CN107400168A - A kind of Chimeric antigen receptor and its application based on CD117 - Google Patents

A kind of Chimeric antigen receptor and its application based on CD117 Download PDF

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CN107400168A
CN107400168A CN201710587317.3A CN201710587317A CN107400168A CN 107400168 A CN107400168 A CN 107400168A CN 201710587317 A CN201710587317 A CN 201710587317A CN 107400168 A CN107400168 A CN 107400168A
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桂思倩
刘昱辰
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Shenzhen Institute Of Immune Gene Therapy
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Abstract

The present invention relates to a kind of Chimeric antigen receptor based on CD117 and its application, the construction method of Chimeric antigen receptor T (CAR T) cell technology specially based on tomour specific target spot CD117 and its application in antineoplaston, the Chimeric antigen receptor are in series including antigen-binding domains, membrane spaning domain, costimulatory signal conducting region and CD3 ζ signal transduction domains;Wherein, the antigen-binding domains combination tumor surface antigen, the tumor surface antigen are CD117.The Chimeric antigen receptor of the present invention to CD117 tumor surface antigens by carrying out specific genetic modification, improved antibody can make antigen-antibody adhesion stronger, it is less likely to occur to be mutated, and there is more preferable effect compared to other Chimeric antigen receptors and other tumour antigens, the expression quantity of target spot is high, so that the immune effect of CAR T cells strengthens, the therapeutic effect of CAR T cells is enhanced.

Description

A kind of Chimeric antigen receptor and its application based on CD117
Technical field
The present invention relates to the cellular immunotherapy field of tumour, more particularly to a kind of Chimeric antigen receptor based on CD117 And its application, the structure of Chimeric antigen receptor T (CAR-T) cell technology specially based on tomour specific target spot CD117 Method and its application in antineoplaston.
Background technology
With the development of tumour immunity theory and clinical technology, Chimeric antigen receptor T cell therapy (Chimeric Antigen receptor T-cell immunotherapy, CAR-T) turn into tumour immunity treatment most promising at present One of method.Typically, Chimeric antigen receptor CAR is by a tumor associated antigen land, extracellular hinge area, trans-membrane region and born of the same parents Interior signal transduction district's groups into.Generally, CAR includes variable (the Single chain fragment of single-chain fragment of antibody Variable, scFv) area or there is specific knot to tumor associated antigen (tumor associated antigen, TAA) Domain is closed, it is coupled by hinge and transmembrane region and the cytoplasmic domains of T cell signal transduction molecule.Most common lymph is thin Born of the same parents' activated partial includes the T cell costimulation domain with T cell effector function triggering (such as CD3 ζ) sections in series.CAR The adoptive immunotherapy of mediation allows the T cell that CAR- is transplanted with non-HLA restrictive ones Direct Recognition target tumour cell TAA.
It is most of with B cell malignant tumour (including B cell acute lymphatic leukemia (B cell acute Lymphocytic leukemia, leukemia, B-ALL) and chronic lymphocytic leukemia (chronic Lymphocytic leukemia, CLL)) patient will be dead due to its disease.A kind of method for treating these patients is logical CAR expression is crossed, the antigen that genetic modification is expressed to target on tumour cell is carried out to T cell.CAR is that to be designed to people white Cellular antigens (human leukocyte antigen, HLA) dependent/non-dependent mode identifies the antigen receptor of cell surface antigen. Attempt to have been achieved for promising success using expression CAR genetically modified cell to treat the patient of these types.
CD19 molecules are to treat the potential target spot of bone-marrow-derived lymphocyte system's tumour, and the focus in CAR researchs, CD19 table It is the CAR targets for safety test accepted extensively up to normal and malignant B cell is confined to.Target the chimeric of CD19 molecules The T cell (CD19CAR-T) of antigen receptor genetic modification is on multiple, intractable B-lineage Acute Lymphocyte Leukemia is treated Immense success is obtained, and in the treatment of the chronic bone-marrow-derived lymphocyte leukaemia of intractable, recurrent and bone-marrow-derived lymphocyte system lymthoma Curative effect is substantially poor.
CN 104788573A disclose know clearly a kind of Chimeric antigen receptor hCD19scFv-CD8 α-CD28-CD3 ζ and its use On the way, the Chimeric antigen receptor is by anti human CD 19 monoclonal antibody HI19a light chains and weight chain variable district (hCD19scFv), people CD8 α Hinge area, people CD28 transmembrane regions and intracellular region and people's CD3 ζ intracellular regions structures in series are formed, and the CD19 in the patent is being carried out After CAR-T cells feedback, CD19 expression quantity can reduce, and easily escape from immunologic mechanism.
Therefore, prepare a kind of Chimeric antigen receptor and can solve the problem that the problem of easily mutation and expression quantity reduce existing for CD19 shows Obtain particularly important.
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 provide a kind of Chimeric antigen receptor and its application based on CD117, and the present invention is prepared chimeric Antigen receptor, so as to improve the immune effect of target spot, enhances CAR-T cells by the way that CD117 target spots are carried out into genetic modification Therapeutic effect.
To use following technical scheme up to this purpose, the present invention:
On the one hand, the present invention provides a kind of Chimeric antigen receptor based on CD117, and the Chimeric antigen receptor includes antigen Binding structural domain, membrane spaning domain, costimulatory signal conducting region and CD3 ζ signal transduction domains are in series;
Wherein, the antigen-binding domains combination tumor surface antigen, the tumor surface antigen are CD117.
In the present invention, by by antigen-binding domains combination tumor surface antigen CD117, then by being fitted together to CD117 Antigen receptor carries out specific human source gene code optimization transformation, so that the combination that tumor surface antigen CD117 can be special There is more preferable effect in the Chimeric antigen receptor of the application, and compared to other Chimeric antigen receptors and other tumour antigens, The expression quantity of target spot is high so that the immune effect enhancing of CAR-T cells.
According to the present invention, single-chain antibody (ScFv) amino acid sequence such as SEQ ID of the CD117 Chimeric antigen receptors Shown in NO.1, single-chain antibody (ScFv) amino acid sequence (SEQ ID NO.1) of the CD117 Chimeric antigen receptors is as follows:
QVQLVQSGAAVKKPGESLKISCKGSGYRFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAGKSIS TAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSSGSTSGSGKPGSSEGSTKGAIQLTQSPSSLSA SVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ FNSYPLTFGGGTKVEIK。
In the present invention, the amino acid of the single-chain antibody (ScFv) of the CD117 Chimeric antigen receptors has carried out specificity and changed Make so that the Ag-Ab adhesion for the antibody that improved sequence is expressed is stronger.
According to the present invention, the tumor surface antigen CD117 also includes the single-stranded of tumor surface antigen CD117 mutant Antibody, amino acid sequence and the ammonia shown in SEQ ID NO.1 of the single-chain antibody of the mutant of the tumor surface antigen CD117 Base acid sequence has more than 90% similarity.
According to the present invention, the membrane spaning domain is CD28 membrane spaning domains and/or CD8 α membrane spaning domains, in some tools In body embodiment, membrane spaning domain can be selected or modified by amino acid substitution.
According to the present invention, the costimulatory signal conducting region is CD28 signal transductions domain, CD27 signal transduction structures In domain and CD137 signal transduction domains any one or at least two combination, preferably CD28 signal transductions domain, The combination of CD137 signal transductions domain and CD27 signal transduction domains, the CD28 signal transductions domain, CD137 letters Number conducting structure domain and/or the arrangement of CD27 signal transduction domains, those skilled in the art can be adjusted as needed, The arrangement different with CD27 signal transduction domains of CD28 signal transductions domain, CD137 signal transductions domain will not be to institute State Chimeric antigen receptor to have an impact, the application preferably uses CD28-CD137-CD27 sequential combination.
According to the present invention, the Chimeric antigen receptor also includes can induce suicide Fusion domain, the inducible suicide Fusion domain is to include the domain of Caspase 9, the amino acid sequence such as SEQ ID of the domain of Caspase 9 Shown in NO.4, the amino acid sequence (SEQ ID NO.4) of the domain of Caspase 9 is as follows:
GSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNA DLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGA LDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSP EDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDL QSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.
According to the present invention, the inducible suicide Fusion domain is mutually gone here and there by 2A sequences and CD3 ζ signal transductions domains Connection, the 2A sequences can make the albumen of the inducible suicide Fusion domain expression and the Chimeric antigen receptor chopping up proteins Open, so that the Chimeric antigen receptor can play a role, and by injecting activator, melt so that can induce suicide Domain activation is closed, so as to cause Chimeric antigen receptor ineffective.
According to the present invention, the Chimeric antigen receptor also includes signal peptide, and the signal peptide is that can instruct chimeric antigen The signal peptide of receptor transmembrane transfer, those skilled in the art can select the conventional signal peptide in this area, the letter as needed Number peptide can be the signal peptide of any one secreted protein gene, and signal peptide of the present invention is Secretory signal peptides, described The amino acid sequence of Secretory signal peptides is as shown in SEQ ID NO.5-6.
Preferably, the Secretory signal peptides are the signal peptide of CD8a genes, and the signal peptide amino acid sequence is such as Shown in SEQ ID NO.5, the amino acid sequence described in the SEQ ID NO.5 is as follows:MALPVTALLLPLALLLHAARP.
Preferably, the Secretory signal peptides are the signal peptide of GMCSFR genes, and the signal peptide amino acid sequence is such as Shown in SEQ ID NO.6, the amino acid sequence described in the SEQ ID NO.6 is as follows:MLLLVTSLLLCELPHPAFLLIP.
The Chimeric antigen receptor of the present invention can also include hinge area, and described hinge area those skilled in the art can basis Actual conditions are selected, and do not do particular determination herein, the presence of hinge area will not be to the property of the Chimeric antigen receptor of the present invention It can have an impact.
According to the present invention, the Chimeric antigen receptor includes signal peptide, antigen-binding domains, altogether membrane spaning domain, thorn Energizing signal conducting region, CD3 ζ signal transductions domain, 2A sequences and inducible suicide Fusion domain are in series.
As optimal technical scheme, the Chimeric antigen receptor is Secretory signal peptides, CD117 antigen binding structures Domain, CD8 α and/or CD28 membrane spaning domains, CD28 signal transductions domain, CD137 signal transductions domain and CD27 signals pass Transduction domain, CD3 ζ signal transductions domain, 2A sequences and the domain of Caspase 9 are in series, and specific arrangement is as follows:
Secretory-CD117-CD28-CD137-CD27-CD3ζ-2A-iCasp9。
According to the present invention, the Chimeric antigen receptor Secretory-CD117-CD28-CD137-CD27-CD3 ζ -2A- ICasp9 amino acid sequence is as shown in SEQ ID NO.2, the amino acid sequence (SEQ ID NO.2) of the Chimeric antigen receptor It is as follows:
MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAAVKKPGESLKISCKGSGYRFTSYWIGWVRQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISAGKSISTAYLQWSSLKASDTAMYYCARHGRGYNGYEGAFDIWGQGTMVTVSSGSTSGSGKP GSSEGSTKGAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGG GSGGGGSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPGGGGSGGGGSTSGGGGSGGGGS VVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKK VDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGS GGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDL TAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFI QACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGS WYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.
According to the present invention, the Chimeric antigen receptor Secretory-CD117-CD28-CD137-CD27-CD3 ζ -2A- ICasp9 nucleotide sequence is as shown in SEQ ID NO.3, the nucleotide sequence (SEQ ID NO.3) of the Chimeric antigen receptor It is as follows:
ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTCCTGCTGATCCCCCAGGTGCAGCT GGTGCAGAGCGGCGCCGCCGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGATTCA CCAGCTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTACCCCGGCGAC AGCGACACCAGATACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGCCGGCAAGAGCATCAGCACCGCCTACCT GCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCAGACACGGCAGAGGCTACAACGGCTACG AGGGCGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCAGCACCAGCGGCAGCGGCAAGCCC GGCAGCAGCGAGGGCAGCACCAAGGGCGCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGA CAGAGTGACCATCACCTGCAGAGCCAGCCAGGGCATCAGCAGCGCCCTGGCCTGGTACCAGCAGAAGCCCGGCAAGG CCCCCAAGCTGCTGATCTACGACGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC ACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTCAACAGCTA CCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGGCCGCCGCCATCGAGGTGATGTACCCCCCCCCCTACC TGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGC CCCAGCAAGCCCTTCTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTT CATCATCTTCTGGGTGAGAAGCAAGAGAAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGACCCG GCCCCACCAGAAAGCACTACCAGCCCTACGCCCCCCCCAGAGACTTCGCCGCCTACAGAAGCGCCAGCGGCGGCGGC GGCAGCGGCGGCGGCGGCAGCCAGAGAAGAAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAGCCCGCCGAGCC CTGCCACTACAGCTGCCCCAGAGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGACTACAGAAAGCCCGAGCCCG CCTGCAGCCCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCACCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GTGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAGGA GGAGGACGGCTGCAGCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAAC CAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGA GATGGGCGGCAAGCCCAGAAGAAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGG CCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACC GCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGAACCAGCGGCAGCGGCGCCACCAACTT CAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGGCGTGCAGGTGGAGACCATCAGCCCCG GCGACGGCAGAACCTTCCCCAAGAGAGGCCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAG GTGGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAGGAGGTGATCAGAGGCTGGGAGGA GGGCGTGGCCCAGATGAGCGTGGGCCAGAGAGCCAAGCTGACCATCAGCCCCGACTACGCCTACGGCGCCACCGGCC ACCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGAGGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGGCGCCATGGTGGGCGCCCTGGAGAGCCTGAGAGGCAACGCCGACCTGGCCTACATCCTGAG CATGGAGCCCTGCGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCAGAGAGAGCGGCCTGAGAACCAGAACCG GCAGCAACATCGACTGCGAGAAGCTGAGAAGAAGATTCAGCAGCCTGCACTTCATGGTGGAGGTGAAGGGCGACCTG ACCGCCAAGAAGATGGTGCTGGCCCTGCTGGAGCTGGCCAGACAGGACCACGGCGCCCTGGACTGCTGCGTGGTGGT GATCCTGAGCCACGGCTGCCAGGCCAGCCACCTGCAGTTCCCCGGCGCCGTGTACGGCACCGACGGCTGCCCCGTGA GCGTGGAGAAGATCGTGAACATCTTCAACGGCACCAGCTGCCCCAGCCTGGGCGGCAAGCCCAAGCTGTTCTTCATC CAGGCCTGCGGCGGCGAGCAGAAGGACCACGGCTTCGAGGTGGCCAGCACCAGCCCCGAGGACGAGAGCCCCGGCAG CAACCCCGAGCCCGACGCCACCCCCTTCCAGGAGGGCCTGAGAACCTTCGACCAGCTGGACGCCATCAGCAGCCTGC CCACCCCCAGCGACATCTTCGTGAGCTACAGCACCTTCCCCGGCTTCGTGAGCTGGAGAGACCCCAAGAGCGGCAGC TGGTACGTGGAGACCCTGGACGACATCTTCGAGCAGTGGGCCCACAGCGAGGACCTGCAGAGCCTGCTGCTGAGAGT GGCCAACGCCGTGAGCGTGAAGGGCATCTACAAGCAGATGCCCGGCTGCTTCAACTTCCTGAGAAAGAAGCTGTTCT TCAAGACCAGCGCCAGCTGA.
In the present invention, the Chimeric antigen receptor also includes promoter, and the promoter is in EF1a, CMV-TAR or CMV Any one or at least two combination.
According to the present invention, described Chimeric antigen receptor is transfected into T cell by its nucleotide sequence encoded and expressed.
According to the present invention, the mode of the transfection is by appointing in viral vector, eukaryon expression plasmid or mRNA sequence A kind of or at least two combinations of anticipating are transfected into T cell, and T cell is transfected into preferably by viral vector.
Preferably, the viral vector is slow virus carrier and/or retroviral vector, preferably slow virus carrier.
Second aspect, the present invention provide a kind of recombinant slow virus, will include Chimeric antigen receptor as described in relation to the first aspect Viral vector and the obtained recombinant slow virus of packaging helper plasmid pNHP and pHEF-VSVG cotransfection mammalian cell.
According to the present invention, the mammalian cell is 293 cells, in 293T cells or TE671 cells any one or extremely Few two kinds combination.
The third aspect, the present invention provide 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 provide 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 the related tumor disease of blood, the related tumor disease of the blood for leukaemia or Lymthoma.
Compared with prior art, the present invention has the advantages that:
(1) Chimeric antigen receptor of the invention is by carrying out specific genetic modification, transformation to CD117 tumor surface antigens Antibody afterwards can make Ag-Ab adhesion stronger;
(2) the specific identification tumor surface antigen CD117 of CD117 Chimeric antigen receptors of the invention energy, CD117 is white Expression quantity is high in blood disease and lymthoma, has more preferable effect compared to other Chimeric antigen receptors and other tumour antigens so that The immune effect enhancing of CAR-T cells, enhance the therapeutic effect of CAR-T cells;
(3) Chimeric antigen receptor of the invention is after CAR-T cell feedbacks are carried out, and tumor surface CD117 expression quantity is not It can reduce, it is not easy to escape from immunologic mechanism, can preferably be treated.
Brief description of the drawings
Fig. 1 is the flow cytometric analysis results figure of CD117 expression quantity in acute T-cell leukemia clinical samples, wherein, Fig. 1 (a) is the flow cytometric analysis results figure of acute T-cell leukemia clinical samples, and Fig. 1 (b) is CD117 in acute T cell Leukaemia's expression quantity;
Fig. 2 is the flow cytometric analysis results figure of CD117 expression quantity in Patients with Acute Myeloid Leukemia sample, wherein, Fig. 2 (a) is the flow cytometric analysis results figure of Patients with Acute Myeloid Leukemia sample, and Fig. 2 (b) is that CD117 is white in acute myeloid Blood disease cell expression quantity;
Fig. 3 is the flow cytometric analysis results figure of tumour cell and CAR-T cell co-cultivations, wherein, Fig. 3 (a) tumours Cell is mixed with T cell, and Fig. 3 (b) tumour cells and Mesothelin CAR-T cell co-cultivations, Fig. 3 (c) tumours are thin Born of the same parents and the CAR-T cell co-cultivations of the application.
Embodiment
Further to illustrate the technological means and its effect of the invention 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
(1) Secretory signal peptides, CD117 antigen-binding domains are synthesized by full genome, CD8 α and/or CD28 across Spanning domain, CD28 signal transductions domain, CD137 signal transductions domain and/or CD27 signal transduction domains, CD3 ζ letters Number conducting structure domain, 2A sequences and the domain of Caspase 9, i.e. Secretory-CD117-CD28-CD137-CD27-CD3 ζ-2A-iCasp9;
The nucleotide sequence SEQ ID NO.3 of the Chimeric antigen receptor are as follows:
ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTCCTGCTGATCCCCCAGGTGCAGCT GGTGCAGAGCGGCGCCGCCGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGATTCA CCAGCTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTACCCCGGCGAC AGCGACACCAGATACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGCCGGCAAGAGCATCAGCACCGCCTACCT GCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCAGACACGGCAGAGGCTACAACGGCTACG AGGGCGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCAGCACCAGCGGCAGCGGCAAGCCC GGCAGCAGCGAGGGCAGCACCAAGGGCGCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGA CAGAGTGACCATCACCTGCAGAGCCAGCCAGGGCATCAGCAGCGCCCTGGCCTGGTACCAGCAGAAGCCCGGCAAGG CCCCCAAGCTGCTGATCTACGACGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC ACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTCAACAGCTA CCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGGCCGCCGCCATCGAGGTGATGTACCCCCCCCCCTACC TGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGC CCCAGCAAGCCCTTCTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTT CATCATCTTCTGGGTGAGAAGCAAGAGAAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGACCCG GCCCCACCAGAAAGCACTACCAGCCCTACGCCCCCCCCAGAGACTTCGCCGCCTACAGAAGCGCCAGCGGCGGCGGC GGCAGCGGCGGCGGCGGCAGCCAGAGAAGAAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAGCCCGCCGAGCC CTGCCACTACAGCTGCCCCAGAGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGACTACAGAAAGCCCGAGCCCG CCTGCAGCCCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCACCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GTGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAGGA GGAGGACGGCTGCAGCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAAC CAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGA GATGGGCGGCAAGCCCAGAAGAAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGG CCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACC GCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGAACCAGCGGCAGCGGCGCCACCAACTT CAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGGCGTGCAGGTGGAGACCATCAGCCCCG GCGACGGCAGAACCTTCCCCAAGAGAGGCCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAG GTGGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAGGAGGTGATCAGAGGCTGGGAGGA GGGCGTGGCCCAGATGAGCGTGGGCCAGAGAGCCAAGCTGACCATCAGCCCCGACTACGCCTACGGCGCCACCGGCC ACCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGAGGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGGCGCCATGGTGGGCGCCCTGGAGAGCCTGAGAGGCAACGCCGACCTGGCCTACATCCTGAG CATGGAGCCCTGCGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCAGAGAGAGCGGCCTGAGAACCAGAACCG GCAGCAACATCGACTGCGAGAAGCTGAGAAGAAGATTCAGCAGCCTGCACTTCATGGTGGAGGTGAAGGGCGACCTG ACCGCCAAGAAGATGGTGCTGGCCCTGCTGGAGCTGGCCAGACAGGACCACGGCGCCCTGGACTGCTGCGTGGTGGT GATCCTGAGCCACGGCTGCCAGGCCAGCCACCTGCAGTTCCCCGGCGCCGTGTACGGCACCGACGGCTGCCCCGTGA GCGTGGAGAAGATCGTGAACATCTTCAACGGCACCAGCTGCCCCAGCCTGGGCGGCAAGCCCAAGCTGTTCTTCATC CAGGCCTGCGGCGGCGAGCAGAAGGACCACGGCTTCGAGGTGGCCAGCACCAGCCCCGAGGACGAGAGCCCCGGCAG CAACCCCGAGCCCGACGCCACCCCCTTCCAGGAGGGCCTGAGAACCTTCGACCAGCTGGACGCCATCAGCAGCCTGC CCACCCCCAGCGACATCTTCGTGAGCTACAGCACCTTCCCCGGCTTCGTGAGCTGGAGAGACCCCAAGAGCGGCAGC TGGTACGTGGAGACCCTGGACGACATCTTCGAGCAGTGGGCCCACAGCGAGGACCTGCAGAGCCTGCTGCTGAGAGT GGCCAACGCCGTGAGCGTGAAGGGCATCTACAAGCAGATGCCCGGCTGCTTCAACTTCCTGAGAAAGAAGCTGTTCT TCAAGACCAGCGCCAGCTGA。
Embodiment 2:Slow virus is packed
(1) 293T cells are cultivated with six orifice plates respectively, 1 × 106Individual cells/well, cultivate 17-18 hours;
(2) the fresh DMEM in 600 μ L/ holes is added, includes 10% FBS;
(3) following reagent is added in sterile centrifugation tube:75 μ L DMEM supernatant, 2.7 μ g auxiliary DNA are taken per hole The pTYF DNA vectors of mixed liquor (1.8 μ g pNHP, 0.5 μ g pHEF-VSV-G, 0.2 μ g pHEF-GFP) and 0.8 μ g, whirlpool Whirlpool vibrates;
(4) add in centrifuge tube and blow and beat 5 times from 7 μ L of every orifice plate center absorption Superfect, be stored at room temperature 7-10 points Clock;
(5) the DNA-Superfect mixed liquors in centrifuge tube are added dropwise in each culture hole, whirlpool is beaten;
(6) 37 DEG C of 3%CO24-5 hours are cultivated in incubator;
(7) nutrient solution of culture medium is siphoned away, culture medium is rinsed with 1.5mL AIM-V, and the AIM-V for adding 1.5mL continues Culture;
(8) culture medium is put back into 3%CO2Overnight incubation in incubator, is transfected 2-3 days mornings with fluorescence microscope Efficiency.
Embodiment 3:The purifying and concentration of slow virus
1) viral purification
Cell fragment is removed by centrifuging (1000g, 5 minutes), vial supernatant is obtained, with one 0.45 micron of low egg White combined filtering device filters vial supernatant, and virus is distributed into aliquot, is stored in -80 DEG C;
Under normal circumstances, in every milliliter of culture medium, transfectional cell can produce 106To 107Transduced unit titrates slow Viral vector.
2) with Centricon filters concentration slow virus carrier
(1) in Biohazard Safety Equipment, Centricon is taken to manage, with 70% alcohol disinfecting 1 time, then with sterile PBS 3 It is secondary;
(2) 18ml vial supernatant is added in each Centricon P-20 screen pipes, then centrifuges 30 under 2500g Minute or until viral volume is reduced to 0.5ml;
(3) screen pipe is shaken, then under 400g, is centrifuged 2 minutes, collects the virus of concentration into collection cups.Finally will Virus in all pipes is focused in a centrifuge tube.
Embodiment 4:The transfection of CAR-T cells
By the T cell after activation with 5 × 10624 orifice plates are inoculated into, add the slow virus of 50 μ l concentration target gene, with The speed of 100g centrifugal force, after room temperature centrifuges 100 minutes, be placed in 37 DEG C of culture 24h, add 1ml containing 2% human serum with it is thin The AIM-V bases of born of the same parents' medium exchange, after cultivating 2-3 days, by cell harvesting and count, with 1 × 10712 orifice plates are inoculated into, cultivate 2-3 My god, carry GFP target cell infections with slow virus carrier and observe cell toxic effect with annexinV/PI decoration methods.
As a result as shown in Figure 1-2, as shown in Fig. 1 (a)-Fig. 1 (b), have in acute T-cell leukemia patient 73% it is swollen Oncocyte altimeter reaches CD117;As shown in Fig. 2 (a)-Fig. 2 (b), in Patients with Acute Myeloid Leukemia, there is 49.2% tumour Cell altimeter reaches CD117.It can obtain, the CD117 that the present invention selects has strong specificity as the target spot of Chimeric antigen receptor, It can fast and accurately identify and kill _ Lymphoid Leukemic Cells.
The Vitro Tumor killing of the CAR-T cells of embodiment 5
(1) the CAR-T cells and tumour cell that compare T cell, Mesothelin CAR T and preparation are co-cultured, are placed in 37 degree of 5%CO2Incubator co-cultures 18h;
(2) the identification killing ability of evaluating in vitro CAR-T cells on cancer cells, tumour cell are that calcein is marked or felt Contaminate LV-GFP;
As a result it is shown from Fig. 3 (a) as can be seen that after tumour cell and T cell co-cultivation as shown in Fig. 3 (a)-Fig. 3 (b) The ratio of tumour cell is 52.7%, from Fig. 3 (b) as can be seen that tumour cell co-cultures with Mesothelin CAR T cells Afterwards, the ratio of shown tumour cell slightly reduces, and is 35.5%, from Fig. 3 (c) as can be seen that tumour cell and the CAR-T prepared After cell co-cultures, the ratio of shown tumour cell is reduced to 16.7%, it is seen that CAR-T cells against tumor prepared by the application Fragmentation effect is best.
In summary, the CD117 tumor surface antigens of Chimeric antigen receptor of the invention are less likely to occur to be mutated, and compare There are more preferable effect, the expression quantity height of target spot so that CAR-T cells are exempted from other Chimeric antigen receptors and other tumour antigens Epidemic disease effect strengthens, and enhances the therapeutic effect of CAR-T cells.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment 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 implemented.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>Immune-gene therapy research institute of Shenzhen
<120>A kind of Chimeric antigen receptor and its application based on CD117
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 248
<212> PRT
<213>Artificial synthesized sequence
<400> 1
Gln Val Gln Leu Val Gln Ser Gly Ala Ala Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Arg Phe Thr Ser Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Gly Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg His Gly Arg Gly Tyr Asn Gly Tyr Glu Gly Ala Phe Asp Ile
100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Ser Thr Ser Gly
115 120 125
Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser Thr Lys Gly Ala Ile Gln
130 135 140
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
145 150 155 160
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala
180 185 190
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
195 200 205
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
210 215 220
Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly
225 230 235 240
Gly Gly Thr Lys Val Glu Ile Lys
245
<210> 2
<211> 1058
<212> PRT
<213>Artificial synthesized sequence
<400> 2
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 Gln Val Gln Leu Val Gln Ser Gly Ala Ala
20 25 30
Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly
35 40 45
Tyr Arg Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly
50 55 60
Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr
65 70 75 80
Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Gly Lys
85 90 95
Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
100 105 110
Thr Ala Met Tyr Tyr Cys Ala Arg His Gly Arg Gly Tyr Asn Gly Tyr
115 120 125
Glu Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
130 135 140
Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser
145 150 155 160
Thr Lys Gly Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
165 170 175
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
180 185 190
Ser Ser Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
195 200 205
Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg
210 215 220
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
225 230 235 240
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser
245 250 255
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ala Ala
260 265 270
Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
275 280 285
Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro
290 295 300
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly
305 310 315 320
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
325 330 335
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
340 345 350
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
355 360 365
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ala Ser Gly Gly
370 375 380
Gly Gly Ser Gly Gly Gly Gly Ser Gln Arg Arg Lys Tyr Arg Ser Asn
385 390 395 400
Lys Gly Glu Ser Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys
405 410 415
Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg
420 425 430
Lys Pro Glu Pro Ala Cys Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly
435 440 445
Gly Ser Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Val
450 455 460
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
465 470 475 480
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
485 490 495
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Gly Ser Gly
500 505 510
Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Val Lys Phe Ser Arg Ser
515 520 525
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
530 535 540
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
545 550 555 560
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
565 570 575
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
580 585 590
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
595 600 605
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
610 615 620
Leu His Met Gln Ala Leu Pro Pro Arg Thr Ser Gly Ser Gly Ala Thr
625 630 635 640
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly
645 650 655
Pro Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
660 665 670
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
675 680 685
Glu Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
690 695 700
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
705 710 715 720
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
725 730 735
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
740 745 750
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Gly
755 760 765
Gly Ser Gly Gly Gly Gly Ser Gly Ala Met Val Gly Ala Leu Glu Ser
770 775 780
Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys
785 790 795 800
Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly
805 810 815
Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg
820 825 830
Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr
835 840 845
Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Arg Gln Asp His
850 855 860
Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln
865 870 875 880
Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys
885 890 895
Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys
900 905 910
Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly
915 920 925
Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu
930 935 940
Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln
945 950 955 960
Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro
965 970 975
Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val
980 985 990
Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp
995 1000 1005
Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu
1010 1015 1020
Leu Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys
1025 1030 1035
Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe
1040 1045 1050
Lys Thr Ser Ala Ser
1055
<210> 3
<211> 3177
<212> DNA
<213>Artificial synthesized sequence
<400> 3
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cttcctgctg 60
atcccccagg tgcagctggt gcagagcggc gccgccgtga agaagcccgg cgagagcctg 120
aagatcagct gcaagggcag cggctacaga ttcaccagct actggatcgg ctgggtgaga 180
cagatgcccg gcaagggcct ggagtggatg ggcatcatct accccggcga cagcgacacc 240
agatacagcc ccagcttcca gggccaggtg accatcagcg ccggcaagag catcagcacc 300
gcctacctgc agtggagcag cctgaaggcc agcgacaccg ccatgtacta ctgcgccaga 360
cacggcagag gctacaacgg ctacgagggc gccttcgaca tctggggcca gggcaccatg 420
gtgaccgtga gcagcggcag caccagcggc agcggcaagc ccggcagcag cgagggcagc 480
accaagggcg ccatccagct gacccagagc cccagcagcc tgagcgccag cgtgggcgac 540
agagtgacca tcacctgcag agccagccag ggcatcagca gcgccctggc ctggtaccag 600
cagaagcccg gcaaggcccc caagctgctg atctacgacg ccagcagcct ggagagcggc 660
gtgcccagca gattcagcgg cagcggcagc ggcaccgact tcaccctgac catcagcagc 720
ctgcagcccg aggacttcgc cacctactac tgccagcagt tcaacagcta ccccctgacc 780
ttcggcggcg gcaccaaggt ggagatcaag gccgccgcca tcgaggtgat gtaccccccc 840
ccctacctgg acaacgagaa gagcaacggc accatcatcc acgtgaaggg caagcacctg 900
tgccccagcc ccctgttccc cggccccagc aagcccttct gggtgctggt ggtggtgggc 960
ggcgtgctgg cctgctacag cctgctggtg accgtggcct tcatcatctt ctgggtgaga 1020
agcaagagaa gcagactgct gcacagcgac tacatgaaca tgacccccag aagacccggc 1080
cccaccagaa agcactacca gccctacgcc ccccccagag acttcgccgc ctacagaagc 1140
gccagcggcg gcggcggcag cggcggcggc ggcagccaga gaagaaagta cagaagcaac 1200
aagggcgaga gccccgtgga gcccgccgag ccctgccact acagctgccc cagagaggag 1260
gagggcagca ccatccccat ccaggaggac tacagaaagc ccgagcccgc ctgcagcccc 1320
ggcggcggcg gcagcggcgg cggcggcagc accagcggcg gcggcggcag cggcggcggc 1380
ggcagcgtgg tgaagagagg cagaaagaag ctgctgtaca tcttcaagca gcccttcatg 1440
agacccgtgc agaccaccca ggaggaggac ggctgcagct gcagattccc cgaggaggag 1500
gagggcggct gcgagctggg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 1560
agcagagtga agttcagcag aagcgccgac gcccccgcct accagcaggg ccagaaccag 1620
ctgtacaacg agctgaacct gggcagaaga gaggagtacg acgtgctgga caagagaaga 1680
ggcagagacc ccgagatggg cggcaagccc agaagaaaga acccccagga gggcctgtac 1740
aacgagctgc agaaggacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 1800
agaagaagag gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggac 1860
acctacgacg ccctgcacat gcaggccctg ccccccagaa ccagcggcag cggcgccacc 1920
aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc catgggcgtg 1980
caggtggaga ccatcagccc cggcgacggc agaaccttcc ccaagagagg ccagacctgc 2040
gtggtgcact acaccggcat gctggaggac ggcaagaagg tggacagcag cagagacaga 2100
aacaagccct tcaagttcat gctgggcaag caggaggtga tcagaggctg ggaggagggc 2160
gtggcccaga tgagcgtggg ccagagagcc aagctgacca tcagccccga ctacgcctac 2220
ggcgccaccg gccaccccgg catcatcccc ccccacgcca ccctggtgtt cgacgtggag 2280
ctgctgaagc tggagggcgg cggcggcagc ggcggcggcg gcagcggcgc catggtgggc 2340
gccctggaga gcctgagagg caacgccgac ctggcctaca tcctgagcat ggagccctgc 2400
ggccactgcc tgatcatcaa caacgtgaac ttctgcagag agagcggcct gagaaccaga 2460
accggcagca acatcgactg cgagaagctg agaagaagat tcagcagcct gcacttcatg 2520
gtggaggtga agggcgacct gaccgccaag aagatggtgc tggccctgct ggagctggcc 2580
agacaggacc acggcgccct ggactgctgc gtggtggtga tcctgagcca cggctgccag 2640
gccagccacc tgcagttccc cggcgccgtg tacggcaccg acggctgccc cgtgagcgtg 2700
gagaagatcg tgaacatctt caacggcacc agctgcccca gcctgggcgg caagcccaag 2760
ctgttcttca tccaggcctg cggcggcgag cagaaggacc acggcttcga ggtggccagc 2820
accagccccg aggacgagag ccccggcagc aaccccgagc ccgacgccac ccccttccag 2880
gagggcctga gaaccttcga ccagctggac gccatcagca gcctgcccac ccccagcgac 2940
atcttcgtga gctacagcac cttccccggc ttcgtgagct ggagagaccc caagagcggc 3000
agctggtacg tggagaccct ggacgacatc ttcgagcagt gggcccacag cgaggacctg 3060
cagagcctgc tgctgagagt ggccaacgcc gtgagcgtga agggcatcta caagcagatg 3120
cccggctgct tcaacttcct gagaaagaag ctgttcttca agaccagcgc cagctga 3177
<210> 4
<211> 423
<212> PRT
<213>Artificial synthesized sequence
<400> 4
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro Met Gly Val Gln Val Glu Thr Ile Ser Pro
20 25 30
Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His
35 40 45
Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val Asp Ser Ser Arg Asp
50 55 60
Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg
65 70 75 80
Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys
85 90 95
Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly
100 105 110
Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys
115 120 125
Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala Met Val
130 135 140
Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu
145 150 155 160
Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe
165 170 175
Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys
180 185 190
Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu Val
195 200 205
Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu
210 215 220
Ala Arg Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu
225 230 235 240
Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr
245 250 255
Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe
260 265 270
Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe
275 280 285
Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala
290 295 300
Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp
305 310 315 320
Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala
325 330 335
Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr
340 345 350
Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr
355 360 365
Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp
370 375 380
Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly
385 390 395 400
Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu
405 410 415
Phe Phe Lys Thr Ser Ala Ser
420
<210> 5
<211> 21
<212> PRT
<213>Artificial synthesized sequence
<400> 5
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 6
<211> 22
<212> PRT
<213>Artificial synthesized sequence
<400> 6
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
20

Claims (10)

1. one kind is based on CD117 Chimeric antigen receptors, it is characterised in that the Chimeric antigen receptor includes antigen binding structure Domain, membrane spaning domain, costimulatory signal conducting region and CD3 ζ signal transduction domains are in series;
Wherein, the antigen-binding domains combination tumor surface antigen, the tumor surface antigen are CD117.
2. Chimeric antigen receptor according to claim 1, it is characterised in that the CD117 Chimeric antigen receptors it is single-stranded Antibody amino acids sequence is as shown in SEQ ID NO.1;
Preferably, the single-chain antibody of the tumor surface antigen CD117 also mutant including tumor surface antigen CD117;
Preferably, the amino acid sequence of the single-chain antibody of the mutant of the tumor surface antigen CD117 and SEQ ID NO.1 institutes The amino acid sequence shown has more than 90% similarity.
3. Chimeric antigen receptor according to claim 1 or 2, it is characterised in that the membrane spaning domain is CD28 cross-films Domain and/or CD8 α membrane spaning domains;
Preferably, the costimulatory signal conducting region is CD28 signal transductions domain, CD137 signal transductions domain or CD27 In signal transduction domain any one or at least two combination, preferably CD28 signal transductions domain, CD137 signals Conducting structure domain and the combination of CD27 signal transduction domains.
4. according to the Chimeric antigen receptor any one of claim 1-3, it is characterised in that the Chimeric antigen receptor is also Including can induce suicide Fusion domain;
Preferably, the inducible suicide Fusion domain is to include the domain of Caspase 9;
Preferably, the amino acid sequence of the domain of Caspase 9 is as shown in SEQ ID NO.4;
Preferably, the inducible suicide Fusion domain is in series by 2A sequences and CD3 ζ signal transduction domains.
5. according to the Chimeric antigen receptor any one of claim 1-4, it is characterised in that the Chimeric antigen receptor bag Include signal peptide, antigen-binding domains, membrane spaning domain, costimulatory signal conducting region, CD3 ζ signal transductions domain, 2A sequences It is in series with inducible suicide Fusion domain;
Preferably, the Chimeric antigen receptor be Secretory signal peptides, CD117 antigen-binding domains, CD8 α and/or CD28 membrane spaning domains, CD28 signal transductions domain, CD137 signal transductions domain and/or CD27 signal transduction domains, CD3 ζ signal transductions domain, 2A sequences and the domain of Caspase 9 are in series.
6. according to the Chimeric antigen receptor any one of claim 1-5, it is characterised in that the Chimeric antigen receptor is Secretory-CD117-CD28-CD137-CD27-CD3ζ-2A-iCasp9;
Preferably, the ammonia of the Chimeric antigen receptor Secretory-CD117-CD28-CD137-CD27-CD3 ζ -2A-iCasp9 Base acid sequence is as shown in SEQ ID NO.2;
Preferably, the core of the Chimeric antigen receptor Secretory-CD117-CD28-CD137-CD27-CD3 ζ -2A-iCasp9 Nucleotide sequence is as shown in SEQ ID NO.3.
7. according to the Chimeric antigen receptor any one of claim 1-6, it is characterised in that described Chimeric antigen receptor It is transfected into T cell and is expressed by the nucleotide sequence of its coding;
Preferably, the mode of the transfection be by any one in viral vector, 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 vector;
Preferably, the viral vector is slow virus carrier and/or retroviral vector, preferably slow virus carrier.
8. a kind of recombinant slow virus, it is characterised in that the Chimeric antigen receptor as any one of claim 1-7 will be included Viral vector and the obtained recombinant slow virus of packaging helper plasmid pNHP and pHEF-VSVG cotransfection mammalian cell;
Preferably, the mammalian cell is 293 cells, any one in 293T cells or TE671 cells or at least two Combination.
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, recombinant slow virus as claimed in claim 8 as any one of claim 1-7 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 the related tumor disease of blood;
Preferably, the related tumor disease of the blood is leukaemia and/or lymthoma.
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CN107974460A (en) * 2017-11-30 2018-05-01 山东兴瑞生物科技有限公司 Chimeric antigen receptor gene for HIV-1, plasmid, T cell, kit and application with the gene
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WO2020108645A1 (en) * 2018-11-30 2020-06-04 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Cd19-and bcma-based combined car-t immunotherapy
WO2023248126A1 (en) * 2022-06-20 2023-12-28 Crispr Therapeutics Ag Chimeric antigen receptor specific to cd117
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