CN109468283A - A kind of immunocyte of the dual Chimeric antigen receptor gene modification based on CD19 and BCMA and its application - Google Patents

A kind of immunocyte of the dual Chimeric antigen receptor gene modification based on CD19 and BCMA and its application Download PDF

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CN109468283A
CN109468283A CN201811458396.9A CN201811458396A CN109468283A CN 109468283 A CN109468283 A CN 109468283A CN 201811458396 A CN201811458396 A CN 201811458396A CN 109468283 A CN109468283 A CN 109468283A
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桂思倩
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Beijing American Kinge Biotech Co Ltd
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Beijing American Kinge Biotech Co Ltd
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Priority to PCT/CN2019/122163 priority patent/WO2020108645A1/en
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Abstract

The immunocyte and its application, the dual Chimeric antigen receptor for the dual Chimeric antigen receptor gene modification based on CD19 and BCMA that the present invention relates to a kind of include Chimeric antigen receptor CD19 and Chimeric antigen receptor BCMA.The Chimeric antigen receptor includes that antigen-binding domains, transmembrane domain, costimulatory signal conducting region and CD3 ζ signal transduction structural domain and inducible suicide Fusion domain are connected in series.Identification the tumor surface antigen CD19 and BCMA of Chimeric antigen receptor energy specificity of the invention, compared to using other single Chimeric antigen receptor T cells, two kinds of antigen targeting CAR-T cells, which are used in combination, makes therapeutic effect more preferable, is not susceptible to CD19 escape, it is easier to alleviate disease.

Description

A kind of dual Chimeric antigen receptor gene modification based on CD19 and BCMA it is immune thin Born of the same parents and its application
Technical field
The present invention relates to the cellular immunotherapy fields of tumour more particularly to a kind of dual chimeric based on CD19 and BCMA The immunocyte of antigen receptor gene modification and its application, it is chimeric specially based on tomour specific target spot CD19 and BCMA The construction method of antigen receptor T (CAR-T) cell technology and its application in antineoplaston.
Background technique
With the development of tumour immunity theory and clinical technology, Chimeric antigen receptor T cell therapy (Chimeric Antigen receptor T-cell immunotherapy, CAR-T) become tumour immunity treatment most promising at present One of method.Generally, Chimeric antigen receptor CAR is by a tumor associated antigen combined area, extracellular hinge area, trans-membrane region and born of the same parents Interior signal transduction district's groups at.In general, the single-chain fragment that CAR includes antibody can be changed (Single chain fragment Variable, scFv) area or to tumor associated antigen (tumor associated antigen, TAA) have specificity knot Structural domain is closed, is coupled by the cytoplasmic domains of hinge and transmembrane region and T cell signal transduction molecule.The most common lymph is thin Born of the same parents' activated partial includes the T cell costimulation structural domain with T cell effector function triggering (such as CD3 ζ) sections in series.CAR The T cell that the adoptive immunotherapy of mediation allows CAR-T to transplant is on non-HLA restrictive one 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.It is logical for treating a kind of method of these patients The expression for crossing CAR carries out genetic modification to T cell to target the antigen expressed on tumour cell.CAR is that be designed to people white Cellular antigens (human leukocyte antigen, HLA) dependent/non-dependent mode identifies the antigen receptor of cell surface antigen. It attempts to have been achieved for promising success using the genetically modified cell of expression CAR to treat the patient of these types.
CD19 molecule is the hot spot treated in the potential target spot of bone-marrow-derived lymphocyte system tumour and CAR research, the table of CD19 It is the CAR target for safety test accepted extensively up to normal and malignant B cell is confined to.Target the chimeric of CD19 molecule The T cell (CD19 CAR-T) of antigen receptor gene modification is on treating multiple, intractable B-lineage Acute Lymphocyte Leukemia 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 obviously poor.
104788573 A of CN has disclosed a kind of Chimeric antigen receptor hCD19scFv-CD8 α-CD28-CD3 ζ and its use On the way, the second generation Chimeric antigen receptor by anti human CD 19 monoclonal antibody HI19a light chain and heavy chain variable region (hCD19scFv), People CD8 α hinge area, people CD28 transmembrane region and intracellular region and people's CD3 ζ intracellular region structures in series are constituted, the CD19 in the patent After carrying out a CAR-T cell and feeding back, the expression quantity of CD19 can be reduced, and be easy to escape from immunologic mechanism, and second generation CART Immune factor storm it is big, have the hidden worry of safety.
In addition, according to this center with the actual count of forth generation CD19 Chimeric antigen receptor treatment lymthoma, in 9 patients In the middle, wherein there is the patient of 4 tumor tissues immunohistochemical staining CD19 strong positives, receiving the treatment of CD19 Chimeric antigen receptor All reach complete incidence graph afterwards, in addition the patient of 5 CD19 weak expressions, only 1 reaches alleviation after the treatment, remaining 2 only Part is alleviated, the stabilization of 1 maintenance disease, 1 disease has progressed, this bearing reaction is single with CD19 is embedding and antigen receptor treatment Predicament.
Therefore, for the recurrence of CD19 feminine gender and the B cell tumour of CD19 low expression, combine another potential chimeric Antigen receptor is able to solve the easily mutation problem low with expression quantity existing for CD19 and is particularly important.In addition to CD19, BCMA is also It is the potentiality target spot for treating malignant B cell tumour, BCMA is a kind of albumen of thick liquid cell selectivity, is found in mature B earliest Lymphocytic cell surface is hardly expressed in other histocytes, but (such as myeloma is thin in the bone-marrow-derived lymphocyte of malignant proliferation Born of the same parents, leukaemia cell) in height express, while its downstream signaling pathway for mediating, to the survival of cell, proliferation, transfer and resistance to Critical effect is played in medicine, these characteristics make it become immunization therapy Huppert's disease and bone-marrow-derived lymphocyte system lymph One target spot of tumor.The studies have shown that BCMA of anti-BCMA antibody clinical trials be evaluated and be confirmed as treatment B cell it is pernicious The good target of tumour.
Therefore, a kind of strong specific, high targeting is found, the Chimeric antigen receptor of CART therapeutic effect can be effectively improved Just it is particularly important.
Summary of the invention
For in current CAR-T technology treatment tumour single targeting long-term effect is not very ideal and tumour micro-loop Border influences the case where CAR-T technology therapeutic effect, and the present invention provides a kind of dual Chimeric antigen receptor based on CD19 and BCMA The immunocyte of gene modification and its application, combined needle has strong special the dual tumor targets of CD19 and BCMA to the present invention for the first time The effect of CART treatment can effectively be improved and be extended to the characteristics of anisotropic, high targeting, for surface antigen CD19 and BCMA Positive leukaemia or B cell lymphoma has better therapeutic effect, is effectively prevented from the escape of missing the target of single target spot.
To achieve this purpose, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of the immune thin of the dual Chimeric antigen receptor gene modification based on CD19 and BCMA Born of the same parents, the dual Chimeric antigen receptor include Chimeric antigen receptor CD19 and Chimeric antigen receptor BCMA.
In the present invention, by by antigen-binding domains combination tumor surface antigen CD19 and BCMA, then pass through slow virus Carrier modifies T cell, so that the combination that tumor surface antigen CD19 and BCMA can be special is in the embedding of the application It closes on antigen receptor, CAR-T cell can eliminate the tumour cell of CD19 and BCMA simultaneously, effectively avoid tumor-cell antigen table It escapes up to reduction, while enhancing the permanent immunity effect of CAR-T cell.
In the present invention, the dual Chimeric antigen receptor can be individual CD19 Chimeric antigen receptor and individual BCMA When Chimeric antigen receptor is used in combination, it is also possible to join CD19 Chimeric antigen receptor and BCMA Chimeric antigen receptor Conjunction is expressed as dual Chimeric antigen receptor, i.e. antigen-binding domains are that can combine tumor surface antigen CD19 and BCMA, this The combination therapy of two kinds of Chimeric antigen receptors either way may be implemented.
According to the present invention, the Chimeric antigen receptor includes antigen-binding domains, transmembrane domain, costimulatory signal biography It leads area, CD3 ζ signal transduction structural domain and can induce suicide Fusion domain and be connected in series.
Preferably, the antigen-binding domains are for the single-chain antibody of tumor surface antigen CD19 and for tumour table The single-chain antibody of face antigen BCMA.
According to the present invention, the T cell Chimerical receptor (CAR) and be directed to CD19 and BCMA antigen that the gene modification is transformed It is described below for the single-chain antibody (scFv) of binding structural domain.
In a specific embodiment, the amino acid sequence tool of the single-chain antibody for tumor surface antigen CD19 There is any one in amino acid sequence shown in (I), (II) or (III):
(I) there is the amino acid sequence as shown in SEQ ID NO.1；
(II) with amino acid sequence shown in SEQ ID NO.1 with >=90%, preferably >=95%, more preferably >=98%, most It is preferred that the amino acid sequence of >=99% homology；
(III) it modified with amino acid sequence shown in SEQ ID NO.1, replace, miss or add one or several ammonia The amino acid sequence that base acid obtains；
The amino acid sequence has the activity of the single-chain antibody for tumor surface antigen CD19；
It is as follows for the amino acid sequence (SEQ ID NO.1) of the single-chain antibody of tumor surface antigen CD19: DIQMTQTT SSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVS WIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWG QGTSVTVSS.
According to the present invention, it is described its have 90% or more homology amino acid sequence or through modification, substitution, lack or add The amino acid sequence for adding one or several amino acid to obtain can be substituted by other single-chain antibodies or humanization CD19 single-chain antibody, Its amino acid mutants still has the function of CD19 single-chain antibody.
In a specific embodiment, the amino acid sequence tool of the single-chain antibody for tumor surface antigen BCMA There is any one in amino acid sequence shown in (I), (II) or (III):
(I) there is the amino acid sequence as shown in SEQ ID NO.2；
(II) with amino acid sequence shown in SEQ ID NO.2 with >=90%, preferably >=95%, more preferably >=98%, most It is preferred that the amino acid sequence of >=99% homology；
(III) it modified with amino acid sequence shown in SEQ ID NO.2, replace, miss or add one or several ammonia The amino acid sequence that base acid obtains；
The amino acid sequence has the activity of the single-chain antibody for tumor surface antigen BCMA；
It is as follows for the amino acid sequence (SEQ ID NO.2) of tumor surface antigen BCMA single-chain antibody:
MLLLVTSLLLCELPHPAFLLIPDIVLTQSPASLAVSLGDRATINCRASESVSVIGAHLIHWYQQKPGQ PPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGS SEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSIQWVRQAPGQGLEWMGWIQTETREPAYAYDFRGRFV FSLDTSVSTAYLQISSLKAEDTAVYYCALDYSYAMDYWGQGTLVTVSS.
According to the present invention, it is described its have 90% or more homology amino acid sequence or through modification, substitution, lack or add The amino acid sequence for adding one or several amino acid to obtain can be substituted by other single-chain antibodies or humanization BCMA single-chain antibody, Its amino acid mutants still has the function of BCMA single-chain antibody.
According to the present invention, the Chimeric antigen receptor by slow virus carrier to T cell carry out genetic modification, CD19 and It is stronger to kill tumor effect in conjunction with tumor surface antigen CD19 and BCMA for the dual CAR-T cell of BCMA.
According to the present invention, the transmembrane domain is CD28 transmembrane domain and/or CD8 α transmembrane domain, in some tools In body embodiment, transmembrane domain can be selected or modified by amino acid substitution.
According to the present invention, the costimulatory signal conducting region is CD28 signal transduction structural domain, CD27 signal transduction structure In domain or CD137 signal transduction structural domain any one or at least two combination, the CD28 signal transduction structural domain, The arrangement of CD27 signal transduction structural domain and CD137 signal transduction structural domain, those skilled in the art can according to need progress Adjustment, the arrangement different with CD137 signal transduction structural domain with CD27 signal transduction structural domain of CD28 signal transduction structural domain is not The Chimeric antigen receptor can be had an impact, the application is combined using the sequence of CD28-CD27.
According to the present invention, the forth generation CAR is to include 9 structure of Caspase with can induce suicide Fusion domain Domain, for amino acid sequence as shown in SEQ ID NO.3, amino acid sequence shown in the SEQ ID NO.3 is as follows:
GSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMV GALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLA LLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQ KDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETL DDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.
According to the present invention, the inducible suicide Fusion domain is mutually gone here and there by 2A sequence with CD3 ζ signal transduction structural domain Connection, the 2A sequence can make the albumen and the Chimeric antigen receptor chopping up proteins of the inducible suicide Fusion domain expression It opens, so that the Chimeric antigen receptor can play a role, and by injection activator, melt so that can induce suicide Structural domain activation is closed, the T cell so as to cause expression Chimeric antigen receptor is dead and ineffective.
According to the present invention, the Chimeric antigen receptor further 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 according to need the signal peptide of selection this field routine, the letter Number peptide is Secretory signal peptide, and the Secretory signal peptide is the signal peptide of GMCSFR gene, and amino acid sequence can With as follows as shown in SEQ ID NO.8: MLLLVTSLLLCELPHPAFLLIP.
Preferably, the Secretory signal peptide is the signal peptide of CD8a gene, the ammonia of the Secretory signal peptide Base acid sequence is as follows as shown in SEQ ID NO.9: MALPVTALLLPLALLLHAARP.
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 do not do particular determination herein, the presence of hinge area will not be to the property of Chimeric antigen receptor of the invention It can have an impact.
According to the present invention, the Chimeric antigen receptor includes signal peptide, antigen-binding domains, transmembrane domain, altogether thorn Energizing signal conducting region, CD3 ζ signal transduction structural domain, 2A sequence and inducible suicide Fusion domain are connected in series.
As optimal technical scheme, the Chimeric antigen receptor is Secretory signal peptide, CD19 antigen-binding domains And/or BCMA antigen-binding domains, CD8 α and/or CD28 transmembrane domain, CD28 extracellular signal conducting structure domain, CD28 Cellular Signaling Transduction Mediated structural domain, CD27 Cellular Signaling Transduction Mediated structural domain, CD3 ζ Cellular Signaling Transduction Mediated structural domain, 2A sequence It is connected in series with FBKP.Casp9 structural domain, specific arrangement is as follows:
Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9；
Secretory-BCMA scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9。
In a specific embodiment, the Chimeric antigen receptor Secretory-CD19 scFv-CD28-CD27- The amino acid sequence of CD3 ζ -2A-FBKP.Casp9 has the amino of 90% or more homology as shown in SEQ ID NO.4 or with it Acid sequence, amino acid sequence shown in the SEQ ID NO.4 are as follows:
MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLE WMGWINTYTGEPTYADAFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSSGSTS GSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIKAAAIEVMYPPPYLDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSASGGGGSGGGGSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPGGGGSGGGG SRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRT FPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGI IPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSN IDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSV EKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLP TPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLF FKTSAS；
In a specific embodiment, the Chimeric antigen receptor Secretory-CD19 scFv-CD28-CD27- The nucleotide sequence of CD3 ζ -2A-FBKP.Casp9 has the nucleosides of 95% or more homology as shown in SEQ ID NO.5 or with it Acid sequence, nucleic acid sequence shown in the SEQ ID NO.5 are as follows:
ATGCTGCTGCTGGTCACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCCGA CATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGC CAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCA GCCGGCTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGATCTGGCACCGACTACAGCCTGACCATCTCCAA CCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACC AAGCTGGAAATCACCGGCAGCACCAGCGGCTCCGGCAAGCCTGGATCTGGCGAGGGCAGCACCAAGGGCGAAGTGA AGCTGCAGGAAAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGAGCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTC CCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGC AGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGT TCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAG CTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTCTCTTCTGCGGCCGCAATTGAAGTTATGTATCCT CCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCC TATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGT AACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACT CCCCGCCGCCCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCG CTAGCGGAGGTGGAGGTTCTGGAGGTGGTGGAAGTCAAAGAAGGAAGTACCGCAGCAACAAAGGAGAATCTCCCGT CGAGCCAGCCGAGCCCTGTCATTATTCATGCCCAAGGGAGGAGGAGGGAAGTACAATCCCAATTCAAGAAGACTAC AGGAAGCCCGAACCTGCATGCAGTCCAGGTGGAGGCGGTTCTGGAGGCGGTGGCTCCCGGGTGAAATTCTCACGGT CTGCAGACGCACCCGCTTACCAGCAAGGCCAGAACCAACTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTA CGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGCACTAGTGGCTCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAAC CCCGGTCCCATGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCT GCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTGGACTCCTCCCGGGACAGAAACAAGCCCTTTAA GTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCC AAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCG TCTTCGATGTGGAGCTTCTAAAACTGGAAGGTGGAGGCGGTTCAGGCGGCGGCGGCAGCGGCGCCATGGTCGGTGC TCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATC AACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGC GTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCT GGAGCTGGCGCGGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGC CACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCA ATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGA CCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCG TTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGT CCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGA CATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAA GGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTAGTTAA.
In a specific embodiment, the Chimeric antigen receptor Secretory-BCMA scFv-CD28-CD27- The amino acid sequence of CD3 ζ -2A-FBKP.Casp9 has the amino of 90% or more homology as shown in SEQ ID NO.6 or with it Acid sequence, amino acid sequence shown in the SEQ ID NO.6 are as follows:
MLLLVTSLLLCELPHPAFLLIPDIVLTQSPASLAVSLGDRATINCRASESVSVIGAHLIHWYQQKPGQ PPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGS SEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSIQWVRQAPGQGLEWMGWIQTETREPAYAYDFRGRFV FSLDTSVSTAYLQISSLKAEDTAVYYCALDYSYAMDYWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGG SRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRT FPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGI IPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSN IDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSV EKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLP TPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLF FKTSAS；
In a specific embodiment, the Chimeric antigen receptor Secretory-BCMA scFv-CD28-CD27- The nucleotide sequence of CD3 ζ -2A-FBKP.Casp9 has the nucleosides of 95% or more homology as shown in SEQ ID NO.7 or with it Acid sequence, nucleic acid sequence shown in the SEQ ID NO.7 are as follows:
ATGCTGCTGTTGGTGACATCTTTGTTGTTGTGCGAATTGCCTCATCCCGCTTTCCTGCTGATTCCCGA CATCGTTCTGACCCAGTCTCCTGCATCTCTCGCCGTTTCTCTCGGTGACCGCGCTACAATCAATTGCAGAGCCTCA GAAAGCGTCTCCGTTATCGGCGCTCACCTGATTCATTGGTATCAACAGAAGCCAGGCCAGCCACCCAAACTGCTCA TCTACCTGGCATCCAACCTGGAAACTGGTGTGCCAGCCAGGTTTAGCGGATCAGGAAGCGGTACTGACTTCACTCT GACTATCTCCTCCCTGCAAGCCGAAGATGCAGCCATCTATTACTGCCTCCAGAGCAGAATCTTCCCTAGAACCTTC GGCCAAGGTACTAAGCTGGAGATTAAAGGCTCCACTAGCGGTTCCGGCAAGCCCGGTTCCAGCGAAGGAAGCACTA AGGGACAGGTCCAGTTGGTGCAGAGCGGATCTGAGCTGAAGAAACCTGGCGCCTCAGTCAAGGTTTCATGTAAGGC ATCCGGATACACTTTCACAGATTATAGCATCCAGTGGGTCCGCCAGGCTCCTGGTCAGGGCTTGGAATGGATGGGT TGGATTCAAACCGAAACCCGCGAGCCTGCCTATGCTTACGACTTCCGCGGAAGATTTGTGTTTAGCCTCGACACTA GCGTGTCTACAGCCTACCTCCAAATCTCATCCCTGAAGGCTGAAGACACCGCAGTTTACTACTGTGCCCTCGACTA CTCCTATGCTATGGATTACTGGGGTCAAGGAACTTTGGTTACTGTGTCTAGCGCGGCCGCAATTGAAGTTATGTAT CCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTC CCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCT AGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATG ACTCCCCGCCGCCCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCT CCGCTAGCGGCGGAGGTGGCTCTGGCGGTGGCGGATCAGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATT CAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAACTGGGCGGCGGCGGCAGCGGAGGCGGTGGCAGCGGCGGCGGCGGCTCTAGAGTGAAGTTCA GCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAG GAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGCACTAGTGGCTCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAA GAAAACCCCGGTCCCATGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCC AGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTGGACTCCTCCCGGGACAGAAACAAGCC CTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAG AGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCA CTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGTGGAGGCGGTTCAGGCGGCGGCGGCAGCGGCGCCATGGT CGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTC ATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGT TGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGC TTTGCTGGAGCTGGCGCGGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAG GCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACA TCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCA GAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCC ACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCT TTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCT GGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCG GTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTA GTTAA.
In the present invention, the Chimeric antigen receptor further includes promoter, and the promoter is in EF1a, CMV-TAR or CMV Any one or at least two combination.
According to the present invention, the Chimeric antigen receptor is transfected into T cell by the nucleic acid sequence of its coding and is expressed.
According to the present invention, the mode of the transfection is by appointing in viral vectors, eukaryon expression plasmid or mRNA sequence A kind of or at least two combinations of anticipating are transfected into T cell, are transfected into T cell preferably by viral vectors.
Preferably, the viral vectors is any one in slow virus carrier or retroviral vector or at least two Combination, preferably slow virus carrier.
Second aspect, the present invention provide a kind of recombinant slow virus, will include dual chimeric antigen as described in relation to the first aspect The recombinant lentiviral that the immunocyte and packaging helper plasmid pNHP and pHEF-VSVG cotransfection mammalian cell of acceptor gene modification obtain Virus.
In the present invention, the recombinant slow virus can effective immunocyte, including T cell can prepare targeting T-cells.
According to the present invention, the mammalian cell is 293 cells, in 293T cell or TE671 cell any one or extremely Few two kinds of combination.
The third aspect, the present invention provide a kind of pharmaceutical composition, comprising dual chimeric antigen as described in relation to the first aspect by The immunocyte of body gene modification and/or the recombinant slow virus as described in second aspect.
Fourth aspect, the present invention provide the immune thin of dual Chimeric antigen receptor gene modification as described in relation to the first aspect Born of the same parents, the recombinant slow virus as described in second aspect or the pharmaceutical composition as described in the third aspect are in preparation Chimeric antigen receptor T Application in cell, immunocompetent cell or tumor therapeutic agent.
In the present invention, the antigen receptor T cell has good targeting, at the same can discharge low dosage it is immune because Son has hypotoxicity reaction property.
Preferably, the tumour is the relevant tumor disease of blood and/or solid tumor, and the tumor disease is selected from but unlimited In leukaemia.
Compared with prior art, the invention has the following beneficial effects:
(1) dual Chimeric antigen receptor of the invention is based on by the way that T cell Chimerical receptor gene is specifically transformed The dual CAR-T cell of CD19 and BCMA is in conjunction with tumor surface antigen CD19 and BCMA, and it is stronger to kill tumor effect, tumor regression Effect becomes apparent；
(2) dual Chimeric antigen receptor of the invention can specificity identification tumor surface antigen CD19 and BCMA, CD19 Expression quantity is high in leukaemia and lymthoma with BCMA, has compared to other Chimeric antigen receptors and other tumour antigens and more pacifies Entirely, more significant effect is not susceptible to CD19 escape, it is easier to reach disease so that the immune effect of CAR-T cell enhances Place, improves the therapeutic effect of disease.
Detailed description of the invention
Fig. 1 is the synthetic gene sequence map of Chimeric antigen receptor CD19 and BCMA of the invention；
Fig. 2 is the principle that Chimeric antigen receptor CD19 and BCMA of the present invention is used in combination；
Fig. 3 be CD19 and BCMA Chimeric antigen receptor T cell Cytotoxicity in vitro B cell lymphoma cell strain streaming as a result, Wherein, Fig. 3 (a) is the experimental result of CD19 CAR-T, and Fig. 3 (b) is the experimental result of BCMA CAR-T；
Fig. 4 is CD19 and BCMA ImmunohistochemistryResults Results, and sample is the DLBCL patient in embodiment；
Fig. 5 is CD19 and BCMA Chimeric antigen receptor T cell combination therapy B cell leukemia/lymthoma clinical trial stream Cheng Tu；
Fig. 6 is combined clinical application CD19 and BCMA Chimeric antigen receptor T cell is treated B cell leukemia/lymthoma and suffered from Person, the CAR gene copy number of patient's vivo detection after feedback.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art, Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from The conventional products of acquisition.
The building of 1 Chimeric antigen receptor of embodiment
(1) by full genome synthesize Secretory signal peptide, CD19 or BCMA antigen-binding domains, CD8 α and/or CD28 transmembrane domain, CD28 signal transduction structural domain, CD27 signal transduction structural domain, CD3 ζ signal transduction structural domain, 2A sequence Column and 9 structural domain of Caspase, as shown in Figure 1, i.e. Secretory-CD19scFv-CD28-CD27-CD3 ζ -2A- FBKP.Casp9 and Secretory-BCMA scFv-CD28-CD27-CD3 ζ -2A-FBKP.Casp9；Particular sequence is as follows:
The nucleotide sequence such as SEQ ID of Secretory-CD19 scFv-CD28-CD27-CD3 ζ -2A-FBKP.Casp9 Shown in NO.5.
The nucleotide sequence such as SEQ ID of Secretory-BCMA scFv-CD28-CD27-CD3 ζ -2A-FBKP.Casp9 Shown in NO.7.
2 slow virus of embodiment packaging
(1) 293T cell is used, is cultivated 17-18 hours；
(2) fresh DMEM is added, includes 10% FBS；
(3) following reagent is added in sterile centrifugation tube: every hole takes DMEM that helper DNA mix (pNHP, pHEF- is added VSV-G) and pTYF DNA vector, vortex oscillation；
(4) centrifuge tube is added to Superfect or any transgenic line, be stored at room temperature 7-10 minutes；
(5) the DNA-Superfect mixed liquor in centrifuge tube is added dropwise in each culture cell, whirlpool is beaten；
(6) 37 DEG C of 3%CO2It is cultivated 4-5 hours in incubator；
(7) culture solution for siphoning away culture medium rinses culture medium with 293 cell culture fluids, and culture solution is added and continues to cultivate；
(8) culture medium is put back into 3%CO2Overnight incubation in incubator, the next morning are transfected with fluorescence microscope Efficiency.
The purifying and concentration of 3 slow virus of embodiment
1) viral purification
By being centrifuged 1000g, 5 minutes removing cell fragments obtain vial supernatant, with one 0.45 micron of low albumen Combined filtering device filters vial supernatant, and virus is distributed into aliquot, is stored in -80 DEG C；
Under normal conditions, in every milliliter of culture medium, transfection cell can produce 106To 107Transduced unit titrates slow Viral vectors.
2) slow virus carrier is concentrated with Centricon or similar filter
(1) vial supernatant is added in Centricon or similar screen pipe, is then centrifuged 30 minutes in 2500g；
(2) screen pipe is shaken, then 400g is centrifuged 2 minutes, collects the virus of concentration into collection cups.Finally by all pipes In virus focus in a centrifuge tube.
The transfection of 4 CAR-T cell of embodiment
By the T cell vaccination after activation into culture dish, the slow virus of concentration target gene is added, with 100g centrifugal force Speed is centrifuged 100 minutes, is placed in 37 DEG C of cultures for 24 hours, and the AIM-V culture medium for having the cell culture factor is added, after culture 2-3 days, Cell is harvested and counted, available CD19 CAR-T cell is become.
Embodiment 5 is killed using the external malignant B cell strain of CD19 and BCMA CAR-T cell
(1) figure it is seen that by CD19 combine BCMA can combination therapy tumour, indicate that the present invention selects double Weight Chimeric antigen receptor can play a significant role to oncotherapy, and prevent CD19 from escaping.
(2) expression of CD19 and BCMA are analyzed with flow cytometer for the cell strain of different B cell tumours, is wrapped Containing leukaemia and myeloma cell strain, as a result as shown in Fig. 3 (a)-Fig. 3 (b), it can be seen that the CD19 in different B cell tumours It is different with BCMA expression degree, B cell tumour can be more effectively directed to by the treatment of joint CD19 and BCMA CART.
(3) identification killing ability of the dual CAR-T cell of evaluating in vitro to target cell: by non-specific T cell, GD2 CD19 CART and BCMA CART cell and expression CD19, BCMA prepared by CAR-T cell and the application but the target for not expressing GD2 Cell, that is, RS4-11 people's acute lymphatic leukaemia cell strain, the myeloma cell strain Molp-2 (T cell: swollen of expression GFP Tumor cell strain=3:1), it is placed in 37 ° of 5%CO2Incubator co-cultures for 24 hours；
(4) after culture 24 hour record survivals RS4-11 cell proportion, as a result as shown in Fig. 3 (a)-Fig. 3 (b).Phase It is all right compared with control group T cell and GD2 CART, CD19 CART, BCMA CART and CD19 CART+BCMA CART of the present invention RS4-11 cell causes significantly to kill effect.After 24 hours apparently, although CD19 CART+BCMA CART killing ability is slightly weaker than CD19 CART is better than BCMA CART.
The clinical application of 6 CD19 and BCMA CAR-T cell of embodiment
Sample: 34 years old women is diagnosed as and diffuses large B cell tumor a year and a half, after multiple chemotherapy, lymthoma encephalic still occurs and invades Violate, continue chemotherapy afterwards, before feedback, cerebrospinal fluid (CSF) streaming shows the big bone-marrow-derived lymphocyte of 16% monoclonal.CD19 CART at present Develop for many years, has had good effect to treatment B acute lymphoblastic tumour, and in the treatment of lymthoma, it has been found that not 85% or more complete incidence graph can be reached as leukaemia, many patients only observe that part is alleviated or only maintains tumour steady It is fixed, then pass through and carry out the mono- target treatment of CD19 CART in this center for the past, and by this center rather than the third-party institution is to it The patient that tumour carries out CD19 immunohistochemical staining counts, and as a result as shown in table 1 below, in 9 patients, there is 4 CD19 The patient of strongly expressed reaches complete incidence graph, in addition the patient of 5 CD19 weak expressions, and only 1 reaches complete incidence graph, other 4 Position only has part to alleviate, and maintains stable disease or progression of disease, it is shown that CD19 expression power affects controlling for CD19 CART Therapeutic effect, for this limitation, we want that combine other target spots treats with CD19 CART.
Table 1
Remarks: n=9 is to contaminate the Lymphoma patients of CD19 according to number in immunohistochemistry table, has only done the mono- target spot of CD19 Patient.
The antigen dyeing identification antigens c ART target spot of 7 tumour cell of embodiment
After the tumor tissues that operation obtains are fixed, block sections are placed on slide, dye to tumour antigen, are made With CD19 and BCMA specific antibody.As shown in figure 4, tumor staining area is as a percentage, intensity is indicated with 0-4+, this case Example tumour expresses antigen intensity CD19,2+, BCMA, 4+, illustrates the CART targeting antigen that two kinds of antigens have all been.Nonspecific antibody Control group (isotype control) dyeing is feminine gender.
Specific clinical workflow figure as shown in figure 5, Case treatment case specific step is as follows, as a result as shown in Figure 6:
(1) the dense white blood cell liquid of patient is collected first, and wherein isolates CD3 positive T cell certainly, in embodiment 4 Method makes CART cell, is divided into two batches, feeds back patient's body, and CD19 CART cell dosage is~1 × 106CART cell is every Kg body weight, BCMA CART cell dosage are calculated as~1 × 106CART cell per kilogram of body weight；
(2) there are the slight CRS phenomenon of low fever He headache after feeding back in three weeks, be most higher than after feeding back the 14th day, in peripheral blood 3.75%CART cell can be measured；The 49th day after feedback, 1.65%CART cell can be also measured in peripheral blood, display CART is thin Born of the same parents are good in internal amplification；74 days after feedback, nuclear magnetic resonance image (MR) shows that thoracolumbar vertebrae section spinal meninges thickened degree subtracts earlier above Gently, strengthen and weaken, the big sheet of chest and back subdermal muscle group is strengthened shadow and is obviously reduced earlier above；
(3) CART copy number can be seen that in peripheral blood and still have in return visit in three months after feeding back, concrete outcome such as Fig. 6 0.11%CART cell has recorded the remission rate and survival day of patient after feeding back by table 2, specific as follows:
Table 2
From table 2 it can be seen that the patient body improves very much, lung inflammation is alleviated substantially, does not depend on oxygen uptake, physiological status Well, temporarily and do not rely on other drugs maintenance.
In conclusion dual Chimeric antigen receptor of the invention can specificity identification tumor surface antigen CD19 and BCMA is used in combination two kinds of CAR-T cells and makes therapeutic effect more compared to other single Chimeric antigen receptor T cells are used It is good, it is not susceptible to CD19 escape, it is easier to allow remission.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field 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., all of which fall within the scope of protection and disclosure of the present invention.
SEQUENCE LISTING
<110>Beijing Meikang Ji Mian Biotechnology Co., Ltd
<120>a kind of immunocyte of dual Chimeric antigen receptor gene modification based on CD19 and BCMA and its application
<130> 2018
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 245
<212> PRT
<213>artificial synthesized sequence
<400> 1
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser
130 135 140
Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu
180 185 190
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
225 230 235 240
Val Thr Val Ser Ser
245
<210> 2
<211> 268
<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 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser
20 25 30
Leu Ala Val Ser Leu Gly Asp Arg Ala Thr Ile Asn Cys Arg Ala Ser
35 40 45
Glu Ser Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu
65 70 75 80
Glu Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr
100 105 110
Tyr Cys Leu Gln Ser Arg Ile Phe Pro Arg Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser
130 135 140
Ser Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln Ser Gly Ser
145 150 155 160
Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
165 170 175
Gly Tyr Thr Phe Thr Asp Tyr Ser Ile Gln Trp Val Arg Gln Ala Pro
180 185 190
Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Gln Thr Glu Thr Arg Glu
195 200 205
Pro Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Val Phe Ser Leu Asp
210 215 220
Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu
225 230 235 240
Asp Thr Ala Val Tyr Tyr Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp
245 250 255
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
260 265
<210> 3
<211> 423
<212> PRT
<213>artificial synthesized sequence
<400> 3
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> 4
<211> 986
<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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu
20 25 30
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
35 40 45
Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly
50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro
65 70 75 80
Thr Tyr Ala Asp Ala Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr
85 90 95
Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser
130 135 140
Gly Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser Thr Lys Gly Asp Ile
145 150 155 160
Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg
165 170 175
Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr
180 185 190
Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
195 200 205
Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
225 230 235 240
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg Glu Val
245 250 255
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ala Ala
260 265 270
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
275 280 285
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
290 295 300
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
305 310 315 320
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
325 330 335
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
340 345 350
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
355 360 365
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ala Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Gln Arg Arg Lys Tyr Arg Ser Asn Lys
385 390 395 400
Gly Glu Ser Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro
405 410 415
Arg Glu Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys
420 425 430
Pro Glu Pro Ala Cys Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly
435 440 445
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
450 455 460
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
465 470 475 480
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
485 490 495
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
500 505 510
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
515 520 525
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
530 535 540
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
545 550 555 560
Arg Thr Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala
565 570 575
Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Val Gln Val Glu Thr
580 585 590
Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys
595 600 605
Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val Asp Ser
610 615 620
Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu
625 630 635 640
Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln
645 650 655
Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly
660 665 670
His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu
675 680 685
Leu Leu Lys Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
690 695 700
Ala Met Val Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala
705 710 715 720
Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn
725 730 735
Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn
740 745 750
Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met
755 760 765
Val Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu
770 775 780
Leu Glu Leu Ala Arg Gln Asp His Gly Ala Leu Asp Cys Cys Val Val
785 790 795 800
Val Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly
805 810 815
Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val
820 825 830
Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys
835 840 845
Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe
850 855 860
Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro
865 870 875 880
Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln
885 890 895
Leu Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser
900 905 910
Tyr Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly
915 920 925
Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His
930 935 940
Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser
945 950 955 960
Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg
965 970 975
Lys Lys Leu Phe Phe Lys Thr Ser Ala Ser
980 985
<210> 5
<211> 2955
<212> DNA
<213>artificial synthesized sequence
<400> 5
atgctgctgc tggtcacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc agcagcctga gcgccagcct gggcgacaga 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aaacccgacg gcaccgtgaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cccagcagat tttctggcag cggatctggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgctac ctacttctgt cagcagggca acaccctgcc ctacaccttc 360
ggcggaggca ccaagctgga aatcaccggc agcaccagcg gctccggcaa gcctggatct 420
ggcgagggca gcaccaaggg cgaagtgaag ctgcaggaaa gcggccctgg cctggtcgcc 480
cctagccaga gcctgtccgt gacctgtacc gtgtccggcg tgtccctgcc cgactacggc 540
gtgtcctgga tcagacagcc ccccagaaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacaa cctactacaa cagcgccctg aagtcccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga cagtctcttc tgcggccgca attgaagtta tgtatcctcc tccttaccta 840
gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 900
cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 960
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccctgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cgctagcgga 1140
ggtggaggtt ctggaggtgg tggaagtcaa agaaggaagt accgcagcaa caaaggagaa 1200
tctcccgtcg agccagccga gccctgtcat tattcatgcc caagggagga ggagggaagt 1260
acaatcccaa ttcaagaaga ctacaggaag cccgaacctg catgcagtcc aggtggaggc 1320
ggttctggag gcggtggctc ccgggtgaaa ttctcacggt ctgcagacgc acccgcttac 1380
cagcaaggcc agaaccaact ctataacgag ctcaatctag gacgaagaga ggagtacgat 1440
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1500
cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1560
attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1620
agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcact 1680
agtggctccg gagccacgaa cttctctctg ttaaagcaag caggagacgt ggaagaaaac 1740
cccggtccca tgggagtgca ggtggaaacc atctccccag gagacgggcg caccttcccc 1800
aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaagtg 1860
gactcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc 1920
cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata 1980
tctccagatt atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact 2040
ctcgtcttcg atgtggagct tctaaaactg gaaggtggag gcggttcagg cggcggcggc 2100
agcggcgcca tggtcggtgc tcttgagagt ttgaggggaa atgcagattt ggcttacatc 2160
ctgagcatgg agccctgtgg ccactgcctc attatcaaca atgtgaactt ctgccgtgag 2220
tccgggctcc gcacccgcac tggctccaac atcgactgtg agaagttgcg gcgtcgcttc 2280
tcctcgctgc atttcatggt ggaggtgaag ggcgacctga ctgccaagaa aatggtgctg 2340
gctttgctgg agctggcgcg gcaggaccac ggtgctctgg actgctgcgt ggtggtcatt 2400
ctctctcacg gctgtcaggc cagccacctg cagttcccag gggctgtcta cggcacagat 2460
ggatgccctg tgtcggtcga gaagattgtg aacatcttca atgggaccag ctgccccagc 2520
ctgggaggga agcccaagct ctttttcatc caggcctgtg gtggggagca gaaagaccat 2580
gggtttgagg tggcctccac ttcccctgaa gacgagtccc ctggcagtaa ccccgagcca 2640
gatgccaccc cgttccagga aggtttgagg accttcgacc agctggacgc catatctagt 2700
ttgcccacac ccagtgacat ctttgtgtcc tactctactt tcccaggttt tgtttcctgg 2760
agggacccca agagtggctc ctggtacgtt gagaccctgg acgacatctt tgagcagtgg 2820
gctcactctg aagacctgca gtccctcctg cttagggtcg ctaatgctgt ttcggtgaaa 2880
gggatttata aacagatgcc tggttgcttt aatttcctcc ggaaaaaact tttctttaaa 2940
acatcagcta gttaa 2955
<210> 6
<211> 986
<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 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser
20 25 30
Leu Ala Val Ser Leu Gly Asp Arg Ala Thr Ile Asn Cys Arg Ala Ser
35 40 45
Glu Ser Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu
65 70 75 80
Glu Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr
100 105 110
Tyr Cys Leu Gln Ser Arg Ile Phe Pro Arg Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser
130 135 140
Ser Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln Ser Gly Ser
145 150 155 160
Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
165 170 175
Gly Tyr Thr Phe Thr Asp Tyr Ser Ile Gln Trp Val Arg Gln Ala Pro
180 185 190
Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Gln Thr Glu Thr Arg Glu
195 200 205
Pro Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Val Phe Ser Leu Asp
210 215 220
Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu
225 230 235 240
Asp Thr Ala Val Tyr Tyr Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp
245 250 255
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Ile
260 265 270
Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly
275 280 285
Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe
290 295 300
Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val
305 310 315 320
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
325 330 335
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
340 345 350
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
355 360 365
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ala Ser Gly Gly Gly Gly
370 375 380
Ser Gly Gly Gly Gly Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu
385 390 395 400
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
405 410 415
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
420 425 430
Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
435 440 445
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
450 455 460
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
465 470 475 480
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
485 490 495
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
500 505 510
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
515 520 525
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
530 535 540
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
545 550 555 560
Arg Thr Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala
565 570 575
Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Val Gln Val Glu Thr
580 585 590
Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys
595 600 605
Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val Asp Ser
610 615 620
Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu
625 630 635 640
Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln
645 650 655
Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly
660 665 670
His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu
675 680 685
Leu Leu Lys Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
690 695 700
Ala Met Val Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala
705 710 715 720
Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn
725 730 735
Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn
740 745 750
Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met
755 760 765
Val Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu
770 775 780
Leu Glu Leu Ala Arg Gln Asp His Gly Ala Leu Asp Cys Cys Val Val
785 790 795 800
Val Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly
805 810 815
Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val
820 825 830
Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys
835 840 845
Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe
850 855 860
Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro
865 870 875 880
Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln
885 890 895
Leu Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser
900 905 910
Tyr Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly
915 920 925
Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His
930 935 940
Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser
945 950 955 960
Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg
965 970 975
Lys Lys Leu Phe Phe Lys Thr Ser Ala Ser
980 985
<210> 7
<211> 2961
<212> DNA
<213>artificial synthesized sequence
<400> 7
atgctgctgt tggtgacatc tttgttgttg tgcgaattgc ctcatcccgc tttcctgctg 60
attcccgaca tcgttctgac ccagtctcct gcatctctcg ccgtttctct cggtgaccgc 120
gctacaatca attgcagagc ctcagaaagc gtctccgtta tcggcgctca cctgattcat 180
tggtatcaac agaagccagg ccagccaccc aaactgctca tctacctggc atccaacctg 240
gaaactggtg tgccagccag gtttagcgga tcaggaagcg gtactgactt cactctgact 300
atctcctccc tgcaagccga agatgcagcc atctattact gcctccagag cagaatcttc 360
cctagaacct tcggccaagg tactaagctg gagattaaag gctccactag cggttccggc 420
aagcccggtt ccagcgaagg aagcactaag ggacaggtcc agttggtgca gagcggatct 480
gagctgaaga aacctggcgc ctcagtcaag gtttcatgta aggcatccgg atacactttc 540
acagattata gcatccagtg ggtccgccag gctcctggtc agggcttgga atggatgggt 600
tggattcaaa ccgaaacccg cgagcctgcc tatgcttacg acttccgcgg aagatttgtg 660
tttagcctcg acactagcgt gtctacagcc tacctccaaa tctcatccct gaaggctgaa 720
gacaccgcag tttactactg tgccctcgac tactcctatg ctatggatta ctggggtcaa 780
ggaactttgg ttactgtgtc tagcgcggcc gcaattgaag ttatgtatcc tcctccttac 840
ctagacaatg agaagagcaa tggaaccatt atccatgtga aagggaaaca cctttgtcca 900
agtcccctat ttcccggacc ttctaagccc ttttgggtgc tggtggtggt tgggggagtc 960
ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 1020
aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc tgggcccacc 1080
cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgctagc 1140
ggcggaggtg gctctggcgg tggcggatca gttgttaaac ggggcagaaa gaaactcctg 1200
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1260
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgggcggcgg cggcagcgga 1320
ggcggtggca gcggcggcgg cggctctaga gtgaagttca gcaggagcgc agacgccccc 1380
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1440
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1500
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1560
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1620
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1680
cgcactagtg gctccggagc cacgaacttc tctctgttaa agcaagcagg agacgtggaa 1740
gaaaaccccg gtcccatggg agtgcaggtg gaaaccatct ccccaggaga cgggcgcacc 1800
ttccccaagc gcggccagac ctgcgtggtg cactacaccg ggatgcttga agatggaaag 1860
aaagtggact cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag 1920
gtgatccgag gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg 1980
actatatctc cagattatgc ctatggtgcc actgggcacc caggcatcat cccaccacat 2040
gccactctcg tcttcgatgt ggagcttcta aaactggaag gtggaggcgg ttcaggcggc 2100
ggcggcagcg gcgccatggt cggtgctctt gagagtttga ggggaaatgc agatttggct 2160
tacatcctga gcatggagcc ctgtggccac tgcctcatta tcaacaatgt gaacttctgc 2220
cgtgagtccg ggctccgcac ccgcactggc tccaacatcg actgtgagaa gttgcggcgt 2280
cgcttctcct cgctgcattt catggtggag gtgaagggcg acctgactgc caagaaaatg 2340
gtgctggctt tgctggagct ggcgcggcag gaccacggtg ctctggactg ctgcgtggtg 2400
gtcattctct ctcacggctg tcaggccagc cacctgcagt tcccaggggc tgtctacggc 2460
acagatggat gccctgtgtc ggtcgagaag attgtgaaca tcttcaatgg gaccagctgc 2520
cccagcctgg gagggaagcc caagctcttt ttcatccagg cctgtggtgg ggagcagaaa 2580
gaccatgggt ttgaggtggc ctccacttcc cctgaagacg agtcccctgg cagtaacccc 2640
gagccagatg ccaccccgtt ccaggaaggt ttgaggacct tcgaccagct ggacgccata 2700
tctagtttgc ccacacccag tgacatcttt gtgtcctact ctactttccc aggttttgtt 2760
tcctggaggg accccaagag tggctcctgg tacgttgaga ccctggacga catctttgag 2820
cagtgggctc actctgaaga cctgcagtcc ctcctgctta gggtcgctaa tgctgtttcg 2880
gtgaaaggga tttataaaca gatgcctggt tgctttaatt tcctccggaa aaaacttttc 2940
tttaaaacat cagctagtta a 2961
<210> 8
<211> 22
<212> PRT
<213>artificial synthesized sequence
<400> 8
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
<210> 9
<211> 21
<212> PRT
<213>artificial synthesized sequence
<400> 9
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

Claims (10)

1. a kind of immunocyte of the dual Chimeric antigen receptor gene modification based on CD19 and BCMA, which is characterized in that described Dual Chimeric antigen receptor includes Chimeric antigen receptor CD19 and Chimeric antigen receptor BCMA.
2. immunocyte according to claim 1, which is characterized in that the Chimeric antigen receptor includes antigen binding structure Domain, transmembrane domain, costimulatory signal conducting region, CD3 ζ signal transduction structural domain and can induce suicide Fusion domain series connection and At；
Preferably, the antigen-binding domains are to resist for the single-chain antibody of tumor surface antigen CD19 and for tumor surface The single-chain antibody of former BCMA.
3. immunocyte according to claim 1 or 2, which is characterized in that the list for tumor surface antigen CD19 The amino acid sequence of chain antibody has any one in amino acid sequence shown in (I), (II) or (III):
(I) there is the amino acid sequence as shown in SEQ ID NO.1；
(II) with amino acid sequence shown in SEQ ID NO.1 with >=90%, preferably >=95%, more preferably >=98%, most preferably The amino acid sequence of >=99% homology；
(III) it modified with amino acid sequence shown in SEQ ID NO.1, replace, miss or add one or several amino acid The amino acid sequence of acquisition；
The amino acid sequence has the activity of the single-chain antibody for tumor surface antigen CD19；
Preferably, the amino acid sequence of the single-chain antibody for tumor surface antigen BCMA has (I), (II) or (III) Shown in any one in amino acid sequence:
(I) there is the amino acid sequence as shown in SEQ ID NO.2；
(II) with amino acid sequence shown in SEQ ID NO.2 with >=90%, preferably >=95%, more preferably >=98%, most preferably The amino acid sequence of >=99% homology；
(III) it modified with amino acid sequence shown in SEQ ID NO.2, replace, miss or add one or several amino acid The amino acid sequence of acquisition；
The amino acid sequence has the activity of the single-chain antibody for tumor surface antigen BCMA.
4. immunocyte according to any one of claim 1-3, which is characterized in that the transmembrane domain be CD28 across Spanning domain and/or CD8 α transmembrane domain；
Preferably, the costimulatory signal conducting region is CD28 signal transduction structural domain, CD27 signal transduction structural domain or CD137 In signal transduction structural domain any one or at least two combination；
Preferably, the inducible suicide Fusion domain is to include 9 structural domain of Caspase；
Preferably, the amino acid sequence of 9 structural domain of Caspase is as shown in SEQ ID NO.3；
Preferably, the inducible suicide Fusion domain is connected in series by 2A sequence and CD3 ζ signal transduction structural domain.
5. immunocyte described in any one of -4 according to claim 1, which is characterized in that the Chimeric antigen receptor includes letter Number peptide, antigen-binding domains, transmembrane domain, costimulatory signal conducting region, CD3 ζ signal transduction structural domain, 2A sequence and can Induction suicide Fusion domain is connected in series；
Preferably, the Chimeric antigen receptor is Secretory signal peptide, CD19 antigen-binding domains and/or BCMA antigen Binding structural domain, CD8 α and/or CD28 transmembrane domain, CD28 signal transduction structural domain, CD27 signal transduction structural domain, CD3 ζ Signal transduction structural domain, 2A sequence and 9 structural domain of Caspase are connected in series；
Preferably, the Chimeric antigen receptor CD19 is Secretory-CD19 scFv-CD28-CD27-CD3 ζ -2A- FBKP.Casp9；The Chimeric antigen receptor BCMA is Secretory-BCMA scFv-CD28-CD27-CD3 ζ -2A- FBKP.Casp9；
Preferably, the amino acid sequence of the Chimeric antigen receptor CD19 as shown in SEQ ID NO.4 or with its have 90% with The amino acid sequence of upper homology；
Preferably, the amino acid sequence of the Chimeric antigen receptor BCMA as shown in SEQ ID NO.6 or with its have 90% with The amino acid sequence of upper homology.
6. immunocyte according to any one of claims 1-5, which is characterized in that the dual Chimeric antigen receptor is logical The nucleic acid sequence for crossing its coding, which is transfected into T cell, to be expressed；
Preferably, the mode of the transfection be by viral vectors, eukaryon expression plasmid or mRNA sequence any one or At least two combination is transfected into T cell, is transfected into T cell preferably by viral vectors；
Preferably, the viral vectors be in slow virus carrier or retroviral vector any one or at least two group It closes, preferably slow virus carrier.
7. a kind of recombinant slow virus, which is characterized in that will be comprising such as dual chimeric antigen of any of claims 1-6 The recombinant lentiviral disease that the immunocyte and packaging helper plasmid pNHP and pHEF-VSVG cotransfection mammalian cell of receptor modification obtain Poison；
Preferably, the mammalian cell is 293 cells, any one in 293T cell or TE671 cell or at least two Combination.
8. a kind of pharmaceutical composition, which is characterized in that the composition includes as of any of claims 1-6 dual The immunocyte and/or recombinant slow virus as claimed in claim 7 of Chimeric antigen receptor modification.
9. such as the immunocyte of dual Chimeric antigen receptor modification of any of claims 1-6, such as claim 7 institute The recombinant slow virus or pharmaceutical composition as claimed in claim 8 stated are thin in preparation Chimeric antigen receptor T cell, immunocompetence Application in born of the same parents or tumor therapeutic agent.
10. application according to claim 9, which is characterized in that the tumour is the relevant tumor disease of blood；
Preferably, the relevant tumor disease of the blood is leukaemia or lymthoma.
CN201811458396.9A 2018-11-30 2018-11-30 A kind of immunocyte of the dual Chimeric antigen receptor gene modification based on CD19 and BCMA and its application Pending CN109468283A (en)

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