CN109485733B - Fully human anti-BCMA chimeric antigen receptor and application thereof - Google Patents
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Abstract
The invention relates to a fully human anti-BCMA chimeric antigen receptor and application thereof, wherein the chimeric antigen receptor comprises an extracellular domain capable of combining with antigen, a transmembrane domain and at least one intracellular domain, wherein the extracellular domain is an anti-BCMA single domain antibody; wherein the fully human anti-BCMA antibody comprises a heavy chain variable region and a light chain variable region of specific sequences. The chimeric antigen receptor has lower immunogenicity, smaller rejection reaction and higher safety, can effectively reduce the solid tumor focus, and effectively improves the treatment effect of tumors.
Description
Technical Field
The invention relates to the field of cell immunotherapy of tumors, in particular to a fully human chimeric antigen receptor (BCMA) and application thereof, and specifically relates to a construction method of a fully human chimeric antigen receptor T (CAR-T) cell technology obtained by screening based on specific target BCMA 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) 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.
The vast majority of patients with B-cell malignancies and multiple myeloma are significant contributors to cancer death. The response of B cell malignancies to various forms of treatment is mixed. The traditional methods for treating B cell malignancies have toxic and side effects, but the immunotherapy using antibodies against CD19, CD20, CD22, CD23, CD52, etc. has general effects. As technology developed, treatment began using cells modified to express Chimeric Antigen Receptors (CARs), but the therapeutic effect of the established antigen binding domains used in CARs was unpredictable, rendering the therapeutic effect unstable.
The use of BCMA antigens as the extracellular domain for the treatment of B-cell related diseases is disclosed, but the antigen binding domain binds too strongly, and CAR T cells induce large-scale cytokine release, resulting in a potentially lethal immune response that is considered a "cytokine storm"; however, if the antigen binding domain binds too weakly, CAR T cells fail to exhibit sufficient therapeutic efficacy in clearing cancer cells. In the prior art, CN 106687483a discloses humanized anti-BCMA chimeric antigen receptor therapy for cancer by administering a method of expressing CAR genetically modified T cells to achieve treatment of diseases associated with expression of B cell maturation antigen protein (BCMA). CN 107207598A discloses BCMA chimeric antigen receptors for use in adoptive T cell therapy of B cell related conditions.
Therefore, the preparation of a fully human BCMA chimeric antigen receptor, which has the advantages of antibody therapy, selects a fully human extracellular domain, can reduce immunogenicity, enables therapeutic procedures to be spared, and necessarily provides a more effective therapeutic option for B cell-related diseases.
Disclosure of Invention
Aiming at the condition that the tumor microenvironment influences the treatment effect of the CAR-T technology in the treatment of tumors by the CAR-T technology at present, the invention provides the fully human anti-BCMA chimeric antigen receptor and the application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a fully human anti-BCMA chimeric antigen receptor comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and at least one intracellular domain, wherein said extracellular domain is a fully human anti-BCMA antibody;
wherein the fully human anti-BCMA antibody comprises:
a) a heavy chain variable region comprising the Complementarity Determining Regions (CDRs) of seq id no:
the amino acid sequence of CDR1 is shown in one of SEQ ID NO.1-4, and specifically as follows:
SEQ ID NO.1：GFTFTNYA；
SEQ ID NO.2：GFTFSSYA；
SEQ ID NO.3：GFTFDDYG；
SEQ ID NO.4：GYTFTANY；
the amino acid sequence of CDR2 is shown in one of SEQ ID NO.5-8, and specifically as follows:
SEQ ID NO.5：ISTGSDTI；
SEQ ID NO.6：ISGSGGST；
SEQ ID NO.7：IKPDGGDK；
SEQ ID NO.8：INPSSGAT；
the amino acid sequence of CDR3 is shown in one of SEQ ID NO.9-12, and specifically as follows:
SEQ ID NO.9：ARGSWVTATTHSENWFDP；
SEQ ID NO.10：ARDFAPAGPDY；
SEQ ID NO.11：ATHHYTSTDYFDYWG；
SEQ ID NO.12：ATSEYGSSSADY；
b) a light chain variable region comprising the Complementarity Determining Regions (CDRs) of the sequences:
the amino acid sequence of CDR1 is shown in one of SEQ ID NO.13-16, and specifically as follows:
SEQ ID NO.13：SLRSYY；
SEQ ID NO.14：NIGTKS；
SEQ ID NO.15：YIGSKS；
SEQ ID NO.16：NIGIRS；
the amino acid sequence of CDR2 is shown in one of SEQ ID NO.17-20, and specifically as follows:
SEQ ID NO.17：DDT；
SEQ ID NO.18：DDD；
SEQ ID NO.19：DDS；
SEQ ID NO.20：DDS；
the amino acid sequence of CDR3 is shown in one of SEQ ID NO.21-24, and specifically as follows:
SEQ ID NO.21：QVWDSINEQVVFGG；
SEQ ID NO.22：QGWDSTTDHVV；
SEQ ID NO.23：QVWDSINDQVV；
SEQ ID NO.24：QVWDSISEVVV.
in the invention, the inventor finds four fully human anti-BCMA single-domain antibodies by screening a fully human scFv phage antibody library, and can reduce immunogenicity, reduce rejection reaction and remarkably improve the treatment effect of the chimeric antigen receptor by combining the antibodies with the extracellular domain of the chimeric antigen receptor.
According to the invention, the amino acid sequence of the heavy chain variable region of the fully human anti-BCMA antibody comprises the amino acid sequence shown in one of SEQ ID NO.25-28, and specifically comprises the following steps:
SEQ ID NO.25：
QVQLQESGGGLVQPGGSLRLSCAASGFTFTNYAMNWVRQAPGKGLEWLSYISTGSDTIYYADSVKGRFTISRDNAKNSLYLQMSSLRDEDTAVYYCARGSWVTATTHSENWFDPWGQGTLVTVSS；
SEQ ID NO.26：
QVQLQESGGGLIEPGGSLRLPCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARDFAPAGPDYWGQGTLVTVSS；
SEQ ID NO.27：
QVQLVESGGSVVRPGGSLRLSCAASGFTFDDYGVSWVRQAPGKGLEGVANIKPDGGDKYYVDSVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCATHHYTSTDYFDYWGLGTLVTVSS；
SEQ ID NO.28：
QVQLQESGAEVKKPGSSVKVSCKASGYTFTANYMRWVRQARGQGLEWMGWINPSSGATNYAQKFQGRVTMTRDTSISTAYMELSSLTSGDTAVYYCATSEYGSSSADYWGQGTLVTVSS.
according to the invention, the amino acid sequence of the light chain variable region of the fully human anti-BCMA antibody comprises the amino acid sequence shown in one of SEQ ID NO.29-32, and specifically comprises the following steps:
SEQ ID NO.29：
NFMLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQERPGQAPVLVVHDDTDRPSGIPERFSGSNSGNTATLTIGRVEAGDEADYYCQVWDSINEQVVFGGGTKLTVLG；
SEQ ID NO.30：
YVLTQPPSVSVAPGQTATISCDGKNIGTKSVHWYQQKPGQAPVLVVYDDDDRPSGIPERFSGSNSGKTATLTISRVEAGDEADYYCQGWDSTTDHVVFGGGTKLTVLG；
SEQ ID NO.31：
NFMLTQPPSVSVAPGQTARITCGGNYIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYFCQVWDSINDQVVFGGGTKLTVLG；
SEQ ID NO.32：
NFMLTQPPSLSVAPGQTARITCGGDNIGIRSVHWYQQKPGQAPVLVVYDDSDRPSAIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSISEVVVFGGGTKLTVLG.
according to the invention, the amino acid sequence of the fully human anti-BCMA antibody comprises a sequence shown in one of SEQ ID NO.33-36, preferably an amino acid sequence shown in SEQ ID NO.34, and specifically comprises the following steps:
T3H-2(SEQ ID NO.33)：
QVQLQESGGGLVQPGGSLRLSCAASGFTFTNYAMNWVRQAPGKGLEWLSYISTGSDTIYYADSVKGRFTISRDNAKNSLYLQMSSLRDEDTAVYYCARGSWVTATTHSENWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQERPGQAPVLVVHDDTDRPSGIPERFSGSNSGNTATLTIGRVEAGDEADYYCQVWDSINEQVVFGGGTKLTVLG；
T3H-5(SEQ ID NO.34)：
QVQLQESGGGLIEPGGSLRLPCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARDFAPAGPDYWGQGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQTATISCDGKNIGTKSVHWYQQKPGQAPVLVVYDDDDRPSGIPERFSGSNSGKTATLTISRVEAGDEADYYCQGWDSTTDHVVFGGGTKLTVLG；
T3Q-14(SEQ ID NO.35)：
QVQLVESGGSVVRPGGSLRLSCAASGFTFDDYGVSWVRQAPGKGLEGVANIKPDGGDKYYVDSVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCATHHYTSTDYFDYWGLGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPPSVSVAPGQTARITCGGNYIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYFCQVWDSINDQVVFGGGTKLTVLG；
T4Q-14(SEQ ID NO.36)：
QVQLQESGAEVKKPGSSVKVSCKASGYTFTANYMRWVRQARGQGLEWMGWINPSSGATNYAQKFQGRVTMTRDTSISTAYMELSSLTSGDTAVYYCATSEYGSSSADYWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPPSLSVAPGQTARITCGGDNIGIRSVHWYQQKPGQAPVLVVYDDSDRPSAIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSISEVVVFGGGTKLTVLG；
note: GGGGSGGGGSGGS is GS linker; VH to VL.
According to the invention, the nucleotide sequence of the fully human anti-BCMA antibody comprises a sequence shown in one of SEQ ID NO.37-40, preferably a nucleotide sequence shown in SEQ ID NO.38, and specifically comprises the following steps:
T3H-2(SEQ ID NO.37)：
CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCACCAACTACGCCATGAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGCTGAGCTACATCAGCACCGGCAGCGACACCATCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAGCAGCCTGAGGGACGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGCTGGGTGACCGCCACCACCCACAGCGAGAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAACTTCATGCTGACCCAGGACCCCGCCGTGAGCGTGGCCCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACAGCCTGAGGAGCTACTACGCCAGCTGGTACCAGGAGAGGCCCGGCCAGGCCCCCGTGCTGGTGGTGCACGACGACACCGACAGGCCCAGCGGCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAACACCGCCACCCTGACCATCGGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTACTGCCAGGTGTGGGACAGCATCAACGAGCAGGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGC；
T3H-5(SEQ ID NO.38)：
CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGATCGAGCCCGGCGGCAGCCTGAGGCTGCCCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCGTGTACCTGCAGATGAACAGCCTGAGGGTGGAGGACACCGCCGTGTACTACTGCGCCAGGGACTTCGCCCCCGCCGGCCCCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGCTACGTGCTGACCCAGCCCCCCAGCGTGAGCGTGGCCCCCGGCCAGACCGCCACCATCAGCTGCGACGGCAAGAACATCGGCACCAAGAGCGTGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTGGTGTACGACGACGACGACAGGCCCAGCGGCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAAGACCGCCACCCTGACCATCAGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTACTGCCAGGGCTGGGACAGCACCACCGACCACGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGC；
T3Q-14(SEQ ID NO.39)：
CAGGTGCAGCTGGTGGAGAGCGGCGGCAGCGTGGTGAGGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGACGACTACGGCGTGAGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCAACATCAAGCCCGACGGCGGCGACAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGGACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCACCCACCACTACACCAGCACCGACTACTTCGACTACTGGGGCCTGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAACTTCATGCTGACCCAGCCCCCCAGCGTGAGCGTGGCCCCCGGCCAGACCGCCAGGATCACCTGCGGCGGCAACTACATCGGCAGCAAGAGCGTGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTGGTGTACGACGACAGCGACAGGCCCAGCGGCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAACACCGCCACCCTGACCATCAGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTTCTGCCAGGTGTGGGACAGCATCAACGACCAGGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGC；
T4Q-14(SEQ ID NO.40)：
CAGGTGCAGCTGCAGGAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGCCAACTACATGAGGTGGGTGAGGCAGGCCAGGGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCAGCAGCGGCGCCACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGGCGACACCGCCGTGTACTACTGCGCCACCAGCGAGTACGGCAGCAGCAGCGCCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAACTTCATGCTGACCCAGCCCCCCAGCCTGAGCGTGGCCCCCGGCCAGACCGCCAGGATCACCTGCGGCGGCGACAACATCGGCATCAGGAGCGTGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTGGTGTACGACGACAGCGACAGGCCCAGCGCCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAACACCGCCACCCTGACCATCAGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTACTGCCAGGTGTGGGACAGCATCAGCGAGGTGGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGC.
according to the invention, the transmembrane domain is a CD28 transmembrane domain and/or a CD8 α transmembrane domain.
According to the invention, the intracellular domain further comprises an intracellular costimulatory signaling domain and/or a CD3 zeta signaling domain.
According to the present invention, the costimulatory signaling domain is any one of or a combination of at least two of the human 4-1BB intracellular domain, the human CD28 intracellular domain, the human CD27 intracellular domain, the human OX40 intracellular domain, the human CD30 intracellular domain, the human CD40 intracellular domain or the human OX40 intracellular domain, preferably the human 4-1BB intracellular domain.
According to the invention, the extracellular domain and the transmembrane domain are connected by a hinge region comprising an IgG1 hinge region and/or a CD8 α hinge region.
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, and the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 α signal peptide or a Secretory signal peptide.
The chimeric antigen receptor of the present invention further comprises a promoter, which can be EFl α or any high expression promoter, and the skilled person can select the promoter according to the actual situation, and the promoter is not particularly limited herein, and the presence of the promoter does not affect the performance of the chimeric antigen receptor of the present invention.
According to the invention, the chimeric antigen receptor comprises a signal peptide, a fully human extracellular domain that binds to BCMA antigen, a hinge region, a transmembrane domain, a costimulatory signaling domain, and a CD3 zeta signaling domain in tandem.
According to the invention, the chimeric antigen receptor is formed by connecting a CD8 α signal peptide, a fully human extracellular domain combined with BCMA antigen, a CD8 α hinge region, a CD8 α transmembrane domain, a 4-1BB signaling domain and a CD3 zeta signaling domain in series.
According to the invention, the amino acid sequence of the chimeric antigen receptor comprises a sequence shown in one of SEQ ID NO.41-44, and the amino acid sequence shown in one of SEQ ID NO.41-44 is specifically as follows:
CAR-T3H-2(SEQ ID NO.41)：
MALPVTALLLPLALLLHAARPQVQLQESGGGLVQPGGSLRLSCAASGFTFTNYAMNWVRQAPGKGLEWLSYISTGSDTIYYADSVKGRFTISRDNAKNSLYLQMSSLRDEDTAVYYCARGSWVTATTHSENWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQERPGQAPVLVVHDDTDRPSGIPERFSGSNSGNTATLTIGRVEAGDEADYYCQVWDSINEQVVFGGGTKLTVLGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；
CAR-T3H-5(SEQ ID NO.42)：
MALPVTALLLPLALLLHAARPQVQLQESGGGLIEPGGSLRLPCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARDFAPAGPDYWGQGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQTATISCDGKNIGTKSVHWYQQKPGQAPVLVVYDDDDRPSGIPERFSGSNSGKTATLTISRVEAGDEADYYCQGWDSTTDHVVFGGGTKLTVLGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；
CAR-T3Q-14(SEQ ID NO.43)：
MALPVTALLLPLALLLHAARPQVQLVESGGSVVRPGGSLRLSCAASGFTFDDYGVSWVRQAPGKGLEGVANIKPDGGDKYYVDSVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCATHHYTSTDYFDYWGLGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPPSVSVAPGQTARITCGGNYIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYFCQVWDSINDQVVFGGGTKLTVLGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；
CAR-T4Q-14(SEQ ID NO.44)：
MALPVTALLLPLALLLHAARPQVQLQESGAEVKKPGSSVKVSCKASGYTFTANYMRWVRQARGQGLEWMGWINPSSGATNYAQKFQGRVTMTRDTSISTAYMELSSLTSGDTAVYYCATSEYGSSSADYWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPPSLSVAPGQTARITCGGDNIGIRSVHWYQQKPGQAPVLVVYDDSDRPSAIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSISEVVVFGGGTKLTVLGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
in a second aspect, a nucleic acid encoding the chimeric antigen receptor of the first aspect.
The nucleic acid is specifically shown as one of SEQ ID NO.45-48, and is specifically as follows:
CAR-T3H-2(SEQ ID NO.45)：
ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCTCAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCACCAACTACGCCATGAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGCTGAGCTACATCAGCACCGGCAGCGACACCATCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAGCAGCCTGAGGGACGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGCTGGGTGACCGCCACCACCCACAGCGAGAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAACTTCATGCTGACCCAGGACCCCGCCGTGAGCGTGGCCCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACAGCCTGAGGAGCTACTACGCCAGCTGGTACCAGGAGAGGCCCGGCCAGGCCCCCGTGCTGGTGGTGCACGACGACACCGACAGGCCCAGCGGCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAACACCGCCACCCTGACCATCGGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTACTGCCAGGTGTGGGACAGCATCAACGAGCAGGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG；
CAR-T3H-5(SEQ ID NO.46)：
ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCTCAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGATCGAGCCCGGCGGCAGCCTGAGGCTGCCCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCGTGTACCTGCAGATGAACAGCCTGAGGGTGGAGGACACCGCCGTGTACTACTGCGCCAGGGACTTCGCCCCCGCCGGCCCCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGCTACGTGCTGACCCAGCCCCCCAGCGTGAGCGTGGCCCCCGGCCAGACCGCCACCATCAGCTGCGACGGCAAGAACATCGGCACCAAGAGCGTGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTGGTGTACGACGACGACGACAGGCCCAGCGGCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAAGACCGCCACCCTGACCATCAGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTACTGCCAGGGCTGGGACAGCACCACCGACCACGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG；
CAR-T3Q-14(SEQ ID NO.47)：
ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCTCAGGTGCAGCTGGTGGAGAGCGGCGGCAGCGTGGTGAGGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGACGACTACGGCGTGAGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCAACATCAAGCCCGACGGCGGCGACAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGGACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCACCCACCACTACACCAGCACCGACTACTTCGACTACTGGGGCCTGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAACTTCATGCTGACCCAGCCCCCCAGCGTGAGCGTGGCCCCCGGCCAGACCGCCAGGATCACCTGCGGCGGCAACTACATCGGCAGCAAGAGCGTGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTGGTGTACGACGACAGCGACAGGCCCAGCGGCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAACACCGCCACCCTGACCATCAGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTTCTGCCAGGTGTGGGACAGCATCAACGACCAGGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG；
CAR-T4Q-14(SEQ ID NO.48)：
ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCTCAGGTGCAGCTGCAGGAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGCCAACTACATGAGGTGGGTGAGGCAGGCCAGGGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCAGCAGCGGCGCCACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGGCGACACCGCCGTGTACTACTGCGCCACCAGCGAGTACGGCAGCAGCAGCGCCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAACTTCATGCTGACCCAGCCCCCCAGCCTGAGCGTGGCCCCCGGCCAGACCGCCAGGATCACCTGCGGCGGCGACAACATCGGCATCAGGAGCGTGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTGGTGTACGACGACAGCGACAGGCCCAGCGCCATCCCCGAGAGGTTCAGCGGCAGCAACAGCGGCAACACCGCCACCCTGACCATCAGCAGGGTGGAGGCCGGCGACGAGGCCGACTACTACTGCCAGGTGTGGGACAGCATCAGCGAGGTGGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG.
in a third aspect, the present invention provides a viral vector comprising the nucleic acid of the second aspect.
According to the invention, the viral vector is a lentiviral vector and/or a retroviral vector, preferably a lentiviral vector.
In a fourth aspect, the invention provides a T cell for expression by transfection of a nucleic acid sequence encoding the chimeric antigen receptor according to the first aspect into the T cell.
According to the invention, the transfection is carried out by transfection of T cells with viral vectors and/or eukaryotic expression plasmids, preferably with viral vectors.
In the invention, the T cell has good target killing effect, can release low-dose immune factors, and has the properties of low toxicity and high immune killing reaction.
In a fifth aspect, the present invention provides a recombinant lentivirus comprising a mammalian cell co-transfected with a viral vector as described in the third aspect and packaging helper plasmids gag/pol, Rev and VSV-G.
According to the present invention, the mammalian cell is any one of 293 cells, 293T cells or 293F cells or a combination of at least two thereof.
In a sixth aspect, the present invention provides a composition comprising a chimeric antigen receptor according to the first aspect and/or a recombinant lentivirus according to the fifth aspect.
In a seventh aspect, the present invention provides a use of the chimeric antigen receptor of the first aspect, the nucleic acid of the second aspect, the viral vector of the third aspect, the T cell of the fourth aspect, the recombinant lentivirus of the fifth aspect or the composition of the sixth aspect for the preparation of a chimeric antigen receptor T cell, an immune cell or in a medicament for the treatment of a tumor.
According to the invention, the tumor is a disease associated with expression of B cell maturation antigen protein, such as multiple myeloma, hodgkin's lymphoma, leukemia or glioblastoma.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, a series of new fully humanized anti-BCMA antibodies can be properly combined with BCMA antigens by screening a large number of BCMA antibodies in an scFv phage antibody library, so that the immunogenicity is reduced, the rejection reaction is reduced, a better treatment effect is achieved, and a more effective treatment selection is provided for B cell related diseases;
(2) the chimeric antigen receptor of the invention has the expression positive rate of 20-50%, has specific killing effect on BCMA positive cells, and has high specificity and good treatment effect.
Drawings
FIG. 1 is a schematic sequence diagram of a chimeric antigen receptor of the present invention;
FIG. 2 is a plasmid map of lentiviral expression vector pLVX-EF1 α -G8CAR-Km, wherein FIG. 2(a) is pCDH-CAR-T3H-2, FIG. 2(b) is pCDH-CAR-T3H-5, FIG. 2(c) is pCDH-CAR-T3Q-14, and FIG. 2(d) is pCDH-CAR-T4Q-14;
FIG. 3 is a graph showing the results of flow-assay of CAR-T expression;
FIG. 4 is a graph showing the results of IFN-. gamma.release from the chimeric antigen receptor of the present invention.
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: screening of scFv sequences against BCMA from fully human scFv phage antibody library
This example was performed by screening scFv sequences for BCMA from a fully human scFv phage library derived from: PhaseII phage library was given as a secondary library from Baiotai (Guangzhou) Biotechnology, Inc.; titers determined prior to screening were: 6.6X 109pfu/ml, total 120. mu.l, library diversity: 1X 109pfu/ml, the specific steps are as follows:
I) culture, expansion and enumeration of K562, K562-BCMA cells
(1) Culture of K562, K562-BCMA cells: culturing with IMDM medium (containing 10% FBS and 1 XPicillin and streptomycin), centrifuging every other day to replace fresh medium, diluting the cell density to 1 × 106/ml and continuing culturing, wherein K562-BCMA cells are screened with puromycin with a final concentration of 10 μ g/ml during culturing;
(2) collection and enumeration of K562, K562-BCMA cells: collecting the K562 and K562-BCMA cell cultures into a 50ml centrifuge tube respectively, centrifuging at 300 Xg for 5min, collecting K562 and K562-BCMA cells, and discarding the supernatant; adding 30ml of autoclaved PBS into each tube, suspending the cells, centrifuging for 5min at 300 Xg, and removing the supernatant; 5ml of autoclaved PBS (containing 10% FBS) was added to each tube, 20. mu.l of each tube was counted by trypan blue staining, and 200. mu.l of each tube was stained with antibody and assayed for BCMA expression by flow.
II) incubation of K562, K562-BCMA cells with phage pools:
(1) preparing 3 sterile 1.5ml EP tubes, adding 1X 107K 562 cells to each tube, adding 40. mu.l PhaseII phage to each tube, diluting to 500. mu.l each tube with PBS (containing 10% FBS), blowing and mixing uniformly with a pipette, and transferring to a 24-well plate, wherein each well is 500. mu.l; placing a 24-hole plate in an incubator at 37 ℃ and 5% CO2 for incubation for 2h, and centrifuging 1500 Xg for 3min after 2h to obtain a supernatant;
(2) preparing 3 sterile 1.5ml EP tubes, adding 1 × 107K 562-BCMA cells into each tube, centrifuging at 1500 × g for 3min, discarding the supernatant, adding the supernatant in the step (1), transferring the resuspended cells into a 24-well plate, each well being 500 μ l, and incubating for 2h at 37 ℃ in an incubator containing 5% CO 2;
(3) the cell pellet was collected by centrifugation at 1500 Xg for 3min, washed with 3ml of pre-cooled PBS, centrifuged at 1500 Xg for 3min to remove supernatant, and repeated 10 times. After the 10 th washing, taking cell sediment, adding 500 mu l of eluent into each tube, carrying out ice bath for 5min, then adding 30 mu l of neutralizing solution, and mixing uniformly.
III) amplification and concentration purification of phages
(1) Directly adding the eluted mixed solution of the cells and the phage into a 2 XYT TG1 bacterial culture solution in a logarithmic growth phase (OD600 is equal to 0.5) of 100ml, standing for 30min at 37 ℃, adding 500 mu l of 20% glucose and 100 mu l of 10mg/ml tetracycline into the culture medium, and performing shake culture for 20h at 37 ℃ and 220rpm in a shaking table;
(2) after 20h of culture, 45 ml/tube of bacterial culture solution is subpackaged in a 50ml centrifuge tube, 10800g is centrifuged for 10min, the precipitate is discarded, 40 ml/tube of supernatant is subpackaged in a new 50ml centrifuge tube, 10ml of PEG8000/NaCl is added into each tube, ice bath is carried out on ice for 2h after inversion and uniform mixing, then 10800g is centrifuged for 10min, the supernatant is discarded, the precipitate is resuspended by 500 mul of PBS, the resuspended liquid is centrifuged for 10min at 11600g, the precipitate is discarded, the supernatant is filtered by a 0.45um filter membrane, 2 mul of concentrated and purified phage is taken for titer determination, and the rest is used for the next round of screening.
IV) monoclonal phage production and Elisa test screening of positive monoclonal samples:
(1) after the 4 th round of selection, 10 μ l of the concentrated and purified phage were serially diluted with PBS, TG1 bacteria in the logarithmic growth phase were infected with each diluted phage solution (200 μ l per tube, OD600 ═ 0.5), TG1 bacteria infected with phage were spread on tetracycline-resistant 2 × YT solid plates, and the plates were inverted in an incubator at 37 ℃ for overnight culture;
(2) randomly selecting 100 monoclonal colonies, respectively inoculating the colonies into 15ml centrifuge tubes containing 5ml of 2 XYT liquid culture medium, performing shake culture at 37 ℃ and 220rpm for 20h, after each monoclonal is preserved, centrifuging at 5000rpm for 10min, and taking supernatant, wherein the supernatant of the culture is used as a sample for Elisa detection;
(3) screening positive monoclonal samples by the Elisa test: dissolving and diluting streptavidin to 10 μ g/ml with 0.1M sodium bicarbonate solution, adding 100 μ l/well into NUNCTM 96-well plate, wrapping the whole plate with Parafilm film, and placing Elisa plate at 4 ℃ overnight; the liquid in the plate is dried off the next day, and the plate is washed for 2 times by using washing liquid and is dried for later use; BCMA protein was diluted to 2. mu.g/ml with 0.1M sodium bicarbonate solution, plated at 50. mu.l/well, coated with Parafilm and the Elisa plate was left at 4 ℃ overnight; drying the liquid in the plate the next day, washing the plate with the washing solution for 5 times, then adding 300 mul of 1 × sealing solution into each hole, sealing for 2h at 37 ℃, drying the liquid in the plate after 2h, washing the plate with the washing solution for 5 times, and drying for later use; sequentially adding 100 mu l of monoclonal phage samples into each hole, wrapping the monoclonal phage samples by a Parafilm film, incubating for 1h at 37 ℃, drying the liquid in the plate for 1h, washing the plate for 5 times by using a washing solution, and drying for later use; adding Anti-M13-HRP antibody into 100 mul of each hole, incubating at 37 ℃ for 1h in the dark, drying the liquid in the plate after 1h, washing the plate for 5 times by using washing liquid, adding TMB developing solution into 100 mul of each hole, incubating at 37 ℃ for 20min in the dark, adding 100 mul of stop solution into each hole after 20min, and reading the light absorption value at 450 nm.
Example 2: sequencing the selected fully human anti-BCMA Single chain antibodies (scFv)
The Elisa test result shows that 4 clones are positive (namely the OD value at 450nm is more than 2 times larger than that of the control group), and after sequencing, the sequences of 4 scFv are shown as SEQ ID NO. 33-36.
Example 3: design of fully human Chimeric Antigen Receptor (CAR) against BCMA
The invention constructs a chimeric antigen receptor of fully human Anti-BCMA, as shown in a schematic diagram in figure 1, the chimeric antigen receptor comprises a signal peptide sequence (Leader) of CD8 α, a single-chain antibody sequence (Anti-BCMA scFv) specifically combined with the BCMA antigen, a Hinge region (Hinge) and Transmembrane region sequence (Transmembrane) of CD8 α, a 4-1BB co-stimulatory domain sequence and a CD3 zeta signaling domain sequence, and the specific partial sequences are as follows:
the amino acid sequence (SEQ ID NO.49) of the CD8 α signal peptide (leader) is MALPVTALLLPLALLLHAARP;
nucleotide sequence of CD8 α signal peptide (leader) (SEQ ID NO. 50):
ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCT；
amino acid sequence of the hinge region (hinge) of CD8 α (SEQ ID NO. 51):
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD；
nucleotide sequence of the hinge region (hinge) of CD8 α (SEQ ID NO. 52):
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT；
the amino acid sequence (SEQ ID NO.53) of the CD8 α transmembrane region (TM) is IYIWAPLAGTCGVLLLSLVITLYC;
nucleotide sequence of CD8 α transmembrane region (TM) (SEQ ID NO. 54):
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC；
amino acid sequence of 4-1BB intracellular co-stimulatory domain (ICD) (SEQ ID NO. 55):
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；
4-1BB Intracellular Costimulatory Domain (ICD) (SEQ ID NO. 56):
AAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTG；
amino acid sequence of the zeta-signaling domain of CD3 (SEQ ID NO. 57):
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；
nucleotide sequence of the CD3 zeta signaling domain (SEQ ID NO. 58):
AGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG.
example 4: construction of chimeric antigen receptor expression vector of fully human anti-BCMA
(1) The 4 nucleotide sequences of the fully human anti-BCMA chimeric antigen receptor in example 3 were synthesized in a whole gene, primers were designed and cloned into PCDH-EF1-MCS lentiviral expression vectors, respectively, and the sequences of the primers were as follows:
upstream primer PCDH-EcoRI (SEQ ID NO. 59):
5-GATCGAATTCGCCGCCACCATGGCACTGCCAGTGACAG-3
downstream primer PCDH-BamHI (SEQ ID NO. 60):
5-GATCGGATCCTTACCTAGGTGGCAGGGCCTGCATGTGC-3
(2) obtaining a gene fragment of a fully human anti-BCMA chimeric antigen receptor by PCR:
the PCR system comprises the following concrete steps:
| assembly | Addition amount (μ l) |
| KOD-Plus- |
1 |
| Form panel | 1(50ng) |
| Primer (10. mu.M) | Each 1.5 |
| 10 |
5 |
| 2mM dNTPs | 5(0.2mM) |
| 25mM MgSO4 | 3 |
| dd H2O | 32 |
| In all | 50 |
The PCR is carried out by adopting the system, and the specific procedures are as follows:
(3) carrying out enzyme digestion treatment on the PCDH-EF1-MCS plasmid and a PCR product, wherein the reaction system is as follows:
① digestion of PCDH-EF1-MCS plasmid:
② digestion of PCR product:
| assembly | Addition amount (μ l) |
| PCR product | 5(1μg) |
| |
1 |
| |
1 |
| 10 Xfast |
2 |
| dd H2O | 11 |
| In all | 20 |
After digestion in a 37 ℃ water bath for 30min, DNA electrophoresis was carried out using 1.5% agarose gel, followed by purification and recovery using a Tiangen agarose gel kit.
(4) The connection of the pCDH-EF1-MCS carrier and the fully human anti-BCMA CAR gene fragment is carried out by the following specific connection system:
| assembly | Addition amount (μ l) |
| PCDH-EF1-MCS vector | 2(50ng) |
| BCMA CAR gene | 10(150ng) |
| |
2 |
| T4DNA ligase (NEB) | 1 |
| dd H2O | 5 |
| In all | 20 |
Ligation was performed at 22 ℃ for 1h, the ligation products were directly transformed into Stbl3 E.coli competent cells, 200. mu.l of the transformation products were spread on ampicillin-resistant LB plates, and the LB plates were cultured in an incubator at 37 ℃ overnight in an inverted manner. Randomly selecting 3 monoclonals the next morning for colony PCR identification, and sending positive clones to sequence.
anti-BCMA chimeric antigen receptor lentiviral expression vector pLVX-EF1 α -BCMA CAR-Km vector map see fig. 2(a) -fig. 2(d) in fig. 2.
Example 5: lentiviral packaging
The lentiviral expression vectors in the examples were each subjected to lentiviral packaging, specifically as follows:
(1) recovering 293T cells: 2 tubes of 293T cells were taken out from the liquid nitrogen tank, thawed in a 37 ℃ water bath with gentle shaking, 4ml of DMEM medium was added to each tube, centrifuged at 300 Xg for 5min at room temperature, and the supernatant was discarded. 1ml of DMEM (10% FBS +1 XPS) was added to each tube to resuspend the cells and transferred to T25 flasks, each supplemented with 5ml of DMEM medium (10% FBS +1 XPS), shaken well and placed at 37 ℃ and 5% CO2Cultured in an incubator.
(2) Passage of 293T cells: culturing for 24h until the cells in the bottle grow full, taking out the T25 bottle, sterilizing with alcohol, and placing into a superclean bench. The culture solution in the bottle is completely sucked by a pipette, 1ml of pancreatin preheated at 37 ℃ is directly added into each T25 bottle, after digestion is carried out for 3min to 5min and partial cells begin to shed, 4ml of DMEM preheated at 37 ℃ (10% FBS) is immediately added, and digestion is stopped after uniform mixing. The cell suspension was pipetted into a 15ml centrifuge tube, centrifuged at 300 Xg for 5min, and after the supernatant had been aspirated, 1ml DMEM (containing 10% FBS +1 XPS) was added to each flask to resuspend the cells. 2T 75 flasks were prepared, each containing 14ml DMEM (10% FBS +1 XPS) and 1ml 293T cell suspension, mixed and incubated at 37 ℃ in a 5% CO2 incubator. After the 293T cells in the T75 bottle were fully grown, the 293T cells were further expanded in a manner of "1-to-3", and 18 bottles of the 293T 75 bottles of the 293T cells were obtained by passage to 3 rd generation.
(3)293T cell plates: the 293T cells after trypsinization are collected into two 50ml centrifuge tubes, centrifugation is carried out for 5min at 300 Xg, after supernatant is completely sucked, 25ml DMEM (containing 10% FBS +1 XPS) is added into each tube to resuspend the cells, the cells are fully mixed, and then 20ul of the cells are taken out to be diluted by 5-10 times and used for trypan blue staining counting. (to ensure accurate counting, the cell clumping rate should be low, if the cell clumping rate is high, then re-mixing and dilution are needed before counting.) 2.7X 108The 293T cells were added to 550ml DMEM (containing 10% FBS +1 XPS) medium, mixed well and poured into 10-layer flasks, covered tightly, and incubated at 37 ℃ with 5% CO2Culturing for 20-24 h in the incubator (the cell density reaches 75-85 percent and transfection begins).
(4) Preparing a transfection reagent: prepare 150 ml centrifuge tube, add 25ml DMEM medium, add 240 ug psPAX2 packaging plasmid, 120ug pMD2.G packaging plasmid and 120ug BCMA chimeric antigen receptor expression plasmid in order, fully mix, then stand for 5 min. 1440. mu.l PEI was added and mixed well and left to stand for 20 min. After 20min, the 25ml transfection reagent was poured into a bottle of 525ml DMEM medium and mixed well for use.
(5) Transfection: taking out the 10-layer bottle from the incubator, and putting the bottle into a super clean bench after alcohol disinfection. The medium in the flask was slowly decanted, then 550ml of DMEM medium containing the transfection reagent was slowly poured into the 10-layer flask, and replaced with fresh DMEM (containing 2% FBS +1 XPS) medium 5 hours after transfection, the cap was closed, and the flask was emptied at 37 ℃ and 5% CO2The culture box is used for culturing for 48 hours.
Example 6: concentration and detection of recombinant lentiviral vectors
(1) Concentration of recombinant lentiviral vectors:
after 48h, the 10-layer bottles are taken out, sterilized by alcohol and then placed in a super clean bench, the virus supernatant in the 10-layer bottles is poured into an autoclaved 500ml blue cap bottle (the volume can be 550ml), and then the virus supernatant is subpackaged into 11 50ml centrifuge tubes, 50ml of each tube, and the centrifugation is carried out for 20min at the temperature of 4 ℃ and 3500 rpm. 2 Millipore sterile filtration devices with 250ml 0.45 μm filters were prepared, the centrifuged viral supernatant was poured into the upper part of the Millipore sterile filtration device, 275ml per vial (275 ml capacity), and the viral supernatant was suction filtered into the lower part of the Millipore sterile filtration device (275 ml capacity) by activating a vacuum suction pump. 8 15ml 100kDa ultrafiltration tubes were prepared, 15ml of filtered viral supernatant was added to each tube and centrifuged at 4000rpm at 4 ℃ for 10 min. The filtrate was discarded, 14ml of the virus supernatant was added to each tube again, and centrifuged at 4000rpm at 4 ℃ for 10 min. Repeating the ultrafiltration concentration process to control the concentration multiple of the virus stock solution to be 50-80 times. 500ul of the virus concentrated solution is subpackaged into a freezing tube and stored at minus 80 ℃ for later use.
(2) Titer detection of recombinant lentiviruses:
① detection of physical titer, the physical titer of the recombinant lentivirus is detected by using (HIV P24) ELISA detection kit, the principle is that the HIV-1P24 protein is detected by double antibody sandwich method, i.e. monoclonal antibody of anti-HIV-1P 24 is coated on a micropore plate, the sample to be detected is added into the coated reaction hole for incubation, if the sample contains HIV-1P24 protein, the protein and the antibody in the hole form antigen-antibody complex, enzyme-labeled anti-HIV-1P 24 monoclonal antibody is added, after incubation, the enzyme-labeled antibody is combined with the antigen on the antigen-antibody complex, and then reacts with substrate for color development, finally sulfuric acid is used for stopping reaction, and OD value is measured by an enzyme-labeling instrument.
② Activity titer test, 293T cells were planted in 24-well plates in the morning, and the plate walls were tapped to spread the cells uniformly, the cell amount per well was 1X 105The volume of DMEM (containing 10% FBS +1 XPS) medium per well was 500. mu.l each. After 7h, 1 tube of virus concentrated solution and 1 tube of virus stock solution are taken out from a refrigerator at the temperature of minus 80 ℃ and are slowly dissolved at the temperature of 4 ℃.4 gradients, namely 1 mul, 2 mul, 4 mul and 8 mul, are set for the virus concentrated solution, and each gradient is provided with two compound wells; the virus stock was set up in 3 gradients, 100. mu.l, 200. mu.l, 400. mu.l, two replicate wells per gradient. And (3) sequentially adding the virus concentrated solution and the virus stock solution (the culture medium with the corresponding volume in each hole is sucked out by a pipette before the virus stock solution is added, so that the final culture medium volume in each hole is 500 mu l after the virus stock solution with the corresponding volume is added) into the corresponding hole of the 24-hole plate, and slightly beating the plate wall to uniformly mix the virus in the culture solution. At 37 ℃ with 5% CO2The culture box is used for culturing for 72 hours. After 72h the 293T cells in each well were digested with pancreatin, antibody stained and incubated at 4 ℃ for 30min, the 293T cells were washed 3 times with PBS buffer (centrifugation at 0.3Xg for 5min each time the supernatant was discarded and 200. mu.l of PBS was added to resuspend the cells). Then, the expression of the BCMA chimeric antigen receptor on the surface of 293T cells is detected in a flow mode, so that the activity titer of the virus is calculated, and the results are shown in the following table 1:
TABLE 1CAR Lentiviral packages and titer detection thereof
| Lentiviral names | Physical titer (Particles/mL) | Activity titer (TU/mL) |
| CAR-T3H2 | 1.2×1010 | 2.5×107 |
| CAR-T3H5 | 0.9×1010 | 2.3×107 |
| CAR-T3Q14 | 1.7×1010 | 2.7×107 |
| CAR-T4Q14 | 1.5×1010 | 2.9×107 |
As can be seen from table 1, CARs constructed using the screened fully human antibody scFv can efficiently package high titers of lentivirus for subsequent infection of T cells to make CAR-T cells.
Example 7: lentiviral infection of T cells
(1)Day 0
Resuscitation and stimulation of T cells culture: taking out a tube of peripheral blood mononuclear cells from liquid nitrogen, taking out the peripheral blood mononuclear cells, and placing the peripheral blood mononuclear cells on dry ice for storage. 30mL of T cell culture medium (containing 250IU/mL IL-2; 2mM GlutaMAX or 2mM L-glutamine; 10mM HEPES; 5% human autologous serum) was dispensed into a 50mL centrifuge tube and placed in a 37 ℃ water bath;
and (3) lightly shaking the frozen cell cryopreservation tube in water bath at 37 ℃ until only one small ice crystal is left, completing cell thawing, disinfecting the outer wall of the frozen cell cryopreservation tube by using 75% alcohol, and putting the frozen cell cryopreservation tube into a super clean bench. Centrifuging at 175 Xg for 10min, collecting T cells, transferring to the T cell culture solution preheated by 37 deg.C water bath, counting cells, and recording concentration;
transfer of T cells toIn 24-well plates, the initial cell mass per well was 1X 106The total volume was 1 ml. 0.5ul of anti-CD3(Miltenyi, cat # 130-. Cells were kept flat at 37 ℃ with 5% CO2Culturing in an incubator.
(2)Day 1
And (4) observation: observing cell state under microscope, keeping the cell flat at 37 deg.C and 5% CO2Culturing in an incubator.
(3)Day 2
Lentivirus transfection: PBMC were cultured without centrifugation with CD3/CD28 and the MOI values for lentiviral vector transduction were calculated based on the initial cell number of Day 0. The frozen lentivirus vector is first thawed in 37 deg.c water bath, and the tube is frozen in water until only one small ice crystal remains. The outer wall of the cell tube is sterilized by 75% alcohol and put into a safety cabinet. All lentiviral vectors were collected in 15mL centrifuge tubes and mixed well. Lentivirus was added according to a preset MOI value (MOI-10-30). Cells were kept flat at 37 ℃ with 5% CO2Culturing in an incubator.
(4)Day 3
Expanding and culturing cells: cells were collected, counted, centrifuged at 175 Xg for 10 min. The supernatant was removed. For example, the number of cells is not less than 0.7X106Perml, diluted to 0.5X 10 with T cell culture medium (containing 250IU/mL IL-2; 2mM GlutaMAX or 2mM L-glutamine; 10mM HEPES; 5% human autologous serum)6and/mL. The number of cells is less than or equal to 0.7x106Per mL, diluted to 0.3X 10 with T cell culture6and/mL. Cells were kept flat at 37 ℃ with 5% CO2Culturing in an incubator.
(5)Day 4-7
Expanding and culturing cells: cells were blown up and counted daily (not centrifuged as much as possible). For example, the number of cells is more than or equal to 0.7x106Per mL, diluted to 0.5X 10 with T cell culture6and/mL. The number of cells is less than or equal to 0.7 multiplied by 106Per mL, diluted to 0.3X 10 with T cell culture6and/mL. Cells were kept flat at 37 ℃ with 5% CO2Culturing in an incubator.
(6)Day 8
Cell testing/cryopreservation: cells were taken, stained, and percent CAR expression was detected. CAR-T expression was detected by flow cytometry using FITC-Labeled HumanBCMA (cat # BCA-HF2H1, Acrobiosystems) for binding to CAR-T, the results are shown in FIG. 3;
from the results of fig. 3, it was shown that the expression positive rate of CAR-T3H2 cells was 23.78%, the expression positive rate of CAR-T3H5 cells was 37.14%, the expression positive rate of CAR-T3Q14 cells was 41.07%, and the expression positive rate of CAR-T4Q14 cells was 47.15%.
Taking cells for cell function experiment, and freezing and storing the rest cells: centrifuge at 175 Xg for 5 minutes and remove the supernatant. Resuspend cells to 1X 10 with ice cold CryoStor10 (final 10% DMSO) or mix 50% PlasmoLayte A + 50% CryoStor10 (final 5% DMSO)6and/mL, subpackaging 1 mL/branch to a freezing tube, transferring to Mr. frost, placing at-80 ℃ for at least 24 hours, and then placing liquid nitrogen for storage.
Example 8: CAR-T functional assay (IFN-. gamma.Release)
The invention uses a Human IFN-gamma ELISA kit (Xinbo splendid) to detect the concentration of IFN-gamma cytokines released by CAR-T cells. CAR-T cells were incubated with target cells K562 and K562-BCMA at a ratio of 1:1 for 12h in 96-well plates, and cell supernatants were aspirated to detect IFN- γ release.
The principle is that a double-antibody sandwich ELISA method is adopted, anti-human IFN-gamma antibody is coated on an ELISA plate, human IFN-gamma in a sample or a standard product is combined with the coated antibody during an experiment, and free components are washed away. Biotinylated anti-human IFN-gamma antibody and horseradish peroxidase-labeled avidin are added in sequence. The anti-human IFN-gamma antibody binds to human IFN-gamma bound to the coating antibody, biotin binds specifically to avidin to form an immune complex, and the free components are washed away. Adding chromogenic substrate (TMB), wherein the TMB is blue under the catalysis of horseradish peroxidase, and becomes yellow after adding stop solution. OD value is measured at the wavelength of 450nm by using an enzyme-labeling instrument, IFN-gamma concentration and OD450 value are in direct proportion, and IFN-gamma concentration in the sample is calculated by drawing a standard curve, and the result is shown in figure 4.
As can be seen from FIG. 4, IFN- γ cytokine levels in the co-culture supernatants of CAR-T3H2 cells, CAR-T3H5 cells, CAR-T3Q14 cells and CAR-T4Q14 cells with BCMA positive cells (K562-BCMA and RPMI 8226) were significantly higher than in the BCMA negative cells (K562 and K562-CD19), especially with CAR-T3H5 cells.
In conclusion, compared with other chimeric antigen receptors and other tumor antigens, the CART has better effect, and the invention finds a novel fully human anti-BCMA antibody which can reduce immunogenicity, reduce rejection reaction, achieve better treatment effect and provide a more effective treatment option for B cell related diseases.
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> Guangzhou Bai-and-Gen-Tech Co Ltd
<120> fully human anti-BCMA chimeric antigen receptor and application thereof
<130>2018
<160>60
<170>PatentIn version 3.3
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Gly Gly Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
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Gly Ser Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro Pro Ser
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<400>36
Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Asn
20 25 30
Tyr Met Arg Trp Val Arg Gln Ala Arg Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Ser Ser Gly Ala Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 7075 80
Met Glu Leu Ser Ser Leu Thr Ser Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ser Glu Tyr Gly Ser Ser Ser Ala Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro Pro Ser Leu
130 135 140
Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asp Asn
145 150 155 160
Ile Gly Ile Arg Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
165 170 175
Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Ala Ile Pro
180 185 190
Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
195 200 205
Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
210 215 220
Asp Ser Ile Ser Glu Val Val Val Phe Gly Gly Gly Thr Lys Leu Thr
225 230235 240
Val Leu Gly
<210>37
<211>747
<212>DNA
<213> artificially synthesized sequence
<400>37
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggctt caccttcacc aactacgcca tgaactgggt gaggcaggcc 120
cccggcaagg gcctggagtg gctgagctac atcagcaccg gcagcgacac catctactac 180
gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa cagcctgtac 240
ctgcagatga gcagcctgag ggacgaggac accgccgtgt actactgcgc caggggcagc 300
tgggtgaccg ccaccaccca cagcgagaac tggttcgacc cctggggcca gggcaccctg 360
gtgaccgtga gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 420
aacttcatgc tgacccagga ccccgccgtg agcgtggccc tgggccagac cgtgaggatc 480
acctgccagg gcgacagcct gaggagctac tacgccagct ggtaccagga gaggcccggc 540
caggcccccg tgctggtggt gcacgacgac accgacaggc ccagcggcat ccccgagagg 600
ttcagcggca gcaacagcgg caacaccgcc accctgacca tcggcagggt ggaggccggc 660
gacgaggccg actactactg ccaggtgtgg gacagcatca acgagcaggt ggtgttcggc 720
ggcggcacca agctgaccgt gctgggc 747
<210>38
<211>726
<212>DNA
<213> artificially synthesized sequence
<400>38
caggtgcagc tgcaggagag cggcggcggc ctgatcgagc ccggcggcag cctgaggctg 60
ccctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gaggcaggcc 120
cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcag cacctactac 180
gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa cagcgtgtac 240
ctgcagatga acagcctgag ggtggaggac accgccgtgt actactgcgc cagggacttc 300
gcccccgccg gccccgacta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 360
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagca gctacgtgct gacccagccc 420
cccagcgtga gcgtggcccc cggccagacc gccaccatca gctgcgacgg caagaacatc 480
ggcaccaaga gcgtgcactg gtaccagcag aagcccggcc aggcccccgt gctggtggtg 540
tacgacgacg acgacaggcc cagcggcatc cccgagaggt tcagcggcag caacagcggc 600
aagaccgcca ccctgaccat cagcagggtg gaggccggcg acgaggccga ctactactgc 660
cagggctggg acagcaccac cgaccacgtg gtgttcggcg gcggcaccaa gctgaccgtg 720
ctgggc 726
<210>39
<211>732
<212>DNA
<213> artificially synthesized sequence
<400>39
caggtgcagc tggtggagag cggcggcagc gtggtgaggc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggctt caccttcgac gactacggcg tgagctgggt gaggcaggcc 120
cccggcaagg gcctggaggg cgtggccaac atcaagcccg acggcggcga caagtactac 180
gtggacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa cagcctgtac 240
ctgcagatgg acagcctgag ggccgaggac accgccgtgt actactgcgc cacccaccac 300
tacaccagca ccgactactt cgactactgg ggcctgggca ccctggtgac cgtgagcagc 360
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcaactt catgctgacc 420
cagcccccca gcgtgagcgt ggcccccggc cagaccgcca ggatcacctg cggcggcaac 480
tacatcggca gcaagagcgt gcactggtac cagcagaagc ccggccaggc ccccgtgctg 540
gtggtgtacg acgacagcga caggcccagc ggcatccccg agaggttcag cggcagcaac 600
agcggcaaca ccgccaccct gaccatcagc agggtggagg ccggcgacga ggccgactac 660
ttctgccagg tgtgggacag catcaacgac caggtggtgt tcggcggcgg caccaagctg 720
accgtgctgg gc 732
<210>40
<211>729
<212>DNA
<213> artificially synthesized sequence
<400>40
caggtgcagc tgcaggagag cggcgccgag gtgaagaagc ccggcagcag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcacc gccaactaca tgaggtgggt gaggcaggcc 120
aggggccagg gcctggagtg gatgggctgg atcaacccca gcagcggcgc caccaactac 180
gcccagaagt tccagggcag ggtgaccatg accagggaca ccagcatcag caccgcctac 240
atggagctga gcagcctgac cagcggcgac accgccgtgt actactgcgc caccagcgag 300
tacggcagca gcagcgccga ctactggggccagggcaccc tggtgaccgt gagcagcggc 360
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcaacttcat gctgacccag 420
ccccccagcc tgagcgtggc ccccggccag accgccagga tcacctgcgg cggcgacaac 480
atcggcatca ggagcgtgca ctggtaccag cagaagcccg gccaggcccc cgtgctggtg 540
gtgtacgacg acagcgacag gcccagcgcc atccccgaga ggttcagcgg cagcaacagc 600
ggcaacaccg ccaccctgac catcagcagg gtggaggccg gcgacgaggc cgactactac 660
tgccaggtgt gggacagcat cagcgaggtg gtggtgttcg gcggcggcac caagctgacc 720
gtgctgggc 729
<210>41
<211>493
<212>PRT
<213> artificially synthesized sequence
<400>41
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 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Thr Asn Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Leu Ser Tyr Ile Ser Thr Gly Ser Asp Thr Ile Tyr
65 7075 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Asp Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Gly Ser Trp Val Thr Ala Thr Thr His
115 120 125
Ser Glu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asn Phe Met Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly
165 170 175
Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr
180 185 190
Ala Ser Trp Tyr Gln Glu Arg Pro Gly Gln Ala Pro Val Leu Val Val
195 200 205
His Asp Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
210 215 220
Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Gly Arg Val Glu Ala
225 230235 240
Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ile Asn Glu
245 250 255
Gln Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
385 390 395400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210>42
<211>486
<212>PRT
<213> artificially synthesized sequence
<400>42
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 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
20 25 30
Ile Glu Pro Gly Gly Ser Leu Arg Leu Pro Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
65 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Asp Phe Ala Pro Ala Gly Pro Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Leu Thr Gln
145 150 155 160
Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile Ser Cys
165 170 175
Asp Gly Lys Asn Ile Gly Thr Lys Ser Val His Trp Tyr Gln Gln Lys
180 185 190
Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Asp Asp Arg Pro
195 200 205
Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Lys Thr Ala
210 215 220
Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr
225 230 235 240
Cys Gln Gly Trp Asp Ser Thr Thr Asp His Val Val Phe Gly Gly Gly
245 250 255
Thr Lys Leu Thr Val Leu Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
340 345 350
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
Gln Ala Leu Pro Pro Arg
485
<210>43
<211>488
<212>PRT
<213> artificially synthesized sequence
<400>43
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 Gln Val Gln Leu Val Glu Ser Gly Gly Ser Val
20 25 30
Val Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Asp Asp Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Gly Val Ala Asn Ile Lys Pro Asp Gly Gly Asp Lys Tyr
65 70 75 80
Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Ser Leu Tyr Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Thr His His Tyr Thr Ser Thr Asp Tyr Phe
115 120 125
Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe Met Leu
145 150 155 160
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile
165 170 175
Thr Cys Gly Gly Asn Tyr Ile Gly Ser Lys Ser Val His Trp Tyr Gln
180 185 190
Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp
195 200 205
Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn
210 215 220
Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp
225 230 235 240
Tyr Phe Cys Gln Val Trp Asp Ser Ile Asn Asp Gln Val Val Phe Gly
245 250 255
Gly Gly Thr Lys Leu Thr Val Leu Gly Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210>44
<211>487
<212>PRT
<213> artificially synthesized sequence
<400>44
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 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val
20 25 30
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
35 40 45
Thr Phe Thr Ala Asn Tyr Met Arg Trp Val Arg Gln Ala Arg Gly Gln
50 55 60
Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Ser Ser Gly Ala Thr Asn
65 70 75 80
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
85 90 95
Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Gly Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Thr Ser Glu Tyr Gly Ser Ser Ser Ala Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe Met Leu Thr
145 150 155 160
Gln Pro Pro Ser Leu Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr
165 170 175
Cys Gly Gly Asp Asn Ile Gly Ile Arg Ser Val His Trp Tyr Gln Gln
180 185 190
Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg
195 200 205
Pro Ser Ala Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr
210 215 220
Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr
225 230 235 240
Tyr Cys Gln Val Trp Asp Ser Ile Ser Glu Val Val Val Phe Gly Gly
245 250 255
Gly Thr Lys Leu Thr Val Leu Gly Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg
485
<210>45
<211>1479
<212>DNA
<213> artificially synthesized sequence
<400>45
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc agctgcagga gagcggcggc ggcctggtgc agcccggcgg cagcctgagg 120
ctgagctgcg ccgccagcgg cttcaccttc accaactacg ccatgaactg ggtgaggcag 180
gcccccggca agggcctgga gtggctgagc tacatcagca ccggcagcga caccatctac 240
tacgccgaca gcgtgaaggg caggttcacc atcagcaggg acaacgccaa gaacagcctg 300
tacctgcaga tgagcagcct gagggacgag gacaccgccg tgtactactg cgccaggggc 360
agctgggtga ccgccaccac ccacagcgag aactggttcg acccctgggg ccagggcacc 420
ctggtgaccg tgagcagcgg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 480
agcaacttca tgctgaccca ggaccccgcc gtgagcgtgg ccctgggcca gaccgtgagg 540
atcacctgcc agggcgacag cctgaggagc tactacgcca gctggtacca ggagaggccc 600
ggccaggccc ccgtgctggt ggtgcacgac gacaccgaca ggcccagcgg catccccgag 660
aggttcagcg gcagcaacag cggcaacacc gccaccctga ccatcggcag ggtggaggcc 720
ggcgacgagg ccgactacta ctgccaggtg tgggacagca tcaacgagca ggtggtgttc 780
ggcggcggca ccaagctgac cgtgctgggc accacgacgc cagcgccgcg accaccaaca 840
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 960
cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaag 1020
agaggcagga agaagctgct gtacatcttc aagcagccct tcatgcgccc cgtgcagaca 1080
acccaggagg aggacggctg cagctgtcgg ttcccagagg aggaggaggg aggatgtgag 1140
ctgagggtga agttttctcg gagcgccgat gcaccagcat atcagcaggg acagaatcag 1200
ctgtacaacg agctgaatct gggcaggcgc gaggagtacg acgtgctgga taagcggaga 1260
ggcagagatc ccgagatggg aggcaagcca aggaggaaga accctcagga gggcctgtat 1320
aatgagctgc agaaggacaa gatggccgag gcctactctg agatcggcat gaagggagag 1380
cggagaaggg gcaagggaca cgatggcctg tatcagggcc tgagcacagc caccaaggac 1440
acctacgatg cactgcacat gcaggccctg ccacctagg 1479
<210>46
<211>1458
<212>DNA
<213> artificially synthesized sequence
<400>46
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc agctgcagga gagcggcggc ggcctgatcg agcccggcgg cagcctgagg 120
ctgccctgcg ccgccagcgg cttcaccttc agcagctacg ccatgagctg ggtgaggcag 180
gcccccggca agggcctgga gtgggtgagc gccatcagcg gcagcggcgg cagcacctac 240
tacgccgaca gcgtgaaggg caggttcacc atcagcaggg acaacgccaa gaacagcgtg 300
tacctgcaga tgaacagcct gagggtggag gacaccgccg tgtactactg cgccagggac 360
ttcgcccccg ccggccccga ctactggggc cagggcaccc tggtgaccgt gagcagcggc 420
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcagctacgt gctgacccag 480
ccccccagcg tgagcgtggc ccccggccag accgccacca tcagctgcga cggcaagaac 540
atcggcacca agagcgtgca ctggtaccag cagaagcccg gccaggcccc cgtgctggtg 600
gtgtacgacg acgacgacag gcccagcggc atccccgaga ggttcagcgg cagcaacagc 660
ggcaagaccg ccaccctgac catcagcagg gtggaggccg gcgacgaggc cgactactac 720
tgccagggct gggacagcac caccgaccac gtggtgttcg gcggcggcac caagctgacc 780
gtgctgggca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960
gtccttctcc tgtcactggt tatcaccctt tactgcaaga gaggcaggaa gaagctgctg 1020
tacatcttca agcagccctt catgcgcccc gtgcagacaa cccaggagga ggacggctgc 1080
agctgtcggt tcccagagga ggaggaggga ggatgtgagc tgagggtgaa gttttctcgg 1140
agcgccgatg caccagcata tcagcaggga cagaatcagc tgtacaacga gctgaatctg 1200
ggcaggcgcg aggagtacga cgtgctggat aagcggagag gcagagatcc cgagatggga 1260
ggcaagccaa ggaggaagaa ccctcaggag ggcctgtata atgagctgca gaaggacaag 1320
atggccgagg cctactctga gatcggcatg aagggagagc ggagaagggg caagggacac 1380
gatggcctgt atcagggcct gagcacagcc accaaggaca cctacgatgc actgcacatg 1440
caggccctgc cacctagg 1458
<210>47
<211>1464
<212>DNA
<213> artificially synthesized sequence
<400>47
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc agctggtgga gagcggcggc agcgtggtga ggcccggcgg cagcctgagg 120
ctgagctgcg ccgccagcgg cttcaccttc gacgactacg gcgtgagctg ggtgaggcag 180
gcccccggca agggcctgga gggcgtggcc aacatcaagc ccgacggcgg cgacaagtac 240
tacgtggaca gcgtgaaggg caggttcacc atcagcaggg acaacgccaa gaacagcctg 300
tacctgcaga tggacagcct gagggccgag gacaccgccg tgtactactg cgccacccac 360
cactacacca gcaccgacta cttcgactac tggggcctgg gcaccctggt gaccgtgagc 420
agcggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcaa cttcatgctg 480
acccagcccc ccagcgtgag cgtggccccc ggccagaccg ccaggatcac ctgcggcggc 540
aactacatcg gcagcaagag cgtgcactgg taccagcaga agcccggcca ggcccccgtg 600
ctggtggtgt acgacgacag cgacaggccc agcggcatcc ccgagaggtt cagcggcagc 660
aacagcggca acaccgccac cctgaccatc agcagggtgg aggccggcga cgaggccgac 720
tacttctgcc aggtgtggga cagcatcaac gaccaggtgg tgttcggcgg cggcaccaag 780
ctgaccgtgc tgggcaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840
gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900
cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960
tgtggggtcc ttctcctgtc actggttatc accctttact gcaagagagg caggaagaag 1020
ctgctgtaca tcttcaagca gcccttcatg cgccccgtgc agacaaccca ggaggaggac 1080
ggctgcagct gtcggttccc agaggaggag gagggaggat gtgagctgag ggtgaagttt 1140
tctcggagcg ccgatgcacc agcatatcag cagggacaga atcagctgta caacgagctg 1200
aatctgggca ggcgcgagga gtacgacgtg ctggataagc ggagaggcag agatcccgag 1260
atgggaggca agccaaggag gaagaaccct caggagggcc tgtataatga gctgcagaag 1320
gacaagatgg ccgaggccta ctctgagatc ggcatgaagg gagagcggag aaggggcaag 1380
ggacacgatg gcctgtatca gggcctgagc acagccacca aggacaccta cgatgcactg 1440
cacatgcagg ccctgccacc tagg 1464
<210>48
<211>1461
<212>DNA
<213> artificially synthesized sequence
<400>48
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc agctgcagga gagcggcgcc gaggtgaaga agcccggcag cagcgtgaag 120
gtgagctgca aggccagcgg ctacaccttc accgccaact acatgaggtg ggtgaggcag 180
gccaggggcc agggcctgga gtggatgggc tggatcaacc ccagcagcgg cgccaccaac 240
tacgcccaga agttccaggg cagggtgacc atgaccaggg acaccagcat cagcaccgcc 300
tacatggagc tgagcagcct gaccagcggc gacaccgccg tgtactactg cgccaccagc 360
gagtacggca gcagcagcgc cgactactgg ggccagggca ccctggtgac cgtgagcagc 420
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcaactt catgctgacc 480
cagcccccca gcctgagcgt ggcccccggc cagaccgcca ggatcacctg cggcggcgac 540
aacatcggca tcaggagcgt gcactggtac cagcagaagc ccggccaggc ccccgtgctg 600
gtggtgtacg acgacagcga caggcccagc gccatccccg agaggttcag cggcagcaac 660
agcggcaaca ccgccaccct gaccatcagc agggtggagg ccggcgacga ggccgactac 720
tactgccagg tgtgggacag catcagcgag gtggtggtgt tcggcggcgg caccaagctg 780
accgtgctgg gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960
ggggtccttc tcctgtcact ggttatcacc ctttactgca agagaggcag gaagaagctg 1020
ctgtacatct tcaagcagcc cttcatgcgc cccgtgcaga caacccagga ggaggacggc 1080
tgcagctgtc ggttcccaga ggaggaggag ggaggatgtg agctgagggt gaagttttct 1140
cggagcgccg atgcaccagc atatcagcag ggacagaatc agctgtacaa cgagctgaat 1200
ctgggcaggc gcgaggagta cgacgtgctg gataagcgga gaggcagaga tcccgagatg 1260
ggaggcaagc caaggaggaa gaaccctcag gagggcctgt ataatgagct gcagaaggac 1320
aagatggccg aggcctactc tgagatcggc atgaagggag agcggagaag gggcaaggga 1380
cacgatggcc tgtatcaggg cctgagcaca gccaccaagg acacctacga tgcactgcac 1440
atgcaggccc tgccacctag g 1461
<210>49
<211>21
<212>PRT
<213> artificially synthesized sequence
<400>49
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210>50
<211>63
<212>DNA
<213> artificially synthesized sequence
<400>50
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cct 63
<210>51
<211>45
<212>PRT
<213> artificially synthesized sequence
<400>51
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210>52
<211>135
<212>DNA
<213> artificially synthesized sequence
<400>52
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210>53
<211>24
<212>PRT
<213> artificially synthesized sequence
<400>53
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210>54
<211>72
<212>DNA
<213> artificially synthesized sequence
<400>54
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210>55
<211>42
<212>PRT
<213> artificially synthesized sequence
<400>55
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210>56
<211>126
<212>DNA
<213> artificially synthesized sequence
<400>56
aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60
acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120
gagctg 126
<210>57
<211>112
<212>PRT
<213> artificially synthesized sequence
<400>57
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg GluGlu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210>58
<211>336
<212>DNA
<213> artificially synthesized sequence
<400>58
agggtgaagt tttctcggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60
tacaacgagc tgaatctggg caggcgcgag gagtacgacg tgctggataa gcggagaggc 120
agagatcccg agatgggagg caagccaagg aggaagaacc ctcaggaggg cctgtataat 180
gagctgcaga aggacaagat ggccgaggcc tactctgaga tcggcatgaa gggagagcgg 240
agaaggggca agggacacga tggcctgtat cagggcctga gcacagccac caaggacacc 300
tacgatgcac tgcacatgcaggccctgcca cctagg 336
<210>59
<211>38
<212>DNA
<213> artificially synthesized sequence
<400>59
gatcgaattc gccgccacca tggcactgcc agtgacag 38
<210>60
<211>38
<212>DNA
<213> artificially synthesized sequence
<400>60
gatcggatcc ttacctaggt ggcagggcct gcatgtgc 38
Claims (26)
1. A fully human anti-BCMA chimeric antigen receptor comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and at least one intracellular domain, wherein said extracellular domain is a fully human anti-BCMA antibody;
the amino acid sequence of the fully human anti-BCMA antibody is shown in one of SEQ ID NO. 33-36.
2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the fully human anti-BCMA antibody is represented by SEQ ID No. 34.
3. The chimeric antigen receptor according to claim 1, wherein the fully human anti-BCMA antibody has a nucleotide sequence as set forth in one of SEQ ID nos. 37 to 40.
4. The chimeric antigen receptor according to claim 2, wherein the fully human anti-BCMA antibody has the nucleotide sequence shown in SEQ ID No. 38.
5. The chimeric antigen receptor according to claim 1, wherein the transmembrane domain is a CD28 transmembrane domain and/or a CD8 α transmembrane domain.
6. The chimeric antigen receptor according to claim 1, wherein the intracellular domain comprises an intracellular costimulatory signaling domain and/or a CD3 zeta signaling domain.
7. The chimeric antigen receptor according to claim 6, wherein the costimulatory signaling domain is any one of or a combination of at least two of the human 4-1BB intracellular domain, the human CD28 intracellular domain, the human CD27 intracellular domain, the human OX40 intracellular domain, the human CD30 intracellular domain, the human CD40 intracellular domain or the human OX40 intracellular domain.
8. The chimeric antigen receptor according to claim 7, wherein the costimulatory signaling domain is the human 4-1BB intracellular domain.
9. The chimeric antigen receptor according to claim 1, wherein the extracellular domain and the transmembrane domain are connected by a hinge region.
10. The chimeric antigen receptor according to claim 9, wherein the hinge region comprises an IgG1 hinge region and/or a CD8 α hinge region.
11. The chimeric antigen receptor according to claim 1, further comprising a signal peptide.
12. The chimeric antigen receptor according to claim 11, wherein the signal peptide is a CD8 α signal peptide or a secretor signal peptide.
13. The chimeric antigen receptor according to any one of claims 1 to 12, wherein the chimeric antigen receptor comprises a signal peptide, a fully human extracellular domain that binds BCMA antigen, a hinge region, a transmembrane domain, a costimulatory signaling domain, and a CD3 zeta signaling domain in tandem.
14. The chimeric antigen receptor according to claim 13, wherein the chimeric antigen receptor is a CD8 α signal peptide, a fully human extracellular domain that binds BCMA antigen, a CD8 α hinge region, a CD8 α transmembrane domain, a 4-1BB signaling domain and a CD3 zeta signaling domain in tandem.
15. The chimeric antigen receptor according to claim 14, wherein the amino acid sequence of the chimeric antigen receptor is as shown in one of SEQ ID nos. 41-44.
16. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1-15.
17. A viral vector comprising the nucleic acid of claim 16.
18. The viral vector according to claim 17, wherein the viral vector is a lentiviral vector and/or a retroviral vector.
19. The viral vector according to claim 18, wherein the viral vector is a lentiviral vector.
20. A T cell expressed by transfection of a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 1-15 into a T cell.
21. The T-cell according to claim 20, wherein the transfection is by transfection into the T-cell with a viral vector and/or a eukaryotic expression plasmid.
22. The T-cell of claim 21, wherein the transfection is by transfection into the T-cell with a viral vector.
23. A recombinant lentivirus comprising a mammalian cell co-transfected with the viral vector of any one of claims 17-19 and a packaging helper plasmid.
24. The recombinant lentivirus of claim 23, wherein the mammalian cell is any one of 293 cells, 293T cells or 293F cells or a combination of at least two thereof.
25. A composition comprising the chimeric antigen receptor of any one of claims 1-15 and/or the recombinant lentivirus of claim 23 or 24.
26. Use of the chimeric antigen receptor of any one of claims 1-15, the recombinant lentivirus of claim 23 or 24 or the composition of claim 25 for the preparation of chimeric antigen receptor T cells or in a medicament for the treatment of B cell malignancies.
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| CN109942709B (en) * | 2019-04-22 | 2019-12-27 | 广州百暨基因科技有限公司 | anti-BCMA single domain antibody and application thereof |
| CN111850013B (en) * | 2019-06-25 | 2021-05-18 | 浙江康佰裕生物科技有限公司 | Chimeric antigen receptor with synergistic co-stimulation receptor and application thereof |
| CN112390894A (en) * | 2019-08-12 | 2021-02-23 | 广东东阳光药业有限公司 | Chimeric antigen receptor and uses thereof |
| KR20220110224A (en) * | 2020-01-03 | 2022-08-05 | 바이오션, 인코포레이티드 | Antibodies that bind BCMA and uses thereof |
| KR102371151B1 (en) * | 2020-03-13 | 2022-03-07 | 주식회사 큐로셀 | Anti-bcma-binding domains, fusion proteins comprising thereof, and compositions comprising thereof |
| CN112851814B (en) * | 2020-03-17 | 2023-07-07 | 西安宇繁生物科技有限责任公司 | Fully human single-chain antibody targeting BCMA and preparation method and application thereof |
| CN112028996B (en) * | 2020-10-30 | 2021-01-22 | 南京北恒生物科技有限公司 | Single domain antibodies targeting BCMA and uses thereof |
| WO2022126416A1 (en) * | 2020-12-16 | 2022-06-23 | 武汉友芝友生物制药股份有限公司 | Anti-bcma antibody, preparation method therefor and application thereof |
| CN112394179B (en) * | 2021-01-18 | 2021-04-09 | 北京百普赛斯生物科技股份有限公司 | FMC63 CAR-T cell immunogenicity ELISA detection kit |
| CN112979808B (en) * | 2021-02-08 | 2022-07-29 | 华道(上海)生物医药有限公司 | Antibody for resisting B cell mature antigen and application thereof |
| CN117924497A (en) * | 2021-03-22 | 2024-04-26 | 浙江纳米抗体技术中心有限公司 | BCMA-targeted nano antibody and application thereof |
| CN115703836A (en) * | 2021-08-10 | 2023-02-17 | 上海恒润达生生物科技股份有限公司 | BCMA (brain cell activating antigen) targeted chimeric antigen receptor based on fully human and murine single-chain antibodies and application thereof |
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| CN111574628B (en) * | 2017-01-23 | 2023-08-08 | 克莱格医学有限公司 | Antibodies targeting BCMA and uses thereof |
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