WO2016150400A1 - 靶向cldn6的免疫效应细胞及其制备方法和应用 - Google Patents

靶向cldn6的免疫效应细胞及其制备方法和应用 Download PDF

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WO2016150400A1
WO2016150400A1 PCT/CN2016/077341 CN2016077341W WO2016150400A1 WO 2016150400 A1 WO2016150400 A1 WO 2016150400A1 CN 2016077341 W CN2016077341 W CN 2016077341W WO 2016150400 A1 WO2016150400 A1 WO 2016150400A1
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cldn6
chimeric antigen
antigen receptor
cells
region
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French (fr)
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王华茂
宋波
蔡秀梅
赵红霞
杨琳琳
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科济生物医药（上海）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of tumor cell therapy, and more particularly, the invention relates to an immune effector cell targeting CLDN6 and a preparation method and application thereof.
  • CTLs cytotoxic lymphocytes
  • TCR T Cell Receptor
  • CAR chimeric antigen receptor
  • MHC major Histocompatibility Complex
  • CAR T lymphocytes are a new immunotherapeutic 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.] .
  • CAR modified NK cells Klingemann H. Challenges of cancer therapy with natural killer cells.
  • 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 extracellular binding regions, transmembrane regions, and intracellular signaling regions.
  • the extracellular region contains a scFv capable of recognizing a tumor-associated antigen
  • a transmembrane region adopts a transmembrane region of a molecule such as CD8, CD28
  • the intracellular signal region adopts an immunoreceptor tyrosine activation motif (ITAM) CD3 ⁇ or Fc ⁇ RI ⁇ and co-stimulation.
  • ITAM immunoreceptor tyrosine activation motif
  • the intracellular signal region of the signaling molecules CD28, CD27, CD137, CD134, and the like.
  • the intracellular signal domain contains only ITAM as the first generation of CAR T lymphocytes, wherein the chimeric antigen receptor portions are linked as follows: scFv-TM-ITAM.
  • This kind of CAR T can stimulate the anti-tumor cytotoxic effect, but the cytokine secretion is relatively small, and can not stimulate the 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.].
  • CD28 or CD137 aka 4-1BB
  • the chimeric antigen receptor moieties were joined as follows: scFv-TM-CD28-ITAM or scFv -TM-/CD137-ITAM.
  • Co-stimulation of B7/CD28 or 4-1BBL/CD137 in the intracellular signal domain causes sustained proliferation of T lymphocytes, and can increase the secretion of cytokines such as IL-2 and IFN- ⁇ by T lymphocytes, and increase CAR T Survival cycle and anti-tumor effect 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.].
  • CAR T lymphocytes have attractive prospects in tumor immunotherapy, their higher risks also need to be considered.
  • specific antigens that can be recognized by the low expression of CAR in certain normal tissues may cause damage to the normal tissues of CAR T lymphocytes expressing the corresponding antigen.
  • CAIX antigen carbonic anhydrase IX
  • the antigen carbonic anhydrase IX (CAIX) expressed on tumor cells of patients with renal cell carcinoma is the first case for the clinical treatment of CAR T lymphocytes. It is also the first case to report the off-target effect of CAR cells. . Patients developed grade 2-4 hepatotoxicity after multiple injections of CAR T lymphocytes.
  • liver bile duct epithelial cells have low expression of CAIX, and the original clinical trial was interrupted while excluding any evaluation of the patient's therapeutic effect [Stoter G. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX : first clinical experience. J clin oncol, 2006, 24(13): e20-e22.; Ngo MC., et al. Ex vivo gene transfer for improved adoptive immunotherapy of cancer. Human Molecular Genetics, 2011, R1-R7].
  • CAR-modified immune effector cells especially T cells
  • the antigenic gene targeted is actually a key choice.
  • CAR CAR-modified immune effector cells
  • the genes are very difficult.
  • tumor-specific antigens are difficult to find and suitable for them.
  • EGFR targets can up-regulate PD-L1 expression (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.
  • a chimeric antigen receptor expressed on the surface of an immune effector cell comprising a sequence-linked: extracellular binding region, a transmembrane region and an intracellular signal a region wherein the extracellular binding region comprises a protein that specifically recognizes CLDN6 (Claudin 6).
  • the immune effector cells comprise: T lymphocytes, NK cells or NKT cells.
  • the protein that specifically recognizes CLDN6 is an antibody or a ligand; preferably, the antibody is a single chain antibody or a domain antibody.
  • the transmembrane region is a sequence comprising a transmembrane region and a hinge region of CD8 or CD28.
  • the intracellular signal region is selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, an intracellular signal sequence of CD134, or a combination thereof.
  • the chimeric antigen receptor comprises a sequence-linked extracellular binding region, a transmembrane region and an intracellular signal region:
  • CD28a a transmembrane region of CD28
  • CD28b an intracellular signal region of CD28 molecule
  • CD3 ⁇ a single-chain antibody that specifically recognizes CLDN6, a transmembrane region of CD28 (CD28a), an intracellular signal region (CD28b) of CD28 molecule, and CD3 ⁇ ; or
  • the chimeric antigen receptor has the amino acid sequence of any one of SEQ ID NOS: 21-24.
  • nucleic acid encoding a chimeric antigen receptor according to any of the preceding claims is provided.
  • the nucleic acid has the nucleotide sequence of any one of SEQ ID NOS: 17-20.
  • an expression vector comprising the nucleic acid of any of the foregoing is provided.
  • the expression vector is derived from the lentiviral plasmid pWPT (or pWPT-eGFP).
  • a virus comprising the vector.
  • the virus e.g., a lentivirus
  • a genetically modified immune effector cell transduced with said nucleic acid, or said expression vector or said virus.
  • a genetically modified immune effector cell having a surface expressing a chimeric antigen receptor, the amino acid sequence of the chimeric antigen receptor being selected from any one of SEQ ID NO: 21-24 The amino acid sequence.
  • the use of the genetically modified immune effector cell for the preparation of a tumor suppressing drug, the tumor being a CLDN6 positive tumor.
  • the CLDN6 positive tumor comprises: ovarian cancer, gastric cancer, lung adenocarcinoma.
  • Figure 3 Schematic representation of a lentiviral expression vector encoding CLDN6.
  • FIG. 1 Western blot analysis 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 individual cell lines to anti-CLDN6 antibodies.
  • Figure 6 Schematic diagram of the sequence of ligation of the various portions of the chimeric antigen receptor.
  • Figure 7 Identification of five kinds of spliced eGFP-F2A-CAR fragments by DNA electrophoresis.
  • Figure 8 Schematic diagram of the construction of the lentiviral vector pWPT-eGFP-F2A-CAR encoding the CAR sequence of the present invention as an example.
  • the inventors have for the first time revealed a CAR-modified immune effector cell based on CLDN6 gene and a preparation method thereof.
  • the present inventors have previously investigated a large number of tumor-specific genes, and found that a considerable part of such genes are also expressed in normal cells of some tissues, and it is difficult to apply to immune effector cell technology of chimeric antigen receptor modification; some tumors
  • the specific gene has better tumor-specific expression characteristics.
  • the CAR-modified immune effector cells based on its design have no tumor cell killing activity or low activity, which may be because the target can induce tumor cell secretion and immune effect.
  • the cell has an inhibitory factor such as PD-L1.
  • CLDN6 The amino acid sequence of Claudin6 (CLDN6) and its gene sequence are disclosed as GenBank accession number NP_067018.1 and NM_021195.3 (serial number: 22 and serial number: 23), or GenBank accession number NP_067018.2 and NM_021195.4 (sequence respectively). No.: 46 and serial number: 47).
  • Claudins are membrane proteins located in the tight junction of the epithelium and the endothelium.
  • CAR T cells have become a potential treatment.
  • many tumors have not been reported on the treatment of CAR T cells, such as gastric cancer.
  • Studies have shown that CLDN6 is a specific marker of gastric tissue.
  • the present inventors have confirmed that CLDN6 is not expressed in most normal tissues but is expressed in some tumors such as ovarian cancer.
  • the current drug candidate for CLDN6 as a therapeutic target is only a monoclonal antibody, and whether it can be successfully used for the corresponding tumor treatment is still unknown. Therefore, it is necessary to find new treatments.
  • the inventors contemplate that if a targeted therapy can be performed with CAR T cells, a new anticancer agent is expected.
  • the CLDN6 antigen is a tight junction protein, and it is not known whether it can be contacted by CAR T cells and trigger the killing of the corresponding target cells.
  • many monoclonal antibodies evolve into single-chain antibodies, which often lose antigen binding activity or specificity.
  • two single chain antibodies AE3-20LH and AE3-20HL
  • CAR T cells composed of these two single chain antibodies retain a selective killing effect on CLDN6 positive cells.
  • the results of the present invention indicate that CLDN6 can indeed be a target for CAR-modified immune effector cells, particularly T cell therapy; CAR T cells against CLDN6 are a new candidate for tumor therapy.
  • CLDN6-targeted CAR-modified T cells do selectively remove CLDN6-positive tumor cells and are not toxic to non-tumor cell 293T cells.
  • the present inventors believe that the corresponding CAR-modified immune effector cells, particularly T cells, should be useful for the treatment of human tumors.
  • the present invention provides a chimeric antigen receptor expressed on the surface of an immune effector cell, the chimeric antigen receptor comprising a sequence-linked: an extracellular binding region, a transmembrane region and an intracellular signal region, wherein the cell
  • the outer binding region comprises a protein that specifically recognizes CLDN6 (claudin 6).
  • CLDN6 claudin 6
  • the chimeric antigen receptor is expressed on the surface of the immune effector cell,
  • the immune effector cells have a highly specific cytotoxic effect on tumor cells highly expressing CLDN6.
  • the extracellular binding region comprises a single chain antibody scFv (CLDN6) that specifically recognizes CLDN6.
  • CLDN6 single chain antibody scFv
  • the extracellular binding region of the above chimeric antigen receptor protein is linked to the transmembrane region of CD8 or CD28 via the CD8 hinge region, and the intracellular signal region is followed by the transmembrane region.
  • the invention also includes nucleic acids encoding the chimeric antigen receptors.
  • the nucleic acid sequence of the invention may be in the form of DNA or in the form of RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • 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 sequences of the chimeric antigen receptor proteins of the present invention may be degenerate, that is, a plurality of degenerate nucleic acid sequences encoding the same amino acid sequence are included in the scope of the present invention. Degenerate nucleic acid codons encoding corresponding amino acids are well known in the art.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the 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.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the antibody specifically recognizing human CLDN6 can be selected from the antibodies disclosed in the prior art, and a plurality of monoclonal antibodies recognizing the C-terminal epitope of CLDN18A2 can be applied to the present invention in a suitable manner, and finally a CAR immune effect having killing activity can be obtained. cell.
  • the antibody of human CLDN6 is the single-chain antibody scFv-CLDN6 (AE3-20LH) or scFv-CLDN6 (AE3-20HL) of the present invention.
  • single-chain antibody (scFv) fragment refers to an antibody fragment defined by a heavy chain variable region (VH) and a light chain variable region which are linked by a linker ( The recombinant protein of VL), the linker associates these two domains to ultimately form an antigen binding site.
  • the size of scFv is typically 1/6 of that of an intact antibody.
  • the single chain antibody is preferably a sequence of one amino acid strand encoded by one nucleotide chain.
  • the single-chain antibodies used in the present invention may be further modified, either singly or in combination, using conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations, and/or other modifications. Methods for introducing such modifications into their DNA sequences based on the amino acid sequence of an antibody are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2002) N.Y. The indicated modifications are preferably carried out at the nucleic acid level.
  • the above single chain antibodies may also include derivatives thereof.
  • the "derivative of an antibody” in the present invention includes, for example, when a derivative of the antibody is obtained by phage display technology, surface plasmon resonance technology such as used in the BIAcore system can be used to increase the efficiency of phage antibody binding to a CLDN6 epitope. (Schier, Human Antibody Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the term "specific recognition" in the present invention means that the bispecific antibody of the present invention does not or substantially does not Any polypeptide other than the target antigen cross-reacts.
  • the degree of specificity can be judged by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.
  • specific recognition is preferably determined by flow cytometry, and the criteria for specific recognition in specific cases can be judged by one of ordinary skill in the art based on the common knowledge in the art.
  • the transmembrane region of the chimeric antigen receptor can be selected from the transmembrane region of a protein such as CD8 or CD28.
  • the human CD8 protein is a heterodimer composed of two chains, ⁇ or ⁇ .
  • the transmembrane region is selected from the transmembrane region of CD8 alpha or CD28.
  • the CD8 ⁇ hinge region is a flexible region, and therefore, CD8 or CD28 and a transmembrane region plus a hinge region are used to link the target recognition domain scFv of the chimeric antigen receptor CAR with the intracellular signal region. .
  • the intracellular signal region can be selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD28, CD137, the intracellular signal region of the CD134 protein, and combinations thereof.
  • the CD3 molecule consists of five subunits, of which the CD3 ⁇ subunit (also known as CD3zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex.
  • CD3 ⁇ Z is a truncated CD3 ⁇ sequence that does not have an ITAM motif and is generally constructed as a negative control in the practice of the present invention.
  • Fc ⁇ RI ⁇ is mainly distributed on the surface of mast cells and basophils, which contains an ITAM motif similar in structure, distribution and function to CD3 ⁇ .
  • CD28, CD137, and CD134 are costimulatory signal molecules, and the co-stimulatory effects of intracellular signal segments upon binding to their respective ligands cause sustained proliferation of immune effector cells (mainly T lymphocytes). It can also increase the levels of cytokines such as IL-2 and IFN- ⁇ secreted by immune effector cells, and increase 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 may be selected from the group consisting of:
  • CD28a in the relevant chimeric antigen receptor protein represents the transmembrane region of the CD28 molecule
  • CD28b represents the intracellular signal region of the CD28 molecule.
  • the various anti-CLDN6 chimeric antigen receptors described above are collectively referred to as scFv (CLDN6)-CAR.
  • the nucleic acid of the invention has the sequence set forth in SEQ ID NOs: 17-20. In another embodiment of the invention, the nucleic acid of the invention is a nucleic acid encoding a chimeric antigen receptor protein having one of SEQ ID NOs: 21-24.
  • the present invention also provides a vector comprising the above nucleic acid encoding a chimeric antigen receptor protein expressed on the surface of an immune effector cell.
  • the vector used in the present invention is a lentiviral plasmid vector pWPT-eGFP. This plasmid belongs to the third generation auto-inactivated lentiviral vector system.
  • the system has three plasmids, namely, the coding protein Gag/Pol, the packaging plasmid psPAX2 encoding Rev protein, and the envelope plasmid PMD2.G encoding VSV-G protein;
  • the vector pWPT-eGFP which can be used for recombinant introduction of a nucleic acid sequence of interest, ie a nucleic acid sequence encoding a CAR.
  • the empty vector pWPT-eGFP (which itself is a mock in subsequent experiments) regulates the expression of enhanced green fluorescent protein (eGFP) by the elongation factor-1 ⁇ (elongation factor-1 ⁇ , EF-1 ⁇ ) promoter. .
  • the recombinant expression vector pWPT-eGFP-F2A-CAR comprising the nucleic acid sequence of interest encoding CAR was obtained by ribosomal skipping sequence 2A (F2A). ) to achieve co-expression of eGFP and CAR.
  • the invention also includes viruses comprising the vectors described above.
  • the virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious virus.
  • Other viruses known in the art that can be used to transduce foreign genes into immune effector cells and their corresponding plasmid vectors can also be used in the present invention.
  • the virus is a lentivirus comprising the above pWPT-eGFP-F2A-CAR recombinant vector (ie containing scFv (CLDN6)-CAR).
  • the present invention also provides a genetically modified immune effector cell transduced with the nucleic acid of the present invention or the above-described recombinant plasmid comprising the nucleic acid of the present invention, or a virus comprising the same.
  • Conventional nucleic acid transduction methods 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 Nucleofector nuclear transfection device developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of the target gene.
  • the transduction efficiency of the transposon system based on Sleeping Beauty system or PiggyBac transposon is much higher than that of ordinary electroporation, and the nucleofector transfection apparatus is combined with the Sleeping Beauty transposon system. It 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.], The method has high transduction efficiency and can achieve targeted integration of the target gene.
  • the transduction method for the immune effector cells that effect the chimeric antigen receptor gene modification is based on a transduction method of a virus such as a retrovirus or a lentivirus.
  • the method has the advantages of high transduction efficiency, stable expression of the exogenous gene, and shortening the time for the cultured immune effector cells to reach the clinical level in vitro.
  • the transduced nucleic acid is expressed on its surface by transcription and translation.
  • the anti-CLDN6 chimeric antigen receptor gene-modified immune effector cells of the present invention have a highly specific tumor cell killing effect (also called cytotoxicity) by in vitro cytotoxicity experiments on various cultured tumor cells.
  • the nucleic acid encoding the chimeric antigen receptor protein of the present invention, the plasmid comprising the nucleic acid, the virus comprising the plasmid, and the transgenic immune effector cell transduced with the nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.
  • the genetically modified immune effector cell of the invention expresses a chimeric antigen receptor on its surface, said chimeric antigen receptor being encoded by a nucleic acid encoding one of SEQ ID NOs: 17-20.
  • the transgenic immune effector cell of the present invention expresses a chimeric antigen receptor on the surface, and the amino acid sequence of the chimeric antigen receptor is selected from one of SEQ ID NOS: 20-24.
  • CAR T targeting CLDN6 In view of the fact that there is no report on CAR T targeting CLDN6, the inventors have successfully found a target gene CLDN6 suitable for CAR T cells from many tumor-related genes, and successfully prepared a CAR T targeting CLDN6. Cells, thus providing a new treatment for ovarian cancer, gastric cancer, lung adenocarcinoma and other tumors.
  • RNA Human normal tissue RNA was purchased from Clontech Laboratories, Inc. Ovarian cancer cells were routinely cultured in a 5% CO 2 incubator at 37 °C. The cells were washed twice with PBS (0.14 M NaCl, 2.7 mM KCl, 10.1 mM Na 2 HPO4, 1.8 mM KH 2 PO 4, pH 7.3).
  • RNA of the well-preserved cells was extracted with Trizol reagent, and 1 ml of Trizol was added to each dish, and thoroughly blown until it was not viscous; 0.2 ml of freshly opened chloroform was added per milliliter of original volume of Trizol, vigorously shaken for 15 sec, and incubated at room temperature for 5 min; Centrifuge at 12,000g for 15min at °C; transfer the colorless supernatant to a new centrifuge tube, add 0.5ml isopropanol per milliliter of original volume Trizol, incubate for 20min at -20°C, centrifuge at 12,000g for 10min at 4°C; Washed with 1 ml of 75% ethanol, centrifuged at 10,000 g for 5 min at 4 ° C; the RNA precipitate was dried at room temperature for 10-20 min, and the precipitate was dissolved in RNase-free H 2 O treated with DEPC. The RNA was quantified by spectrophotometry.
  • Reverse transcription 1 ⁇ l of total cellular RNA (1 ⁇ g/ ⁇ l) and Oligo dT (10 ⁇ M) 1 ⁇ l were mixed, and incubated at 72 ° C for 5 min, immediately placed on ice for 5 min; 15 ⁇ l of reverse transcription mixture (Nuclease-free H2O 6.1 ⁇ l, 5 ⁇ RT Buffer 4 ⁇ l, 25 mM MgCl 2 2.4 ⁇ l, 10 mM dNTPs 1 ⁇ l, RNase Ribonuclease Inhibitor 0.5 ⁇ l, Improm-IITM Reverse Transcriptase 1 ⁇ l), incubation at 25 ° C for 5 min, incubation at 42 ° C for 1 h; incubation at 70 ° C for 15 min to terminate the reaction.
  • reverse transcription mixture Nuclease-free H2O 6.1 ⁇ l, 5 ⁇ RT Buffer 4 ⁇ l, 25 mM MgCl 2 2.4 ⁇ l, 10 mM
  • RT-PCR using the above reverse transcribed cDNA as a template, PCR was carried out with primers CLDN6-F: GGAATCGAGATCCTGGGAGT, CLDN6-R: ATGAAAGCGGTCA, PCR reaction system: 1 ⁇ l template, 2.5 ⁇ l 2 mM dNTP, 2.5 ⁇ l 10 ⁇ buffer, primers L ⁇ l, 5U Taq enzyme, supplemented with 25 ⁇ l with H 2 O. PCR reaction conditions: 94 ° C for 3 min; 94 ° C for 30 sec, 56 ° C for 30 sec, 72 ° C for 40 sec, 35 cycles; 72 ° C for 10 min; 4 ° C storage.
  • ⁇ -actin was amplified as an internal reference, and ⁇ -actin primer: ⁇ -actin-F: 5'-TCCTCCCTGGAGAAGAGCTA-3', ⁇ -actin-R: 5'-GTACTTGCGCTCAGGAGGAG-3'.
  • Amplification conditions 94 ° C for 3 min; 94 ° C for 30 sec, 58 ° C for 30 sec, 72 ° C for 30 sec, 30 cycles; 72 10 min ° C.
  • the fragment size was observed by 2% agarose gel electrophoresis.
  • RT-PCR results showed that no amplification of CLDN6 gene was detected in normal tissues. Only A2780 cells in ovarian cancer cells amplified positive bands, while positive bands were not amplified in Hey cells.
  • Antibodies against CLDN6 although available in the prior art, often affect their antigen binding activity, even specificity, if they evolve directly from monoclonal antibodies to single chain antibodies.
  • the inventors synthesized scFv-CLDN6 (64) (SEQ ID NO: 1 (nucleotide), 2 (amino acid)), scFv-CLDN6 (AE3-20LH) (SEQ ID NO) by PCR-based gene synthesis technology.
  • V5-scFv-CLDN6(64)-Fc was obtained.
  • V5-scFv-CLDN6(AE3-20LH)-Fc and V5-scFv-CLDN6(AE3-20HL)-Fc eukaryotic expression plasmids.
  • the above expression plasmids were transfected into well-preserved HEK-293F cells, cultured continuously for 7 days at 37 ° C, 5% CO 2 , 125 rpm shaker, centrifuged at 4000 rpm for 10 min, the precipitate was removed, and the supernatant was collected and filtered through a 0.45 ⁇ m filter.
  • the processed sample was affinity-purified with a protein A (purchased from GE) affinity column to finally obtain a purified single-chain antibody-Fc fusion protein scFv-CLDN6(64)-Fc, scFv-CLDN6(AE3-20LH)-Fc. , scFv-CLDN6 (AE3-20HL)-Fc, the identification results are shown in Figure 2.
  • the CLDN6 gene fragment (SEQ ID NO: 7 (nucleotide), 8 (amino acid) was synthesized by PCR-based gene synthesis method, and double-digested with MluI/SalI (purchased from NEB) to obtain T4 DNA ligase (purchased From NEB), the plasmid vector pWPT (purchased from Shanghai Ruijin Biotechnology Co., Ltd.), which was also digested with MluI/SalI, was ligated and transformed into the host strain TOP10, and the clone was picked to identify positive clones by PCR and confirmed by sequencing. Lentiviral plasmid pWPT-CLDN6 ( Figure 3).
  • Packaging of lentivirus 293T cells (7 ⁇ 10 5 /well) were seeded in 6-well plates with a cell abundance of about 70% to 80%, and the cell culture medium was 2 ml of antibiotic-free DMEM + 10% FBS. The next day, transfection was performed using Lipofectamine TM 2000 to prepare a liposome/DNA mixture: A solution: 2 ⁇ g of pWPT-CLDN6 plasmid, 1.5 ⁇ g of pAX2 plasmid, 0.6 ⁇ g of pMD2.G plasmid, and dissolved in 100 ⁇ l of serum-free DMEM. Mix gently in the solution.
  • Preparation of solution B 10 ⁇ l of Lipofectamine TM 2000 was dissolved in 100 ⁇ l of serum-free DMEM medium, gently mixed, and incubated at room temperature for 5 min. Mix liquid A and liquid B gently and let stand for 20 minutes at room temperature. The original cell culture medium in the 6-well plate was discarded, and a new 1.8 ml antibiotic-free DMEM medium containing 2% FBS was added. The A/B mixed droplets were then added to the cell culture dish and gently mixed by pipetting. The culture was continued for 72 h at 37 ° C, 5% CO 2 , and the supernatant virus solution was collected.
  • the collected CLDN6 virus solution was separately added to HEK-293T cells plated in a 6 cm plate, and after 72 hours, the cells were collected and lysed using a cell lysate.
  • 40 ⁇ g of the lysed collected cell protein was subjected to SDS-PAGE gel electrophoresis, and then the gel was immunoblotted and stained with a mouse anti-flag antibody (purchased from Shanghai Ruijin Biotechnology Co., Ltd.). After washing with PBS, it was incubated with horseradish peroxidase-labeled goat anti-mouse antibody (purchased from Shanghai Ruijin Biotechnology Co., Ltd.), developed with ECL reagent, and finally developed.
  • the single-chain antibody scFv-CLDN6 (64), scFv-CLDN6 (AE3-20LH) and scFv-CLDN6 (AE3-20HL) were each analyzed with the following cell lines by fluorescence activated cell sorter (FACS) (BD, FACSCalibur). Binding ability.
  • 293T, 293T-CLDN6 and ovarian cancer cell line A2780 tumor cells in logarithmic growth phase were inoculated into 6 cm dishes, inoculated with a cell density of about 90%, and cultured overnight at 37 °C.
  • the cells were digested with 10 mM EDTA, and the cells were collected by centrifugation at 200 g ⁇ 5 min.
  • the cells were resuspended in 1% of calf serum-containing phosphate buffer (NBS PBS) at a concentration of 1 ⁇ 10 6 to 1 ⁇ 10 7 /mL, and added to a flow-type dedicated tube in an amount of 100 ul / tube.
  • NBS PBS calf serum-containing phosphate buffer
  • test antibody scFv-CLDN6 (64), scFv-CLDN6 (AE3-20LH) and scFv-CLDN6 (AE3-20HL), respectively, and use PBS as a negative control.
  • the final concentration of the antibody is 10 ⁇ g/ml. Add 100ul. Ice bath, 45 minutes.
  • Example 3 Construction of a lentiviral plasmid expressing a chimeric antigen receptor protein encoded by a nucleic acid of the present invention and virus packaging
  • Table 1 explains the order of attachment of the various portions of the chimeric antigen receptor of the present invention, which can also be seen in Figure 6.
  • V5-scFv-CLDN6(AE3-20LH)-Fc plasmid as a template, the primer pair primer (SEQ ID NO: 9, comprising the sequence of Part 2A) and the reverse primer (SEQ ID NO: 10, including the part) CD8hinge sequence), PCR amplification obtained scFv (CLDN6-AE3-20LH); similarly, using V5-scFv-CLDN6 (AE3-20HL)-Fc plasmid as template, primer pair positive primer (SEQ ID NO: 11) The sequence comprising part 2A) and the reverse primer (SEQ ID NO: 12, comprising a partial CD8hinge sequence) were PCR amplified to obtain scFv (CLDN6-AE3-20HL).
  • the nucleic acid sequence of the anti-CLDN6 chimeric antigen receptor protein other than scFv is SEQ ID NO: 20 as disclosed in Patent Application No. 201310164725.X, 201310108532.2, respectively.
  • , 23 is a template obtained by PCR.
  • the eGFP-F2A sequence was obtained by PCR amplification of the primer pair (SEQ ID NO: 13, 14) using the plasmid of SEQ ID NO: 20 contained in Patent Application No. 201310164725.X as a template.
  • the CD137-CD3 ⁇ (28BBZ) nucleic acid fragment was subjected to three-segment splicing and PCR as shown in Fig. 6.
  • the splicing conditions were: pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: 68 °C, 140s, 5 cycles, then total extension 68 ° C, 10 min, supplement DNA polymerase and forward primer (SEQ ID NO: 13) and reverse primer (SEQ ID NO: 16) and PCR amplification 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, then total extension 68 ° C, 10 min.
  • the fragments obtained by amplification are called (Table 2):
  • eGFP-F2A-scFv-CLDN6-AE3-20HL-28BBZ SEQ ID NO: 20.
  • the following vector system for constructing the lentiviral plasmid vector of the present invention belongs to the third generation auto-inactivated lentiviral vector system, which has three plasmids, the coding protein Gag/Pol, and the packaging plasmid psPAX2 encoding the Rev protein. From the addgene); the envelope plasmid PMD2.G (purchased from addgene) encoding the VSV-G protein and the recombinant expression vector encoding the gene of interest CAR based on the empty vector pWPT-eGFP (purchased from addgene).
  • the promoter of elongation factor-1 ⁇ can regulate the expression of enhanced green fluorescent protein (eGFP) in empty vector.
  • eGFP enhanced green fluorescent protein
  • F2A food and mouth disease virus
  • F2A is a core sequence of 2A (or "self-cleaving polypeptide 2A") from foot-and-mouth disease virus. It has a "self-shearing" function of 2A and can achieve co-expression of upstream and downstream genes.
  • 2A has an effective and feasible strategy for constructing gene therapy polycistronic vectors due to its high shear efficiency, high balance of upstream and downstream gene expression and short self-sequence.
  • the sequence is used to achieve co-expression of the target gene with GFP or eGFP, and the expression of CAR can be detected indirectly by detecting GFP or eGFP.
  • This example constructs a lentiviral expression vector in which eGFP linked to F2A is co-expressed with a specific CAR, 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 component following F2A is abbreviated as CAR) is digested by MluI and SalI restriction enzymes, and ligated into the same double digestion.
  • pWPT vector a lentiviral vector expressing each chimeric antigen receptor is constructed.
  • the successfully constructed vector can be prepared for lentiviral packaging after MluI and SalI digestion and sequencing.
  • eGFP-F2A-CAR is transcribed into one mRNA, but is finally translated into two peptide chains, eGFP and anti-CLDN6 chimeric antigen receptor, in which the anti-CLDN6 chimeric antigen receptor will be localized under the guidance of CD8 ⁇ signal peptide.
  • CD8 ⁇ signal peptide In the cell membrane on.
  • the obtained carrier containing each of the following CARs is as follows (the component following F2A can be simply referred to as CAR):
  • 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.
  • HEK-293T cells (ATCC: CRL-11268) cultured to the 6th to 10th passages were inoculated at a density of 6 ⁇ 10 6 in a 10 cm culture dish, and cultured overnight at 37 ° C, 5% CO 2 for transfection.
  • the medium was DMEM (purchased from PAA) containing 10% fetal bovine serum (purchased from PAA).
  • PEI polyethyleneimine, purchased from Polysciences
  • transfection complex 4.4 1.6 ml of the transfection complex was added dropwise to HEK-293T cells. After 4-5 h, the transfected 293T cells were exchanged with 2% FBS DMEM.
  • the transfection efficiency (ie, the proportion of cells showing green fluorescence) was observed on the next day of transfection, and the positive transfection efficiency of ⁇ 80% was successful in the transfection experiment.
  • the virus was collected by filtration using a 0.45 ⁇ m filter (purchased from Millipore), and then centrifuged at 28000 rpm for 2 hours at 4 ° C using a Beckman Optima L-100XP ultracentrifuge, the supernatant was discarded, and the precipitate was centrifuged for 1/10.
  • ⁇ 1/50 stock volume of Quantum 007 medium (purchased from PAA) was resuspended and stored at -80 °C in 100 ⁇ L/tube for virus titration or infection of T lymphocytes.
  • 293T cells were seeded at 1 ⁇ 10 5 /mL in 96-well culture plates, 100 ⁇ L/well, 37 ° C, 5% CO 2 , and the culture was DMEM containing 10% fetal bovine serum.
  • 50 ⁇ L/well of the culture supernatant was discarded, 50 ⁇ L/well of the above fresh culture solution was added, and polybrene was added at a final concentration of 6 ⁇ g/mL, and incubated at 37 ° C, 5% CO 2 for 30 min.
  • eGFP was detected by flow cytometry, and the number of cells with a positive rate of 5-20% was appropriate.
  • the titer (U/mL) positive rate ⁇ dilution factor ⁇ 100 ⁇ 10 4 was calculated.
  • the titer of the virus containing the mock empty vector control and each eGFP-F2A-CAR packaged by the PEI transfection method was about 2 to 4 ⁇ 10 6 U/mL, and the virus titer measured after concentration was about It is 2 to 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 peripheral blood of healthy people by density gradient centrifugation. Peripheral blood mononuclear cells were obtained by CTL cell magnetic beads (purchased from Stem Cell Technologies) negative sorting method to obtain CTL. The sorted CTL cells were subjected to flow cytometry to detect the purity of CTL cells, and the positive rate of CTL cells was ⁇ 95%.
  • the Quantum 007 lymphocyte culture medium (purchased from PAA) was added at a density of about 1 ⁇ 10 6 /mL, and magnetic beads coated with anti-CD3 and CD28 antibodies were added at a cell:magnetic bead ratio of 1:1 (Invitrogen)
  • the company and the recombinant human IL-2 (purchased from Shanghai Huaxin Biotech Co., Ltd.) with a final concentration of 300 U/mL were stimulated for 24 h.
  • CTL cells were then infected with the above recombinant lentivirus at MOI ⁇ 5. The infected cells were passaged every other day at a density of 5 ⁇ 10 5 /mL, and a recombinant human IL-2 having a final concentration of 300 U/mL was supplemented in the lymphocyte culture solution.
  • Infected CTL cells were tested for expression of different chimeric antigen receptors by flow cytometry on day 8 of culture. Since eGFP was co-expressed with CAR, positive cells detecting eGFP were positive cells expressing chimeric antigen receptors.
  • the positive rate of virus-infected CTL cells expressing different chimeric antigen receptors using uninfected T lymphocytes as a negative control is shown in Table 3. The positive rate results indicate that a certain positive rate of CAR + CTL cells can be obtained by a method of lentiviral infection.
  • CTL cells After infecting the virus packed with different chimeric antigen receptors, CTL cells were subcultured, counted, and supplemented with IL-2 at a cell density of 5 ⁇ 10 5 /ml every other day (final concentration was 300U/ml), about 20-40 times amplification on the 11th day of culture, indicating that CTL cells expressing different chimeric antigen receptors can be expanded in vitro, providing a follow-up in vitro toxicity test and in vivo test. Guarantee.
  • the CTL of CAR + has a target ratio of 3:1, 1:1, and 1:3, respectively, and the number of target cells is 10,000/well, corresponding to effector cells according to different target-to-target ratios. Five replicate wells were set for each group, and the average of five replicate wells was taken. The detection time is 18h.
  • Each experimental group each target cell + CTL expressing different chimeric antigen receptors
  • Control group 1 The maximum release of LDH from target cells
  • Control group 2 The target cells spontaneously release LDH
  • Control group 3 Effector cells spontaneously released LDH.
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. This method is based on the colorimetric detection method and can replace the 51 Cr release method. CytoTox The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released when cells are lysed and released in much the same way as 51 Cr is released in radioactive analysis. The released LDH medium supernatant can be detected by a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is directly proportional to the number of cells lysed. Refer specifically to the instructions for the CytoTox 96 non-radioactive cytotoxicity test kit.
  • INT tetrazolium salt
  • the cytotoxicity calculation formula is:
  • the CTL and CLDN6- expressing the chimeric antigen receptor (fused to express the single-chain antibody CLDN6 (AE3-20LH) or CLDN6 (AE3-20HL)) CLDN6-Z CAR +
  • the CBB of 28BBZ CAR + had a significant killing effect on 293T cells with high expression of CLDN6, but no killing of CLDN6-negative 293T cells, indicating that they can selectively kill cells expressing CLDN6.
  • the CTL expressing the chimeric antigen receptor CLDN6-Z CAR + and the CTL of CLDN6-28BBZ CAR + of the present invention can also significantly kill the ovarian cancer cell A2780 which endogenously expresses CLDN6 (see Table 4 and Table 5). ), and the effect of the target-target gradient-dependent effect-target ratio is higher, and the cytotoxic effect is stronger.
  • the data of the target-to-dependent ratio further shows that the CTL pair expressing the anti-CLDN6 chimeric antigen receptor of the present invention is high. Specific cytotoxic effects of cells expressing CLDN6.
  • CTLs transfected with a virus containing a mock plasmid (carrying scFv-CLDN6- ⁇ Z) as a negative control showed very low cytotoxic effects against the above three cell lines highly expressing CLDN6.
  • the cytotoxicity of the cell line highly expressing CLDN6 and the cytotoxicity data of the CTL expressing the anti-CLDN6 chimeric antigen receptor of the present invention showed very significant differences.

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Abstract

提供了一种靶向CLDN6 的CAR修饰的免疫效应细胞及其制备方法和应用，所述免疫效应细胞适用于研制嵌合抗原受体(CAR)修饰的免疫效应细胞的靶基因CLDN6，可治疗卵巢癌、胃癌和肺腺癌。

Description

靶向CLDN6的免疫效应细胞及其制备方法和应用 技术领域
本发明属于肿瘤细胞治疗领域，更具体地，本发明涉及靶向CLDN6的免疫效应细胞及其制备方法和应用。
背景技术
免疫效应细胞在肿瘤免疫应答中的作用日益受到重视。基于免疫效应细胞的过继性免疫治疗在部分肿瘤中取得了一定的效果，并且该种免疫治疗方法可以克服抗体治疗的上述缺陷，但在大多数肿瘤的疗效仍不能令人满意[Grupp SA，et al.Adoptive cellular therapy.Curr Top Microbiol Immunol.，2011；344:149-72.]。近年来，根据细胞毒性T淋巴细胞(cytotoxic lymphocyte，CTL)对靶细胞的识别特异性依赖于T淋巴细胞受体(T Cell Receptor，TCR)的发现，将针对肿瘤细胞相关抗原的抗体的scFv与T淋巴细胞受体的CD3ζ或FcεRIγ等胞内信号激活基序融合成嵌合抗原受体(Chimeric antigen receptor，CAR)，并将其通过如慢病毒感染等方式基因修饰在T淋巴细胞表面。这种CAR T淋巴细胞能够以主要组织兼容性复合物(Major Histocompatibility Complex，MHC)非限制性方式选择性地将T淋巴细胞定向到肿瘤细胞并特异性地杀伤肿瘤。CAR T淋巴细胞是肿瘤免疫治疗领域的一个新的免疫治疗策略[Schmitz M，et al.Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer.J Biomed Biotechnol，2010，doi:10.1155/2010/956304.]。此外，CAR修饰的NK细胞(Klingemann H.Challenges of cancer therapy with natural killer cells.
Cytotherapy.2014 Dec 18.pii:S1465-3249(14)00791-9)或者NKT细胞也在临床前研究中展示了良好的抗肿瘤活性(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)。
嵌合抗原受体包括胞外结合区，跨膜区和胞内信号区。通常胞外区包含能够识别肿瘤相关抗原的scFv，跨膜区采用CD8，CD28等分子的跨膜区，胞内信号区采用免疫受体酪氨酸活化基序(ITAM)CD3ζ或FcεRIγ及共刺激信号分子CD28、CD27、CD137、CD134等的胞内信号区。
胞内信号区仅包含ITAM的为第一代CAR T淋巴细胞，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-ITAM。该种CAR T可以激发抗肿瘤的细胞毒性效应，但是细胞因子分泌比较少，并且在体内不能激发持久的抗肿瘤效应[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.]。
随后发展的第二代CAR T淋巴细胞加入了CD28或CD137(又名4-1BB)的胞内信号区，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28 -ITAM或scFv-TM-/CD137-ITAM。胞内信号区发生的B7/CD28或4-1BBL/CD137共刺激作用引起T淋巴细胞的持续增殖，并能够提高T淋巴细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR T在体内的存活周期和抗肿瘤效果[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.]。
近些年发展的第三代CAR T淋巴细胞，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28-CD137-ITAM或scFv-TM-CD28-CD134-ITAM，进一步提高了CAR T在体内的存活周期和其抗肿瘤效果[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.]。
尽管CAR T淋巴细胞在肿瘤免疫治疗中具有诱人的前景，但其较高风险亦需要考虑。比如，由于某些/种正常组织低表达CAR所能识别的特异性抗原可能造成CAR T淋巴细胞对表达相应抗原的正常组织的损伤。如，针对肾细胞癌患者肿瘤细胞上表达的抗原碳酸酐酶IX(CAIX)是第一个用于临床的CAR T淋巴细胞过继治疗的案例，也是第一个报道含CAR细胞的脱靶效应的案例。病人在多次输入CAR T淋巴细胞后出现2-4级肝毒性。分析原因为肝胆管上皮细胞低表达CAIX，原临床试验被迫中断同时排除了病人治疗效果的任何评价[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]。另外，CAR中过多的共刺激信号会降低效应细胞激活所需的阈值，使得基因修饰的T淋巴细胞在低水平抗原或没有抗原触发的条件下也可能会被活化，导致大量细胞因子的释放以致可能引发所谓的“细胞因子风暴”。这种信号外漏(signal leakage)会导致脱靶细胞毒性，从而产生非特异性的组织损伤。例如，在采用针对Her2的第三代CAR临床治疗一个具有肝和肺转移的晚期结肠癌患者的过程中由于正常肺组织中低表达Her2而引发所谓的“细胞因子风暴”致病人猝死[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.]。
设计CAR修饰的免疫效应细胞，特别T细胞时，所针对的抗原基因实际上是一种关键性的选择，鉴于体内基因表达的复杂性以及各种不可控因素，选择到一个合适的用于CAR的基因是非常困难的。并且，很多肿瘤特异性的抗原，很难找到针对其的且适 合于构建CAR修饰的免疫效应细胞的特异性分子。此外，由于不同的靶点可能会对免疫效应细胞产生不一样的影响，如EGFR靶点可以上调PD-L1的表达(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-1pathway contributes to immune escape in EGFR-driven lung tumors.Cancer Discov.2013；3(12):1355-63.)，从而可能会影响效应T细胞的杀伤作用(Moon EK，Wang LC，Dolfi DV，Wilson CB，Ranganathan R，Sun J，Kapoor V，Scholler J，PuréE，Milone MC，June CH，Riley JL，Wherry EJ，Albelda SM.Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors.Clin Cancer Res.2014 Aug 15；20(16):4262-73.)。因此，不同的靶点选择，可能会导致所制备的CAR修饰的免疫细胞不一样的抗肿瘤活性。由于生物学的复杂性和不可预测性，这些都是需要实验去验证。
发明内容
本发明的目的在于提供靶向CLDN6的免疫效应细胞及其制备方法和应用。
在本发明的第一方面，提供表达于免疫效应细胞表面的嵌合抗原受体(CAR)，所述的嵌合抗原受体包含顺序连接的：胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含特异性识别CLDN6(Claudin 6)的蛋白。
在一个优选例中，所述的免疫效应细胞包括：T淋巴细胞，NK细胞或NKT细胞。
在另一优选例中，所述的特异性识别CLDN6的蛋白是抗体或配体；较佳地，所述的抗体是单链抗体或结构域抗体。
在另一优选例中，所述的跨膜区是包含CD8或CD28的跨膜区和铰链区的序列。
在另一优选例中，所述的胞内信号区选自：CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134的胞内信号区序列，或其组合。
在另一优选例中，所述的嵌合抗原受体包括如下的顺序连接的胞外结合区，跨膜区和胞内信号区：
特异性识别CLDN6的单链抗体、CD8和CD3ζ；
特异性识别CLDN6的单链抗体、CD8、CD137和CD3ζ；
特异性识别CLDN6的单链抗体、CD28分子的跨膜区(CD28a)、CD28分子的胞内信号区(CD28b)和CD3ζ；或
特异性识别CLDN6的单链抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
在另一优选例中，所述的嵌合抗原受体具有SEQ ID NO:21～24任一所述的氨基酸序列。
在本发明的另一方面，提供编码前面任一所述的嵌合抗原受体的核酸。
在一个优选例中，所述的核酸具有SEQ ID NO:17～20任一所述的核苷酸序列。
在本发明的另一方面，提供一种表达载体，其包含前面任一所述的核酸。
在一个优选例中，所述的表达载体来源于慢病毒质粒pWPT(或pWPT-eGFP)。
在本发明的另一方面，提供一种病毒，所述的病毒(如慢病毒)包含所述载体。
前面任一所述的嵌合抗原受体、或所述的核酸、或所述的表达载体、或所述的病毒的用途，用于制备靶向CLDN6的基因修饰的免疫效应细胞。
在本发明的另一方面，提供一种基因修饰的免疫效应细胞，其转导有所述的核酸，或所述的表达载体或所述的病毒。
在本发明的另一方面，提供一种基因修饰的免疫效应细胞，其表面表达一种嵌合抗原受体，所述嵌合抗原受体的氨基酸序列选自SEQ ID NO:21-24任一所述的氨基酸序列。
在本发明的另一方面，提供所述的基因修饰的免疫效应细胞的用途，用于制备抑制肿瘤的药物，所述的肿瘤是CLDN6阳性的肿瘤。
在另一优选例中，所述的CLDN6阳性的肿瘤包括：卵巢癌、胃癌、肺腺癌。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、RT-PCR检测各组织及各细胞中CLDN6基因表达情况。
图2、抗CLDN6的单链抗体纯化电泳结果。
图3、编码CLDN6的慢病毒表达载体示意图。
图4、Western blot检测CLDN6的稳定表达。293T指的是HEK-293T；293T-CLDN6指的是转染了CLDN6的HEK-293T细胞。
图5、流式细胞学分析各个细胞系与抗CLDN6抗体的结合情况。
图6、嵌合抗原受体各部分的连接顺序的示意图。
图7、五种拼接好的eGFP-F2A-CAR片段DNA电泳鉴定图。
图8、作为示例的本发明的包含编码CAR序列的慢病毒载体pWPT-eGFP-F2A-CAR的结构示意图。
具体实施方式
本发明人经过广泛而深入的研究，首次揭示了一种基于CLDN6基因的CAR修饰的免疫效应细胞及其制备方法。
CLDN6基因
本发明人前期考察了许多种肿瘤特异性基因，发现这类基因中相当大一部分也表达于部分组织的正常细胞中，较难以应用于嵌合抗原受体修饰的免疫效应细胞技术；有的肿瘤特异性基因具有较好的肿瘤特异性表达特性，但是，基于其设计的CAR修饰的免疫效应细胞没有肿瘤细胞杀伤活性或者活性很低，这可能是因为该靶点可以引发肿瘤细胞分泌对免疫效应细胞起抑制作用的因子如PD-L1。
经过反复考察和筛选，本发明人找到了CLDN6基因作为设计CAR的靶基因。Claudin6(CLDN6)的氨基酸序列及其基因序列，分别公开为GenBank登录号NP_067018.1及NM_021195.3(序列号:22及序列号:23)、或者GenBank登录号NP_067018.2及NM_021195.4(序列号:46及序列号:47)。Claudins是位于上皮与内皮的紧密连接的膜蛋白。
目前CAR T细胞已经成为一种潜在的治疗手段。但是很多肿瘤尚没有有关CAR T细胞治疗的报道，如胃癌。已有研究表明，CLDN6是胃组织的特异标志物。本发明人研究证实，CLDN6确实在绝大多数正常组织中不表达，而在一些肿瘤如卵巢癌中表达。但是，目前关于CLDN6作为治疗靶点的候选药物只有单克隆抗体，且它是否能成功用于相应肿瘤治疗尚不得而知。因此，有必要寻找新的治疗手段。考虑到CLDN6的组织特异性，本发明人设想如果能够用CAR T细胞进行靶向治疗，有望可以得到一种新的抗癌制剂。但是，公知CLDN6抗原是一个紧密连接蛋白，是否能够被CAR T细胞接触并引发对相应靶细胞的杀伤尚不得而知。此外，由于蛋白质空间构象是牵一发而动全身，很多单抗演化成单链抗体往往丧失了抗原结合活性或特异性。幸运地，本发明人发现有两个单链抗体(AE3-20LH和AE3-20HL)具有良好的抗原结合特异性。本发明人的进一步研究表明，由这两个单链抗体所构成的CAR T细胞保留了对CLDN6阳性细胞的选择性杀伤作用。本发明的结果表明，CLDN6确实可以成为CAR修饰的免疫效应细胞，特别是T细胞治疗的靶点；针对CLDN6的CAR T细胞是一种肿瘤治疗候选新手段。
进一步研究证明，CLDN6靶向的CAR修饰的T细胞确实可以选择性地清除CLDN6阳性的肿瘤细胞，而对非肿瘤细胞293T细胞没有毒性。本发明人认为，相应的CAR修饰的免疫效应细胞，特别是T细胞应该可以用于人体肿瘤的治疗。
嵌合抗原受体及其编码核酸
本发明提供了一种表达于免疫效应细胞表面的嵌合抗原受体，所述的嵌合抗原受体包含顺序连接的：胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含特异性识别CLDN6(claudin 6)的蛋白。将该嵌合抗原受体表达于免疫效应细胞的表面，可使 得免疫效应细胞对高表达CLDN6的肿瘤细胞具有高度特异性的细胞毒性作用。
作为本发明的优选方式，所述胞外结合区包含特异性识别CLDN6的单链抗体scFv(CLDN6)。上述嵌合抗原受体蛋白的胞外结合区通过CD8铰链区与CD8或者CD28的跨膜区相连接，跨膜区后紧接胞内信号区。
本发明也包括编码所述嵌合抗原受体的核酸。本发明的核酸序列可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明的编码嵌合抗原受体蛋白氨基酸序列的核酸密码子可以是简并的，即编码同一氨基酸序列的多种简并核酸序列都包含在本发明的范围之中。编码对应氨基酸的简并核酸密码子是本领域公知的。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
特异性识别人CLDN6的抗体可以选用现有技术中公开的抗体，多种识别CLDN18A2的C末端表位的单克隆抗体可以以合适的方式运用于本发明，最后可以获得具有杀伤活性的CAR免疫效应细胞。作为本发明的特别优选方式，所述的人CLDN6的抗体是本发明的单链抗体scFv-CLDN6(AE3-20LH)或scFv-CLDN6(AE3-20HL)。
本发明中使用的术语“单链抗体(scFv)片段”指的是通过如下定义的抗体片段，其是包含通过接头(linker)连接的重链可变区(VH)和轻链可变区(VL)的重组蛋白，接头使得这两个结构域相关联，以最终形成抗原结合位点。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。本发明使用的单链抗体可单独或联合使用本领域已知的常规技术，例如氨基酸缺失、插入、取代、增加、和/或重组以及/或其它修饰方法作进一步修饰。根据一种抗体的氨基酸序列在其DNA序列中引入这种修饰的方法对本领域技术人员来说是众所周知的；见例如，Sambrook，分子克隆：实验手册，Cold Spring Harbor Laboratory(2002)N.Y.。所指的修饰优选在核酸水平上进行。上述单链抗体还可以包括其衍生物。本发明中“抗体的衍生物”包括例如当通过噬菌体展示技术获得所述抗体的衍生物时，可使用如BIAcore系统中使用的表面等离子共振技术来增加与CLDN6抗原表位结合的噬菌体抗体的效率(Schier，人抗体杂交瘤7(1996)，97-105；Malmborg，免疫学方法杂志183(1995)，7-13)。还包括，例如WO 89/09622中描述的嵌合抗体的产生的方法，EP-A10239400和WO90/07861中描述的人源化抗体产生的方法，WO91/10741，WO94/02602和WO96/33735中有描述的产生异种抗体例如小鼠中的人抗体的方法所产生的抗体的衍生物。
本发明的术语“特异性识别”的意思是本发明的双特异性抗体不与或基本上不与 目标抗原以外的任意多肽交叉反应。其特异性的程度可以通过免疫学技术来判断，包括但不限于免疫印迹，免疫亲和层析，流式细胞分析等。在本发明中，特异性识别优选通过流式细胞技术来确定，而具体情况下特异性识别的标准可由本领域一般技术人员根据其掌握的本领域常识来判断。
嵌合抗原受体的跨膜区可以选自CD8或CD28等蛋白的跨膜区。人CD8蛋白是个异二聚体，由αβ或者γδ两条链组成。在本发明的一个实施方案中，跨膜区选自CD8α或者CD28的跨膜区。此外，CD8α铰链区(hinge)是一个柔性区域，因此，CD8或CD28和跨膜区加上铰链区被用于将嵌合抗原受体CAR的靶点识别结构域scFv和胞内信号区连接起来。
胞内信号区可以选自CD3ζ，FcεRIγ，CD28，CD137，CD134蛋白的胞内信号区，及其组合。CD3分子由五个亚单位组成，其中CD3ζ亚单位(又称CD3zeta，简称Z)含有3个ITAM基序，该基序是TCR-CD3复合体中重要的信号转导区。CD3δZ是截短的不具有ITAM基序的CD3ζ序列，在本发明实践中一般作为阴性对照的构建。FcεRIγ主要分布在肥大细胞和嗜碱性粒细胞表面，其含有一个ITAM基序，在结构、分布及功能上与CD3ζ类似。此外如前所述，CD28，CD137，CD134是共刺激信号分子，在与各自配体结合后其胞内信号区段产生的共刺激作用引起免疫效应细胞(主要是T淋巴细胞)的持续增殖，并能够提高免疫效应细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR免疫效应细胞在体内的存活周期和抗肿瘤效果。
本发明的核酸所编码的抗CLDN6嵌合抗原受体蛋白可以选自按如下方式顺序连接：
scFv(CLDN6)-CD8-CD3ζ，
scFv(CLDN6)-CD8-CD137-CD3ζ，
scFv(CLDN6)-CD28a-CD28b-CD3ζ，
scFv(CLDN6)-CD28a-CD28b-CD137-CD3ζ，
及其组合，其中相关嵌合抗原受体蛋白中CD28a代表CD28分子的跨膜区，CD28b代表CD28分子的胞内信号区。上述各种抗CLDN6嵌合抗原受体统称为scFv(CLDN6)-CAR。
在本发明的一个实施方案中，本发明的核酸具有如SEQ ID NO:17～20所述的序列。在本发明的另一个实施方案中，本发明的核酸是编码具有如SEQ ID NO:21-24之一的嵌合抗原受体蛋白的核酸。
表达载体及细胞
本发明还提供了包含上述编码表达于免疫效应细胞表面的嵌合抗原受体蛋白的核酸的载体。在一个具体实施方案中，本发明使用的载体是一种慢病毒质粒载体 pWPT-eGFP。该质粒属于第三代自灭活慢病毒载体系统，该系统共有三个质粒即编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2；编码VSV-G蛋白的包膜质粒PMD2.G；及空载体pWPT-eGFP，其可以用于重组引入目的核酸序列，即编码CAR的核酸序列。空载体pWPT-eGFP(其本身为后续试验中的mock)中由延长因子-1 α(elongation factor-1α，EF-1α)启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达。而包含编码CAR的目的核酸序列的重组表达载体pWPT-eGFP-F2A-CAR是通过由来自口蹄疫病毒(food-and-mouth disease virus，FMDV)的核糖体跳跃序列(ribosomal skipping sequence 2A)(简称F2A)实现eGFP与CAR的共表达的。
本发明还包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其它可用于将外源基因转导入免疫效应细胞的病毒及其对应的质粒载体也可用于本发明。
在本发明的一个实施方案中，所述病毒是包含上述pWPT-eGFP-F2A-CAR重组载体(即含有scFv(CLDN6)-CAR)的慢病毒。
本发明还提供了基因修饰的免疫效应细胞，其被转导有本发明的核酸或被转导有本发明的上述包含所述含有该核酸的重组质粒，或包含该质粒的病毒。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。基于非病毒的转导方法包括电穿孔法和转座子法。近期Amaxa公司研发的Nucleofector核转染仪能够直接将外源基因导入细胞核获得目的基因的高效转导。另外，基于睡美人转座子(Sleeping Beauty system)或PiggyBac转座子等转座子系统的转导效率较普通电穿孔有较大提高，将nucleofector转染仪与睡美人转座子系统联合应用已有报道[Davies JK.，et al.Combining CD19redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res，2010，70(10):OF1-10.]，该方法既具有较高的转导效率又能够实现目的基因的定点整合。在本发明的一个实施方案中，实现嵌合抗原受体基因修饰的免疫效应细胞的转导方法是基于病毒如逆转录病毒或慢病毒的转导方法。该方法具有转导效率高，外源基因能够稳定表达，且可以缩短体外培养免疫效应细胞到达临床级数量的时间等优点。在该转基因免疫效应细胞表面，转导的核酸通过转录、翻译表达在其表面。通过对各种不同的培养的肿瘤细胞进行体外细胞毒实验证明，本发明的抗CLDN6嵌合抗原受体基因修饰的免疫效应细胞具有高度特异性的肿瘤细胞杀伤效果(亦称细胞毒性)。因此本发明的编码嵌合抗原受体蛋白的核酸，包含该核酸的质粒，包含该质粒的病毒和转导有上述核酸，质粒或病毒的转基因免疫效应细胞可以有效地用于肿瘤的免疫治疗。
在一个实施方案中，本发明的基因修饰的免疫效应细胞，其表面表达一种嵌合抗原受体，所述嵌合抗原受体由SEQ ID NO:17-20之一的核酸编码表达。在另一个实施方案 中，本发明的转基因免疫效应细胞表面表达一种嵌合抗原受体，所述嵌合抗原受体的氨基酸序列选自SEQ ID NO:20-24之一。
鉴于目前尚没有关于靶向CLDN6的CAR T的报道，本发明人首次从诸多肿瘤相关基因中成功地找到了一种适用于CAR T细胞的靶基因CLDN6，并成功制备了靶向CLDN6的CAR T细胞，从而为卵巢癌、胃癌、肺腺癌等肿瘤提供一种全新的治疗手段。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编着，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、CLDN6在各种正常组织及肿瘤细胞的表达检测
细胞总RNA的提取：人正常组织RNA购自Clontech Laboratories，Inc。卵巢癌细胞于5％CO2，37℃培养箱中常规培养。PBS(0.14M NaCl，2.7mM KCl，10.1mM Na2HPO4，1.8mM KH2PO4，pH 7.3)洗细胞2次。用Trizol试剂抽提生长状态良好的细胞总RNA，每皿细胞加入1ml Trizol，充分吹打至不粘稠；按每毫升原始体积Trizol加0.2ml新开封的氯仿，剧烈振荡15sec，室温孵育5min；4℃，12,000g离心15min；将无色上清液移入新的离心管中，按每毫升原始体积Trizol加0.5ml异丙醇，-20℃孵育20min，12,000g 4℃离心10min；倒掉上清，用1ml 75％乙醇洗涤，4℃，10,000g离心5min；室温干燥RNA沉淀10-20min，用DEPC处理后的无RNA酶H2O溶解沉淀。分光光度计法定量RNA。
反转录：细胞总RNA 1μl(1μg/μl)和Oligo dT(10μM)1μl混合，72℃孵育5min后，立刻置于冰上5min；加入15μl反转录混合液(Nuclease-free H2O 6.1μl，5×RT Buffer 4μl，25mM MgCl2 2.4μl，10mM dNTPs 1μl，RNase Ribonuclease Inhibitor 0.5μl，Improm-IITM Reverse Transcriptase 1μl)，25℃孵育5min，42℃孵育1h；70℃孵育15min终止反应。
RT-PCR：以上述反转录的cDNA为模板，以引物CLDN6-F：GGAATGCAGATCCTGGGAGT，CLDN6-R：ATGAAAGCGGTCA进行PCR，PCR反应体系：1μl模板，2.5μl 2mM dNTP，2.5μl 10×buffer，引物各lμl，5U Taq酶，以H2O补足25μl。PCR反应条件：94℃3min；94℃30sec，56℃30sec，72℃40sec，35个循环；72℃10min；4℃保存。同时扩增β-actin作为内参，β-actin引物：β-actin-F:5’-TCCTCCCTGGAGAAGAGCTA-3’，β-actin-R:5’-GTACTTGCGCTCAGGAGGAG-3’。扩增条件：94℃3min；94℃30sec，58℃30sec，72℃30sec，30个循环；72 10min℃。 通过2％琼脂糖凝胶电泳观察片段大小。
如图1，RT-PCR结果显示，正常组织中均未检测到CLDN6基因的扩增，卵巢癌细胞中只有A2780细胞扩增出阳性条带，而Hey细胞中未扩增出阳性条带。
实施例2、抗CLDN6的单链抗体的表达
抗CLDN6的抗体尽管现有技术中已有，但是如果从单克隆抗体直接演化成单链抗体，往往会影响其抗原结合活性，甚至特异性。通过基于PCR搭桥的基因合成技术，本发明人合成了scFv-CLDN6(64)(SEQ ID NO:1(核苷酸)，2(氨基酸))、scFv-CLDN6(AE3-20LH)(SEQ ID NO:3(核苷酸)，4(氨基酸))、scFv-CLDN6(AE3-20HL)(SEQ ID NO:5(核苷酸)，6(氨基酸))单链抗体序列，通过NheI/BamHI(购自NEB)双酶切，以T4 DNA连接酶(购自NEB)于同样以NheI/BamHI双酶切载体质粒pCMV-V5-Fc(该载体在多克隆位点下游融合表达人抗体Fc片段，以下简称V5-Fc，购自上海锐劲生物技术有限公司)连接并转化于宿主菌TOP10中，挑取克隆通过PCR鉴定阳性克隆并通过测序确认，分别获得V5-scFv-CLDN6(64)-Fc、V5-scFv-CLDN6(AE3-20LH)-Fc和V5-scFv-CLDN6(AE3-20HL)-Fc真核表达质粒。
将上述表达质粒分别转染生长良好的HEK-293F细胞，37℃，5％CO2，125rpm摇床连续培养7天，4000rpm离心10min，去除沉淀，收集上清，并用0.45μm滤膜过滤，将处理好的样品以protein A(购自GE)亲和柱进行亲和纯化，最终获得纯化的单链抗体-Fc融合蛋白scFv-CLDN6(64)-Fc、scFv-CLDN6(AE3-20LH)-Fc、scFv-CLDN6(AE3-20HL)-Fc，鉴定结果如图2。
实施例3、CLDN6的稳定表达细胞系的构建
1、外源表达CLDN6的细胞系构建
通过基于PCR搭桥的基因合成方法，合成CLDN6基因片段(SEQ ID NO:7(核苷酸)，8(氨基酸))，通过MluI/SalI(购自NEB)双酶切，以T4DNA连接酶(购自NEB)于同样以MluI/SalI双酶切的质粒载体pWPT(购自上海锐劲生物技术有限公司)连接并转化于宿主菌TOP10中，挑取克隆通过PCR鉴定阳性克隆并通过测序确认，获得慢病毒质粒pWPT-CLDN6(图3)。
慢病毒的包装：将293T细胞(7×105/孔)接种于6孔板，约70％～80％细胞丰度，细胞培养液为2ml无抗生素的DMEM+10％FBS。次日，使用LipofectamineTM2000进行转染，配制脂质体/DNA混合物：A液配制：将pWPT-CLDN6质粒2μg，pAX2质粒1.5μg，pMD2.G质粒0.6μg，溶解于100μl的无血清DMEM培养液中，轻轻混匀。B液配制：将10μl LipofectamineTM2000溶解于100μl的无血清DMEM培养液中，轻轻混匀，室温孵育5min。将A液与B液轻轻混合，室温放置20min后。将6孔板内细胞原培养液 弃去，加入新的1.8ml无抗生素的含2％FBS的DMEM培养液。然后将A/B混合液滴加入到细胞培养皿中，轻轻吹打混匀。37℃，5％CO2继续培养72h，收集上清病毒液。
将上述收集的CLDN6病毒液分别加至铺于6cm平板内的HEK-293T细胞中，72h后收集细胞，使用细胞裂解液进行裂解。将裂解收集的细胞蛋白取40μg进行SDS-PAGE凝胶电泳，随后对凝胶进行免疫印迹，并用小鼠抗flag抗体(购自上海锐劲生物技术有限公司)染色。PBS洗涤后，与辣根过氧化物酶标记的羊抗小鼠抗体(购自上海锐劲生物技术有限公司)孵育后使用ECL试剂显色，最后进行显影。
Western blot结果显示，在转染了CLDN6的HEK-293T细胞(即293T-CLDN6)中可检测到分子量大小约25KD的条带，而在未转染的空细胞中未检测到相应条带(图4)，说明外源表达CLDN6的细胞系构建成功。
2、流式细胞学分析各个细胞系与抗CLDN6抗体的结合情况实验步骤
通过荧光激活细胞分选仪(FACS)(BD公司，FACSCalibur)分析单链抗体scFv-CLDN6(64)，scFv-CLDN6(AE3-20LH)和scFv-CLDN6(AE3-20HL)各自与下列细胞系的结合能力。
具体方法如下：
1).取对数生长期的293T，293T-CLDN6以及卵巢癌细胞系A2780肿瘤细胞接种到6cm平皿中，接种细胞密度约为90％，37℃孵箱过夜培养。
2).使用10mM的EDTA消化细胞，200g×5min离心收集细胞。以1×106～1×107/mL的浓度重悬于1％含小牛血清的磷酸盐缓冲液(NBS PBS)中，按100ul/管的量加入流式专用管中。
3).200g×5min离心，弃上清。
4).分别加入待测抗体scFv-CLDN6(64)，scFv-CLDN6(AE3-20LH)和scFv-CLDN6(AE3-20HL)，同时以PBS作为阴性对照，抗体终浓度为10μg/ml，每管加入100ul。冰浴，45分钟。
5).每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。
6).弃上清，加入1:50稀释的FITC荧光标记的羊抗人抗体(来自上海康成生物工程有限公司)，每管加入100ul。冰浴，45分钟。
7).每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。
8).弃上清，重悬于300ul 1％NBS PBS中，流式细胞仪检测。
9).应用流式细胞仪数据分析软件WinMDI 2.9分析数据。
结果如图5，流式细胞分析结果表明，单链抗体scFv-64无论与CLDN6阴性细胞293T，还是与CLDN6稳定表达的293T-CLDN6以及内源表达CLDN6的A2780细胞均不结合。而单链抗体scFv-CLDN6(AE3-20LH)和scFv-CLDN6(AE3-20HL)可以特异识别 CLDN6稳定表达的293T-CLDN6细胞，但是与CLDN6阴性的293T细胞不结合，表明这两个单链抗体可以特异识别CLDN6。此外这两个单链抗体也可以特异识别内源性表达CLDN6的A2780细胞。
实施例3、表达本发明核酸编码的嵌合抗原受体蛋白的慢病毒质粒的构建及病毒包装
表1解释了本发明示例的嵌合抗原受体各部分的连接顺序，该连接还可参见图6中所示。
表1
1、核酸片段的扩增
(1)scFv(CLDN6-AE3-20LH，CLDN6-AE3-20HL)序列的扩增
以V5-scFv-CLDN6(AE3-20LH)-Fc质粒为模板，采用的引物对正向引物(SEQ ID NO:9，包含部分2A的序列)和反向引物(SEQ ID NO:10，包含部分CD8hinge序列)，PCR扩增获得scFv(CLDN6-AE3-20LH)；同样的，以V5-scFv-CLDN6(AE3-20HL)-Fc质粒为模板，采用的引物对正向引物(SEQ ID NO:11，包含部分2A的序列)和反向引物(SEQ ID NO:12，包含部分CD8hinge序列)PCR扩增获得scFv(CLDN6-AE3-20HL)。
(2)嵌合抗原受体其它部分的核酸序列
抗CLDN6嵌合抗原受体蛋白的除scFv(CLDN6-AE3-20LH，CLDN6-AE3-20HL)外其它部分的核酸序列分别以专利申请号为201310164725.X，201310108532.2中公开的序列SEQ ID NO:20，23为模板通过PCR方式获得。具体地，其中eGFP-F2A序列以专利申请号201310164725.X中所载的SEQ ID NO:20质粒为模板，以引物对(SEQ ID NO:13，14)进行PCR扩增获得。CD8-CD3ζ(Z)和CD28a-CD28b-CD137-CD3ζ(28BBZ)，分别以scFv(GPC3)-CD8-CD3ζ(申请专利201310164725.X中SEQ ID NO:20)和scFv(GPC3)-CD28a-CD28b-CD137-CD3ζ(申请专利201310164725.X中SEQ ID NO:23)为 模板，采用引物对(SEQ ID NO:15，16)通过PCR扩增获得CD8-CD3ζ(Z)和CD28a-CD28b-CD137-CD3ζ(28BBZ)片段。
2、核酸片段的拼接
分别将如前述获得的eGFP-F2A核酸片段，与等摩尔的scFv(CLDN6-AE3-20LH)或scFv(CLDN6-AE3-20HL)核酸片段以及等摩尔的CD8-CD3ζ(Z)或CD28a-CD28b-CD137-CD3ζ(28BBZ)核酸片段，按图6所示进行三片段拼接并PCR，拼接条件为：预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行5个循环，然后总延伸68℃，10min，补充DNA聚合酶及正向引物(SEQ ID NO:13)和反向引物(SEQ ID NO:16)后PCR扩增30个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行30个循环，然后总延伸68℃，10min。扩增获得的片段分别称为(表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)。
拼接好的eGFP-F2A-CAR片段的DNA电泳鉴定结果如图7。
表2、本发明中的序列
Figure PCTCN2016077341-appb-000002
Figure PCTCN2016077341-appb-000003
3、慢病毒质粒载体的构建
作为示例，以下构建本发明的慢病毒质粒载体使用的载体系统属于第三代自灭活慢病毒载体系统，该系统共有三个质粒即编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2(购自addgene)；编码VSV-G蛋白的包膜质粒PMD2.G(购自addgene)及基于空载体pWPT-eGFP(购自addgene)的编码目的基因CAR的重组表达载体。
在空载体pWPT-eGFP中，自带的延长因子-1α(elongation factor-1α，EF-1α)的启动子可调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达，在空载体中插入本实施例前述构建的构建体后，形成编码目的基因CAR的重组表达载体，其中通过来自口蹄疫病毒的核糖体跳跃序列(food and mouth disease virus，FMDV，ribosomal skipping sequence，F2A)实现eGFP与目的基因CAR的共表达。F2A是来自口蹄疫病毒的2A(或称为“自剪切多肽2A”)的一段核心序列，具备2A的“自剪切”功能，可以实现上游和下游基因共表达。2A由于其剪切效率高、上下游基因表达平衡性高及自身序列短小的优点为构建基因治疗多顺反子载体提供了一种有效的可行策略。尤其在基于嵌合抗原受体基因修饰T淋巴细胞的免疫治疗中多应用该序列实现目的基因与GFP或者eGFP的共表达，通过检测GFP或者eGFP即可间接检测CAR的表达。
本实施例构建了由F2A相连的eGFP与特异性CAR共表达的慢病毒表达载体，统称为pWPT-eGFP-F2A-CAR(图8)。上述步骤2中获得的目的基因eGFP-F2A-CAR(参见实施例3中的2，F2A后面的组件简称为CAR)通过MluI和SalI限制性内切酶双酶切，连入同样双酶切的pWPT载体中，从而构建表达各嵌合抗原受体的慢病毒载体。构建成功的载体经MluI和SalI酶切鉴定及序列测定正确后，可以准备用于慢病毒包装。如前所述，eGFP-F2A-CAR转录为一条mRNA，但最终翻译为eGFP和抗CLDN6嵌合抗原受体两条肽链，其中在CD8α信号肽的引导下抗CLDN6嵌合抗原受体将定位在细胞膜 上。
得到的含有各目的CAR的载体如下(F2A后面的组件可简称为CAR)：
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。
通过以上构建，分别可获得四个eGFP-F2A-CAR多肽序列，称为：
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、质粒转染293T包装慢病毒
以6×106的密度接种培养至第6～10代的HEK-293T细胞(ATCC：CRL-11268)于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染。培养基为含10％胎牛血清(购自PAA公司)的DMEM(购自PAA公司)。
转染的步骤如下：
4.1A液配制：将10μg mock对照或10μg的各目的基因质粒pWPT-eGFP-F2A-CAR，分别与7.5μg包装质粒PAX2：和3μg包膜质粒pMD2.G，溶入800μL的无血清DMEM培养液中，混匀。
4.2B液配制：将60μg PEI(聚乙烯亚胺，购自Polysciences公司)溶解于800μL的无血清DMEM培养液中，轻轻混匀，室温孵育5min。
4.3转染复合物的形成：将A液加入B液中轻轻混合，加入后立即涡旋混合或轻轻混匀，室温下孵育20min。
4.4将转染复合物1.6ml滴加入HEK-293T细胞中，4-5h小时后，用2％FBS的DMEM培基给转染的293T细胞换液。
在转染次日观察转染效率(即呈绿色荧光的细胞比例)，～80％的阳性转染效率即为转染实验成功。在转染72h后，使用0.45μm滤器(购自Millipore公司)过滤收集病毒，然后采用Beckman Optima L-100XP超速离心机28000rpm，4℃离心2小时，弃离心上清，离心所得沉淀用1/10～1/50原液体积的Quantum 007培养液(购自PAA公司)进行重悬，以100μL/管分装冻存于-80℃，以待病毒滴定或感染T淋巴细胞。
5、测定包装有mock或者eGFP-F2A-CAR的慢病毒滴度
第一天，以1×105/mL接种293T细胞于96孔培养板，100μL/孔，37℃，5％CO2 培养，培养液为含10％胎牛血清的DMEM。第二天，弃50μL/孔培养上清，补加50μL/孔新鲜上述培养液，并含终浓度为6μg/mL的polybrene，37℃，5％CO2孵育30min。加10μL/孔的病毒原液或1μL/孔的病毒浓缩液，5倍稀释，4个梯度，两个复孔，37℃，5％CO2培养。感染48h后，流式细胞仪检测eGFP，以阳性率为5～20％的细胞数为宜，计算滴度(U/mL)＝阳性率×稀释倍数×100×104。PEI转染法包装的上述包含mock即空载体对照和各eGFP-F2A-CAR的病毒的滴度均为约2～4×106U/mL的水平，经浓缩后所测的病毒滴度约为2～4×107U/mL。
实施例4、重组慢病毒感染CTL细胞
由健康人外周血通过密度梯度离心法获得人外周血单个核细胞(上海市血液中心提供)，外周血单个核细胞通过CTL细胞磁珠(购自Stem Cell Technologies)负性分选方法获得CTL，分选后的CTL细胞进行流式细胞检测CTL细胞的纯度，以CTL细胞的阳性率≥95％为宜进行下一步操作。以约1×106/mL密度加入Quantum 007淋巴细胞培养基液(购自PAA公司)培养并以细胞：磁珠比例为1:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen公司)和终浓度300U/mL的重组人IL-2(购自上海华新生物高技术有限公司)刺激培养24h。然后以MOI≈5用上述重组慢病毒感染CTL细胞。感染后的细胞每隔一天采用5×105/mL的密度进行传代，同时在淋巴细胞培养液中补加终浓度300U/mL的重组人IL-2。
感染的CTL细胞在培养第8天时通过流式细胞检测各不同嵌合抗原受体表达，由于eGFP与CAR共表达，检测eGFP的阳性细胞即为表达嵌合抗原受体的阳性细胞。以未感染的T淋巴细胞作为阴性对照，表达不同嵌合抗原受体的病毒感染CTL细胞其阳性率如表3所示。该阳性率结果表明，通过慢病毒感染的方法能够获得一定阳性率的CAR+CTL细胞。
表3
Figure PCTCN2016077341-appb-000004
CTL细胞在分别感染包装有不同嵌合抗原受体的病毒后，以细胞密度为5×105/ml 隔天传代培养、计数、并对传代的细胞培养液补加IL-2(终浓度为300U/ml)，培养第11天约有20～40倍的扩增，表明表达不同嵌合抗原受体的CTL细胞在体外能够进行一定数量的扩增，为后续体外毒性试验及体内试验提供了保证。
实施例5、表达嵌合抗原受体的T淋巴细胞的体外毒性效果实验
体外毒性实验使用的材料如下：
如表4所示的293T和卵巢癌细胞作为靶细胞，效应细胞为如实施例4所验证的体外培养12天的FACS检测嵌合抗原受体表达的阳性细胞记为嵌合抗原受体阳性(CAR+)的CTL，效靶比视情况分别为3:1，1:1和1:3，靶细胞数量为10000个/孔，根据不同效靶比对应效应细胞。各组均设5个复孔，取5个复孔的平均值。检测时间为第18h。
其中各实验组和各对照组如下：
各实验组：各靶细胞+表达不同嵌合抗原受体的CTL，
对照组1：靶细胞最大释放LDH，
对照组2：靶细胞自发释放LDH，
对照组3：效应细胞自发释放LDH。
检测方法：采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。该方法是基于比色法的检测方法，可替代51Cr释放法。CytoTox 
Figure PCTCN2016077341-appb-000005
检测定量地测量乳酸脱氢酶(LDH)。LDH是一种稳定的胞质酶，在细胞裂解时会释放出来，其释放方式与51Cr在放射性分析中的释放方式基本相同。释放出的LDH培养基上清中，可通过30分钟偶联的酶反应来检测，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
细胞毒性计算公式为：
Figure PCTCN2016077341-appb-000006
具体如表4和表5所示，本发明的表达嵌合抗原受体(融合表达了单链抗体CLDN6(AE3-20LH)或者CLDN6 (AE3-20HL))CLDN6-Z CAR+的CTL和CLDN6-28BBZ CAR+的CTL对高表达CLDN6的293T细胞有明显的杀伤作用，而对CLDN6阴性的293T细胞没有杀伤，表明它们可以选择性地杀伤表达CLDN6的细胞。此外，本发明的表达嵌合抗原受体CLDN6-Z CAR+的CTL和CLDN6-28BBZ CAR+的CTL也可以对内源表达CLDN6的卵巢癌细胞A2780具有显着的杀伤(见表4和表5)，并呈现效靶比梯度依赖性即效靶比越高细胞毒性作用越强。
效靶比依赖性的数据进一步显示表达本发明的抗CLDN6嵌合抗原受体的CTL对高 表达CLDN6的细胞的特异性细胞毒性作用。
相比较而言，被含有mock质粒(携带scFv-CLDN6-δZ)的病毒转染的作为阴性对照的CTL显示对上述三种高表达CLDN6的细胞系的细胞毒性作用均非常低。其对高表达CLDN6的细胞系的细胞毒性与表达本发明的抗CLDN6嵌合抗原受体的CTL的细胞毒性数据呈现出非常显著的差异。
以上结果表明，选择针对CLDN6的单链抗体所构建的嵌合抗原受体，能够选择性地杀伤高表达CLDN6的靶细胞。此外，从细胞毒性数据来看，CLDN6-28BBZ的CAR T比CLDN6-Z的CART对表达CLDN6的细胞毒性更强。
表4、融合表达单链抗体CLDN6 (AE3-20LH)的CAR T细胞的细胞毒性
Figure PCTCN2016077341-appb-000007
表5、融合表达单链抗体CLDN6 (AE3-20HL)的CAR T细胞的细胞毒性
Figure PCTCN2016077341-appb-000008
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 表达于免疫效应细胞表面的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包含顺序连接的：胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含特异性识别CLDN6的蛋白。
  2. 如权利要求所述1的嵌合抗原受体，其特征在于，所述的免疫效应细胞包括：T淋巴细胞，NK细胞或NKT细胞。
  3. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的特异性识别CLDN6的蛋白是抗体或配体；较佳地，所述的抗体是单链抗体或结构域抗体。
  4. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的跨膜区是包含CD8或CD28的跨膜区和铰链区的序列。
  5. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的胞内信号区选自：CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134的胞内信号区序列，或其组合。
  6. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包括如下的顺序连接的胞外结合区，跨膜区和胞内信号区：
    特异性识别CLDN6的单链抗体、CD8和CD3ζ；
    特异性识别CLDN6的单链抗体、CD8、CD137和CD3ζ；
    特异性识别CLDN6的单链抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
    特异性识别CLDN6的单链抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
  7. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体具有SEQ ID NO:21～24任一所述的氨基酸序列。
  8. 编码权利要求1-7任一所述的嵌合抗原受体的核酸。
  9. 如权利要求8所述的核酸，其特征在于，所述的核酸具有SEQ ID NO:17～20任一所述的核苷酸序列。
  10. 一种表达载体，其特征在于，其包含权利要求8-9任一所述的核酸。
  11. 如权利要求10所述的表达载体，其特征在于，所述的表达载体来源于慢病毒质粒pWPT。
  12. 一种病毒，其特征在于，所述的病毒包含权利要求10或11所述载体。
  13. 权利要求1-7任一所述的嵌合抗原受体、或权利要求8或9所述的核酸、或权利要求10或11所述的表达载体、或权利要求12所述的病毒的用途，用于制备靶向CLDN6的基因修饰的免疫效应细胞。
  14. 一种基因修饰的免疫效应细胞，其特征在于，其转导有权利要求8或9所述的核酸，或权利要求10或11所述的表达载体或权利要求12所述的病毒。
  15. 一种基因修饰的免疫效应细胞，其特征在于，其表面表达一种嵌合抗原受体，所述嵌合抗原受体的氨基酸序列选自SEQ ID NO:21-24任一所述的氨基酸序列。
  16. 权利要求14或15所述的基因修饰的免疫效应细胞的用途，其特征在于，用于制备抑制肿瘤的药物，所述的肿瘤是CLDN6阳性的肿瘤。
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