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CN106146666B - Target the immune effector cell and its preparation method and application of CLDN6 - Google Patents

Target the immune effector cell and its preparation method and application of CLDN6 Download PDF

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CN106146666B
CN106146666B CN201510136749.3A CN201510136749A CN106146666B CN 106146666 B CN106146666 B CN 106146666B CN 201510136749 A CN201510136749 A CN 201510136749A CN 106146666 B CN106146666 B CN 106146666B
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cldn6
cell
chimeric antigen
antigen receptor
immune effector
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CN106146666A (en
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王华茂
宋波
蔡秀梅
赵红霞
杨琳琳
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Keji Biomedical (shanghai) Co Ltd
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Abstract

The present invention relates to the immune effector cells and its preparation method and application of targeting CLDN6 (CLDN6).The present inventor has been successfully found a kind of target gene CLDN6 of immune effector cell for being suitable for developing Chimeric antigen receptor (CAR) modification from many tumor-related genes for the first time, and it is successfully prepared the immune effector cell of the CAR modification of targeting CLDN6, to provide a kind of completely new treatment means for tumours such as oophoroma, gastric cancer, adenocarcinomas of lung.

Description

Target the immune effector cell and its preparation method and application of CLDN6
Technical field
The invention belongs to tumour cell therapy fields, more particularly it relates to target the immune effector cell of CLDN6 And its preparation method and application.
Background technique
Effect of the immune effector cell in tumor immune response is paid more and more attention.Adopting based on immune effector cell Property immunization therapy achieves certain effect in Partial tumors, and this kind of immunotherapy method can overcome Antybody therapy Drawbacks described above, but in still unsatisfactory [Grupp SA, the et al.Adoptive cellular of the curative effect of most of tumours Therapy.Curr Top Microbiol Immunol., 2011；344:149-72.].In recent years, it is drenched according to cytotoxic T Bar cell (cytotoxic lymphocyte, CTL) depends on t lymphocyte receptor (T to the identification specificity of target cell Cell Receptor, TCR) discovery, will be for the scFv and t lymphocyte receptor of the antibody of tumor cell associated antigen The intracellular signals such as CD3 ζ or Fc ε RI γ activation motif be fused into Chimeric antigen receptor (Chimeric antigen receptor, CAR), and by it by the modes such as such as slow-virus infection gene modification on T lymphocyte surface.This CAR T lymphocyte energy It is enough to be selected with Major Histocompatibility compound (Major Histocompatibility Complex, MHC) non-limiting way Selecting property T lymphocyte is directed to tumour cell and specifically kills tumour.CAR T lymphocyte is immunotherapy of tumors The new Immunotherapy Strategy of one of field [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, NK cell (the Klingemann H.Challenges of cancer of CAR modification therapy with natural killer cells.
Cytotherapy.2014Dec 18.pii:S1465-3249 (14) 00791-9) or NKT cell also in clinic Illustrated in preceding research good anti-tumor activity (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 receptor includes extracellular combined area, transmembrane region and intracellular signal area.Usual extracellular region includes that can identify The scFv of tumor associated antigen, transmembrane region use CD8, the equimolecular transmembrane region of CD28, and intracellular signal area uses immunity receptor junket Propylhomoserin activation motifs (ITAM) CD3 ζ or Fc ε RI γ and costimulatory signal molecule CD28, CD27, CD137, CD134's etc. is intracellular Signaling zone.
Intracellular signal area only includes that ITAM is first generation CAR T lymphocyte, and wherein Chimeric antigen receptor each section is pressed Following form connection: scFv-TM-ITAM.This kind of CAR T can excite antitumoral cytotoxic effect, but cell factor It secretes fewer, and lasting anti-tumor effect [Zhang T. et al.Chimeric NKG2D- cannot be excited in vivo 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 second generation CAR T lymphocyte then developed joined the intracellular signal of CD28 or CD137 (also known as 4-1BB) Area, wherein Chimeric antigen receptor each section is connected by following form: scFv-TM-CD28-ITAM or scFv-TM-/CD137- ITAM.B7/CD28 the or 4-1BBL/CD137 costimulation effect that intracellular signal area occurs causes the continuous proliferation of T lymphocyte, And it can be improved the level of the T lymphocyte secretion cell factors such as IL-2 and IFN-γ, while improving the survival of CAR T in vivo Period and antitumous effect [Dotti G. et al. CD28costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma Patients. J Clin Invest, 2011,121 (5): 1822-1826.].
The third generation CAR T lymphocyte developed in recent years, wherein Chimeric antigen receptor each section is connected by following form: ScFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM further improves CAR T depositing in vivo Period living and its antitumous effect [Carpenito C., et al. Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137domains. PNAS, 2009,106 (9): 3360-3365.].
Although CAR T lymphocyte has tempting prospect in immunotherapy of tumors, high risk also needs to examine Consider.For example, since certain/kind of normal tissue low expression CAR specific antigen that can be identified may cause CAR T lymphocyte Damage to the normal tissue of expression corresponding antigens.Such as, for the antigen carbonic anhydride expressed on renal cell carcinoma patients tumour cell Enzyme IX (CAIX) be first for clinical CAR T lymphocyte adopt treatment case and first report containing CAR The case of the undershooting-effect of cell.There are 2-4 grades of hepatotoxicity wind agitation after repeatedly input CAR T lymphocyte in patient.Analyzing reason is Hepatic duct epithelial cell low expression CAIX, former clinical test are forced to interrupt while eliminating any evaluation of Case treatment effect [Stoter G. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX:first clinical experience. J clin oncol, 2006,24 (13): e20-e22.；Ngo MC., et al. Ex vivo gene transfer for improved adoptive immunotherapy of cancer. Human Molecular Genetics, 2011, R1-R7].In addition, costimulatory signal excessive in CAR can reduce effector cell Threshold value needed for activation, so that the T lymphocyte of gene modification is in low-level antigen or does not have antigen can also under conditions of triggering It can be activated, cause being released so that for large amount of cell factor that may cause so-called " cytokine storm ".Outside this signal Leakage (signal leakage) will lead to cytotoxicity of missing the target, to generate nonspecific tissue damage.For example, using needle Due to normal lung during to third generation CAR clinical treatment one of the Her2 Advanced Colon Cancer patient with liver and Lung metastases Low expression Her2 in tissue and cause so-called " cytokine storm " cause a disease people die suddenly [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.]。
The immune effector cell for designing CAR modification, when special T cell, targeted antigen gene is actually a kind of pass The selection of keyness, complexity and various uncontrollable factors in view of gene expression in vivo choose one suitably for CAR Gene be very difficult.Also, the antigen of many tumour-specifics is difficult to find for it and is suitable for constructing CAR The specific molecular of the immune effector cell of modification.Further, since different target spots may generate not immune effector cell The same influence, as EGFR target spot can raise 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. Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov.2013；3 (12): 1355-63.), Consequently, it is possible to influential effect T cell lethal effect (Moon EK, Wang LC, Dolfi DV, Wilson CB, Ranganathan R, Sun J, Kapoor V, Scholler J, Pur é E, 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.).Therefore, different target spot Selection may result in the different anti-tumor activity of immunocyte of prepared CAR modification.Due to the complexity of biology And unpredictability, these are all to need to test to go to verify.
Summary of the invention
The purpose of the present invention is to provide the immune effector cells and its preparation method and application of targeting CLDN6.
In the first aspect of the present invention, the Chimeric antigen receptor (CAR) for being expressed in immune effector cell surface is provided, it is described Chimeric antigen receptor include be linked in sequence: extracellular combined area, transmembrane region and intracellular signal area, wherein the extracellular combined area Albumen comprising specific recognition CLDN6 (Claudin 6).
In a preferred embodiment, the immune effector cell includes: T lymphocyte, NK cell or NKT cell.
In another preferred example, the albumen of the specific recognition CLDN6 is antibody or ligand；Preferably, described Antibody is single-chain antibody or domain antibodies.
In another preferred example, the transmembrane region is the sequence of the transmembrane region comprising CD8 or CD28 and hinge area.
In another preferred example, the intracellular signal area is selected from: CD3 ζ, Fc ε RI γ, CD27, CD28, CD137, The intracellular signal region sequence of CD134, or combinations thereof.
In another preferred example, the Chimeric antigen receptor includes the extracellular combined area of following sequential connection, cross-film Area and intracellular signal area:
Single-chain antibody, CD8 the and CD3 ζ of specific recognition CLDN6；
Single-chain antibody, CD8, CD137 and CD3 ζ of specific recognition CLDN6；
The single-chain antibody of specific recognition CLDN6, the transmembrane region (CD28a) of CD28 molecule, CD28 molecule intracellular signal Area (CD28b) and CD3 ζ；Or
The single-chain antibody of specific recognition CLDN6, the transmembrane region of CD28 molecule, the intracellular signal area of CD28 molecule, CD137 With CD3 ζ.
In another preferred example, the Chimeric antigen receptor has any amino acid in NO:21~24 SEQ ID Sequence.
In another aspect of this invention, the nucleic acid of any Chimeric antigen receptor in coding front is provided.
In a preferred embodiment, the nucleic acid has any nucleotide sequence in NO:17~20 SEQ ID.
In another aspect of this invention, a kind of expression vector is provided, it includes any nucleic acid in front.
In a preferred embodiment, the expression vector derives from slow virus plasmid pWPT (or pWPT-eGFP).
In another aspect of this invention, a kind of virus is provided, the virus (such as slow virus) includes the carrier.
Any Chimeric antigen receptor in front or the nucleic acid or the expression vector or the virus Purposes, be used to prepare targeting CLDN6 gene modification immune effector cell.
In another aspect of this invention, a kind of immune effector cell of gene modification is provided, transduction has the nucleic acid, Or the expression vector or the virus.
In another aspect of this invention, a kind of immune effector cell of gene modification is provided, surface expression one kind is chimeric Antigen receptor, the amino acid sequence of the Chimeric antigen receptor are selected from any amino acid sequence of SEQ ID NO:21-24.
In another aspect of this invention, the purposes of the immune effector cell of the gene modification is provided, suppression is used to prepare The drug of tumour processed, the tumour are the tumours of the CLDN6 positive.
In another preferred example, the tumour of the CLDN6 positive includes: oophoroma, gastric cancer, adenocarcinoma of lung.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.
Detailed description of the invention
CLDN6 expression conditions in each tissue of Fig. 1, RT-PCR detection and each cell.
The single-chain antibody purifying electrophoresis result of Fig. 2, anti-CLDN 6.
Fig. 3, the Lentiviral schematic diagram for encoding CLDN6.
Fig. 4, Western blot detect stablizing for CLDN6 and express.293T refers to HEK-293T；What 293T-CLDN6 referred to It is the HEK-293T cell for having transfected CLDN6.
Fig. 5, flow cyctometry analyze the combination situation of each cell line and anti-CLDN 6 antibody.
Fig. 6, Chimeric antigen receptor each section the order of connection schematic diagram.
Fig. 7, five kinds of eGFP-F2A-CAR piece segment DNA electrophoretic identifications spliced.
Fig. 8, as the exemplary slow virus carrier pWPT-eGFP-F2A-CAR's comprising coding CAR sequence of the invention Structural schematic diagram.
Specific embodiment
The present inventor after extensive and in-depth study, discloses a kind of exempting from for CAR modification based on CLDN6 gene for the first time Epidemic disease effector cell and preparation method thereof.
CLDN6 gene
Many kinds of tumor-specific genes of the present inventor's primary-stage survey find in this genoid a large portion also table Up in the normal cell of portion of tissue, it is more difficult to be applied to the immune effector cell technology of Chimeric antigen receptor modification；Have Tumor-specific genes have preferable tumor specific expression characteristic, still, the immunological effect of the CAR modification based on its design Cell does not have tumor cytotoxicity activity or activity very low, this may be because the target spot can trigger tumor cell secretion pair Immune effector cell plays the factor such as PD-L1 of inhibiting effect.
By inspecting and screening repeatedly, the present inventor has found target gene of the CLDN6 gene as design CAR. The amino acid sequence and its gene order of Claudin6 (CLDN6), be disclosed as respectively GenBank accession number NP_067018.1 and NM_021195.3 (sequence number: 22 and sequence number: 23) or GenBank accession number NP_067018.2 and NM_021195.4 (sequence number: 46 and sequence number: 47).Claudins is the close-connected memebrane protein positioned at epithelium and endothelium.
CAR T cell has become a kind of potential treatment means at present.But much tumour is still thin without related CAR T The report of born of the same parents' treatment, such as gastric cancer.Existing research shows that CLDN6 is the specific markers of gastric tissue.The present inventor, which studies, to be confirmed, CLDN6 is not expressed in most normal tissues really, and is expressed in some tumours such as oophoroma.But at present about CLDN6 only has monoclonal antibody as the drug candidate of therapy target, and whether it can be used successfully to corresponding oncotherapy still not It obtains and knows.Therefore, it is necessary to find new treatment means.In view of the tissue specificity of CLDN6, inventors have contemplated that if energy Enough CAR T cells carry out targeted therapy, are expected to the new anticancer agent of available one kind.But known CLDN6 antigen is one A tight junction protein, if can be contacted by CAR T cell and cause it is still unknown to the killing of corresponding target cell.This Outside, since protein conformation is to pull one hair and move the whole body, many monoclonal antibodies are evolved into single-chain antibody and often lose antigen knot Close activity or specificity.Fortunately, the inventors discovered that there are two single-chain antibody (AE3-20LH and AE3-20HL) to have well Antigen-binding specificity.The present inventor's further study showed that, the CAR T cell that is made of the two single-chain antibodies is protected The selective killing effect to CLDN6 positive cell is stayed.It is of the invention the result shows that, CLDN6 can become CAR really and modify Immune effector cell, especially T cell treatment target spot；CAR T cell for CLDN6 is that a kind of oncotherapy is candidate new Means.
Further research has shown that the T cell of the CAR modification of CLDN6 targeting removes to the property of can choose the CLDN6 positive really Tumour cell, and there is no toxicity to non-tumor cell 293T cell.Inventors believe that the immune effect of corresponding CAR modification Answer cell, especially T cell that should can be used for the treatment of human tumor.
Chimeric antigen receptor and its code nucleic acid
The present invention provides a kind of Chimeric antigen receptor for being expressed in immune effector cell surface, the chimeric antigen by Body includes to be linked in sequence: extracellular combined area, transmembrane region and intracellular signal area, wherein the extracellular combined area includes that specificity is known The albumen of other CLDN6 (claudin 6).The Chimeric antigen receptor is expressed in the surface of immune effector cell, may make immune Effector cell has the cytotoxic effect of high degree of specificity to the tumour cell of high expression CLDN6.
As preferred embodiment of the invention, the extracellular combined area includes the single-chain antibody scFv of specific recognition CLDN6 (CLDN6).The extracellular combined area of above-mentioned Chimeric antigen receptor albumen passes through the transmembrane region phase of CD8 hinge area and CD8 or CD28 Connection, immediately intracellular signal area after transmembrane region.
The present invention also includes the nucleic acid for encoding the Chimeric antigen receptor.Nucleic acid sequence of the invention can be DNA form Or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand. DNA can be coding strand or noncoding strand.The nucleic acid code of encoding chimeric antigen receptor protein amino acid sequence of the invention It can be degeneracy, that is, a variety of degeneracy nucleic acid sequences encoded with amino acid sequence are included among the scope of the present invention. It is well known in the art for encoding the degeneracy nucleic acid code of orresponding amino acid.The invention further relates to the variations of above-mentioned polynucleotides Body, coding have the polypeptide of identical amino acid sequence or the segment of polypeptide, analogs and derivatives with the present invention.This multicore glycosides The variant of acid can be the variant that the allelic variant naturally occurred or non-natural occur.These nucleotide variants include Substitution variants, Deletion variants and insertion variant.As known in the art, allelic variant is replacing for a polynucleotides Change form, it may be substitution, missing or the insertion of one or more nucleotide, but not from substantially change its encode it is more The function of peptide.
The antibody of specific recognition people CLDN6 can select antibody disclosed in the prior art, a variety of identification CLDN18A2 The monoclonal antibody of C-terminal epitope can apply to the present invention in an appropriate manner, can finally obtain with killing activity CAR immune effector cell.As particularly preferred mode of the invention, the antibody of the people CLDN6 is of the invention single-stranded Antibody scFv-CLDN6 (AE3-20LH) or scFv-CLDN6 (AE3-20HL).
Term used in the present invention " single-chain antibody (scFv) segment " refers to through such as undefined antibody fragment, It is the recombinant protein comprising heavy chain variable region (VH) and light chain variable region (VL) by connector (linker) connection, connector makes The two structural domains are associated, to ultimately form antigen binding site.Be typically of size of a complete antibody 1/6 of scFv. Single-chain antibody is preferably the amino acid chain sequence encoded by a nucleotide chain.The single-chain antibody that the present invention uses can be independent Or routine techniques known in the art is used in combination, such as amino acid deletions, insertion, substitution, increase, and/or recombination and/or Other method of modifying make further modification.This modification is introduced in its DNA sequence dna according to a kind of amino acid sequence of antibody Method is well known to those skilled in the art；See for example, Sambrook, molecular cloning: laboratory manual, Cold Spring Harbor Laboratory(2002)N.Y..Signified modification carries out preferably in nucleic acid level.It is above-mentioned single-stranded anti- Body can also include its derivative." derivative of antibody " includes for example when obtaining institute by display technique of bacteriophage in the present invention When stating the derivative of antibody, it is anti-with CLDN6 to increase that the surface plasma resonance technology as used in BIAcore system can be used Efficiency (Schier, human antibody hybridoma 7 (1996), the 97-105 for the phage antibody that former epitope combines；Malmborg is immunized Method magazine 183 (1995), 7-13).Further include, for example, chimeric antibody described in WO 89/09622 generation method, Humanized antibody described in EP-A10239400 and WO90/07861 generate method, WO91/10741, WO94/02602 and The derivative of antibody caused by the method for the human antibody in generation xenoantibody such as mouse being described in WO96/33735.
Term " specific recognition " of the invention mean bispecific antibody of the invention not with or substantially not with Any polypeptide cross reaction other than target antigen.Its specificity degree can be judged by immunological technique, including but It is not limited to immunoblotting, immunoaffinity chromatography, flow cytometry etc..In the present invention, specific recognition preferably passes through streaming Cell technology determines, and the standard of specific recognition can be grasped by persons skilled in the art according to it under concrete condition Common sense in the field judges.
The transmembrane region of Chimeric antigen receptor can be selected from the transmembrane region of the albumen such as CD8 or CD28.People's CD8 albumen is a different two Aggressiveness is made of two chains of α β or γ δ.In one embodiment of the invention, transmembrane region is selected from CD8 α or CD28 Transmembrane region.In addition, CD8 α hinge area (hinge) is a flexible region, therefore, CD8 or CD28 and transmembrane region add hinge area It is used to the target spot of Chimeric antigen receptor CAR identifying that structural domain scFv and intracellular signal area are connected.
Intracellular signal area can be selected from CD3 ζ, the intracellular signal area of Fc ε RI γ, CD28, CD137, CD134 albumen, and its Combination.CD3 molecule is made of five subunits, and wherein 3 ITAM bases are contained in CD3 ζ subunit (also known as CD3 zeta, abbreviation Z) Sequence, the motif are signal transduction areas important in TCR-CD3 complex.CD3 δ Z is the truncated CD3 ζ without ITAM motif Sequence, in the practice of the present invention generally as the building of negative control.Fc ε RI γ is mainly distributed on mast cell and basophilla grain Cell surface contains an ITAM motif, similar with CD3 ζ in structure, distribution and function.Further, as described above, CD28, CD137, CD134 are costimulatory signal molecules, are acted in the costimulation generated with its intracellular signal section after respective ligand binding Cause the continuous proliferation of immune effector cell (mainly T lymphocyte), and can be improved immune effector cell secretion IL-2 and The level of the cell factors such as IFN-γ, while improving the time to live and antitumous effect of CAR immune effector cell in vivo.
The anti-CLDN 6 Chimeric antigen receptor albumen of encoded by nucleic acid of the invention can be selected from sequentially to be connected as follows It connects:
ScFv (CLDN6)-CD8-CD3 ζ,
ScFv (CLDN6)-CD8-CD137-CD3 ζ,
ScFv (CLDN6)-CD28a-CD28b-CD3 ζ,
ScFv (CLDN6)-CD28a-CD28b-CD137-CD3 ζ,
And combinations thereof, wherein CD28a represents the transmembrane region of CD28 molecule, CD28b generation in related Chimeric antigen receptor albumen The intracellular signal area of table CD28 molecule.Above-mentioned various anti-CLDN 6 Chimeric antigen receptors are referred to as scFv (CLDN6)-CAR.
In one embodiment of the invention, nucleic acid of the invention has the sequence as described in NO:17~20 SEQ ID Column.In another embodiment of the present invention, nucleic acid of the invention is coding with the embedding of one of such as SEQ ID NO:21-24 Close the nucleic acid of antigen receptor albumen.
Expression vector and cell
The present invention also provides the Chimeric antigen receptor albumen that immune effector cell surface is expressed in comprising above-mentioned coding The carrier of nucleic acid.In a specific embodiment, the carrier that the present invention uses is a kind of slow virus plasmid vector pWPT- eGFP.The plasmid belongs to the third generation from slow virus carrier system is inactivated, and there are three plasmids to encode Protein G ag/ altogether for the system Pol, the packaging plasmid psPAX2 for encoding Rev albumen；Encode the envelope plasmid PMD2.G of vesicular stomatitis virus-G protein；And empty carrier pWPT- EGFP can be used for recombinating introducing purpose nucleic acid sequence, that is, encode the nucleic acid sequence of CAR.Empty carrier pWPT-eGFP (its As the mock in follow-up test) in by -1 α of elongation factor (elongation factor-1 α, EF-1 α) promoter regulation increase The expression of strong type green fluorescent protein (enhanced green fluorescent protein, eGFP).And include coding CAR The recombinant expression carrier pWPT-eGFP-F2A-CAR of purpose nucleic acid sequence be by by from foot and mouth disease virus (food-and- Mouth disease virus, FMDV) ribosomal skip sequence (ribosomal skipping sequence 2A) (letter Claim F2A) realize the coexpression of eGFP and CAR.
The invention also includes the viruses comprising above-mentioned carrier.Virus of the invention includes the infectious disease of tool after packaging Poison also includes the virus to be packaged for being packaged as having infectious viral institute essential component.Known in the art its Its viral and its corresponding plasmid vector into immune effector cell that can be used for transduceing foreign gene can also be used for the present invention.
In one embodiment of the invention, the virus is comprising above-mentioned pWPT-eGFP-F2A-CAR recombinant vector The slow virus of (containing scFv (CLDN6)-CAR).
The present invention also provides the immune effector cells of gene modification, have by the nucleic acid for transduceing of the invention or by transduction The above-mentioned recombinant plasmid containing the nucleic acid comprising described in of the invention, or the virus comprising the plasmid.The nucleic acid of this field routine Transduction method may be used to the present invention including non-viral and viral transduction method.Include based on non-viral transduction method Electroporation and transposons method.The Nucleofector nuclear transfection instrument of recent Amaxa company research and development can be directly by foreign gene Import the high efficiency transduction that nucleus obtains target gene.In addition, being based on sleeping beauty transposon stand (Sleeping Beauty System) or the more common electroporation of transduction efficiency of the Transposon Systems such as PiggyBac transposons improves a lot, will [Davies JK., et has been reported in nucleofector transfection instrument and the system combined application of sleeping beauty transposon stand al.Combining CD19 redirection and alloanergization to generate tumor-specific Human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res, 2010,70 (10): OF1-10.], this method not only transduction efficiency with higher but also can be realized the site-directed integration of target gene. In one embodiment of the invention, the transduction method for realizing the immune effector cell of Chimeric antigen receptor gene modification is base In virus such as retrovirus or the transduction method of slow virus.This method has transduction efficiency high, and foreign gene can stablize table It reaches, and the advantages that in vitro culture immune effector cell reaches the time of clinical number of stages can be shortened.In the immune effect of the transgenosis Cell surface is answered, the nucleic acid of transduction is by transcription, accurate translation on its surface.Pass through the tumour cell to a variety of different cultures Cell in vitro poison is carried out it is demonstrated experimentally that the immune effector cell of anti-CLDN 6 Chimeric antigen receptor gene modification of the invention has The tumor cytotoxicity effect (also known as cytotoxicity) of high degree of specificity.Therefore encoding chimeric antigen receptor protein of the invention Nucleic acid, the plasmid comprising the nucleic acid, virus and transduction comprising the plasmid have above-mentioned nucleic acid, and plasmid or viral transgenosis are immune Effector cell can be efficiently used for the immunization therapy of tumour.
In one embodiment, the immune effector cell of gene modification of the invention, a kind of inosculating antibody of surface expression Original receptor, the Chimeric antigen receptor are expressed by the nucleic acid encode of one of SEQ ID NO:17-20.In another embodiment In, a kind of Chimeric antigen receptor of transgenosis immune effector cell surface expression of the invention, the amino of the Chimeric antigen receptor Acid sequence is selected from one of SEQ ID NO:20-24.
In view of at present still not about targeting CLDN6 CAR T report, the present inventor is for the first time from many tumor-related It has been successfully found a kind of target gene CLDN6 suitable for CAR T cell because in, and has been successfully prepared the CAR T of targeting CLDN6 Cell, to provide a kind of completely new treatment means for tumours such as oophoroma, gastric cancer, adenocarcinomas of lung.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is compiled, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.
Embodiment 1, CLDN6 are in various normal tissues and the detection of expression of tumour cell
The extraction of cell total rna: Normal human tissue RNA is purchased from Clontech Laboratories, Inc.Ovarian cancer cell In 5%CO2, routine culture in 37 DEG C of incubators.PBS (0.14M NaCl, 2.7mM KCl, 10.1mM Na2HPO4,1.8mM KH2PO4, pH 7.3) it washes cell 2 times.The good cell total rna of growth conditions is extracted with Trizol reagent, every ware cell is added 1ml Trizol is sufficiently blown and beaten to not sticky；By the chloroform that every milliliter of initial volume Trizol adds 0.2ml newly to break a seal, acutely shake 15sec is swung, 5min is incubated at room temperature；4 DEG C, 12,000g centrifugation 15min；Colorless supernatant liquid is moved into new centrifuge tube, by every milli It rises initial volume Trizol and adds 0.5ml isopropanol, -20 DEG C of incubations 20min, 12,000g 4 DEG C of centrifugation 10min；Supernatant is outwelled, is used 75% ethanol washing of 1ml, 4 DEG C, 10,000g centrifugation 5min；Drying at room temperature RNA precipitate 10-20min, with DEPC treated nothing RNA enzyme H2O dissolution precipitating.Spectrophotometer standard measure RNA.
Reverse transcription: 1 μ l of cell total rna (1 μ g/ μ l) and 1 μ l of Oligo dT (10 μM) is mixed, and after 72 DEG C of incubation 5min, is stood It is placed in 5min on ice quarter；Be added 15 μ l reverse transcription mixed liquors (6.1 μ l, 5 × RT Buffer of Nuclease-free H2O, 4 μ l, 25mM MgCl22.4 μ l, 10mM dNTPs 1 μ l, RNase Ribonuclease Inhibitor0.5 μ l, Improm-IITM 1 μ l of Reverse Transcriptase), 25 DEG C of incubations 5min, 42 DEG C of incubation 1h；70 DEG C of incubation 15min terminate reaction.
RT-PCR: using the cDNA of above-mentioned reverse transcription as template, with primer CLDN6-F:GGAATGCAGATCCTGGGAGT, CLDN6-R:ATGAAAGCGGTCA carries out PCR, PCR reaction system: 1 μ l template, 2.5 μ l 2mM dNTP, and 2.5 μ l 10 × Buffer, each l μ l, 5U Taq enzyme of primer, with H2O supplies 25 μ l.PCR reaction condition: 94 DEG C of 3min；94 DEG C of 30sec, 56 DEG C 30sec, 72 DEG C of 40sec, 35 circulations；72℃10min；4 DEG C of preservations.β-actin is expanded simultaneously as internal reference, and β-actin draws Object: β-actin-F:5 '-TCCTCCCTGGAGAAGAGCTA-3 ', β-actin-R:5 '-GTACTTGCGCTCAGGAGGAG-3 '. Amplification condition: 94 DEG C of 3min；94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 30sec, 30 circulations；7210min℃.Pass through 2% fine jade Sepharose electrophoresis observes clip size.
Such as Fig. 1, RT-PCR is the results show that be not detected the amplification of CLDN6 gene, in ovarian cancer cell in normal tissue Only A2780 cell amplifies positive band, and does not amplify positive band in Hey cell.
Embodiment 2, anti-CLDN 6 single-chain antibody expression
The antibody of anti-CLDN 6 although have in the prior art, if be directly evolved into from monoclonal antibody single-stranded anti- Body often will affect its antigen-binding activity, or even specificity.The gene synthesis technology put up a bridge by based on PCR, the present inventor ScFv-CLDN6 (64) (SEQ ID NO:1 (nucleotide), 2 (amino acid)), scFv-CLDN6 (AE3-20LH) (SEQ are synthesized ID NO:3 (nucleotide), 4 (amino acid)), scFv-CLDN6 (AE3-20HL) (SEQ ID NO:5 (nucleotide), 6 (amino Acid)) single chain antibody sequence, by NheI/BamHI (being purchased from NEB) double digestion, with T4 DNA ligase (being purchased from NEB) in same With NheI/BamHI double digestion vector plasmid pCMV-V5-Fc, (carrier is in multiple cloning sites downstream amalgamation and expression human antibody Fc piece Section, hereinafter referred to as V5-Fc are purchased from Shanghai Rui Jing Bioisystech Co., Ltd) it connects and converts in host strain TOP10, picking Clone identifies positive colony by PCR and is confirmed by sequencing, obtains V5-scFv-CLDN6 (64)-Fc, V5-scFv- respectively CLDN6 (AE3-20LH)-Fc and V5-scFv-CLDN6 (AE3-20HL)-Fc eukaryon expression plasmid.
Above-mentioned expression plasmid is transfected into well-grown HEK-293F cell respectively, 37 DEG C, 5%CO2, 125rpm shaking table company Continuous culture 7 days, 4000rpm are centrifuged 10min, and removal precipitating collects supernatant, and with 0.45 μm of membrane filtration, the sample that will be handled well Product carry out affinity purification, the final single-chain antibody-Fc fusion protein for obtaining purifying with protein A (being purchased from GE) affinity column ScFv-CLDN6 (64)-Fc, scFv-CLDN6 (AE3-20LH)-Fc, scFv-CLDN6 (AE3-20HL)-Fc, qualification result is such as Fig. 2.
Embodiment 3, CLDN6 stable expression cell line building
1, the cell line building of heterogenous expression CLDN6
The method for synthesizing gene put up a bridge by based on PCR, and synthesis CLDN6 genetic fragment (SEQ ID NO:7 (nucleotide), 8 (amino acid)), by MluI/SalI (being purchased from NEB) double digestion, with T4 DNA ligase (being purchased from NEB) in equally with MluI/ The plasmid vector pWPT (being purchased from Shanghai Rui Jing Bioisystech Co., Ltd) of SalI double digestion is connected and is converted in host strain TOP10 In, picked clones are identified positive colony by PCR and are confirmed by sequencing, obtain slow virus plasmid pWPT-CLDN6 (Fig. 3).
The packaging of slow virus: by 293T cell (7 × 105/ hole) it is inoculated in 6 orifice plates, about 70%~80% Cell abundance, carefully Born of the same parents' culture solution is the DMEM+10%FBS of 2ml antibiotic-free.Next day uses LipofectamineTM2000 are transfected, and are prepared Liposome/DNA mixture: A liquid is prepared: molten by 2 μ g, pAX2 plasmid of pWPT-CLDN6 plasmid, 1.5 μ g, pMD2.G plasmid, 0.6 μ g Solution mixes gently in the serum-free DMEM culture solution of 100 μ l.B liquid is prepared: by 10 μ l LipofectamineTM2000 dissolutions It in the serum-free DMEM culture solution of 100 μ l, mixes gently, is incubated at room temperature 5min.A liquid and B liquid are gently mixed, are placed at room temperature for After 20min.6 orifice plates inner cell original fluid is discarded, the DMEM culture containing 2%FBS of new 1.8ml antibiotic-free is added Liquid.Then A/B mixed liquor is added dropwise in Tissue Culture Dish, gently piping and druming mixes.37 DEG C, 5%CO2Continue to cultivate 72h, receive Collect viral supernatant liquid.
The CLDN6 virus liquid of above-mentioned collection is added to respectively in the HEK-293T cell being laid in 6cm plate, is received after 72h Collect cell, is cracked using cell pyrolysis liquid.40 μ g are taken to carry out PAGE gel electrophoresis the cell protein that cracking is collected, Immunoblotting then is carried out to gel, and is dyed with the anti-flag antibody of mouse (being purchased from Shanghai Rui Jing Bioisystech Co., Ltd). After PBS washing, incubated with the goat anti-mouse antibody (being purchased from Shanghai Rui Jing Bioisystech Co., Ltd) of horseradish peroxidase-labeled ECL reagent colour development is used after educating, and is finally developed.
Western blot in the HEK-293T cell (i.e. 293T-CLDN6) for having transfected CLDN6 the results show that can examine The band of molecular size range about 25KD is measured, and respective strap (Fig. 4) is not detected in the ghost of untransfected, illustrates external source The cell line of expression CLDN6 constructs successfully.
2, flow cyctometry analyzes the combination situation experimental procedure of each cell line and anti-CLDN 6 antibody
Single-chain antibody scFv- is analyzed by fluorescence-activated cell sorter (FACS) (BD company, FACSCalibur) CLDN6 (64), scFv-CLDN6 (AE3-20LH) and scFv-CLDN6 (AE3-20HL) are respectively and the combination energy of following cell lines Power.
The specific method is as follows:
1) 293T of logarithmic growth phase, 293T-CLDN6 and ovarian cancer cell line A2780 tumor cell inoculation arrive In 6cm plate, inoculating cell density is about 90%, and 37 DEG C of incubators are incubated overnight.
2) uses the EDTA vitellophag of 10mM, and cell is collected by centrifugation in 200g × 5min.With 1 × 106~1 × 107/ mL's Concentration is resuspended in 1% phosphate buffer (NBS PBS) containing calf serum, and streaming dedicated pipe is added by the amount of 100ul/ pipe In.
3) .200g × 5min is centrifuged, and abandons supernatant.
4) is separately added into test antibodies scFv-CLDN6 (64), scFv-CLDN6 (AE3-20LH) and scFv-CLDN6 (AE3-20HL), while using PBS as negative control, 100ul is added in the final concentration of 10 μ g/ml of antibody, every pipe.Ice bath, 45 points Clock.
5) 2ml 1%NBS PBS is added in the every pipe of, is centrifuged with 200g × 5min, totally two times.
6) abandons supernatant, and the goat anti-human antibody of the diluted FITC fluorescent marker of 1:50 is added (from upper Haikang at bioengineering Co., Ltd), 100ul is added in every pipe.Ice bath, 45 minutes.
7) 2ml 1%NBS PBS is added in the every pipe of, is centrifuged with 200g × 5min, totally two times.
8) abandons supernatant, is resuspended in 300ul 1%NBS PBS, flow cytomery.
9) application Flow cytometry data analysis software WinMDI 2.9 analyzes data.
As a result such as Fig. 5, flow cytometric analysis results show single-chain antibody scFv-64 and CLDN6 negative cells 293T, or do not combined with the CLDN6 A2780 cell for stablizing the 293T-CLDN6 and endogenous expression CLDN6 of expression.And it is single Chain antibody scFv-CLDN6 (AE3-20LH) and scFv-CLDN6 (AE3-20HL) can stablize expression with specific recognition CLDN6 293T-CLDN6 cell, but do not combined with the 293T cell of CLDN6 feminine gender, show that the two single-chain antibodies can be with specific recognition CLDN6.Furthermore the two single-chain antibodies can also be with the A2780 cell of specific recognition endogenous expression CLDN6.
The building and virus of the slow virus plasmid of the Chimeric antigen receptor albumen of embodiment 3, expression nucleic acid encode of the present invention Packaging
Table 1 explains the order of connection of the exemplary Chimeric antigen receptor each section of the present invention, and the connection is referring also in Fig. 6 It is shown.
Table 1
1, the amplification of nucleic acid fragment
(1) amplification of scFv (CLDN6-AE3-20LH, CLDN6-AE3-20HL) sequence
Using V5-scFv-CLDN6 (AE3-20LH)-Fc plasmid as template, primer pair forward primer (the SEQ ID of use NO:9, the sequence comprising part 2A) and reverse primer (SEQ ID NO:10 includes part CD8 hinge sequence), PCR amplification It obtains scFv (CLDN6-AE3-20LH)；Likewise, using V5-scFv-CLDN6 (AE3-20HL)-Fc plasmid as template, use (SEQ ID NO:12 includes part for primer pair forward primer (SEQ ID NO:11, the sequence comprising part 2A) and reverse primer CD8 hinge sequence) PCR amplification acquisition scFv (CLDN6-AE3-20HL).
(2) nucleic acid sequence of Chimeric antigen receptor other parts
Removing for anti-CLDN 6 Chimeric antigen receptor albumen is other outside scFv (CLDN6-AE3-20LH, CLDN6-AE3-20HL) Partial nucleic acid sequence is respectively 201310164725.X with number of patent application, sequence SEQ disclosed in 201310108532.2 ID NO:20,23 are obtained for template by PCR mode.Specifically, wherein eGFP-F2A sequence with number of patent application Contained SEQ ID NO:20 plasmid is template in 201310164725.X, carries out PCR with primer pair (SEQ ID NO:13,14) Amplification obtains.CD8-CD3 ζ (Z) and CD28a-CD28b-CD137-CD3 ζ (28BBZ), respectively with scFv (GPC3)-CD8-CD3 ζ (applying for a patent SEQ ID NO:20 in the 201310164725.X) and (Shen scFv (GPC3)-CD28a-CD28b-CD137-CD3 ζ Please SEQ ID NO:23 in patent 201310164725.X) it is template, PCR is passed through using primer pair (SEQ ID NO:15,16) Amplification obtains CD8-CD3 ζ (Z) and CD28a-CD28b-CD137-CD3 ζ (28BBZ) segment.
2, the splicing of nucleic acid fragment
Respectively by the eGFP-F2A nucleic acid fragment of such as aforementioned acquisition, with equimolar scFv (CLDN6-AE3-20LH) or ScFv (CLDN6-AE3-20HL) nucleic acid fragment and equimolar CD8-CD3 ζ (Z) or CD28a-CD28b-CD137-CD3 ζ (28BBZ) nucleic acid fragment carries out three fragment assemblies and PCR as shown in Figure 6, splices condition are as follows: initial denaturation: 94 DEG C, 4min；Become Property: 94 DEG C, 40s；Annealing: 60 DEG C, 40s；Extend: 68 DEG C, 140s, carrying out 5 circulations, then 68 DEG C of overall elongation, 10min is mended Archaeal dna polymerase and forward primer (SEQ ID NO:13) and reverse primer (SEQ ID NO:16) PCR amplification 30 circulations afterwards are filled, Amplification condition is initial denaturation: 94 DEG C, 4min；Denaturation: 94 DEG C, 40s；Annealing: 60 DEG C, 40s；Extend: 68 DEG C, 140s, carrying out 30 A circulation, then 68 DEG C of overall elongation, 10min.The segment that amplification obtains is referred to as (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 electroresis appraisal result such as Fig. 7 for the eGFP-F2A-CAR segment spliced.
Sequence in table 2, the present invention
3, the building of slow virus plasmid vector
As an example, constructing the carrier system that slow virus plasmid vector of the invention uses below belongs to the third generation from inactivation Slow virus carrier system, the system are altogether the packaging plasmid for encoding Protein G ag/Pol, encoding Rev albumen there are three plasmid PsPAX2 (is purchased from addgene)；It encodes the envelope plasmid PMD2.G (being purchased from addgene) of vesicular stomatitis virus-G protein and is based on empty carrier The recombinant expression carrier of the coding target gene CAR of pWPT-eGFP (being purchased from addgene).
In empty carrier pWPT-eGFP, included -1 α of elongation factor (elongation factor-1 α, EF-1 α's) is opened The expression of the controllable enhanced green fluorescence protein of mover (enhanced green fluorescent protein, eGFP), After being inserted into the construct of the aforementioned building of the present embodiment in empty carrier, the recombinant expression carrier of coding target gene CAR is formed, In by ribosomal skip sequence from foot and mouth disease virus (food and mouth disease virus, FMDV, Ribosomal skipping sequence, F2A) realize eGFP and target gene CAR coexpression.F2A is from aftosa One section of core sequence of the 2A (or being " self cleavage polypeptide 2A ") of virus, has " self cleavage " function of 2A, may be implemented Trip and downstream gene coexpression.2A is since its shear efficiency is high, upstream and downstream gene expression balance is high and own sequence is short and small Advantage provides a kind of effective possible strategy for building gene therapy polycistronic vector.Especially it is being based on Chimeric antigen receptor The coexpression for mostly realizing target gene and GFP or eGFP in the immunization therapy of gene modification T lymphocyte using the sequence, leads to The expression of CAR can be detected indirectly by crossing detection GFP or eGFP.
The present embodiment is constructed by the Lentiviral of the F2A eGFP being connected and specific C AR coexpression, is referred to as PWPT-eGFP-F2A-CAR (Fig. 8).The target gene eGFP-F2A-CAR obtained in above-mentioned steps 2 is (referring in embodiment 3 2, F2A subsequent components are referred to as CAR) by MluI and SalI restriction enzymes double zyme cutting, it is connected into same double digestion In pWPT carrier, to construct the slow virus carrier for expressing each Chimeric antigen receptor.Successful carrier is constructed through MluI and SalI After digestion identification and sequencing are correct, it can prepare to pack for slow virus.As previously mentioned, eGFP-F2A-CAR is transcribed into one MRNA, but two peptide chains of eGFP and anti-CLDN 6 Chimeric antigen receptor finally are translated as, wherein under the guidance of CD8 alpha signal peptide Anti-CLDN 6 Chimeric antigen receptor will be located on cell membrane.
The obtained carrier containing each purpose CAR is following (the subsequent component of F2A may be simply referred to as 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。
By constructing above, it can get four eGFP-F2A-CAR polypeptide sequences respectively, referred to as:
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 293T packs slow virus
With 6 × 106Density inoculated and cultured to the 6th~10 generation HEK-293T cell (ATCC:CRL-11268) in 10cm In culture dish, 37 DEG C, 5%CO2Overnight incubation prepares for transfecting.Culture medium is containing 10% fetal calf serum (being purchased from PAA company) DMEM (be purchased from PAA company).
The step of transfection, is as follows:
4.1A liquid is prepared: 10 μ g mock control or each target gene plasmid pWPT-eGFP-F2A-CAR of 10 μ g divide Not with 7.5 μ g packaging plasmid PAX2: and 3 μ g envelope plasmid pMD2.G, dissolves in the serum-free DMEM culture solution of 800 μ L, mix.
4.2B liquid is prepared: 60 μ g PEI (polyethyleneimine is purchased from Polysciences company) are dissolved in the nothing of 800 μ L It in serum DMEM culture solution, mixes gently, is incubated at room temperature 5min.
The formation of 4.3 transfection composites: A liquid is added in B liquid and is gently mixed, vortex mixed or is gently mixed immediately after addition It is even, it is incubated for 20min at room temperature.
4.4 are added dropwise to transfection composite 1.6ml in HEK-293T cell, after 4-5h hours, are trained with the DMEM of 2%FBS Base changes liquid to the 293T cell of transfection.
Transfection next day observation transfection efficiency (being in the cell proportion of green fluorescence) ,~80% Positive transfections efficiency is For transfection experiment success.After transfecting 72h, virus is collected by filtration using 0.45 μm of filter (being purchased from Millipore company), then Using Beckman Optima L-100XP ultracentrifuge 28000rpm, 4 DEG C are centrifuged 2 hours, abandon centrifugation supernatant, centrifugation gained 007 culture solution of Quantum (being purchased from PAA company) for precipitating 1/10~1/50 stoste volume is resuspended, with 100 μ L/ pipe point Dress freezes in -80 DEG C, to titration of virus or infection T lymphocyte.
5, measurement is packaged with the slow virus titre of mock or eGFP-F2A-CAR
First day, with 1 × 105/ mL is inoculated with 293T cell in 96 well culture plates, and 100 holes μ L/, 37 DEG C, 5%CO2 is cultivated, Culture solution is the DMEM containing 10% fetal calf serum.Second day, 50 hole μ L/ culture supernatants are abandoned, the 50 fresh above-mentioned cultures in the hole μ L/ are added Liquid, and the polybrene containing final concentration of 6 μ g/mL, 37 DEG C, 5%CO2 is incubated for 30min.Add the virus stock solution used or 1 μ in 10 holes μ L/ The viral concentration liquid in the hole L/, 5 times of dilutions, 4 gradients, two multiple holes, 37 DEG C, 5%CO2Culture.After infecting 48h, fluidic cell Instrument detects eGFP, is advisable with the cell number that positive rate is 5~20%, and calculating titre (U/mL)=positive rate × extension rate × 100×104.The titre of the above-mentioned virus comprising mock, that is, empty vector control and each eGFP-F2A-CAR of PEI infection protocol packaging It is about 2~4 × 106The level of U/mL, the virus titer surveyed after concentrated is about 2~4 × 107U/mL。
Embodiment 4, recombinant slow virus infect CTL cell
Human peripheral blood single nucleus cell (Shanghai City Blood Center is obtained by density-gradient centrifugation method by healthy human peripheral blood There is provided), peripheral blood mononuclear cells passes through CTL cell magnetic bead (being purchased from Stem Cell Technologies) negativity method for separating CTL is obtained, the CTL cell after sorting carries out the purity of FCM analysis CTL cell, with positive rate >=95% of CTL cell It is advisable and carries out next step operation.With about 1 × 106It is (public purchased from PAA that 007 lymphocytes culture medium liquid of Quantum is added in/mL density Department) it cultivates and with cell: magnetic bead ratio is magnetic bead (the Invitrogen public affairs that 1:1 was added while being coated with AntiCD3 McAb and CD28 antibody Department) and recombinant human il-2's (be purchased from Huaxin Advanced Biotechnical Co., Ltd., Shanghai) stimulation of final concentration 300U/mL cultivate for 24 hours.So CTL cell is infected with the above-mentioned recombinant slow virus of MOI ≈ 5 afterwards.Metainfective cell every other day uses 5 × 105The density of/mL It is passed on, while adding the recombinant human il-2 of final concentration 300U/mL in lymphocyte culture medium.
The CTL cell of infection is expressed when cultivating the 8th day by the variant Chimeric antigen receptor of FCM analysis, due to EGFP and CAR is co-expressed, and the positive cell for detecting eGFP is the positive cell for expressing Chimeric antigen receptor.With the T being uninfected by It is as shown in table 3 to express the virus infection CTL cell of different Chimeric antigen receptors its positive rate as negative control for lymphocyte. The positive rate the result shows that, the CAR of certain positive rate can be obtained by the method for slow-virus infection+CTL cell.
Table 3
CTL cell is after infection is packaged with the virus of different Chimeric antigen receptors respectively, with cell density for 5 × 105/ml Secondary culture, counting simultaneously add IL-2 (final concentration of 300U/ml) cell culture fluid of passage every other day, cultivate the 11st day about There is 20~40 times of amplification, shows that the CTL cell for expressing different Chimeric antigen receptors is able to carry out a certain number of expansions in vitro Increase, provides guarantee for subsequent in vitro toxicity test and in vivo studies.
The in vitro toxicity effect experiment of embodiment 5, the T lymphocyte of expression Chimeric antigen receptor
The material that in vitro toxicity experiment uses is as follows:
293T and ovarian cancer cell as shown in table 4 is verified external as target cell, effector cell for such as embodiment 4 The positive cell of the FACS detection Chimeric antigen receptor expression of culture 12 days is denoted as the Chimeric antigen receptor positive (CAR+) CTL, Target is imitated than being optionally respectively 3:1,1:1 and 1:3, target cell numbers are 10000/hole, according to different effect targets than corresponding effect Cell.Each group is all provided with 5 multiple holes, takes the average value of 5 multiple holes.Detection time is 18h.
Wherein each experimental group and each control group are as follows:
Each experimental group: the CTL of the different Chimeric antigen receptors of each target cell+expression,
Control group 1: target cell maximum discharges LDH,
Control group 2: the spontaneous release LDH of target cell,
Control group 3: the spontaneous release LDH of effector cell.
Detection method: it is carried out using 96 non-radioactive cell toxicity detection kit (Promega company) of CytoTox.It should Method is the detection method based on colorimetric method, alternative51Cr method for releasing.Detection quantitatively measures lactic dehydrogenase Enzyme (LDH).LDH is a kind of stable cytoplasm enzyme, can be released in cell cracking, delivery mode with51Cr is in radioactivity Delivery mode in analysis is essentially identical.In the LDH culture medium supernatant released, it can be examined by the enzyme reaction of coupling in 30 minutes It surveys, LDH can make a kind of tetrazolium salts (INT) be converted into red formazan (formazan) in enzyme reaction.The red product of generation Amount it is directly proportional to the cell number of cracking.Referring in particular to 96 non-radioactive cell toxicity detection kit specification of CytoTox.
Cytotoxicity calculation formula are as follows:
Specifically as shown in table 4 and table 5, expression Chimeric antigen receptor of the invention (amalgamation and expression single-chain antibody CLDN6 (AE3-20LH) or CLDN6 (AE3-20HL)) CLDN6-Z CAR+CTL and CLDN6-28BBZ CAR+CTL to height express The 293T cell of CLDN6 has apparent lethal effect, and does not kill to the 293T cell of CLDN6 feminine gender, shows that they can be with The selectively cell of killing expression CLDN6.In addition, expression Chimeric antigen receptor CLDN6-Z CAR of the invention+CTL and CLDN6-28BBZ CAR+CTL can also have to the ovarian cancer cell A2780 of endogenous expression CLDN6 significant killing (see Table 4 and table 5), and present effect target imitated than gradient dependence target than more high cell toxicity effect it is stronger.
Effect target further displays the CTL for expressing anti-CLDN 6 Chimeric antigen receptor of the invention to high than the data of dependence Express the specific cytotoxicity effect of the cell of CLDN6.
In comparison, negative control is used as by the virus transfection containing mock plasmid (carrying scFv-CLDN6- δ Z) CTL show that the cytotoxic effect of the cell line to expression CLDN6 above-mentioned three kinds high is very low.It expresses CLDN6 to height The cytotoxicity of cell line and the cell toxicity data of CTL of expression anti-CLDN 6 Chimeric antigen receptor of the invention show The difference of highly significant.
The above result shows that Chimeric antigen receptor constructed by single-chain antibody of the selection for CLDN6, it can be selectively The target cell of the high expression CLDN6 of killing.In addition, from the point of view of cell toxicity data, the CAR T ratio CLDN6-Z's of CLDN6-28BBZ CART is stronger to the cytotoxicity of expression CLDN6.
The cytotoxicity of the CAR T cell of table 4, amalgamation and expression single-chain antibody CLDN6 (AE3-20LH)
The cytotoxicity of the CAR T cell of table 5, amalgamation and expression single-chain antibody CLDN6 (AE3-20HL)
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (15)

1. being expressed in the Chimeric antigen receptor on immune effector cell surface, which is characterized in that the Chimeric antigen receptor includes It is linked in sequence: extracellular combined area, transmembrane region and intracellular signal area, wherein the extracellular combined area includes specific recognition The albumen of CLDN6；Wherein, the extracellular combined area is single-chain antibody, amino acid sequence such as SEQ ID NO:4 or SEQ ID Shown in NO:6.
2. Chimeric antigen receptor as described in claim 1, which is characterized in that the immune effector cell includes: that T lymph is thin Born of the same parents, NK cell or NKT cell.
3. Chimeric antigen receptor as described in claim 1, which is characterized in that the transmembrane region includes CD8 or CD28 The sequence of transmembrane region and hinge area.
4. Chimeric antigen receptor as described in claim 1, which is characterized in that the intracellular signal area is selected from: CD3 ζ, Fc ε The intracellular signal region sequence of RI γ, CD27, CD28, CD137, CD134, or combinations thereof.
5. Chimeric antigen receptor as described in claim 1, which is characterized in that the Chimeric antigen receptor includes following suitable The extracellular combined area of sequence connection, transmembrane region and intracellular signal area:
Single-chain antibody, CD8 the and CD3 ζ of specific recognition CLDN6；
Single-chain antibody, CD8, CD137 and CD3 ζ of specific recognition CLDN6；
The single-chain antibody of specific recognition CLDN6, the transmembrane region of CD28 molecule, the intracellular signal area of CD28 molecule and CD3 ζ；Or
The single-chain antibody of specific recognition CLDN6, the transmembrane region of CD28 molecule, the intracellular signal area of CD28 molecule, CD137 and CD3ζ。
6. Chimeric antigen receptor as described in claim 1, which is characterized in that the Chimeric antigen receptor has SEQ ID Amino acid sequence shown in NO:21~24 are any.
7. encoding the nucleic acid of any Chimeric antigen receptor of claim 1-6.
8. nucleic acid as claimed in claim 7, which is characterized in that the nucleic acid has NO:17~20 SEQ ID any shown Nucleotide sequence.
9. a kind of expression vector, which is characterized in that it includes any nucleic acid of claim 7-8.
10. expression vector as claimed in claim 9, which is characterized in that the expression vector derives from slow virus plasmid pWPT。
11. a kind of virus, which is characterized in that the virus includes the carrier of claim 9 or 10.
12. nucleic acid or claim 9 described in claim the 1-6 any Chimeric antigen receptor or claim 7 or 8 Or the purposes of virus described in expression vector described in 10 or claim 11, it is used to prepare the gene modification of targeting CLDN6 Immune effector cell.
13. a kind of immune effector cell of gene modification, which is characterized in that its transduce have the right to require 7 or 8 described in nucleic acid, Or virus described in expression vector described in claim 9 or 10 or claim 11.
14. a kind of immune effector cell of gene modification, which is characterized in that a kind of Chimeric antigen receptor of its surface expression, it is described The amino acid sequence of Chimeric antigen receptor be selected from SEQ ID NO:21-24 it is any shown in amino acid sequence.
15. the purposes of the immune effector cell of gene modification described in claim 13 or 14, which is characterized in that be used to prepare suppression The drug of tumour processed, the tumour are the tumours of the CLDN6 positive.
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Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116999572A (en) 2017-09-29 2023-11-07 第一三共株式会社 Antibody-drug conjugate, pharmaceutical composition and application thereof in preparation of drugs for treating tumors
EP3747433A4 (en) 2018-02-02 2021-12-22 Cafa Therapeutics Limited Combination of cellular immunotherapy
EP3778649A4 (en) 2018-03-09 2022-05-04 CRAGE medical Co., Limited Method and composition for treating tumors
WO2019210863A1 (en) 2018-05-03 2019-11-07 科济生物医药（上海）有限公司 Immune effector cell and use thereof
AU2019271819A1 (en) 2018-05-15 2021-01-14 Crage Medical Co., Limited Genetically engineered cell and application thereof
CN112930199B (en) 2018-07-24 2024-08-13 恺兴生命科技(上海)有限公司 Method for treating tumor by immune effector cells
EP3892333A4 (en) 2018-12-07 2022-11-30 CRAGE medical Co., Limited Tumor combined immunotherapy
KR20210126008A (en) 2019-01-07 2021-10-19 카르스젠 테라퓨틱스 컴퍼니, 리미티드 Combination of Cellular Immunotherapy
CN113490689B (en) 2019-02-01 2024-06-04 恺兴生命科技(上海)有限公司 TCR fusion proteins and cells expressing TCR fusion proteins
CN110172449A (en) * 2019-06-04 2019-08-27 上海交通大学医学院附属新华医院 A kind of leukaemia cell's excretion body and its preparation method and application
US20230310600A1 (en) 2020-08-07 2023-10-05 Crage Medical Co., Limited Engineered cells and method for engineering cells
AR124414A1 (en) 2020-12-18 2023-03-22 Century Therapeutics Inc CHIMERIC ANTIGEN RECEPTOR SYSTEM WITH ADAPTABLE RECEPTOR SPECIFICITY
US20240181055A1 (en) 2021-04-08 2024-06-06 Crage Medical Co., Limited Cellular immunotherapy use
WO2023274303A1 (en) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Chimeric polypeptide for regulating cell physiological activity
WO2024088325A1 (en) * 2022-10-25 2024-05-02 科济生物医药（上海）有限公司 Antibody and use thereof
CN116003629B (en) * 2022-11-11 2023-10-20 汕头普罗凯融生物医药科技有限公司 MUC 1-targeted chimeric antigen receptor and modified NK cells thereof
CN116064622B (en) * 2023-02-13 2023-10-13 优睿赛思(武汉)生物科技有限公司 Preparation and application of Claudin6-mCherry reporter gene CHO-K1 stable transgenic cell strain

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596981A (en) * 2011-04-08 2014-02-19 美国卫生和人力服务部 Anti-epidermal growth factor receptor variant iii chimeric antigen receptors and use of same for the treatment of cancer
WO2015014375A1 (en) * 2013-07-30 2015-02-05 Biontech Ag Tumor antigens for determining cancer therapy

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG187457A1 (en) * 2008-01-11 2013-02-28 Univ Tokyo Anti-cldn6 antibody
PL3026064T3 (en) * 2011-05-13 2019-05-31 Ganymed Pharmaceuticals Gmbh Antibodies for treatment of cancer expressing claudin 6
WO2014145252A2 (en) * 2013-03-15 2014-09-18 Milone Michael C Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
CN104140974B (en) * 2013-05-08 2017-09-29 科济生物医药（上海）有限公司 Encode the nucleic acid of the Chimeric antigen receptor albumen of GPC 3 and express the T lymphocytes of the Chimeric antigen receptor albumen of GPC 3
US20170335281A1 (en) * 2014-03-15 2017-11-23 Novartis Ag Treatment of cancer using chimeric antigen receptor
PT3514172T (en) * 2014-04-01 2020-04-21 Tron Translationale Onkologie An Der Univ Der Johannes Gutenberg Univ Mainz Gemeinnuetzige Gmbh Claudin-6-specific immunoreceptors and t cell epitioes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596981A (en) * 2011-04-08 2014-02-19 美国卫生和人力服务部 Anti-epidermal growth factor receptor variant iii chimeric antigen receptors and use of same for the treatment of cancer
WO2015014375A1 (en) * 2013-07-30 2015-02-05 Biontech Ag Tumor antigens for determining cancer therapy

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