CN110951689A - CD19 and CD30 based double chimeric antigen receptor gene modified immune cell and application thereof - Google Patents

CD19 and CD30 based double chimeric antigen receptor gene modified immune cell and application thereof Download PDF

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CN110951689A
CN110951689A CN201811458441.0A CN201811458441A CN110951689A CN 110951689 A CN110951689 A CN 110951689A CN 201811458441 A CN201811458441 A CN 201811458441A CN 110951689 A CN110951689 A CN 110951689A
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chimeric antigen
antigen receptor
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李昱琛
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Beijing Meikang Geno Immune Biotechnology Co ltd
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Beijing Meikang Geno Immune Biotechnology Co ltd
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Priority to PCT/CN2019/122160 priority patent/WO2020108642A1/en
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Abstract

The invention relates to a CD19 and CD 30-based double chimeric antigen receptor gene modified immune cell and application thereof, wherein the double chimeric antigen receptor comprises a chimeric antigen receptor CD19 and a chimeric antigen receptor CD 30. The chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain, in tandem with an inducible suicide fusion domain. The chimeric antigen receptor can specifically recognize tumor surface antigens CD19 and CD30, and compared with other single chimeric antigen receptor T cells, two antigens are combined to target CAR-T cells to negative relapse of CD19, and B cell tumors with low expression of CD19 have better treatment effect and are easier to relieve diseases.

Description

CD19 and CD30 based double chimeric antigen receptor gene modified immune cell and application thereof
Technical Field
The invention relates to the field of cell immunotherapy of tumors, in particular to an immune cell based on gene modification of a dual chimeric antigen receptor of CD19 and CD30 and application thereof, and specifically relates to a construction method of a chimeric antigen receptor T (CAR-T) cell technology based on tumor specific targets of CD19 and CD30 and application thereof in anti-tumor therapy.
Background
With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) is one of the most promising tumor immunotherapy. Generally, a chimeric antigen receptor CAR consists of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Typically, the CAR comprises a Single chain fragment variable (scFv) region of an antibody or a binding domain specific for a Tumor Associated Antigen (TAA), which is coupled to the cytoplasmic domain of a T cell signaling molecule by a hinge and a transmembrane region. The most common lymphocyte activation moiety comprises a T cell costimulatory domain in tandem with a T cell effector function triggering (e.g., CD3 ζ) moiety. CAR-mediated adoptive immunotherapy allows CAR-transplanted T cells to directly recognize TAAs on target tumor cells in a non-HLA-restricted manner.
Most patients with B-cell malignancies, including B-cell acute lymphocytic leukemia (leukamia, B-ALL) and Chronic Lymphocytic Leukemia (CLL), will die as a result of their disease one method of treating these patients is to genetically modify T cells to target antigens expressed on tumor cells through the expression of CARs.
The CD19 molecule is a potential target for treatment of B lymphocyte lineage tumors and is also a hotspot in CAR research, and expression of CD19 is restricted to normal and malignant B cells and is a widely accepted CAR target for safety testing. Chimeric antigen receptor gene-modified T cells targeting the CD19 molecule (CD19CAR-T) have had great success in treating multiple, refractory acute B-lymphocyte leukemia, but have limited efficacy in the treatment of refractory, relapsed chronic B-lymphocyte leukemia and B-lymphocyte lineage lymphoma.
CN 104788573A discloses a chimeric antigen receptor hCD19scFv-CD8 α -CD28-CD3 zeta and the application thereof, the second generation chimeric antigen receptor is composed of anti-human CD19 monoclonal antibody HI19a light chain and heavy chain variable region (hCD19scFv), human CD8 α hinge region, human CD28 transmembrane region and intracellular region, and human CD3 zeta intracellular region which are connected in series, after the CD19 in the patent is subjected to CAR-T cell transfusion once, the expression level of CD19 is reduced, the immune mechanism is easy to escape, and the immune factor of the second generation CART is large in storm and has safety concern.
Therefore, it is important that the combination of another potential chimeric antigen receptor can solve the problems of easy mutation and low expression of CD19 aiming at CD19 negative recurrence and B cell tumor with low expression of CD 19. In addition to CD19, CD30 is also a potential target for the treatment of malignant B-cell tumors, and CD30 is mainly expressed in hodgkin's lymphoma and some non-hodgkin's lymphoma (including anaplastic large cell lymphoma, ALCL, mediastinal large B-cell lymphoma, PMBCL). CD30 is rarely expressed in normal tissues and is an ideal therapeutic target for the diagnosis and prognosis of a variety of lymphomas.
Therefore, it is important to find a chimeric antigen receptor which has strong specificity and high targeting property and can effectively improve the CART treatment effect.
Disclosure of Invention
Aiming at the situations that the single-targeting long-term effect in the current CAR-T technology for treating tumors is not ideal and the tumor microenvironment influences the CAR-T technology treatment effect, the invention provides a CD19 and CD 30-based double chimeric antigen receptor gene modified immune cell and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a CD19 and CD 30-based dual chimeric antigen receptor genetically modified immune cell, the dual chimeric antigen receptor comprising a chimeric antigen receptor CD19 and a chimeric antigen receptor CD 30.
According to the invention, the antigen binding domain is combined with tumor surface antigens CD19 and CD30, and then T cells are modified by a lentiviral vector, so that the tumor surface antigens CD19 and CD30 can be specifically combined on the chimeric antigen receptor of the invention, and the CAR-T cells can simultaneously eliminate tumor cells of CD19 and CD30, thereby effectively avoiding escape caused by reduced expression of the tumor cell antigens and simultaneously enhancing the long-term immune effect of the CAR-T cells.
In the present invention, the dual chimeric antigen receptor may be a CD19 chimeric antigen receptor alone and a CD30 chimeric antigen receptor alone, or may be a CD19 chimeric antigen receptor and a CD30 chimeric antigen receptor jointly expressed as a dual chimeric antigen receptor, that is, the antigen binding domain is capable of binding to the tumor surface antigens CD19 and CD30, and both cases can realize the combined therapy of two chimeric antigen receptors.
According to the present invention, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, and an inducible suicide fusion domain in tandem.
Preferably, the antigen binding domains are a single chain antibody directed to the tumor surface antigen CD19 and a single chain antibody directed to the tumor surface antigen CD 30.
According to the present invention, the genetically modified T-cell chimeric receptor (CAR) and single chain antibody (scFv) against CD19 and CD30 antigen binding domains are exemplified as follows.
In a specific embodiment, the amino acid sequence of the single chain antibody against the tumor surface antigen CD19 has any one of the amino acid sequences shown in (I), (II) or (III):
(I) has an amino acid sequence shown as SEQ ID NO. 1;
(II) an amino acid sequence having a homology of 90% or more, preferably 95% or more, more preferably 98% or more, most preferably 99% or more with the amino acid sequence shown in SEQ ID NO. 1;
(III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown in SEQ ID NO. 1;
the amino acid sequence has the activity of a single-chain antibody against the tumor surface antigen CD 19;
the amino acid sequence of a single chain antibody against the tumor surface antigen CD19 (SEQ ID NO.1) is as follows: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS are provided.
According to the invention, the amino acid sequence with more than 90% of homology or the amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids can be replaced by other single-chain antibodies or humanized CD19 single-chain antibodies, and the amino acid mutants thereof still have the functions of the CD19 single-chain antibody.
In a specific embodiment, the amino acid sequence of the single chain antibody against the tumor surface antigen CD30 has any one of the amino acid sequences shown in (I), (II) or (III):
(I) has an amino acid sequence shown as SEQ ID NO. 2;
(II) an amino acid sequence having a homology of not less than 90%, preferably not less than 95%, more preferably not less than 98%, most preferably not less than 99% with the amino acid sequence represented by SEQ ID NO. 2;
(III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown in SEQ ID NO. 2;
the amino acid sequence has the activity of a single-chain antibody against the tumor surface antigen CD 30;
the amino acid sequence of the single-chain antibody against the tumor surface antigen CD30 (SEQ ID NO.2) is as follows:
QIQLVQSGAEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGQGTKVEIK.
according to the invention, the amino acid sequence with more than 90% of homology or the amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids can be replaced by other single-chain antibodies or humanized CD30 single-chain antibodies, and the amino acid mutants thereof still have the functions of the CD30 single-chain antibody.
According to the invention, the chimeric antigen receptor is used for carrying out gene modification on T cells through a lentiviral vector, and the CD19 and CD30 double CAR-T cells are combined with tumor surface antigens CD19 and CD30, so that the tumor killing effect is stronger.
According to the present invention, the transmembrane domain is a CD28 transmembrane domain and/or a CD8 α transmembrane domain, which may be selected or modified by amino acid substitutions in some embodiments.
According to the present invention, the co-stimulatory signaling region is any one or combination of at least two of CD28 signaling domain, CD27 signaling domain or CD137 signaling domain, the arrangement of the CD28 signaling domain, CD27 signaling domain and CD137 signaling domain can be adjusted as required by those skilled in the art, the different arrangements of CD28 signaling domain and CD27 signaling domain and CD137 signaling domain do not affect the chimeric antigen receptor, and the sequential combination of CD28-CD27 is adopted in the present application.
According to the invention, the fourth generation CAR has an inducible suicide fusion domain and comprises a caspase 9 domain, the amino acid sequence of the fourth generation CAR is shown as SEQ ID NO.3, and the amino acid sequence shown as SEQ ID NO.3 is as follows:
GSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.
according to the invention, the inducible suicide fusion domain is concatenated with the CD3 zeta signaling domain via a 2A sequence, the 2A sequence cleaving the protein expressed by the inducible suicide fusion domain from the chimeric antigen receptor protein, thereby enabling the chimeric antigen receptor to function, and by injecting an activator, thereby enabling activation of the inducible suicide fusion domain, thereby causing death and loss of function of the T cell expressing the chimeric antigen receptor.
According to the present invention, the chimeric antigen receptor further comprises a signal peptide, wherein the signal peptide is a signal peptide capable of directing transmembrane transfer of the chimeric antigen receptor, and a person skilled in the art can select a signal peptide conventional in the art according to needs, wherein the signal peptide is a secretor signal peptide, the secretor signal peptide is a signal peptide of GMCSFR gene, and the amino acid sequence of the signal peptide is shown as the following SEQ ID No. 8: MLLLVTSLLLCELPHPAFLLIP are provided.
Preferably, the secretor signal peptide is a signal peptide of the CD8a gene, and the amino acid sequence of the secretor signal peptide is shown as SEQ ID No.9 as follows: MALPVTALLLPLALLLHAARP are provided.
The chimeric antigen receptor of the present invention may further include a hinge region, which may be selected by those skilled in the art according to practical circumstances, and is not particularly limited herein, and the presence of the hinge region does not affect the performance of the chimeric antigen receptor of the present invention.
According to the present invention, the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, a 2A sequence, and an inducible suicide fusion domain in tandem.
Preferably, the chimeric antigen receptor is a secretor signal peptide, a CD19 antigen binding domain and/or a CD30 antigen binding domain, a CD8 α and/or a CD28 transmembrane domain, a CD28 extracellular signaling domain, a CD28 intracellular signaling domain, a CD27 intracellular signaling domain, a CD3 ζ intracellular signaling domain, a 2A sequence and a fbkp. casp9 domain which are connected in series, and specifically arranged as follows:
Secretory-CD19scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9；
Secretory-CD30scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9。
in a specific embodiment, the amino acid sequence of the chimeric antigen receptor Secretory-CD19scFv-CD28-CD27-CD3 ζ -2A-FBKP.Casp9 is shown in SEQ ID No.4 or an amino acid sequence having more than 90% homology thereto, and the amino acid sequence shown in SEQ ID No.4 is as follows:
MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADAFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS；
in a specific embodiment, the nucleotide sequence of the chimeric antigen receptor Secretory-CD19scFv-CD28-CD27-CD3 ζ -2A-FBKP.Casp9 is shown as SEQ ID No.5 or a nucleotide sequence having more than 95% homology thereto, and the nucleic acid sequence shown as SEQ ID No.5 is as follows:
ATGCTGCTGCTGGTCACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGATCTGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCAGCACCAGCGGCTCCGGCAAGCCTGGATCTGGCGAGGGCAGCACCAAGGGCGAAGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGAGCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTCTCTTCTGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGCTAGCGGAGGTGGAGGTTCTGGAGGTGGTGGAAGTCAAAGAAGGAAGTACCGCAGCAACAAAGGAGAATCTCCCGTCGAGCCAGCCGAGCCCTGTCATTATTCATGCCCAAGGGAGGAGGAGGGAAGTACAATCCCAATTCAAGAAGACTACAGGAAGCCCGAACCTGCATGCAGTCCAGGTGGAGGCGGTTCTGGAGGCGGTGGCTCCCGGGTGAAATTCTCACGGTCTGCAGACGCACCCGCTTACCAGCAAGGCCAGAACCAACTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCACTAGTGGCTCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTGGACTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGTGGAGGCGGTTCAGGCGGCGGCGGCAGCGGCGCCATGGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCGGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTAGTTAA.
in a specific embodiment, the amino acid sequence of the chimeric antigen receptor Secretory-CD30scFv-CD28-CD27-CD3 ζ -2A-FBKP.Casp9 is shown in SEQ ID No.6 or an amino acid sequence having more than 90% homology thereto, and the amino acid sequence shown in SEQ ID No.6 is as follows:
MLLLVTSLLLCELPHPAFLLIPQIQLVQSGAEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGQGTKVEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS；
in a specific embodiment, the nucleotide sequence of the chimeric antigen receptor Secretory-CD30scFv-CD28-CD27-CD3 ζ -2A-FBKP.Casp9 is shown as SEQ ID No.7 or a nucleotide sequence having 95% or more homology thereto, and the nucleic acid sequence shown as SEQ ID No.7 is as follows:
ATGCTGCTCCTGGTTACTTCTCTCCTGCTCTGCGAACTGCCTCACCCTGCCTTTCTGCTCATCCCTCAGATCCAGCTCGTGCAGTCAGGAGCCGAAGTGAAGAAGCCAGGAGCCTCTGTCAAGGTGTCTTGCAAGGCCTCAGGCTACACTTTCACAGACTACTATATCACATGGGTCAGGCAGGCACCAGGCCAAGGGCTGGAGTGGATGGGCTGGATCTATCCTGGAAGTGGAAATACAAAGTACAATGAGAAATTCAAAGGAAGGGTGACTATGACCAGAGACACATCCATTTCAACTGCCTACATGGAACTCTCACGCCTGCGCAGTGATGATACCGCTGTGTATTACTGCGCAAACTATGGCAACTATTGGTTTGCCTACTGGGGGCAGGGCACCCTGGTCACTGTTTCTAGCGGTTCAACCTCCGGAAGCGGTAAACCTGGCTCCAGCGAGGGAAGCACTAAAGGCGACATTGTGATGACCCAAAGCCCTGATAGCCTGGCAGTCTCTCTGGGAGAGAGAGCCACCATTAACTGCAAGGCTTCTCAGTCCGTTGACTTTGATGGAGATTCATACATGAATTGGTATCAGCAGAAGCCTGGGCAACCACCAAAGCTGCTGATCTATGCTGCTTCAAACCTGGAGTCAGGTGTCCCAGACAGGTTCAGCGGTTCTGGATCAGGAACCGATTTCACACTCACTATCAGCTCCCTCCAAGCCGAGGATGTCGCTGTGTATTACTGTCAGCAGAGTAATGAAGATCCCTGGACCTTTGGTCAAGGAACCAAGGTGGAGATCAAAGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGCTAGCGGAGGTGGAGGTTCTGGAGGTGGTGGAAGTCAAAGAAGGAAGTACCGCAGCAACAAAGGAGAATCTCCCGTCGAGCCAGCCGAGCCCTGTCATTATTCATGCCCAAGGGAGGAGGAGGGAAGTACAATCCCAATTCAAGAAGACTACAGGAAGCCCGAACCTGCATGCAGTCCAGGTGGAGGCGGTTCTGGAGGCGGTGGCTCCCGGGTGAAATTCTCACGGTCTGCAGACGCACCCGCTTACCAGCAAGGCCAGAACCAACTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCACTAGTGGCTCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTGGACTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGTGGAGGCGGTTCAGGCGGCGGCGGCAGCGGCGCCATGGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCGGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTAGTTAA.
in the invention, the chimeric antigen receptor also comprises a promoter, wherein the promoter is any one or the combination of at least two of EF1a, CMV-TAR or CMV.
According to the invention, the chimeric antigen receptor is expressed by transfection of its encoded nucleic acid sequence into a T cell.
According to the invention, the transfection is by transfection into T cells by any one of or a combination of at least two of viral vectors, eukaryotic expression plasmids or mRNA sequences, preferably by viral vectors.
Preferably, the viral vector is any one of a lentiviral vector or a retroviral vector or a combination of at least two, preferably a lentiviral vector.
In a second aspect, the present invention provides a recombinant lentivirus obtained by co-transfecting an immune cell comprising the double chimeric antigen receptor gene modification according to the first aspect with a packaging helper plasmid pNHP and pHEF-VSVG into a mammalian cell.
In the invention, the recombinant lentivirus can effectively immunize cells, including T cells, and can prepare target T cells.
According to the present invention, the mammalian cell is any one of 293 cell, 293T cell or TE671 cell or a combination of at least two thereof.
In a third aspect, the present invention provides a pharmaceutical composition comprising an immune cell genetically modified with a dual chimeric antigen receptor according to the first aspect and/or a recombinant lentivirus according to the second aspect.
In a fourth aspect, the present invention provides the use of the dual chimeric antigen receptor genetically modified immune cell of the first aspect, the recombinant lentivirus of the second aspect, or the pharmaceutical composition of the third aspect, for the preparation of a chimeric antigen receptor T cell, an immunocompetent cell, or a medicament for the treatment of a tumor.
In the invention, the antigen receptor T cell has good targeting effect, can release low-dose immune factors and has low toxicity reaction property.
Preferably, the tumor is a blood-related tumor disease selected from, but not limited to, leukemia and/or a solid tumor.
Compared with the prior art, the invention has the following beneficial effects:
(1) the dual chimeric antigen receptor of the invention is characterized in that the T cell chimeric receptor gene is specifically modified, and the dual CAR-T cell based on CD19 and CD30 is combined with tumor surface antigens CD19 and CD30, so that the tumor killing effect is stronger, and the tumor reduction effect is more obvious;
(2) the dual chimeric antigen receptor can specifically recognize tumor surface antigens CD19 and CD30, CD19 and CD30 have high expression in leukemia and lymphoma, compared with other chimeric antigen receptors and other tumor antigens, the dual chimeric antigen receptor has safer and more obvious effects, two CAR-T cells are combined to be used for CD19 negative relapse, and B cell tumors with low expression of CD19 enable the treatment effect to be better and enable the disease to be more easily relieved.
Drawings
FIG. 1 is a synthetic gene sequence map of the chimeric antigen receptors of the invention, CD19 and CD 30;
FIG. 2 is a schematic representation of the combined use of the chimeric antigen receptors of the invention, CD19 and CD 30;
FIG. 3 is a flow chart of the clinical experiments of chimeric antigen receptor T cells of CD19 and CD 30;
FIG. 4 shows the expression intensity and ratio of CD19 and CD30 chimeric antigen receptors in tumor tissues of lymphoma patients by IHC staining;
FIG. 5 is a comparison of imaging before and after reinfusion of an example of a PMBCL patient treated with a combination of clinical CD19 and CD30 chimeric antigen receptor T cells;
FIG. 6 shows the clinical combination of CD19 and CD30 chimeric antigen receptor T cells in patient 1 to treat B cell malignancies after reinfusion of the patient with the CAR gene copy number;
FIG. 7 shows the CAR gene copy number measured in patient 2 after reinfusion of patients treated with B cell malignancies with the combined clinical use of CD19 and CD30 chimeric antigen receptor T cells.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
EXAMPLE 1 construction of chimeric antigen receptors
(1) Secretory signal peptide, CD19 or CD30 antigen binding domain, CD8 α and/or CD28 transmembrane domain, CD28 signaling domain, CD27 signaling domain, CD3 zeta signaling domain, 2A sequence and caspase 9 domain are synthesized by whole gene, as shown in FIG. 1, namely Secretory-CD19scFv-CD28-CD27-CD3 zeta-2A-FBKP. Casp9 and Secretory-CD30scFv-CD28-CD27-CD 3-2A-FBKP. Casp9, and the specific sequences are as follows:
the nucleotide sequence of Secretory-CD19scFv-CD28-CD27-CD3 zeta-2A-FBKP.Casp9 is shown in SEQ ID NO. 5.
The nucleotide sequence of Secretory-CD30scFv-CD28-CD27-CD3 zeta-2A-FBKP.Casp9 is shown in SEQ ID NO. 7.
Example 2 Lentiviral packaging
(1) 293T cells are used for culturing for 17-18 hours;
(2) adding fresh DMEM containing 10% FBS;
(3) the following reagents were added to sterile centrifuge tubes: DMEM was added to the helper DNA mix (pNHP, pHEF-VSV-G) and pTYF DNA vector per well and vortexed;
(4) adding Superfect or any transgenic material into a centrifuge tube, and standing for 7-10 minutes at room temperature;
(5) dropwise adding the DNA-Superfect mixed solution in the centrifugal tube into each cultured cell, and uniformly swirling;
(6)37℃3％CO2culturing in an incubator for 4-5 hours;
(7) sucking away the culture solution of the culture medium, flushing the culture medium by using 293 cell culture solution, and adding the culture solution for continuous culture;
(8) the medium was returned to 3% CO2The cells were incubated overnight in an incubator and the next morning the transfection efficiency was observed using a fluorescence microscope.
Example 3 purification and concentration of lentivirus
1) Virus purification
Removing cell debris by centrifugation at 1000g for 5 minutes to obtain viral supernatant, filtering the viral supernatant with a 0.45 micron low protein binding filter, portioning the virus and storing at-80 deg.C;
generally, transfected cells can produce 10 cells per ml of medium6To 107Transduction unit titrated lentiviral vectors.
2) Concentration of lentiviral vectors with Centricon or similar filters
(1) Adding virus supernatant to a Centricon or similar filter tube, and then centrifuging at 2500g for 30 minutes;
(2) the filter tube was shaken and then centrifuged at 400g for 2 minutes and the concentrated virus was collected in a collection cup. Finally, the viruses in all tubes are collected in a centrifuge tube.
Example 4 transfection of CAR-T cells
Inoculating the activated T cells into a culture dish, adding lentivirus of a concentrated target gene, centrifuging for 100 minutes at the speed of 100g of centrifugal force, culturing at 37 ℃ for 24 hours, adding AIM-V culture medium with cell culture factors, culturing for 2-3 days, and harvesting and counting the cells to obtain the usable CD19CAR-T cells and CD30CAR-T cells.
Example 5 clinical application of CD19 and CD30CAR-T cells
As can be seen from the combined schematic diagram of FIG. 2, the tumor can be treated by combining CD19 with CD30, which shows that the dual chimeric antigen receptor selected by the invention can play a significant role in tumor treatment, especially for the tumor with weak CD19 expression, and effectively prevent CD19 from escaping.
FIG. 3 is a flow chart of clinical trials, including the following steps:
(1) patients are first evaluated in hospitals and laboratories and then autologous or donor leukocytes are collected after the conditions of group entry are met. Following standard protocols, CD3 positive T cells were selected from PBMC, activated and transfected with 4SCAR19 and 4SCAR30 to generate CD19CAR-T cells and CD30CAR-T cells. Patients were pretreated with cyclophosphamide and fludarabine prior to infusion. The average number of cells infused was 1X 10 per kg body weight6A cell. The quality of leukocytes and CAR-T cells, gene transfection efficiency, CAR-T cell expansion and number of effective CAR-T cell infusions were evaluated and recorded.
(2) After the patient returned CAR-T cells, their immune factor storm and CAR copy number were closely monitored over a month. Long-term clinical and laboratory assessments can lead to conclusions about the toxicity of the treatment and the ultimate therapeutic effect. Toxicity assessments were in accordance with the National Cancer Institute's Common genetics Criteria for Adverse Events (National Cancer Institute Adverse event Common Terminology Criteria) (CTCAE v 4.03).
The specific clinical results are as follows:
FIG. 4 shows the intensity and proportion of CD19 and CD30 expression in tumor tissues of a lymphoma patient by IHC staining. In the figure, the expression intensity of CD19 in the tumor tissue of the patient is 2.5+, the expression area is about 60%, the expression intensity of CD30 is 1+, and the expression area is about 60%. The expression area of CD30 in lymphoma tissues is wide, and the CD30 is a good auxiliary target point.
Example 7 clinical application of CD19 and CD30CAR-T cells actual examples
Sample preparation: PMBCL patients, male 31 years old. The focus involves the thoracic and precordial lymph nodes.
FIG. 5 is a comparison of imaging before and after reinfusion of an example of a PMBCL patient treated with a combination of clinical CD19 and CD30 chimeric antigen receptor T cells. It can be seen that the density shadow of the flaky soft tissue in the front upper mediastinum of the patient before the reinfusion is shown, the size is about 6.3cm multiplied by 2.2cm, the strengthening scan is strengthened, the density shadow of the flaky soft tissue in the front upper mediastinum is shown 20 days after the reinfusion, the size of the maximum layer is about 4.7cm multiplied by 1.6cm, the strengthening scan is strengthened, and compared with the density shadow before the reinfusion, the focus is reduced.
Sample preparation: b cell malignancy, Male
FIGS. 6-7 show the copy number of the CAR gene detected in peripheral blood of patients after reinfusion, in two patients treated with B cell malignancies with the combined clinical use of CD19 and CD30 chimeric antigen receptor T cells. The copy numbers of both patient 1CD19 and CD30 peaked at day 14. The copy numbers of patient 2CD19 and CD30 peaked at day 7.
Clinical combined use of CD19 and CD30 chimeric antigen receptor T cells for treatment of 4 cases of B cell malignancies, with 1 complete remission and 2 partial remissions as shown in table 1:
TABLE 14 CD19+ CD30CART remission table for PMBCL patients
Figure BDA0001888176640000201
In conclusion, the dual chimeric antigen receptor of the invention can specifically recognize the tumor surface antigens CD19 and CD30, and after one CAR-T cell transfusion, the expression level of CD19 is reduced and the antigen easily escapes from immune mechanisms in CD 19. The combined use of two CAR-T cells against CD19 negative relapse, and CD19 underexpressed B cell tumors made treatment more effective and more susceptible to disease remission.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Beijing Meikang Ji exempt Biotech Co., Ltd
<120> CD19 and CD30 based double chimeric antigen receptor gene modified immune cell and application thereof
<130>2018
<160>9
<170>PatentIn version 3.3
<210>1
<211>245
<212>PRT
<213> artificially 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
115120 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>246
<212>PRT
<213> artificially synthesized sequence
<400>2
Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
15 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser
115 120 125
Ser Glu Gly Ser Thr Lys Gly Asp Ile Val Met Thr Gln Ser Pro Asp
130 135 140
Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala
145 150 155 160
Ser Gln Ser Val Asp Phe Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln
165170 175
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn
180 185 190
Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
210 215 220
Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Trp Thr Phe Gly Gln Gly
225 230 235 240
Thr Lys Val Glu Ile Lys
245
<210>3
<211>423
<212>PRT
<213> artificially 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
5055 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
210215 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 375380
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> artificially 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
8590 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
245250 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
405410 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 570575
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 730735
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> artificially 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>985
<212>PRT
<213> artificially 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 Gln Ile 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 Asp Tyr Tyr Ile Thr Trp Val Arg Gln Ala Pro Gly
50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr
65 70 75 80
Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr
85 90 95
Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly
130 135 140
Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser Thr Lys Gly Asp Ile Val
145 150 155 160
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala
165 170 175
Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Phe Asp Gly Asp Ser
180 185 190
Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
195 200 205
Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
225 230 235 240
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro
245 250 255
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 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 Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly
385 390 395 400
Glu Ser Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg
405 410 415
Glu Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro
420 425 430
Glu Pro Ala Cys Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
435 440 445
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
450 455 460
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
465 470 475 480
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
485 490 495
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
500 505 510
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
515 520 525
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
530 535 540
ThrLys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
545 550 555 560
Thr Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly
565 570 575
Asp Val Glu Glu Asn Pro Gly Pro Met Gly Val Gln Val Glu Thr Ile
580 585 590
Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val
595 600 605
Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val Asp Ser Ser
610 615 620
Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val
625 630 635 640
Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg
645 650 655
Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His
660 665 670
Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu
675 680 685
Leu Lys Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala
690 695 700
Met Val GlyAla Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr
705 710 715 720
Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val
725 730 735
Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile
740 745 750
Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val
755 760 765
Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu
770 775 780
Glu Leu Ala Arg Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val
785 790 795 800
Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala
805 810 815
Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn
820 825 830
Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu
835 840 845
Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu
850 855 860
Val Ala Ser Thr SerPro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu
865 870 875 880
Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu
885 890 895
Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr
900 905 910
Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser
915 920 925
Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser
930 935 940
Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val
945 950 955 960
Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys
965 970 975
Lys Leu Phe Phe Lys Thr Ser Ala Ser
980 985
<210>7
<211>2958
<212>DNA
<213> artificially synthesized sequence
<400>7
atgctgctcc tggttacttc tctcctgctc tgcgaactgc ctcaccctgc ctttctgctc 60
atccctcaga tccagctcgt gcagtcagga gccgaagtga agaagccagg agcctctgtc 120
aaggtgtctt gcaaggcctc aggctacact ttcacagact actatatcac atgggtcagg 180
caggcaccag gccaagggct ggagtggatg ggctggatct atcctggaag tggaaataca 240
aagtacaatg agaaattcaa aggaagggtg actatgacca gagacacatc catttcaact 300
gcctacatgg aactctcacg cctgcgcagt gatgataccg ctgtgtatta ctgcgcaaac 360
tatggcaact attggtttgc ctactggggg cagggcaccc tggtcactgt ttctagcggt 420
tcaacctccg gaagcggtaa acctggctcc agcgagggaa gcactaaagg cgacattgtg 480
atgacccaaa gccctgatag cctggcagtc tctctgggag agagagccac cattaactgc 540
aaggcttctc agtccgttga ctttgatgga gattcataca tgaattggta tcagcagaag 600
cctgggcaac caccaaagct gctgatctat gctgcttcaa acctggagtc aggtgtccca 660
gacaggttca gcggttctgg atcaggaacc gatttcacac tcactatcag ctccctccaa 720
gccgaggatg tcgctgtgta ttactgtcag cagagtaatg aagatccctg gacctttggt 780
caaggaacca aggtggagat caaagcggcc 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
ggaggtggag gttctggagg tggtggaagt caaagaagga agtaccgcag caacaaagga 1200
gaatctcccg tcgagccagc cgagccctgt cattattcat gcccaaggga ggaggaggga 1260
agtacaatcc caattcaaga agactacagg aagcccgaac ctgcatgcag tccaggtgga 1320
ggcggttctg gaggcggtgg ctcccgggtg aaattctcac ggtctgcaga cgcacccgct 1380
taccagcaag gccagaacca actctataac gagctcaatc taggacgaag agaggagtac 1440
gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1500
aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1560
gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1620
ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1680
actagtggct ccggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa 1740
aaccccggtc ccatgggagt gcaggtggaa accatctccc caggagacgg gcgcaccttc 1800
cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga tggaaagaaa 1860
gtggactcct cccgggacag aaacaagccc tttaagttta tgctaggcaa gcaggaggtg 1920
atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc caaactgact 1980
atatctccag attatgccta tggtgccact gggcacccag gcatcatccc accacatgcc 2040
actctcgtct tcgatgtgga gcttctaaaa ctggaaggtg gaggcggttc aggcggcggc 2100
ggcagcggcg ccatggtcgg tgctcttgag agtttgaggg gaaatgcaga tttggcttac 2160
atcctgagca tggagccctg tggccactgc ctcattatca acaatgtgaa cttctgccgt 2220
gagtccgggc tccgcacccg cactggctcc aacatcgact gtgagaagtt gcggcgtcgc 2280
ttctcctcgc tgcatttcat ggtggaggtg aagggcgacc tgactgccaa gaaaatggtg 2340
ctggctttgc tggagctggc gcggcaggac cacggtgctc tggactgctg cgtggtggtc 2400
attctctctc acggctgtca ggccagccac ctgcagttcc caggggctgt ctacggcaca 2460
gatggatgcc ctgtgtcggt cgagaagatt gtgaacatct tcaatgggac cagctgcccc 2520
agcctgggag ggaagcccaa gctctttttc atccaggcct gtggtgggga gcagaaagac 2580
catgggtttg aggtggcctc cacttcccct gaagacgagt cccctggcag taaccccgag 2640
ccagatgcca ccccgttcca ggaaggtttg aggaccttcg accagctgga cgccatatct 2700
agtttgccca cacccagtga catctttgtg tcctactcta ctttcccagg ttttgtttcc 2760
tggagggacc ccaagagtgg ctcctggtac gttgagaccc tggacgacat ctttgagcag 2820
tgggctcact ctgaagacct gcagtccctc ctgcttaggg tcgctaatgc tgtttcggtg 2880
aaagggattt ataaacagat gcctggttgc tttaatttcc tccggaaaaa acttttcttt 2940
aaaacatcag ctagttaa 2958
<210>8
<211>22
<212>PRT
<213> artificially 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> artificially 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 CD19 and CD 30-based dual chimeric antigen receptor genetically modified immune cell, wherein the dual chimeric antigen receptor comprises a chimeric antigen receptor CD19 and a chimeric antigen receptor CD 30.
2. The immune cell of claim 1, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, and an inducible suicide fusion domain in tandem;
preferably, the antigen binding domains are a single chain antibody directed to the tumor surface antigen CD19 and a single chain antibody directed to the tumor surface antigen CD 30.
3. The immune cell of claim 1 or 2, wherein the amino acid sequence of the single chain antibody against the tumor surface antigen CD19 has any one of the amino acid sequences shown in (I), (II), or (III):
(I) has an amino acid sequence shown as SEQ ID NO. 1;
(II) an amino acid sequence having a homology of 90% or more, preferably 95% or more, more preferably 98% or more, most preferably 99% or more with the amino acid sequence shown in SEQ ID NO. 1;
(III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown in SEQ ID NO. 1;
the amino acid sequence has the activity of a single-chain antibody against the tumor surface antigen CD 19;
preferably, the amino acid sequence of the single-chain antibody against the tumor surface antigen CD30 has any one of the amino acid sequences shown in (I), (II) or (III):
(I) has an amino acid sequence shown as SEQ ID NO. 2;
(II) an amino acid sequence having a homology of not less than 90%, preferably not less than 95%, more preferably not less than 98%, most preferably not less than 99% with the amino acid sequence represented by SEQ ID NO. 2;
(III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown in SEQ ID NO. 2;
the amino acid sequence has the activity of a single chain antibody against the tumor surface antigen CD 30.
4. The immune cell of any one of claims 1-3, wherein the transmembrane domain is a CD28 transmembrane domain and/or a CD8 α transmembrane domain;
preferably, the co-stimulatory signaling region is any one of, or a combination of at least two of, a CD28 signaling domain, a CD27 signaling domain, or a CD137 signaling domain;
preferably, the inducible suicide fusion domain is a caspase 9-containing domain;
preferably, the amino acid sequence of the caspase 9 domain is shown as SEQ ID NO. 3;
preferably, the inducible suicide fusion domain is in tandem with the CD3 zeta signaling domain via the 2A sequence.
5. The immune cell of any one of claims 1-4, wherein the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, a 2A sequence, and an inducible suicide fusion domain in tandem;
preferably, the chimeric antigen receptor is formed by connecting a secretor signal peptide, a CD19 antigen binding domain and/or a CD30 antigen binding domain, a CD8 α and/or a CD28 transmembrane domain, a CD28 signaling domain, a CD27 signaling domain, a CD3 zeta signaling domain, a 2A sequence and a caspase 9 domain in series;
preferably, the chimeric antigen receptor CD19 is secretor-CD 19scFv-CD28-CD27-CD3 ζ -2A-fbkp. casp 9; the chimeric antigen receptor CD30 is Secretory-CD30scFv-CD28-CD27-CD3 zeta-2A-FBKP.Casp9;
preferably, the amino acid sequence of the chimeric antigen receptor CD19 is shown in SEQ ID NO.4 or an amino acid sequence with more than 90% homology with the amino acid sequence;
preferably, the amino acid sequence of the chimeric antigen receptor CD30 is shown in SEQ ID NO.6 or an amino acid sequence with more than 90% homology with the same.
6. The immune cell of any one of claims 1-5, wherein the dual chimeric antigen receptor is expressed by transfection into a T cell of a nucleic acid sequence encoding the dual chimeric antigen receptor;
preferably, the transfection is by transfection into T cells by any one or a combination of at least two of a viral vector, a eukaryotic expression plasmid or an mRNA sequence, preferably by transfection into T cells by a viral vector;
preferably, the viral vector is any one of a lentiviral vector or a retroviral vector or a combination of at least two, preferably a lentiviral vector.
7. A recombinant lentivirus obtained by co-transfecting a mammalian cell with the packaging helper plasmids pNHP and pHEF-VSVG a recombinant lentivirus comprising the dual chimeric antigen receptor modified immune cell of any one of claims 1-6;
preferably, the mammalian cell is any one of 293 cell, 293T cell or TE671 cell or a combination of at least two thereof.
8. A pharmaceutical composition comprising the dual chimeric antigen receptor genetically modified immune cell of any one of claims 1-6 and/or the recombinant lentivirus of claim 7.
9. Use of the dual chimeric antigen receptor genetically modified immune cell of any one of claims 1 to 6, the recombinant lentivirus of claim 7 or the pharmaceutical composition of claim 8 for the preparation of a chimeric antigen receptor T cell, an immunocompetent cell or a medicament for the treatment of tumors.
10. The use of claim 9, wherein the tumor is a blood-related neoplastic disease;
preferably, the blood-related neoplastic disease is leukemia or lymphoma.
CN201811458441.0A 2018-11-30 2018-11-30 CD19 and CD30 based double chimeric antigen receptor gene modified immune cell and application thereof Pending CN110951689A (en)

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