CN106749675A - A kind of recombinant slow virus and its application - Google Patents

A kind of recombinant slow virus and its application Download PDF

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CN106749675A
CN106749675A CN201611226888.6A CN201611226888A CN106749675A CN 106749675 A CN106749675 A CN 106749675A CN 201611226888 A CN201611226888 A CN 201611226888A CN 106749675 A CN106749675 A CN 106749675A
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intracellular
chimeric antigen
gpc3
antigen receptor
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Shenzhen In Vivo Biological Medicine Technology Co Ltd
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Abstract

The present invention relates to the cellular immunotherapy field of tumour, more particularly to a kind of recombinant slow virus and its application, the recombinant slow virus include Chimeric antigen receptor, and the Chimeric antigen receptor mainly includes that signal peptide, antigen recognizing district, transmembrane region, intracellular costimulatory signal transduction domain and CD3 ζ signal transductions domain are in series；Wherein, the intracellular costimulatory signal transduction domain mainly includes people's TLR2 intracellular regions.GPC3 CAT T cells prepared by the present invention have strong cell killing effect to HCC, and expression Th1 cell factors high, high degree stimulates the Tumor cytotoxicity that non-CAR T cells cause, can effectively prevent the escape and potential recurrence of GPC3 tumour cells from threatening, and express the maximum killing tumor cell of T cell energy of this Chimeric antigen receptor, the extent of injury very little of normal tissue, can break through tumour immunity and suppress microenvironment, so as to have more preferable curative effect to solid tumor.

Description

A kind of recombinant slow virus and its application
Technical field
The present invention relates to the cellular immunotherapy field of tumour, more particularly to a kind of recombinant slow virus and its application, specifically It is the construction method of Chimeric antigen receptor T (CAR-T) cell technology based on tomour specific target spot GPC3 and its anti-swollen Application in knurl treatment.
Background technology
GPC3 (Glypican-3, glypican-3) assignment of genes gene mapping take part in thin in human chromosome X26.10 Born of the same parents' division, growth, development and cell are to life processes such as the reactions of growth factor.The function of GPC3 is expected in vivo Substantial connection is distributed with, in the embryonic period, embryonic phase of human body, GPC3 has expression in placenta, embryonic liver, embryo lung and embryo kidney.Into In Normal human tissue, do not expressed in brain, liver, spleen, stomach, intestines etc., only have extremely low water at positions such as ovary, lung and kidneys Flat expression (Hsu HC et al., 1997Cancer Reseach).Genetic GPC3 gene mutations missing, Hedgehog Signal path loses negative regulation and control, cause Simpson-Golabi-Behmel (SGBS) syndrome (Capurro MI et al., 2008Dev Cell).SGBS syndromes are a kind of X-linkage hereditary diseases, are mainly characterized by macroglossia, refer to (toe), rib more And deformity of spine, Tendon defection etc., while the dangerous increase that embryonal tumors occur.
Research report, GPC3 is in liver cancer (Capurro M et al., 2003Gastroenterology), clear-cell carcinoma (Valsechi MC et al., 2014BMC Cancer), oophoroma (Maeda D et al., 2009Mod Pathol), lung Cancer (Lin Q et al., 2012Mod Pathol), melanoma (Nakatsura T et al., 2004 Clin Cancer Res specificity overexpression in).GPC3 is tumor associated antigen, is produced by the autocrine and paracrine that raise Wnt signal paths Thing, stimulates liver cancer cell growth (Lai JP et al., 2010 Hepatology).Meanwhile, GPC3 or one is potential swollen Knurl suppressor.In ovarian cancer cell line, GPC3 gene expressions can usually be methylated silence (Lin H et al., 1999Cancer Res).By gene expression analysis, find GPC3 in adenocarcinoma of lung (relative to normal lung carcinoma cell), smoking people Expression (Kim H et al., 2003Am J Respir Cell Mol high in group's (relative to non-smokers) lung tissue Biol).GPC3 expression high in ductal adenocarcinoma of pancreas, (relative to normal pancreatic tissue), has with tumorigenesis, prognosis Close, be the candidate markers (Yao H et al., 2016Cancer Biomark) of ductal adenocarcinoma of pancreas diagnosis.
Used as the molecular marker of detection early liver cancer, GPC3 specificity in liver cancer is in expression status high, be not true to type Be can't detect in hyperplastic nodule and other tumours；And sensitiveness is high, can be detected early stage liver cancer occurs.GPC3-AS1 (The lncRNA glypican 3antisense transcript 1), i.e. GPC3 antisense transcripts non-coding RNA long, Significantly high expression in liver cancer, and relevant (the Zhu XT such as its expression and GPC3 expression, tumour growth, vessel invasion, prognosis et al.,2016FEBS J)。
GPC3 participates in the inflammatory reaction in tumor tissues, and GPC3 can raise F4/80+CD86+ macrophage quantity, cause GPC3 specific C D8+T cell immune responses, extension survival time of mice (Luo C et al., 2014Oncol Rep).Pass through GPC3 polypeptide vaccines, the lethal effect of activation CTL cells expression HCC high to GPC3, treatment hepatocarcinoma patient faces in the second phase Bed experiment (Sawada Y et al., 2016Oncoimmunology).Clinical research is reported, using GPC3 specific dendritics Shape cell vaccine treats liver cancer, and the identification for activating GPC3+ HCCs kills (Tada F et al., 2012Int J Oncol).Relative to costimulating factor combination CD28, CD28 joint 41BB, GPC3-41BB Chimeric antigen receptors T cell has more Strong amplification and antitumor potential (Li W et al., 2016Hum Gene Ther).
The target spot with GPC3 as tumour, is mainly adopted by GPC3 peptide fragments vaccine, selectively targeted GPC3 in the prior art Property immune, Chimeric antigen receptor T cell etc..And GPC3 vaccines mainly can play certain work in tumor prevention and tumour early stage With and when often liver cancer is made a definite diagnosis all being liver cancer middle and advanced stage, GPC3 vaccines often DeGrain.GPC3 antigentic specificities DC-CIK immunocytes are to the internal identification fragmentation effect of tumour cell than relatively low, and immunologic cellular activity is weaker.
The A of CN 102180969 disclose a kind of resisting liver cancer activity monoclonal antibody and its application.The monoclonal antibody, be The monoclonal antibody that can be combined with GPC3 antigen epitope specificities；The amino acid sequence of the GPC3 epitopes is certainly GenBank accession Number are the aminoterminal end 359-580 amino acids residues of AAA98132.1.The invention system For high-titer, the anti-human GPC3-C Dan Ke of mouse high special and that there is cytotoxicity with immunocompetence and to tumour cell Grand antibody.GPC3 monoclonal antibodies can effectively suppress liver cancer cell growth and migration, however, GPC3 antibody easily forms resistance, And GPC3 negative HCC cell evasions can be caused, bury the threat of tumor recurrence.
The A of CN 105949324 disclose Chimeric antigen receptor of targeting GPC3 and application thereof, and the Chimeric antigen receptor contains The leader peptide of the CD8 antigens being sequentially connected, resisting GPC 3 single-chain antibody, people CD8 α hinge areas, people CD8 α transmembrane regions, people's 41BB intracellulars The fragment of the III containing ectodomain and ectodomain IV of area, people's CD3 ζ intracellular regions and optional EGFR sequences.The inosculating antibody Former Targeting Effect is not very good, and tumor microenvironment can influence its therapeutic effect.
The content of the invention
It is not very good for being targetted in current CAR-T technologies treatment tumour, and tumor microenvironment influence CAR-T technologies The situation of therapeutic effect, the present invention provides a kind of recombinant slow virus and its application, and GPC3CAT T cells prepared by the present invention are to liver Cancer cell has strong cell killing effect, and expression Th1 cell factors high, and high degree stimulates non-CAR T cells to cause Tumor cytotoxicity, can effectively prevent GPC3- tumour cells escape and potential recurrence threaten.
It is that, up to this purpose, the present invention uses following technical scheme：
On the one hand, the present invention provides a kind of Chimeric antigen receptor, and the Chimeric antigen receptor mainly includes signal peptide, antigen Cog region, transmembrane region, intracellular costimulatory signal transduction domain and CD3 ζ signal transductions domain are in series；
Wherein, the intracellular costimulatory signal transduction domain mainly includes people's TLR2 intracellular regions.
In the present invention, by introducing people TLR2 (TOLL LIKE RECEPTOR2) intracellular signal in GPC3 CAR molecules Domain, the GPC3 CAR T cells being built into find that Th1 types are partial in the secretion of its immune cell factor, have to HCC Strong cell killing effect, and expression Th1 cell factors high, high degree stimulate the tumor-killing that non-CAR T cells cause Effect, can effectively prevent the escape and potential recurrence of GPC3- HCCs from threatening.
According to the present invention, the amino acid sequence of the people TLR2 intracellular regions as shown in SEQ ID NO.1, the people TLR2 born of the same parents The nucleotide sequence of inner region is as shown in SEQ ID NO.2.
The amino acid sequence (SEQ ID NO.1) of the people TLR2 intracellular regions is as follows：
QAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFV LSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLR AAIKS.
The nucleotide sequence (SEQ ID NO.2) of the people TLR2 intracellular regions is as follows：
5-CAGGCCAAAAGGAAGCCCAGGAAAGCTCCCAGCAGGAACATCTGCTATGATGCATTTGTTTCTTACA GTGAGCGGGATGCCTACTGGGTGGAGAACCTTATGGTCCAGGAGCTGGAGAACTTCAATCCCCCCTTCAAGTTGTGT CTTCATAAGCGGGACTTCATTCCTGGCAAGTGGATCATTGACAATATCATTGACTCCATTGAAAAGAGCCACAAAAC TGTCTTTGTGCTTTCTGAAAACTTTGTGAAGAGTGAGTGGTGCAAGTATGAACTGGACTTCTCCCATTTCCGTCTTT TTGATGAGAACAATGATGCTGCCATTCTCATTCTTCTGGAGCCCATTGAGAAAAAAGCCATTCCCCAGCGCTTCTGC AAGCTGCGGAAGATAATGAACACCAAGACCTACCTGGAGTGGCCCATGGACGAGGCTCAGCGGGAAGGATTTTGGGT AAATCTGAGAGCTGCGATAAAGTCC-3.
Chimeric antigen receptor of the invention can also include hinge area, and described hinge area those skilled in the art can basis Actual conditions are selected, and particular determination is not done herein, and the presence of hinge area will not be to the property of Chimeric antigen receptor of the invention Influence can be produced.
According to the present invention, Chimeric antigen receptor (the Chimeric Antigen of the selectively targeted GPC3 of overexpression Receptor, CAR) T cell, can specific recognition GPC3+Tumour antigen, killing tumor cell, with significantly extending out in vivo Increase and tumor-killing effect, the antigen recognizing district is the region of specific recognition combination GPC3 antigens, and preferably resisting GPC 3 resists Body extracellular fragment.
According to the present invention, the signal peptide is that can to instruct the signal peptide of Chimeric antigen receptor transmembrane process be all feasible , those skilled in the art can as needed select the conventional signal peptide in this area, the signal peptide behaviour IgM signal peptides or People's CD8 alpha signal peptides, are preferably people's IgM signal peptides.
Preferably, described transmembrane region behaviour CD28 transmembrane regions or people's CD8 transmembrane regions, is preferably people's CD28 transmembrane regions.
According to the present invention, the intracellular costimulatory signal transduction domain in addition to the intracellular regions of TLR2 containing someone, also including people CD28 intracellular regions, people 4-1BB intracellular regions, people CD27 intracellular regions, people CD28 intracellular regions, people OX40 intracellular regions, people CD30 intracellular regions, People CD40 intracellular regions, people PD-1 intracellular regions, people ICOS intracellular regions, human lymphocyte function correlation antigen 1 intracellular region, people CD2 born of the same parents In inner region, people CD7 intracellular regions, people LIGHT intracellular regions, people NKG2C intracellular regions, human B 7-H 3 intracellular region or people's CD83 intracellular regions Any one or at least two combination, it is preferably people 4-1BB intracellular regions or CD28 intracellular regions that the combination for example can be, In the present invention, the complete intracellular costimulatory signal transduction domain behaviour CD28 intracellular regions or people 4-1BB intracellular regions and people TLR2 intracellular regions are in series.
Used as optimal technical scheme, the Chimeric antigen receptor is by people IgM signal peptides, resisting GPC 3 antibody, people's CD28 cross-films Area, people 4-1BB intracellular regions, people TLR2 intracellular regions and people's CD3 ζ signal transductions domain are in series, i.e. IgM-GPC3-4-1BB-CD3 ξ-TLR2(GTBBξ)；Chimeric antigen receptor is by people IgM signal peptides, resisting GPC 3 antibody, people CD28 transmembrane regions and intracellular region, people TLR2 intracellular regions and people's CD3 ζ signal transductions domain are in series, i.e. IgM-GPC3-CD28-CD3 ξ-TLR2 (GT28 ξ).
Inventor has found that the Chimeric antigen receptor is by people IgM signal peptides, resisting GPC 3 antibody, people CD28 transmembrane regions, people CD28 or people 4-1BB intracellular regions, people TLR2 intracellular regions and people's CD3 ζ signal transductions domain are in series, and its identification to tumour is killed Effect preferably, can target GPC3- tumour cells, effectively suppress liver cancer growth.
Preferably, the amino acid sequence such as SEQ ID of the Chimeric antigen receptor IgM-GPC3-CD28-CD3 ξ-TLR2 Shown in NO.3, the amino acid sequence (SEQ ID NO.3) of the Chimeric antigen receptor GT28 ζ gene chemical synthesis sequences is as follows：
MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQ QSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYME LRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRD FIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKI MNTKTYLEWPMDEAQREGFWVNLRAAIKS.
Preferably, the Chimeric antigen receptor preferably, the Chimeric antigen receptor IgM-GPC3-41BB-CD3 ξ-TLR2 Amino acid sequence as shown in SEQ ID NO.4, the amino acid sequence of the Chimeric antigen receptor GTBB ζ gene chemical synthesis sequences (SEQ ID NO.4) is as follows：
MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQ QSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYME LRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPS RNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWC KYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS.
Preferably, described Chimeric antigen receptor is transfected into T cell by its nucleotide sequence for encoding and expressed.
Preferably, the mode of the transfection is by any one in viral vectors, eukaryon expression plasmid or mRNA sequence Plant or at least two combination is transfected into T cell, T cell is transfected into preferably by viral vectors.
Preferably, the viral vectors is any one in slow virus carrier, adenovirus vector or retroviral vector Kind or at least two combination, preferably slow virus carrier.
Second aspect, the present invention provides a kind of recombinant slow virus, by comprising Chimeric antigen receptor as described in relation to the first aspect Viral vectors and the packaging recombinant slow virus that obtain of helper plasmid pMD2.G and psPAX2 cotransfection mammalian cell.
The third aspect, the present invention provides a kind of composition, and the composition includes chimeric antigen as described in relation to the first aspect Acceptor and/or the recombinant slow virus as described in second aspect.
Fourth aspect, the present invention provides Chimeric antigen receptor as described in relation to the first aspect, the restructuring as described in second aspect Slow virus or the composition as described in the third aspect are preparing Chimeric antigen receptor T cell and its answering in anti-tumor medicine With.
Preferably, the tumour is GPC3 positive tumors.
According to the present invention, the Chimeric antigen receptor can specific recognition GPC3+Tumour antigen, contains GPC3+Tumour antigen Tumour cell can all be identified and treat, the GPC3 positive tumors include liver cancer, oophoroma, lung cancer, melanoma or kidney In cell cancer any one or at least two combination, wherein, in the oophoroma, lung cancer, melanoma or clear-cell carcinoma All contain GPC3+Tumour antigen, it can be recognized that its mechanism is similar with liver cancer by the Chimeric antigen receptor, the inosculating antibody Original receptor can also treat oophoroma, lung cancer, melanoma or clear-cell carcinoma, and the GPC3 positive tumors are preferably liver cancer.
Compared with prior art, the present invention has the advantages that：
(1) Chimeric antigen receptor of the invention has efficient tumour identification lethal effect, can activate non-CAR T cells, body The tumour immunity effect of the tumour immunity relevant cell such as interior T, NK, macrophage, targets GPC3- tumour cells, and anti-curing oncoma is exempted from Epidemic disease is escaped and recurrence is threatened；
(2) Chimeric antigen receptor of the invention is partial to the secretion of Th1 cytokines, can positive feedback activation CAR T tumours Immunological effect, the tumour immunity that can also to a certain extent weaken tumor microenvironment suppresses；
(3) present invention expresses the maximum killing tumor cell of T cell energy of this Chimeric antigen receptor, normal tissue Extent of injury very little, tumour immunity can be broken through and suppress microenvironment, so as to having more preferable curative effect to solid tumor.
Brief description of the drawings
Fig. 1 is the synthetic gene sequence collection of illustrative plates of the Chimeric antigen receptor in the embodiment of the present invention 1；
Fig. 2 is that CAR GT28 ξ slow-virus infections efficiency of the present invention and CAR T cells are expanded；
Effectively identification kills GPC3 to Fig. 3 CAR GT28 ξ T cells of the present invention in vitro+HCC, wherein, Fig. 3 (A) is pin To GPC3+Huh7-GL HCCs, Fig. 3 (B) is for GPC3+HepG2-GL HCCs,It is Mock-Car T,It is GPC3-Car T；
Fig. 4 is the security verification of CAR-GT28 ξ T cells of the present invention,It is Mock-Car T,It is GPC3- Car T；
Fig. 5 suppresses GPC3 in vitro for CAR GT28 ξ T cells of the present invention+Liver cancer growth, wherein, Fig. 5 (A) is GPC3+'s Huh7-GL HCCs, Fig. 5 (B) is GPC3+HepG2-GL HCCs,It is Mock-Car T,It is GPC3- Car T；
Fig. 6 is the GT28 ξ CAR T cells secretion Th1 cytokines of GPC3 antigen-specific activations of the present invention, wherein, Fig. 6 (A) is the IL-2 factors of secretion, and Fig. 6 (B) is the INF- γ factors of secretion, and " " is Mock-Car T, and " ■ " is GPC3- Car T；
Fig. 7 is CAR-GTBB ξ of the present invention, CAR-GT28 ξ, CAR-G28BB ξ T cells to GPC3+HepG2-GL liver cancer it is thin The specific recognition of born of the same parents is killed.
Specific embodiment
Further to illustrate technological means and its effect that the present invention is taken, below in conjunction with accompanying drawing and by specific real Mode is applied to further illustrate technical scheme, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art, Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from The conventional products of acquisition.
Embodiment 1：The structure of Chimeric antigen receptor slow virus carrier
(1) people IgM signal peptides, resisting GPC 3 antibody extracellular fragment, people CD28 transmembrane regions, people's 41BB intracellulars are synthesized by full genome Area, people TLR2 intracellular regions and people's CD3 ζ signal transductions domain, i.e. CAR-GPC3-41BB-CD3 ξ-TLR2 (CAR-GTBB ξ), full genome Synthesis people IgM signal peptides, resisting GPC 3 antibody extracellular fragment, people CD28 transmembrane regions and intracellular region, people TLR2 intracellular regions and people CD3 ζ letter Number transduction domain, i.e. CAR-GPC3-CD28-CD3 ξ-TLR2 (CAR-GT28 ξ), its sequence is as shown in SEQ ID NO.4, then full base Because synthesis CAR-CD19-CD28-CD3 ξ-TLR2 (Mock) nucleotide sequences are used as negative control, CAR-GPC3-41BB-CD28- CD3 ξ (CAR-G28BB ξ) nucleotide sequences are as its gene order collection of illustrative plates of positive control as shown in figure 1, the gene C end of synthesis is containing limit Property restriction endonuclease Pme1 restriction enzyme sites processed and its protection base and N-terminal Spe1 containing restriction enzyme restriction enzyme sites and its protection alkali Base；
(2) respectively by restriction enzyme Pme1 and Spe1 double digestion, gel electrophoresis is reclaimed and obtained containing cohesive end Synthetic DNA fragment CAR-GTBB ξ, CAR-GT28 ξ, CAR-G28BB ξ and Mock, by CAR-GTBB ξ, CAR-GT28 ξ, CAR- The linearisation DNA of G28BB ξ, Mock and the pWPXLd-eGFP slow virus carriers containing cohesive end；
(3) by T4DNA ligases (Invitrogent companies), the pWPXLd-2A-EGFP that will be linearized is sticky with band The DNA fragmentation CAR-GTBB ξ of the synthesis of end, CAR-GT28 ξ, Mock are attached, and obtain CAR plasmid transfer vectors pWPXLd-CAR-GTBBξ-2A-EGFP、pWPXLd-CAR-GT28ξ-2A-EGFP、pWPXLd-CAR-G28BBξ-2A-EGFP、 pWPXLd-Mock-2A-EGFP。
Embodiment 2：GT28 ξ CAR slow virus is packed
(1) 293T cells are cultivated in 10cm culture dishes, culture medium is：DMEM high glucose mediums+10%FBS (tire ox bloods Clearly)+1% dual anti-(100 × Pen .- Strep mixed solution)；
(2) when the 293T cell densities in 150mm culture dishes reach 80-90%, culture medium is changed：DMEM high glucose mediums + 1%FBS+1% is dual anti-；
(3) medium culture is changed after 2-6 hours, with PEI respectively by pWPXLd-CAR-EGFP plasmids (pWPXLd-CAR- GTBBξ-2A-EGFP、pWPXLd-CAR-GT28ξ-2A-EGFP、pWPXLd-CAR-G28BBξ-2A-EGFP、pWPXLd-Mock- 2A-EGFP) enter 293T cells with slow virus packaging helper plasmid pMD2.G, psPAX2 co-transduction respectively, add reagent and agent Amount is as follows：
Reagent Dosage
PWPXLd-CAR-EGFP plasmids 9μg
PMD2.G helper plasmids 3μg
psPAX2 12μg
PEI 72μg
(4) respectively at 24,48 and 72 hours after conversion, culture medium supernatant is collected, and add fresh culture：DMEM sugar high Culture medium+1%FBS+1% is dual anti-；
(5) culture medium supernatant is collected and finished, after supernatant 2500g is centrifuged 0.5 hour；
(6) centrifugation supernatant is taken, it is small using Ultracentrifuge 28000rpm centrifugations 1.5 after being filtered with 0.45um filters When；
(7) after ultracentrifugation, supernatant is gently removed, adds 200ul PBS, be placed in 4 DEG C and dissolve for 12-16 hours, obtained final product CAR slow virus；
(8) after virolysis, collect viral solution and be sub-packed in PCR pipe, freeze stand-by in -80 DEG C.
Embodiment 3：The structure of CAR T cells
(1) human T cell isolates and purifies：The single core isolated by Ficoll density gradient methods in blood of human body is thin Born of the same parents, after removing red blood cell through erythrocyte cracked liquid cracking, then go out T cell by MACS Pan-T magnetic bead sortings；
(2) the T cell culture medium that will be sorted out：AIM-V culture medium+5%FBS+ penicillin 100U/ml+ streptomysins 0.1mg/ml is diluted to cell concentration 2.5 × 106Individual/mL is stand-by；
(3) stimulate T cell by being coated with the magnetic bead (Germany U.S. day Ni) of CD2, CD3, CD28 antibody, that is, be coated with magnetic bead and T Cell is with 1:2 ratios mix, and T cell final densities should be 5 × 106Individual/mL/cm2.After mixing, 37 DEG C, 5%CO are placed in2Culture Case culture stimulates 48 hours；
(4) slow-virus transfection T cell：Magnetic bead in the T cell-magnetic bead mixed liquor that will be activated is removed by magnetic fields, 300g is centrifuged 5min, removes supernatant, resuspended with fresh culture, is separately added into expression CAR and GFP (blank) slow virus (disease Malicious addition is MOI=10) after, polybrene the and 300IU/mL IL-2 of 8 μ g/mL are added, it is placed in 37 DEG C, 5%CO2Culture After case culture 24h, 300g centrifugation 5min remove supernatant, resuspended with the fresh culture containing 300IU/mL IL-2, obtain final product overexpression The T cell of CAR plasmids；
(5) CAR T cells amplification：CAR T cell density is maintained 1 × 106Individual/mL or so, it is every to carry out once within 2-3 days Half amount changes liquid, after two weeks, amplifiable 100 times of CAR T cell numbers, and GFP positive cell is the successful cell of transfection, and GFP is positive Ratio detected by streaming, that is, obtain CAR-GTBB ξ T, CAR-GT28 ξ T, CAR-G28BB ξ T, Mock T cell or feminine gender The ratio of (unprocessed) T born of the same parents is compareed, as a result as shown in Figure 2, it can be seen that add the Chimeric antigen receptor T of TLR2 intracellular regions thin Born of the same parents have transfection efficiency higher, and T cell can be promoted preferably to expand.
Embodiment 4：The structure of the CAR T cells of other intracellular costimulatory signal transduction domains
(1) synthesize people's CD8 alpha signals peptide, resisting GPC 3 antibody extracellular fragment, people CD28 transmembrane regions, intracellular by full genome to pierce altogether Energizing signal transduction domain, people TLR2 intracellular regions and people's CD3 ζ signal transductions domain, wherein intracellular costimulatory signal transduction domain behaviour CD27 Intracellular region, people CD28 intracellular regions, people OX40 intracellular regions, people CD30 intracellular regions, people CD40 intracellular regions, people PD-1 intracellular regions, people ICOS intracellular regions, human lymphocyte function correlation antigen 1 intracellular region, people CD2 intracellular regions, people CD7 intracellular regions, people's LIGHT intracellulars Any one in area, people NKG2C intracellular regions, human B 7-H 3 intracellular region or people CD83, synthesizes 14 Chimeric antigen receptors altogether, closes Into the gene C end restriction enzyme sites of Pme1 containing restriction enzyme and its digestions of Spe1 containing restriction enzyme of protection base and N-terminal Site and its protection base；
(2) respectively by restriction enzyme Pme1 and Spe1 double digestion, gel electrophoresis is reclaimed and obtained containing cohesive end The linearisation DNA of synthetic DNA fragment and the pWPXLd-eGFP slow virus carriers containing cohesive end；
(3) by T4 DNA ligases (Invitrogent companies), the pWPXLd-2A-EGFP that will be linearized is sticky with band The DNA fragmentation connection of the synthesis of end, obtains CAR plasmid transfer vectors.
The packaging step of slow virus can be prepared not with embodiment 2, the construction step of CAR T cells with embodiment 3 With 14 CAR T cells of intracellular costimulatory signal transduction domain.
Embodiment 5：The Vitro Tumor of CAR-GT28 ξ T cells is killed
(1) CAR-GT28 ξ T, Mock T cells for preparing embodiment 2 and GPC3+HCC is respectively with 1：3、1： 1、3：1、5：1 ratio mixes, and is added in the U-shaped plate in 96 holes, and every group sets 3 multiple holes, after 250g centrifugations 5min, is placed in 37 degree 5% CO2Incubator co-cultures 18h；
(2) evaluating in vitro CAR-GT28 ξ T cells are to GPC3+The identification killing ability of HCC, tumour cell is selected respectively With the Bel7402 of Huh7-GL, HepG2-GL with luciferase；
(3) the quantitative killing-efficiency appraisal procedure of luciferase (Luciferase)：CAR T cells are co-cultured with tumour cell (experimental comparison group is individually cultivated for tumour cell) 18 hours afterwards, adds the fluorescein in 100 μ l/ holes in 96 porocyte culture plates Zymolyte (1 ×), by the resuspended mixing of cell, determines RLU (relative light unit) by multi-function microplate reader immediately, Minute is set to 1 second.
Killing ratio computing formula：100% × (control wells reading-experimental port reading)/control wells reading (is not added with cell Blank group number-reading can be ignored)；
Shown in its result such as Fig. 3 (A)-Fig. 3 (B), as a result show, CAR-GT28 ξ T cells are to GPC3+Huh7-GL, HepG2-GL HCCs have significant Cytotoxicity in vitro efficiency.
CAR-G28BB ξ T cells are to GPC3+Huh7-GL, HepG2-GL HCC fragmentation effect and CAR-GT28 ξ T cell is similar to, although and selecting 14 CAR-T cells of other intracellular costimulatory signal transduction domains to GPC3+Huh7-GL, HepG2-GL HCCs have fragmentation effect, but effect is not so good as CAR-G28BB ξ T cells and CAR-GT28 ξ T cells.
Embodiment 6：CAR T cell safety testings
(1) CAR-GT28 ξ T, Mock T cells and GPC3- tumour cells for preparing embodiment 2 are respectively with 1：3、1：1、 3：1、5：1 ratio mixes, and is added in the U-shaped plate in 96 holes, and every group sets 3 multiple holes, after 250g centrifugations 5min, is placed in 37 degree of 5%CO2 Incubator co-cultures 18h；
(2) evaluating in vitro CAR-GT28 ξ T cells recognize lethal effect, GPC3- to the non-specific of GPC3- tumour cells Tumour cell selects Lu-csf-1s of the A549-GL with luciferase respectively；
(3) the quantitative killing-efficiency appraisal procedure of luciferase (Luciferase)：CAR T cells are co-cultured with tumour cell (experimental comparison group is individually cultivated for tumour cell) 18 hours afterwards, adds the fluorescein in 100 μ l/ holes in 96 porocyte culture plates Zymolyte (1 ×), by the resuspended mixing of cell, determines RLU (relative light unit) by multi-function microplate reader immediately, Minute is set to 1 second.
Killing ratio computing formula：100% × (control wells reading-experimental port reading)/control wells reading (is not added with cell Blank group number-reading can be ignored)；
As a result its result as shown in figure 4, show, CAR-GT28 ξ T cells are not non-to the A549-GL lung carcinoma cells of GPC3- Specific killing effect.
CAR-G28BB ξ T cells are to the A549-GL lung carcinoma cells of GPC3- also without non-specificity lethal effect.
Embodiment 7：The in-vivo tumour of CAR GT28 ξ T cells is killed
(1) it is 1 × 10 by cell number5GPC3+HCC is implanted into NOD/SCID IL2rg-/- immunodeficient mouse In vivo, liver cancer xenograft mouse model is built；
(2) CAR-GT28 ξ T cells are assessed in vivo to GPC3+The inhibitory action of liver cancer cell growth, tumour cell is selected respectively With the Bel7402 of Huh7-GL, HepG2-GL with luciferase；
(3) after tumour transplatation 7,14 days, respectively liver cancer xenograft mouse model medium sized vein injection cell number be 2 × 106 CAR-GT28 ξ T, Mock T cells, totally two experimental groups, five repetitions of every group of setting；
(3) in the 7th, 10,13,16,19,21 days after tumour transplatation, two experimental mices are measured respectively with slide measure Hypodermic tumour block size, and record, draw tumor growth curve figure；
Shown in result such as Fig. 5 (A)-Fig. 5 (B), as a result show, CAR-GT28 ξ T cells can recognize that kill GPC3+ in vivo HCC Huh7-GL, HepG2-GL, effectively suppress liver cancer growth.
CAR-G28BB ξ T cells also can in vivo can recognize that killing GPC3+ HCC Huh7-GL, HepG2-GL, effectively Suppress liver cancer growth.
Embodiment 8：The CAR GT28 ξ T cell cytokine secretions of activation
(1) CAR-GT28 ξ T, Mock T cell (negative control, non-specificity targeting GPC3) for preparing embodiment 2 with GPC3+HCC is respectively with 1：1 ratio mixes, and is added in the U-shaped plate in 96 holes, and every group sets 3 multiple holes, 250g centrifugations 5min Afterwards, 37 degree of 5%CO are placed in2Incubator co-cultures 18h；
(2) GPC3 in cytokine secretion assessment+Tumour cell is respectively from Huh7-GL, HepG2-GL band luciferase Bel7402, GPC3- tumour cells select human lung cancer cell lines of the A549-GL with luciferase；
(3) after co-culturing 18h, the co-cultivation supernatant of different experiments group is taken, detects that the Th1 types in supernatant are thin by ELISA The expression of intracellular cytokine (IL2 and IFN γ)；
Shown in result such as Fig. 6 (A)-Fig. 6 (B), as a result show, specific activation of the CAR-GT28 ξ T cells in GPC3 antigens Under, cell factor IL-2, the expression of IFN γ are significantly higher than Mock T cells.
Embodiment 9：The Vitro Tumor of the CAR-GPC3 T cells of different intracellular regions is killed
(1) CAR-GTBB ξ, CAR-GT28 ξ, CAR-G28BB ξ (positive control), the Mock (feminine genders for preparing embodiment 2 Control) T cell and GPC3+HCC is respectively with 1：3、1：1、3：1 ratio mixes, and is added in the U-shaped plate in 96 holes, and every group sets 3 multiple holes, after 250g centrifugations 5min, are placed in 37 degree of 5%CO2Incubator co-cultures 18h；
(2) evaluating in vitro CAR-GT28 ξ T cells are to GPC3+The identification killing ability of HCC, tumour cell is selected respectively With Bel7402s of the HepG2-GL with luciferase；
(3) the quantitative killing-efficiency appraisal procedure of luciferase (Luciferase)：CAR T cells are co-cultured with tumour cell (experimental comparison group is individually cultivated for tumour cell) 18 hours afterwards, adds the fluorescein in 100 μ L/ holes in 96 porocyte culture plates Zymolyte (1 ×), by the resuspended mixing of cell, determines RLU (relative light unit) by multi-function microplate reader immediately, Minute is set to 1 second.
Killing ratio computing formula：100% × (control wells reading-experimental port reading)/control wells reading (is not added with cell Blank group number-reading can be ignored)；
Its result as shown in fig. 7, as a result show, to the fragmentation effect CAR-GTBB ξ of HepG2>CAR-GT28ξ>CAR- G28BB ξ, have obvious lethal effect relative to control group Mock, show that costimulatory signal molecule TLR2 intracellular regions can be carried effectively Specific recognition lethal effect of the Chimeric antigen receptor T cell high to tumour cell.
In sum, Chimeric antigen receptor of the invention is partial to the secretion of Th1 cytokines, can positive feedback activation CAR T tumour immunity effects, the tumour immunity that can also to a certain extent weaken tumor microenvironment suppresses, and the present invention expresses this inosculating antibody The maximum killing tumor cell of T cell energy of original receptor, the injury very little of normal tissue can break through tumour immunity suppression Microenvironment, so as to have more preferable curative effect to solid tumor.
Applicant states that the present invention illustrates method detailed of the invention by above-described embodiment, but the present invention not office It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Art Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
SEQUENCE LISTING
<110>Vivo biodistribution Pharmaceutical Technology Co., Ltd of Shenzhen
<120>A kind of recombinant slow virus and its application
<130> 2016
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 159
<212> PRT
<213>Artificial synthesized sequence
<400> 1
Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr
1 5 10 15
Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn
20 25 30
Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys
35 40 45
Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile
50 55 60
Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu
65 70 75 80
Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His
85 90 95
Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu
100 105 110
Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg
115 120 125
Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala
130 135 140
Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser
145 150 155
<210> 2
<211> 477
<212> DNA
<213>Artificial synthesized sequence
<400> 2
caggccaaaa ggaagcccag gaaagctccc agcaggaaca tctgctatga tgcatttgtt 60
tcttacagtg agcgggatgc ctactgggtg gagaacctta tggtccagga gctggagaac 120
ttcaatcccc ccttcaagtt gtgtcttcat aagcgggact tcattcctgg caagtggatc 180
attgacaata tcattgactc cattgaaaag agccacaaaa ctgtctttgt gctttctgaa 240
aactttgtga agagtgagtg gtgcaagtat gaactggact tctcccattt ccgtcttttt 300
gatgagaaca atgatgctgc cattctcatt cttctggagc ccattgagaa aaaagccatt 360
ccccagcgct tctgcaagct gcggaagata atgaacacca agacctacct ggagtggccc 420
atggacgagg ctcagcggga aggattttgg gtaaatctga gagctgcgat aaagtcc 477
<210> 3
<211> 645
<212> PRT
<213>Artificial synthesized sequence
<400> 3
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser
20 25 30
Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
35 40 45
Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
50 55 60
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
65 70 75 80
Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val
100 105 110
Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly
115 120 125
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
145 150 155 160
Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His
180 185 190
Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Ile Glu Val Met Tyr
260 265 270
Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
275 280 285
Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser
290 295 300
Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
305 310 315 320
Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys
325 330 335
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
340 345 350
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
355 360 365
Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
Gln Ala Leu Pro Pro Arg Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro
485 490 495
Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp
500 505 510
Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu Glu Asn Phe Asn
515 520 525
Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe Ile Pro Gly Lys
530 535 540
Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys Ser His Lys Thr
545 550 555 560
Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr
565 570 575
Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala
580 585 590
Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln
595 600 605
Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu
610 615 620
Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp Val Asn Leu Arg
625 630 635 640
Ala Ala Ile Lys Ser
645
<210> 4
<211> 687
<212> PRT
<213>Artificial synthesized sequence
<400> 4
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser
20 25 30
Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
35 40 45
Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
50 55 60
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
65 70 75 80
Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val
100 105 110
Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly
115 120 125
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
145 150 155 160
Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His
180 185 190
Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Ile Glu Val Met Tyr
260 265 270
Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
275 280 285
Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser
290 295 300
Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
305 310 315 320
Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys
325 330 335
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
340 345 350
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
355 360 365
Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
370 375 380
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
385 390 395 400
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
405 410 415
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
420 425 430
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
435 440 445
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
450 455 460
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
465 470 475 480
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
485 490 495
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
500 505 510
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
515 520 525
Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr
530 535 540
Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn
545 550 555 560
Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys
565 570 575
Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile
580 585 590
Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu
595 600 605
Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His
610 615 620
Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu
625 630 635 640
Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg
645 650 655
Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala
660 665 670
Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser
675 680 685

Claims (10)

1. a kind of Chimeric antigen receptor, it is characterised in that the Chimeric antigen receptor mainly include signal peptide, antigen recognizing district, Transmembrane region, intracellular costimulatory signal transduction domain and CD3 ζ signal transductions domain are in series；
Wherein, the intracellular costimulatory signal transduction domain mainly includes people's TLR2 intracellular regions.
2. Chimeric antigen receptor according to claim 1, it is characterised in that the amino acid sequence of the people TLR2 intracellular regions As shown in SEQ ID NO.1；
Preferably, the nucleotide sequence of the people TLR2 intracellular regions is as shown in SEQ ID NO.2.
3. Chimeric antigen receptor according to claim 1 and 2, it is characterised in that the antigen recognizing district is known for specificity Not Jie He GPC3 antigens region, preferably resisting GPC 3 antibody extracellular fragment.
4. the Chimeric antigen receptor according to any one of claim 1-3, it is characterised in that the signal peptide behaviour IgM Signal peptide or people's CD8 alpha signal peptides, are preferably people's IgM signal peptides；
Preferably, described transmembrane region behaviour CD28 transmembrane regions or people's CD8 transmembrane regions, is preferably people's CD28 transmembrane regions.
5. the Chimeric antigen receptor according to any one of claim 1-4, it is characterised in that the intracellular costimulatory signal Transduction domain also includes people CD28 intracellular regions, people 4-1BB intracellular regions, people CD27 intracellular regions, people CD28 intracellular regions, people's OX40 intracellulars Area, people CD30 intracellular regions, people CD40 intracellular regions, people PD-1 intracellular regions, people ICOS intracellular regions, human lymphocyte function related antigen 1 intracellular region, people CD2 intracellular regions, people CD7 intracellular regions, people LIGHT intracellular regions, people NKG2C intracellular regions, human B 7-H 3 intracellular region or people In CD83 intracellular regions any one or at least two combination, be preferably people 4-1BB intracellular regions or CD28 intracellular regions；
Preferably, the intracellular costimulatory signal transduction domain behaviour 41BB intracellular regions or people CD28 intracellular regions and people's TLR2 intracellular regions It is in series.
6. the Chimeric antigen receptor according to any one of claim 1-5, it is characterised in that the Chimeric antigen receptor by People IgM signal peptides, resisting GPC 3 antibody, people CD28 transmembrane regions and intracellular region, people TLR2 intracellular regions and people's CD3 ζ signal transductions domain string Join the IgM-GPC3-CD28-CD3 ξ-TLR2 Chimeric antigen receptors for constituting；
Preferably, the amino acid sequence of the Chimeric antigen receptor IgM-GPC3-CD28-CD3 ξ-TLR2 such as SEQ ID NO.3 institutes Show；
Preferably, the Chimeric antigen receptor is by people IgM signal peptides, resisting GPC 3 antibody, people CD28 transmembrane regions, people's 41BB intracellulars Area, people TLR2 intracellular regions and people's CD3 ζ signal transductions domain IgM-GPC3-41BB-CD3 ξ-TLR2 chimeric antigens in series are received Body；
Preferably, the amino acid sequence of the Chimeric antigen receptor IgM-GPC3-41BB-CD3 ξ-TLR2 such as SEQ ID NO.4 institutes Show.
7. the Chimeric antigen receptor according to any one of claim 1-6, it is characterised in that described Chimeric antigen receptor It is transfected into T cell by its nucleotide sequence for encoding and is expressed；
Preferably, the mode of the transfection be by any one in viral vectors, eukaryon expression plasmid or mRNA sequence or At least two combination is transfected into T cell, and T cell is transfected into preferably by viral vectors；
Preferably, the viral vectors be slow virus carrier, adenovirus vector or retroviral vector in any one or At least two combination, preferably slow virus carrier.
8. a kind of recombinant slow virus, it is characterised in that by comprising the Chimeric antigen receptor as any one of claim 1-7 Viral vectors and the packaging recombinant slow virus that obtain of helper plasmid pMD2.G and psPAX2 cotransfection mammalian cell.
9. a kind of composition, it is characterised in that the composition includes the chimeric antigen as any one of claim 1-7 Acceptor and/or recombinant slow virus as claimed in claim 8.
10. Chimeric antigen receptor as any one of claim 1-7, recombinant slow virus as claimed in claim 8 or Composition as claimed in claim 9 is preparing Chimeric antigen receptor T cell and its application in anti-tumor medicine；
Preferably, the tumour is GPC3 positive tumors；
Preferably, the GPC3 positive tumors are any one in liver cancer, oophoroma, lung cancer, melanoma or clear-cell carcinoma Or at least two combination, preferably liver cancer.
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