off-target cytotoxicity, resulting in nonspecific tissue damage. For example, in 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" caused the sudden death of the patient due to the low expression of Her2 in normal lung tissue[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.].

When designing CAR-modified immune effector cells, especially T cells, the antigen gene targeted is actually a key choice. In view of the complexity of gene expression in vivo and various uncontrollable factors, it is necessary to select a suitable one for CAR Genetics are very difficult. Moreover, it is difficult to find specific molecules for many tumor-specific antigens that are suitable for constructing CAR-modified immune effector cells. In addition, since different targets may have different effects on immune effector cells, for example, EGFR targets can upregulate the expression of PD-L1 (Akbay EA, Koyama S, Carretero J, Altabef A, Tchaicha JH, Christensen CL, Mikse OR, Cherniack AD, Beauchamp EM, Pugh TJ, Wilkerson MD, Fecci PE, Butaney M, Reibel JB, Soucheray M, Cohoon TJ, Janne PA, Meyerson M, Hayes DN, Shapiro GI, Shimamura T, Sholl LM, Rodig SJ, Freeman GJ, Hammerman PS, Dranoff G, Wong KK. Activation of the PD-1 pathway contributes toimmune escape in EGFR-driven lung tumors. Cancer Discov. 2013; 3(12):1355-63.), which may affect the effect T Cell killing effect (Moon EK, Wang LC, Dolfi DV, Wilson CB, Ranganathan R, Sun J, Kapoor V, Scholler J, Puré E, Milone MC, June CH, Riley JL, Wherry EJ, Albelda SM.Multifactorial T- cell hypofunction that is reversiblecan limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res. 2014 Aug 15; 20(16):4262-73.). Therefore, different target selection may lead to different anti-tumor activities of the prepared CAR-modified immune cells. Due to the complexity and unpredictability of biology, these require experimental verification.

Contents of the invention

The purpose of the present invention is to provide immune effector cells targeting CLDN6 and its preparation method and application.

In the first aspect of the present invention, there is provided a chimeric antigen receptor (CAR) expressed on the surface of immune effector cells, said chimeric antigen receptor comprising sequentially linked: an extracellular binding region, a transmembrane region and an intracellular signal region, wherein the extracellular binding region comprises a protein that specifically recognizes CLDN6 (Claudin 6).

In a preferred example, the immune effector cells include: T lymphocytes, NK cells or NKT cells.

In another preferred example, the protein that specifically recognizes CLDN6 is an antibody or a ligand; preferably, the antibody is a single-chain antibody or a domain antibody.

In another preferred example, the transmembrane region is a sequence comprising the transmembrane region and hinge region of CD8 or CD28.

In another preferred example, the intracellular signal region is selected from: CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134 intracellular signal region sequence, or a combination thereof.

In another preferred example, the chimeric antigen receptor includes the following sequentially connected extracellular binding region, transmembrane region and intracellular signal region:

Single-chain antibodies that specifically recognize CLDN6, CD8 and CD3ζ;

Single-chain antibodies that specifically recognize CLDN6, CD8, CD137 and CD3ζ;

A single-chain antibody that specifically recognizes CLDN6, the transmembrane region (CD28a) of the CD28 molecule, the intracellular signaling region (CD28b) of the CD28 molecule, and CD3ζ; or

The single-chain antibody specifically recognizes CLDN6, the transmembrane region of CD28 molecule, the intracellular signal region of CD28 molecule, CD137 and CD3ζ.

In another preferred example, the chimeric antigen receptor has the amino acid sequence of any one of SEQ ID NO: 21-24.

In another aspect of the present invention, there is provided a nucleic acid encoding any one of the aforementioned chimeric antigen receptors.

In a preferred example, the nucleic acid has the nucleotide sequence of any one of SEQ ID NO: 17-20.

In another aspect of the present invention, there is provided an expression vector comprising any one of the aforementioned nucleic acids.

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

In another aspect of the present invention, a virus (such as a lentivirus) comprising the vector is provided.

The use of any one of the aforementioned chimeric antigen receptors, or the nucleic acid, or the expression vector, or the virus is used to prepare genetically modified immune effector cells targeting CLDN6.

In another aspect of the present invention, a genetically modified immune effector cell transduced with the nucleic acid, the expression vector or the virus is provided.

In another aspect of the present invention, a genetically modified immune effector cell 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 of SEQ ID NO:21-24 The amino acid sequence described.

In another aspect of the present invention, the application of the genetically modified immune effector cells is provided for preparing a drug for suppressing tumors, and the tumors are CLDN6-positive tumors.

In another preferred example, the CLDN6-positive tumors include: ovarian cancer, gastric cancer, and lung adenocarcinoma.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. RT-PCR detection of CLDN6 gene expression in various tissues and cells.

Figure 2. Electrophoresis results of purification of single-chain antibody against CLDN6.

Fig. 3. Schematic diagram of lentiviral expression vector encoding CLDN6.

Figure 4. Western blot detection of stable expression of CLDN6. 293T refers to HEK-293T; 293T-CLDN6 refers to HEK-293T cells transfected with CLDN6.

Figure 5. Flow cytometry analysis of the binding of various cell lines to anti-CLDN6 antibodies.

Figure 6. Schematic diagram of the linkage sequence of each part of the chimeric antigen receptor.

Figure 7. DNA electrophoresis identification diagram of five spliced eGFP-F2A-CAR fragments.

Fig. 8 is a schematic structural diagram of the lentiviral vector pWPT-eGFP-F2A-CAR containing the coding sequence of the present invention as an example.

Detailed ways

After extensive and in-depth research, the inventors revealed for the first time a CAR-modified immune effector cell based on the CLDN6 gene and a preparation method thereof.

CLDN6 gene

The inventors investigated many kinds of tumor-specific genes in the early stage, and found that a considerable part of these genes are also expressed in normal cells of some tissues, and it is difficult to apply the technology of immune effector cells modified by chimeric antigen receptors; some tumors Specific genes have good tumor-specific expression characteristics, but CAR-modified immune effector cells designed based on them have no tumor cell killing activity or very low activity, which may be because the target can trigger tumor cells to secrete immune effector cells. Cell inhibitory factors such as PD-L1.

After repeated investigation and screening, the inventors found the CLDN6 gene as the target gene for designing CAR. The amino acid sequence and gene sequence of Claudin6 (CLDN6) are disclosed as GenBank accession numbers NP_067018.1 and NM_021195.3 (sequence number: 22 and sequence number: 23), or GenBank accession numbers NP_067018.2 and NM_021195.4 (sequence No.: 46 and Serial No.: 47). Claudins are membrane proteins located in the tight junctions of epithelium and endothelium.

At present, CAR T cells have become a potential therapeutic method. However, there are no reports on CAR T cell therapy for many tumors, such as gastric cancer. Studies have shown that CLDN6 is a specific marker of gastric tissue. The inventor's research confirmed that CLDN6 is indeed not expressed in most normal tissues, but expressed in some tumors such as ovarian cancer. However, the current candidate drug for CLDN6 as a therapeutic target is only a monoclonal antibody, and whether it can be successfully used in the corresponding tumor treatment is still unknown. Therefore, it is necessary to find new treatments. Considering the tissue specificity of CLDN6, the inventors imagine that if CAR T cells can be used for targeted therapy, a new anticancer agent can be obtained. However, it is well known that the CLDN6 antigen is a tight junction protein, and whether it can be contacted by CAR T cells and trigger the killing of the corresponding target cells is still unknown. In addition, because the spatial conformation of proteins affects the whole body, many monoclonal antibodies evolve into single-chain antibodies and often lose their antigen-binding activity or specificity. Fortunately, the inventors found that there are two single chain antibodies (AE3-20LH and AE3-20HL) with good antigen-binding specificity. Further studies by the inventors have shown that the CAR T cells composed of these two single-chain antibodies retain the selective killing effect on CLDN6-positive cells. The results of the present invention show that CLDN6 can indeed become the target of CAR-modified immune effector cells, especially T cell therapy; CAR T cells targeting CLDN6 are a new candidate for tumor therapy.

Further studies proved that CLDN6-targeted CAR-modified T cells could indeed selectively eliminate CLDN6-positive tumor cells without toxicity to non-tumor cells 293T cells. The inventors believe that the corresponding CAR-modified immune effector cells, especially T cells, should be used for the treatment of human tumors.

Chimeric antigen receptor and its encoding nucleic acid

The present invention provides a chimeric antigen receptor expressed on the surface of immune effector cells, the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region and an intracellular signal region connected in sequence, wherein the cell The outer binding region contains a protein that specifically recognizes CLDN6 (claudin 6). Expressing the chimeric antigen receptor on the surface of the immune effector cells can enable the immune effector cells to have highly specific cytotoxic effects on tumor cells that highly express CLDN6.

As a preferred mode of the present invention, the extracellular binding region comprises a single-chain antibody scFv (CLDN6) that specifically recognizes CLDN6. The extracellular binding region of the chimeric antigen receptor protein is connected to the transmembrane region of CD8 or CD28 through the hinge region of CD8, and the transmembrane region is immediately followed by the intracellular signal region.

The invention also includes nucleic acids encoding said chimeric antigen receptors. A nucleic acid sequence of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The nucleic acid codon 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 all included in the scope of the present invention. The degenerate nucleic acid codons encoding the 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 polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. 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 an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

Antibodies that specifically recognize human CLDN6 can be selected from antibodies disclosed in the prior art, and a variety of monoclonal antibodies that recognize the C-terminal epitope of CLDN18A2 can be used in the present invention in an appropriate manner, and finally a CAR immune effect with killing activity can be obtained cell. As a particularly preferred embodiment of the present invention, the antibody against human CLDN6 is the single-chain antibody scFv-CLDN6 (AE3-20LH) or scFv-CLDN6 (AE3-20HL) of the present invention.

The term "single-chain antibody (scFv) fragment" used in the present invention refers to an antibody fragment as defined below, which is composed of a heavy chain variable region (VH) and a light chain variable region ( VL) of the recombinant protein, the linker allows the two domains to associate to ultimately form the antigen-binding site. The size of scFv is generally 1/6 of a whole antibody. A single-chain antibody is preferably a sequence of one amino acid chain encoded by one nucleotide chain. The single-chain antibody used in the present invention can be further modified by 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, eg, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2002) N.Y. The modifications referred to are preferably carried out at the nucleic acid level. The above-mentioned single-chain antibody may also include derivatives thereof. "Derivatives of antibodies" in the present invention include, for example, when the derivatives of the antibodies are obtained by phage display technology, surface plasmon resonance technology such as that used in BIAcore system can be used to increase the efficiency of phage antibody binding to CLDN6 epitope (Schier, Human Antibody Hybridoma 7 (1996), 97-105; Malmborg, J Immunol Methods 183 (1995), 7-13). Also included are, for example, methods for the production of chimeric antibodies as described in WO 89/09622, methods for the production of humanized antibodies as described in EP-A10239400 and WO90/07861, in WO91/10741, WO94/02602 and WO96/33735 Derivatives of antibodies produced by the described methods for producing xenogeneic antibodies, eg, human antibodies in mice.

The term "specific recognition" in 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 judged by immunological techniques, including but not limited to western blotting, immunoaffinity chromatography, flow cytometry and the like. In the present invention, the specific recognition is preferably determined by flow cytometry, and in specific cases, the standard of specific recognition can be judged by those of ordinary skill in the art based on their common knowledge in the field.

The transmembrane region of the chimeric antigen receptor can be selected from the transmembrane region of proteins such as CD8 or CD28. Human CD8 protein is a heterodimer composed of two chains, αβ or γδ. In one embodiment of the invention, the transmembrane region is selected from the transmembrane region of CD8α or CD28. In addition, the CD8α hinge region (hinge) 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 chimeric antigen receptor CAR and the intracellular signal region .

The intracellular signaling region may be selected from CD3ζ, FcεRIγ, CD28, CD137, intracellular signaling region of CD134 protein, and combinations thereof. The CD3 molecule consists of five subunits, among which the CD3ζ subunit (also known as CD3 zeta, referred to as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. CD3δZ is a truncated CD3ζ sequence without an ITAM motif, which is generally used as a negative control construction in the practice of the present invention. FcεRIγ is mainly distributed on the surface of mast cells and basophils, and it contains an 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 effect produced by their intracellular signal segments after binding to their respective ligands causes the continuous proliferation of immune effector cells (mainly T lymphocytes), And it can increase the level of cytokines secreted by immune effector cells such as IL-2 and IFN-γ, and at the same time improve the survival cycle and anti-tumor effect of CAR immune effector cells in vivo.

The anti-CLDN6 chimeric antigen receptor protein encoded by the nucleic acid of the present invention can be selected from sequential connection in the following manner:

scFv(CLDN6)-CD8-CD3ζ,

scFv(CLDN6)-CD8-CD137-CD3ζ,

scFv(CLDN6)-CD28a-CD28b-CD3ζ,

scFv(CLDN6)-CD28a-CD28b-CD137-CD3ζ,

And the combination thereof, wherein CD28a in the relevant chimeric antigen receptor protein represents the transmembrane region of CD28 molecule, and CD28b represents the intracellular signal region of CD28 molecule. The above-mentioned various anti-CLDN6 chimeric antigen receptors are collectively referred to as scFv(CLDN6)-CAR.

In one embodiment of the present invention, the nucleic acid of the present invention has a sequence as described in SEQ ID NO: 17-20. 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 NO: 21-24.

Expression Vectors and Cells

The present invention also provides a vector comprising the above-mentioned nucleic acid encoding the chimeric antigen receptor protein expressed on the surface of immune effector cells. 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. There are three plasmids in this system, namely the coding protein Gag/Pol, the packaging plasmid psPAX2 coding Rev protein; the envelope plasmid PMD2.G coding VSV-G protein; and empty The vector pWPT-eGFP can be used for recombinantly introducing the target nucleic acid sequence, that is, the nucleic acid sequence encoding CAR. The expression of enhanced green fluorescent protein (eGFP) was regulated by the elongation factor-1α (elongation factor-1α, EF-1α) promoter in the empty vector pWPT-eGFP (itself a mock in subsequent experiments). The recombinant expression vector pWPT-eGFP-F2A-CAR containing the target nucleic acid sequence encoding CAR is obtained by the ribosomal skipping sequence (ribosomal skipping sequence 2A) (referred to as F2A) from the food-and-mouth disease virus (FMDV). ) to achieve co-expression of eGFP and CAR.

The present invention also includes viruses comprising the vectors described above. The viruses of the present invention include packaged infectious viruses, as well as viruses to be packaged that contain the components necessary for packaging as infectious viruses. Other viruses and their corresponding plasmid vectors known in the art that can be used to transfer foreign genes into immune effector cells can also be used in the present invention.

In one embodiment of the present invention, the virus is a lentivirus comprising the above pWPT-eGFP-F2A-CAR recombinant vector (ie comprising scFv(CLDN6)-CAR).

The present invention also provides genetically modified immune effector cells, 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 based transduction methods include electroporation and transposon methods. Recently, the Nucleofector nucleofector developed by Amaxa can directly introduce exogenous genes into the nucleus to obtain high-efficiency transduction of target genes. In addition, the transduction efficiency of transposon systems based on Sleeping Beauty system or PiggyBac transposon has been greatly improved compared with ordinary electroporation. The combined application of nucleofector transfection instrument and Sleeping Beauty transposon system has been It has been reported [Davies JK., etal.Combining CD19 redirection and alloanergization to generate tumor-specifichuman T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res, 2010, 70(10):OF1-10.], the method both It has high transduction efficiency and can realize the site-specific integration of the target gene. In one embodiment of the present invention, the method of transduction of immune effector cells to achieve chimeric antigen receptor genetic modification is based on a virus such as retrovirus or lentivirus. This method has the advantages of high transduction efficiency, stable expression of exogenous genes, and can shorten the time for culturing immune effector cells in vitro to reach clinical-grade quantities. On the surface of the transgenic immune effector cells, the transduced nucleic acid is expressed on the surface through transcription and translation. In vitro cytotoxicity experiments on various cultured tumor cells prove that the anti-CLDN6 chimeric antigen receptor gene-modified immune effector cells of the present invention have highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid encoding chimeric antigen receptor protein of the present invention, the plasmid containing the nucleic acid, the virus containing the plasmid, and the transgenic immune effector cells transduced with the above nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.

In one embodiment, the genetically modified immune effector cell of the present invention expresses a chimeric antigen receptor on its surface, and the chimeric antigen receptor is expressed by the nucleic acid of one of SEQ ID NO: 17-20. In another embodiment, a chimeric antigen receptor is expressed on the surface of the transgenic immune effector cell of the present invention, and the amino acid sequence of the chimeric antigen receptor is selected from one of SEQ ID NO: 20-24.

In view of the fact that there is no report on CAR T targeting CLDN6, the inventors successfully found a target gene CLDN6 suitable for CAR T cells from many tumor-related genes for the first time, and successfully prepared CAR T targeting CLDN6 cells, thus providing a new treatment method for ovarian cancer, gastric cancer, lung adenocarcinoma and other tumors.

Below in conjunction with specific embodiment, further illustrate the present invention. 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 method that does not indicate specific conditions in the following examples, usually according to the conditions described in J. Sambrook et al. edited, Molecular Cloning Experiment Guide, the third edition, Science Press, 2002 according to conventional conditions, or according to the conditions described in the manufacture conditions recommended by the manufacturer.

Example 1, CLDN6 expression detection in various normal tissues and tumor cells

Extraction of total cellular RNA: Human normal tissue RNA was purchased from Clontech Laboratories, Inc. Ovarian cancer cells were routinely cultured in a 5% CO ₂ , 37°C incubator. The cells were washed twice with PBS (0.14M NaCl, 2.7mM KCl, 10.1mM Na ₂ HPO4, 1.8mM KH ₂ PO4, pH 7.3). Use Trizol reagent to extract the total RNA of cells in good growth state, add 1ml Trizol to each plate of cells, and pipette until it is not viscous; add 0.2ml freshly opened chloroform to each ml of the original volume of Trizol, shake vigorously for 15sec, and incubate at room temperature for 5min; 4 Centrifuge at 12,000g for 15min at 12,000g; transfer the colorless supernatant to a new centrifuge tube, add 0.5ml of isopropanol per ml of the original volume of Trizol, incubate at -20°C for 20min, centrifuge at 12,000g at 4°C for 10min; discard the supernatant , wash with 1ml of 75% ethanol, centrifuge at 10,000g for 5min at 4°C; dry the RNA pellet at room temperature for 10-20min, and dissolve the pellet with RNase-free H ₂ O after DEPC treatment. Quantification of RNA by spectrophotometry.

Reverse transcription: Mix 1 μl of total cellular RNA (1 μg/μl) and 1 μl of Oligo dT (10 μM), incubate at 72°C for 5 minutes, and immediately place on ice for 5 minutes; add 15 μl of reverse transcription mixture (Nuclease-free H2O 6.1 μl, 5×RT Buffer 4μl, 25mM MgCl ₂ 2.4μl, 10mM dNTPs 1μl, RNase Ribonuclease Inhibitor 0.5μl, Improm-IITM Reverse Transcriptase 1μl), incubate at 25°C for 5min, at 42°C for 1h; incubate at 70°C for 15min to terminate the reaction.

RT-PCR: Use the above reverse-transcribed cDNA as a template and primers CLDN6-F: GGAATGCAGATCCTGGGAGT, CLDN6-R: ATGAAAGCGGTCA for PCR. PCR reaction system: 1 μl template, 2.5 μl 2mM dNTP, 2.5 μl 10×buffer, each primer 1 μl, 5U Taq enzyme, make up 25 μl with H ₂ O. PCR reaction conditions: 94°C for 3min; 35 cycles of 94°C for 30sec, 56°C for 30sec, and 72°C for 40sec; 72°C for 10min; store at 4°C. Simultaneously amplify β-actin as an internal reference, β-actin primers: β-actin-F: 5'-TCCTCCCTGGAGAAGAGCTA-3', β-actin-R: 5'-GTACTTGCGCTCAGGAGGAG-3'. Amplification conditions: 94°C for 3min; 94°C for 30sec, 58°C for 30sec, 72°C for 30sec, 30 cycles; 7210min°C. Fragment sizes were observed by 2% agarose gel electrophoresis.

As shown in Figure 1, RT-PCR results showed that no amplification of CLDN6 gene was detected in normal tissues, and only A2780 cells amplified positive bands in ovarian cancer cells, while no positive bands were amplified in Hey cells.

Embodiment 2, the expression of the anti-CLDN6 single-chain antibody

Although anti-CLDN6 antibodies are available in the prior art, if they are directly evolved from monoclonal antibodies to single-chain antibodies, their antigen-binding activity and even specificity will often be affected. Through gene synthesis technology based on PCR bridging, the inventors synthesized scFv-CLDN6(64) (SEQ ID NO: 1 (nucleotide), 2 (amino acid)), scFv-CLDN6 (AE3-20LH) (SEQ ID NO: 3 (nucleotide), 4 (amino acid)), scFv-CLDN6 (AE3-20HL) (SEQ ID NO: 5 (nucleotide), 6 (amino acid)) single chain antibody sequence, through NheI/BamHI (purchased from NEB) double enzyme digestion, T4 DNA ligase (purchased from NEB) was also used to NheI/BamHI double enzyme digestion 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 Co., Ltd.) was ligated and transformed into the host strain TOP10, and the positive clones were picked and identified by PCR and confirmed by sequencing to obtain V5-scFv-CLDN6(64)-Fc, V5 - scFv-CLDN6(AE3-20LH)-Fc and V5-scFv-CLDN6(AE3-20HL)-Fc eukaryotic expression plasmids.

The above expression plasmids were transfected into well-growing HEK-293F cells, cultured continuously for 7 days at 37°C, 5% CO ₂ , 125rpm shaker, centrifuged at 4000rpm for 10min, removed the precipitate, collected the supernatant, and filtered it with a 0.45μm filter membrane. The processed samples were affinity purified with a protein A (purchased from GE) affinity column to obtain purified single-chain antibody-Fc fusion proteins scFv-CLDN6(64)-Fc, scFv-CLDN6(AE3-20LH)-Fc , scFv-CLDN6(AE3-20HL)-Fc, the identification results are shown in Figure 2.

Embodiment 3, the construction of the stable expression cell line of CLDN6

1. Construction of cell lines exogenously expressing CLDN6

The CLDN6 gene fragment (SEQ ID NO: 7 (nucleotide), 8 (amino acid)) was synthesized by the gene synthesis method based on PCR bridging, and was digested by MluI/SalI (purchased from NEB) double enzymes, and T4 DNA ligase ( (purchased from NEB) was ligated to the plasmid vector pWPT (purchased from Shanghai Ruijin Biotechnology Co., Ltd.) that was also digested with MluI/SalI and transformed into the host strain TOP10, and the positive clones were identified by PCR and confirmed by sequencing. The lentiviral plasmid pWPT-CLDN6 was obtained (Fig. 3).

Lentivirus packaging: 293T cells (7×10 ⁵ /well) were inoculated in a 6-well plate with a cell abundance of about 70% to 80%, and the cell culture medium was 2ml of antibiotic-free DMEM+10%FBS. The next day, use Lipofectamine ^TM 2000 for transfection, and prepare liposome/DNA mixture: Solution A preparation: 2 μg of pWPT-CLDN6 plasmid, 1.5 μg of pAX2 plasmid, 0.6 μg of pMD2.G plasmid, dissolved in 100 μl of serum-free DMEM for culture solution, mix gently. Solution B preparation: Dissolve 10 μl Lipofectamine ^TM 2000 in 100 μl serum-free DMEM culture medium, mix gently, and incubate at room temperature for 5 minutes. Gently mix liquid A and liquid B, and let stand at room temperature for 20 minutes. The original culture medium of the cells in the 6-well plate was discarded, and a new 1.8 ml DMEM culture medium containing 2% FBS without antibiotics was added. Then add the A/B mixture droplet into the cell culture dish, and gently blow and mix. 37°C, 5% CO ₂ continued to culture for 72h, and the supernatant virus liquid was collected.

The CLDN6 virus liquid collected above was added to the HEK-293T cells spread on a 6cm flat plate, and the cells were collected after 72 hours and lysed with cell lysate. 40 μg of the collected cell protein was subjected to SDS-PAGE gel electrophoresis, followed by immunoblotting on the gel and stained with mouse anti-flag antibody (purchased from Shanghai Ruijin Biotechnology Co., Ltd.). After washing with PBS, incubate with horseradish peroxidase-labeled goat anti-mouse antibody (purchased from Shanghai Ruijin Biotechnology Co., Ltd.), develop color with ECL reagent, and finally develop.

The results of Western blot showed that a band with a molecular weight of about 25KD could be detected in HEK-293T cells transfected with CLDN6 (ie, 293T-CLDN6), while no corresponding band was detected in untransfected empty cells (Fig. 4), indicating that the cell line exogenously expressing CLDN6 was successfully constructed.

2. Flow cytometry analysis of the binding of each cell line to the anti-CLDN6 antibody experimental steps

Analysis of single-chain antibody scFv-CLDN6 (64), scFv-CLDN6 (AE3-20LH) and scFv-CLDN6 (AE3-20HL) with the following cell lines by fluorescence activated cell sorter (FACS) (BD company, FACSCalibur) binding ability.

The specific method is as follows:

1). The 293T, 293T-CLDN6 and ovarian cancer cell line A2780 tumor cells in the logarithmic growth phase were inoculated into a 6cm plate with a cell density of about 90%, and cultured overnight in a 37°C incubator.

2). Use 10mM EDTA to digest the cells, and collect the cells by centrifugation at 200g×5min. Resuspend in 1% calf serum-containing phosphate buffered saline (NBS PBS) at a concentration of 1×10 ⁶ -1×10 ⁷ /mL, and add 100 ul/tube into a special flow tube.

3). Centrifuge at 200g×5min, discard the supernatant.

4). Add the antibodies to be tested scFv-CLDN6(64), scFv-CLDN6(AE3-20LH) and scFv-CLDN6(AE3-20HL), and use PBS as a negative control at the same time, the final concentration of the antibody is 10μg/ml, each tube Add 100ul. Ice bath, 45 minutes.

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

6). Discard the supernatant, add FITC fluorescently labeled goat anti-human antibody (from Shanghai Kangcheng Bioengineering Co., Ltd.) diluted 1:50, and add 100ul to each tube. Ice bath, 45 minutes.

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

8). The supernatant was discarded, resuspended in 300ul 1% NBS PBS, and detected by flow cytometry.

9). The flow cytometry data analysis software WinMDI 2.9 was used to analyze the data.

The results are shown in Figure 5. The results of flow cytometry analysis showed that the single-chain antibody scFv-64 did not bind to 293T CLDN6-negative cells, 293T-CLDN6 stably expressing CLDN6, or A2780 cells endogenously expressing CLDN6. However, scFv-CLDN6 (AE3-20LH) and scFv-CLDN6 (AE3-20HL) can specifically recognize 293T-CLDN6 cells stably expressing CLDN6, but do not bind to CLDN6-negative 293T cells, indicating that the two scFvs Can specifically recognize CLDN6. In addition, these two scFvs can also specifically recognize A2780 cells endogenously expressing CLDN6.

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

Table 1 explains the linkage sequence of each part of the exemplary chimeric antigen receptor of the present invention, which linkage can also be seen in FIG. 6 .

Table 1

1. Amplification of nucleic acid fragments

(1) Amplification of scFv (CLDN6-AE3-20LH, CLDN6-AE3-20HL) sequence

Using the V5-scFv-CLDN6(AE3-20LH)-Fc plasmid as a template, the primer pair used was forward primer (SEQ ID NO:9, comprising the sequence of part 2A) and reverse primer (SEQ ID NO:10, comprising part of CD8 hinge sequence), PCR amplification obtained scFv (CLDN6-AE3-20LH); similarly, using the V5-scFv-CLDN6 (AE3-20HL)-Fc plasmid as a template, the primer pair used was the forward primer (SEQ ID NO: 11 , including the sequence of part 2A) and reverse primer (SEQ ID NO: 12, including part of the CD8 hinge sequence) PCR amplification to obtain scFv (CLDN6-AE3-20HL).

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

The nucleic acid sequences of the other parts of the anti-CLDN6 chimeric antigen receptor protein except scFv (CLDN6-AE3-20LH, CLDN6-AE3-20HL) are respectively the sequence SEQ ID NO: 20 disclosed in the patent application number 201310164725.X, 201310108532.2, 23 templates were obtained by PCR. Specifically, the sequence of eGFP-F2A is obtained by PCR amplification using a primer pair (SEQ ID NO: 13, 14) using the plasmid SEQ ID NO: 20 contained in Patent Application No. 201310164725.X as a template. CD8-CD3ζ(Z) and CD28a-CD28b-CD137-CD3ζ(28BBZ), respectively as scFv(GPC3)-CD8-CD3ζ (SEQ ID NO: 20 in application patent 201310164725.X) and scFv(GPC3)-CD28a-CD28b -CD137-CD3ζ (SEQ ID NO:23 in the patent application 201310164725.X) was used as a template, and CD8-CD3ζ(Z) and CD28a-CD28b-CD137- CD3ζ(28BBZ) fragment.

2. Splicing of nucleic acid fragments

The eGFP-F2A nucleic acid fragment obtained as described above was mixed with equimolar scFv (CLDN6-AE3-20LH) or scFv (CLDN6-AE3-20HL) nucleic acid fragment and equimolar CD8-CD3ζ(Z) or CD28a-CD28b- CD137-CD3ζ(28BBZ) nucleic acid fragments were spliced into three fragments and performed PCR as shown in Figure 6. The splicing conditions were: pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s; annealing: 60°C, 40s; extension: 68°C ℃, 140s, 5 cycles, and then total extension 68 ℃, 10min, complement DNA polymerase and forward primer (SEQ ID NO:13) and reverse primer (SEQ ID NO:16) after PCR amplification for 30 cycles The amplification conditions are 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 amplified fragments are respectively called (Table 2):

eGFP-F2A-scFv-CLDN6-AE3-20LH-Z (SEQ ID NO: 17),

eGFP-F2A-scFv-CLDN6-AE3-20LH-28BBZ (SEQ ID NO: 18),

eGFP-F2A-scFv-CLDN6-AE3-20HL-Z (SEQ ID NO: 19),

eGFP-F2A-scFv-CLDN6-AE3-20HL-28BBZ (SEQ ID NO: 20).

The DNA electrophoresis identification results of the spliced eGFP-F2A-CAR fragments are shown in Figure 7.

Table 2, sequences in the present invention

3. Construction of lentiviral plasmid vector

As an example, the vector system used to construct the lentiviral plasmid vector of the present invention below belongs to the third-generation self-inactivating lentiviral vector system, and the system has three plasmids in total, namely the packaging plasmid psPAX2 encoding protein Gag/Pol and encoding Rev protein (purchased from addgene); the envelope plasmid PMD2.G encoding 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).

In the empty vector pWPT-eGFP, the self-contained elongation factor-1α (elongation factor-1α, EF-1α) promoter can regulate the expression of enhanced green fluorescent protein (eGFP), in the empty vector After inserting the construct previously constructed in this example, a recombinant expression vector encoding the target gene CAR is formed, in which eGFP and the target gene are linked by a ribosomal skipping sequence (food and mouth disease virus, FMDV, ribosomal skipping sequence, F2A) from the foot-and-mouth disease virus. Co-expression of gene CAR. F2A is a core sequence of 2A (or "self-cleaving polypeptide 2A") from foot-and-mouth disease virus. It has the "self-cleaving" function of 2A and can realize the co-expression of upstream and downstream genes. 2A provides an effective and feasible strategy for the construction of polycistronic vectors for gene therapy due to its advantages of high shear efficiency, high balance of upstream and downstream gene expression, and short sequence. 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.

In this example, a lentiviral expression vector co-expressed by F2A-linked eGFP and specific CAR was constructed, collectively referred to as pWPT-eGFP-F2A-CAR ( FIG. 8 ). The target gene eGFP-F2A-CAR obtained in the above step 2 (see 2 in Example 3, the module behind F2A is referred to as CAR) is double-digested by MluI and SalI restriction endonucleases, and connected into the same double-digested pWPT vector to construct 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 correctly, it can be prepared for lentiviral packaging. As mentioned above, eGFP-F2A-CAR is transcribed into one mRNA, but finally translated into two peptide chains of eGFP and anti-CLDN6 chimeric antigen receptor, in which the anti-CLDN6 chimeric antigen receptor will be localized under the guidance of CD8α signal peptide on the cell membrane.

The obtained vectors containing the CARs of each purpose are as follows (the components behind F2A can be referred to as CAR for short):

pWPT-eGFP-F2A-scFv(CLDN6)-AE3-20LH-Z;

pWPT-eGFP-F2A-scFv(CLDN6)-AE3-20LH-28BBZ;

pWPT-eGFP-F2A-scFv(CLDN6)-AE3-20HL-Z;

pWPT-eGFP-F2A-scFv(CLDN6)-AE3-20HL-28BBZ.

Through the above construction, four eGFP-F2A-CAR polypeptide sequences can be obtained respectively, called:

eGFP-F2A-scFv-CLDN6-AE3-20LH-Z (SEQ ID NO: 21);

eGFP-F2A-scFv-CLDN6-AE3-20LH-28BBZ (SEQ ID NO: 22);

eGFP-F2A-scFv-CLDN6-AE3-20HL-Z (SEQ ID NO: 23);

eGFP-F2A-scFv-CLDN6-AE3-20HL-28BBZ (SEQ ID NO: 24).

4. Plasmid transfection with 293T packaging lentivirus

Inoculate HEK-293T cells (ATCC: CRL-11268) from passage 6 to passage 10 (ATCC: CRL-11268) at a density of 6×10 ⁶ in a 10 cm dish, and culture overnight at 37°C in 5% CO ₂ for transfection. The medium is DMEM (purchased from PAA Company) containing 10% fetal bovine serum (purchased from PAA Company).

The steps of transfection are as follows:

4.1A solution preparation: Dissolve 10μg of mock control or 10μg of each target gene plasmid pWPT-eGFP-F2A-CAR with 7.5μg of packaging plasmid PAX2: and 3μg of envelope plasmid pMD2.G, respectively, into 800μL of serum-free DMEM culture medium medium, mix well.

4. Preparation of Solution 2B: Dissolve 60 μg PEI (polyethyleneimine, purchased from Polysciences) in 800 μL serum-free DMEM culture solution, mix gently, and incubate at room temperature for 5 minutes.

4.3 Formation of transfection complex: add solution A to solution B and mix gently, vortex or mix gently immediately after adding, and incubate at room temperature for 20 minutes.

4.4 Add 1.6ml of the transfection complex dropwise into the HEK-293T cells, and after 4-5 hours, replace the medium of the transfected 293T cells with 2% FBS in DMEM.

The transfection efficiency (that is, the proportion of cells showing green fluorescence) was observed on the second day after transfection, and the transfection experiment was successful if the positive transfection efficiency was ~80%. After 72 hours of transfection, use a 0.45 μm filter (purchased from Millipore Company) to filter and collect the virus, then use a Beckman Optima L-100XP ultracentrifuge at 28,000 rpm, centrifuge at 4°C for 2 hours, discard the centrifuged supernatant, and centrifuge the obtained precipitate with 1/10 Quantum 007 culture solution (purchased from PAA Company) was resuspended at ~1/50 volume of the original solution, and frozen in 100 μL/tube at -80°C, pending virus titration or infection of T lymphocytes.

5. Determination of the titer of lentivirus packaged with mock or eGFP-F2A-CAR

On the first day, 293T cells were inoculated in 96-well culture plate at 1×10 ⁵ /mL, 100 μL/well, 37°C, 5% CO2 culture, and the culture medium was DMEM containing 10% fetal bovine serum. On the next day, discard 50 μL/well of the culture supernatant, add 50 μL/well of fresh above-mentioned culture solution containing polybrene with a final concentration of 6 μg/mL, and incubate at 37°C with 5% CO2 for 30 minutes. Add 10 μL/well of virus stock solution or 1 μL/well of virus concentrate, 5-fold dilution, 4 gradients, two duplicate wells, 37 ° C, 5% CO ₂ culture. 48 hours after infection, eGFP was detected by flow cytometry, and the number of cells with a positive rate of 5-20% was suitable, and the calculated titer (U/mL) = positive rate × dilution factor × 100 × 10 ⁴ . The titers of the above-mentioned viruses containing the mock vector control and each eGFP-F2A-CAR packaged by the PEI transfection method were at a level of about 2 to 4×10 ⁶ U/mL, and the titers of the viruses measured after concentration were about It is 2～4×10 ⁷ U/mL.

Example 4, Recombinant lentivirus infection of CTL cells

Human peripheral blood mononuclear cells (provided by Shanghai Blood Center) were obtained from the peripheral blood of healthy people by density gradient centrifugation, and CTL were obtained from peripheral blood mononuclear cells by CTL cell magnetic beads (purchased from Stem Cell Technologies). The sorted CTL cells are subjected to flow cytometry to detect the purity of the CTL cells, and it is advisable to proceed to the next step with the positive rate of the CTL cells ≥ 95%. Add Quantum 007 lymphocyte culture medium (purchased from PAA Company) at a density of about 1×10 ⁶ /mL for culture, and add magnetic beads coated with anti-CD3 and CD28 antibodies (Invitrogen company) and recombinant human IL-2 (purchased from Shanghai Huaxin Biotech Co., Ltd.) at a final concentration of 300 U/mL were stimulated for 24 h. Then the CTL cells were infected with the above-mentioned recombinant lentivirus at MOI ≈ 5. The infected cells were subcultured at a density of 5×10 ⁵ /mL every other day, and at the same time, recombinant human IL-2 with a final concentration of 300 U/mL was added to the lymphocyte culture medium.

The expression of different chimeric antigen receptors was detected by flow cytometry on the 8th day of culture of the infected CTL cells. Since eGFP and CAR were co-expressed, the positive cells detected for eGFP were positive cells expressing chimeric antigen receptors. Using uninfected T lymphocytes as a negative control, the positive rates of virus-infected CTL cells expressing different chimeric antigen receptors are shown in Table 3. The result of the positive rate indicates that CAR ⁺ CTL cells with a certain positive rate can be obtained by lentiviral infection.

table 3

After CTL cells were respectively infected with viruses packaged with different chimeric antigen receptors, they were subcultured every other day at a cell density of 5×10 ⁵ /ml, counted, and IL-2 was added to the subcultured cell culture medium (final concentration: 300U/ml), there was about 20-40 times expansion on the 11th day of culture, indicating that CTL cells expressing different chimeric antigen receptors can undergo a certain amount of expansion in vitro, which provides a basis for subsequent in vitro toxicity tests and in vivo tests. ensure.

Example 5. In vitro toxicity effect experiment of T lymphocytes expressing chimeric antigen receptors

The materials used in the in vitro toxicity test are as follows:

293T and ovarian cancer cells as shown in Table 4 are used as target cells, and the effector cells are positive cells detected by FACS of 12 days of in vitro culture as verified in Example 4, and the expression of chimeric antigen receptor is recorded as positive for chimeric antigen receptor ( CAR ⁺ ) CTL, the effector-to-target ratio is 3:1, 1:1 and 1:3 depending on the situation, and the number of target cells is 10,000/well, and the corresponding effector cells are based on different effector-target ratios. Each group had 5 replicate holes, and the average value of the 5 replicate holes was taken. The detection time is the 18th hour.

Wherein each experimental group and each control group are as follows:

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

Control group 1: Target cells release LDH maximally,

Control group 2: Target cells release LDH spontaneously,

Control group 3: Effector cells release LDH spontaneously.

Detection method: CytoTox 96 non-radioactive cytotoxicity detection kit (Promega Company) was used. This method is a detection method based on colorimetry, which can replace the ⁵¹ Cr release method. The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis in much the same way as ⁵¹ Cr is released in radioactive assays. The released LDH medium 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 produced is directly proportional to the number of cells lysed. For details, refer to the instructions of the CytoTox 96 non-radioactive cytotoxicity detection kit.

The formula for calculating cytotoxicity is:

Specifically, as shown in Table 4 and Table 5, the CTL and CLDN6- expressing chimeric antigen receptor (fused to express single-chain antibody CLDN6 (AE3-20LH) or CLDN6 (AE3-20HL)) CLDN6-Z CAR ⁺ and CLDN6- 28BBZ CAR ⁺ CTLs had a significant killing effect on 293T cells with high expression of CLDN6, but had no killing effect on CLDN6-negative 293T cells, indicating that they could selectively kill CLDN6-expressing cells. In addition, the CTLs expressing the chimeric antigen receptor CLDN6-Z CAR ⁺ and the CTLs CLDN6-28BBZ CAR ⁺ of the present invention can also have significant killing effects on ovarian cancer cells A2780 endogenously expressing CLDN6 (see Table 4 and Table 5 ), and showed a gradient-dependent effect-to-target ratio. The higher the target-to-effect ratio, the stronger the cytotoxic effect.

The data of effect-target ratio dependence further showed that the CTL expressing the anti-CLDN6 chimeric antigen receptor of the present invention has specific cytotoxic effect on cells highly expressing CLDN6.

In comparison, the negative control CTL transfected with a virus containing a mock plasmid (carrying scFv-CLDN6-δZ) showed very low cytotoxic effects on the above three cell lines with high expression of CLDN6. Its cytotoxicity to cell lines highly expressing CLDN6 is significantly different from the cytotoxicity data of CTLs expressing the anti-CLDN6 chimeric antigen receptor of the present invention.

The above results show that the chimeric antigen receptor constructed by selecting the single-chain antibody against CLDN6 can selectively kill the target cells with high expression of CLDN6. In addition, from the cytotoxicity data, the CAR T of CLDN6-28BBZ is more toxic to the cells expressing CLDN6 than the CAR T of CLDN6-Z.

Table 4. Cytotoxicity of CAR T cells fused to express single-chain antibody CLDN6 (AE3-20LH)

Table 5. Cytotoxicity of CAR T cells fused to express single-chain antibody CLDN6 (AE3-20HL)

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 (15)

1. A chimeric antigen receptor expressed on the surface of immune effector cells, characterized in that the chimeric antigen receptor comprises sequentially connected: an extracellular binding region, a transmembrane region and an intracellular signal region, wherein the cell The extracellular binding region includes a protein that specifically recognizes CLDN6; wherein, the extracellular binding region is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO:4 or SEQ ID NO:6. 2. The chimeric antigen receptor according to claim 1, wherein the immune effector cells include: T lymphocytes, NK cells or NKT cells. 3. The chimeric antigen receptor according to claim 1, wherein 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, wherein the intracellular signal region is selected from the group consisting of CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134 intracellular signal region sequences, or a combination thereof . 5. The chimeric antigen receptor according to claim 1, characterized in that, said chimeric antigen receptor comprises an extracellular binding region, a transmembrane region and an intracellular signal region connected in sequence as follows: Single-chain antibodies that specifically recognize CLDN6, CD8 and CD3ζ; Single-chain antibodies that specifically recognize CLDN6, CD8, CD137 and CD3ζ; A single-chain antibody that specifically recognizes CLDN6, the transmembrane region of CD28 molecule, the intracellular signal region of CD28 molecule and CD3ζ; or The single-chain antibody specifically recognizes CLDN6, the transmembrane region of CD28 molecule, the intracellular signal region of CD28 molecule, CD137 and CD3ζ. 6. The chimeric antigen receptor according to claim 1, characterized in that, the chimeric antigen receptor has the amino acid sequence shown in any one of SEQ ID NO: 21-24. 7. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1-6. 8. The nucleic acid according to claim 7, wherein the nucleic acid has the nucleotide sequence shown in any one of SEQ ID NO: 17-20. 9. An expression vector, characterized in that it comprises the nucleic acid according to any one of claims 7-8. 10. The expression vector according to claim 9, wherein the expression vector is derived from lentiviral plasmid pWPT. 11. A virus, characterized in that the virus comprises the vector according to claim 9 or 10. 12. The chimeric antigen receptor described in any one of claims 1-6, or the nucleic acid described in claim 7 or 8, or the expression vector described in claim 9 or 10, or the virus described in claim 11 The method is used for preparing genetically modified immune effector cells targeting CLDN6. 13. A genetically modified immune effector cell, characterized in that it is transduced with the nucleic acid according to claim 7 or 8, or the expression vector according to claim 9 or 10, or the virus according to claim 11. 14. A genetically modified immune effector cell, characterized in that it expresses a chimeric antigen receptor on its surface, and the amino acid sequence of the chimeric antigen receptor is selected from any of SEQ ID NO:21-24 amino acid sequence. 15. The use of the genetically modified immune effector cells according to claim 13 or 14, characterized in that it is used for the preparation of drugs for suppressing tumors, and the tumors are CLDN6-positive tumors.