CN109651511A - A kind of Chimeric antigen receptor and its application targeting BCMA - Google Patents

A kind of Chimeric antigen receptor and its application targeting BCMA Download PDF

Info

Publication number
CN109651511A
CN109651511A CN201811598951.8A CN201811598951A CN109651511A CN 109651511 A CN109651511 A CN 109651511A CN 201811598951 A CN201811598951 A CN 201811598951A CN 109651511 A CN109651511 A CN 109651511A
Authority
CN
China
Prior art keywords
chimeric antigen
antigen receptor
cell
domain
bcma
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811598951.8A
Other languages
Chinese (zh)
Other versions
CN109651511B (en
Inventor
李光超
罗敏
曾剑华
郭锦涛
莫文俊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Bio Gene Technology Co Ltd
Original Assignee
Guangzhou Bio Gene Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Bio Gene Technology Co Ltd filed Critical Guangzhou Bio Gene Technology Co Ltd
Priority to CN201811598951.8A priority Critical patent/CN109651511B/en
Publication of CN109651511A publication Critical patent/CN109651511A/en
Application granted granted Critical
Publication of CN109651511B publication Critical patent/CN109651511B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a kind of targeting BCMA Chimeric antigen receptor, the Chimeric antigen receptor includes can be in conjunction with the extracellular domain, transmembrane domain and at least one intracellular domain of antigen, wherein the extracellular domain is anti-BCMA single domain antibody；Wherein, the amino acid sequence of the anti-BCMA single domain antibody is selected from: (a) amino acid sequence as shown in SEQ ID NO.1；Or, (b) amino acid sequence as shown in SEQ ID NO.1 is formed by one or more replacing, adding or deleting for amino acid, can specific bond in Chimeric antigen receptor, have in conjunction with BCMA and inducing T cell signal transduction function variant.Chimeric antigen receptor of the invention has smaller clinical side effects and higher safety, can effectively reduce solid tumor stove, effectively improve the therapeutic effect of tumour.

Description

A kind of Chimeric antigen receptor and its application targeting BCMA
Technical field
The present invention relates to the cellular immunotherapy field of tumour more particularly to it is a kind of target BCMA Chimeric antigen receptor and Its apply, the construction method of Chimeric antigen receptor T (CAR-T) cell technology specially based on special target spot BCMA and its Application in antineoplaston.
Background technique
With the development of tumour immunity theory and clinical technology, Chimeric antigen receptor T cell therapy (Chimeric Antigen receptor T-cell immunotherapy, CAR-T) become tumour immunity treatment most promising at present One of method.Generally, Chimeric antigen receptor CAR is by a tumor associated antigen combined area, extracellular hinge area, trans-membrane region and born of the same parents Interior signal transduction district's groups at.In general, CAR includes single-chain fragment variable region (the Single chain fragment of antibody Variable, scFv) or to tumor associated antigen (tumor associated antigen, TAA) have specificity combination Structural domain is coupled by the cytoplasmic domains of hinge and transmembrane region and T cell signal transduction molecule.The most common lymphocyte Activated partial includes the T cell costimulation structural domain with T cell effector function triggering (such as CD3 ζ) sections in series.CAR is situated between The T cell that the adoptive immunotherapy led allows CAR- to transplant is on non-HLA restrictive one Direct Recognition target tumour cell TAA。
It is the significant contribution person of cancer mortality that the overwhelming majority, which has the patient of B cell malignant tumour and Huppert's disease,.B Cell malignancies mix the response of various form of therapy.Treat B cell malignant tumour conventional method all have toxicity and Side effect, and the antibody such as anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD52 is used to carry out immunization therapy, effect is general. With the development of technology, the cell of expression Chimeric antigen receptor (CAR) modification is begun to use to be treated, but used in CAR Given antigen binding structural domain therapeutic effect be it is unpredictable, cause therapeutic effect unstable.
Had openly as extracellular domain for treating B cell related disease using BCMA antigen, but antigen binding structure Domain combines too strong, then the CAR induced t cell mass cell factor discharges, leads to the possibility for being considered as " cytokine storm " Lethal immune response；But if antigen-binding domains combination is too weak, then CAR T cell can not be in terms of removing cancer cell Sufficient therapeutic efficiency is presented.And in the prior art, 106687483 A of CN discloses the anti-BCMA Chimeric antigen receptor of humanization and controls Cancer is treated, by the method for the T cell of application expression CAR genetic modification, to realize treatment and B cell maturation antigen protein (BCMA) the relevant disease of expression.107207598 A of CN discloses BCMA Chimeric antigen receptor, is used for B cell related diseases The application of the adoptive T cell therapy of condition.
Therefore, prepare a kind of Chimeric antigen receptor for targeting BCMA, in addition to having the advantages that Antybody therapy, select one with Antigen-binding domains combine moderate antibody, must can provide B cell related disease one more effective therapeutic choice.
Summary of the invention
CAR-T technology is influenced for not very ideal and tumor microenvironment is targeted in current CAR-T technology treatment tumour The case where therapeutic effect, the present invention provides a kind of Chimeric antigen receptor and its application for targeting BCMA, prepared by the present invention chimeric Antigen receptor obtains the Chimeric antigen receptor of targeting BCMA by screening, enhances the therapeutic effect of CAR-T cell.
To achieve this purpose, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of Chimeric antigen receptor for targeting BCMA, the born of the same parents comprising that can combine antigen Extracellular portion, transmembrane domain and at least one intracellular domain, wherein the extracellular domain is anti-BCMA single domain antibody；
Wherein, the amino acid sequence of the anti-BCMA single domain antibody is selected from:
(a) amino acid sequence as shown in SEQ ID NO.1；Alternatively,
(b) the the replacing, adding or deleting by one or more amino acid of the amino acid sequence as shown in SEQ ID NO.1 And formed, can specific bond in Chimeric antigen receptor, have in conjunction with BCMA and inducing T cell signal transduction function change Body.
In the present invention, can inventor by affinity, be combined by screening to a large amount of BCMA antibody with Fc sections of people Analysis, has found an anti-BCMA single domain antibody, the extracellular domain of Chimeric antigen receptor is incorporated in by it, can significantly be mentioned The therapeutic effect of high Chimeric antigen receptor.
In a specific embodiment, amino acid sequence shown in the SEQ ID NO.1 is as follows: qvqlvesgggl vqaggslrlscaasgrtfsdhtlgwfrqapgkerefvgaiswsggstyyadsvsgrftisrdkakntgylqmnslk pedtavyycaaaddrysdyrywgqgtqvtvss.
According to the present invention, the amino acid sequence as shown in SEQ ID NO.1 taking by one or more amino acid Amino acid sequence shown in generation, addition or missing and the amino acid sequence and SEQ ID NO.1 that are formed is at least 90% same Property, preferably 95%, the more preferably variant of 98% identity.
In a specific embodiment, the variant and SEQ ID NO.1 have 92% identity, still have energy Enough specific bonds have the function of to combine BCMA and inducing T cell signal transduction in Chimeric antigen receptor.
In a specific embodiment, the variant and SEQ ID NO.1 have 94% identity, still have energy Enough specific bonds have the function of to combine BCMA and inducing T cell signal transduction in Chimeric antigen receptor.
In a specific embodiment, the variant and SEQ ID NO.1 have 95% identity, still have energy Enough specific bonds have the function of to combine BCMA and inducing T cell signal transduction in Chimeric antigen receptor.
In a specific embodiment, the variant and SEQ ID NO.1 have 96% identity, still have energy Enough specific bonds have the function of to combine BCMA and inducing T cell signal transduction in Chimeric antigen receptor.
In a specific embodiment, the variant and SEQ ID NO.1 have 98% identity, still have energy Enough specific bonds have the function of to combine BCMA and inducing T cell signal transduction in Chimeric antigen receptor.
In a specific embodiment, the variant and SEQ ID NO.1 have 99% identity, still have energy Enough specific bonds have the function of to combine BCMA and inducing T cell signal transduction in Chimeric antigen receptor.
According to the present invention, the nucleotide sequence of the anti-BCMA single domain antibody includes to have as shown in SEQ ID NO.2 Sequence, nucleotide sequence shown in SEQ ID NO.2 are specific as follows: caggtgcagctggtagagtctgggggaggattggt gcaggctgggggctctctgagactctcctgtgcagcctctggacgtaccttcagtgaccataccctgggctggttc cgccaggctcccgggaaggagcgtgagtttgtaggagctatttcctggagtggtggtagcacatactatgcagact ccgtgagcggccgattcaccatctctcgagacaaggccaagaacacgggctatctgcaaatgaacagcctgaaacc tgaggacacggccgtttattactgtgcagcagccgacgatcgctatagtgactatcgctactggggccaggggacc caggtcaccgtctcctca.
According to the present invention, the nucleotide sequence of the anti-BCMA single domain antibody includes and sequence shown in SEQ ID NO.2 With at least 85%, for example, can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, preferably 90%, the variant of more preferable 95% identity, the albumen of expression has can Specific bond has the function of to combine BCMA and inducing T cell signal transduction in Chimeric antigen receptor.
According to the present invention, the transmembrane domain is CD28 transmembrane domain and/or CD8 α transmembrane domain.
According to the present invention, the intracellular domain further includes costimulatory signal transduction domain and/or CD3 ζ signal transduction intracellular Domain.
According to the present invention, costimulatory signal conducting structure domain behaviour 4-1BB intracellular region, people CD28 intracellular region, people In CD27 intracellular region, people OX40 intracellular region, people CD30 intracellular region, people CD40 intracellular region or people's OX40 intracellular region any one or At least two combination is preferably people's 4-1BB intracellular region.
According to the present invention, the extracellular domain with transmembrane domain is connected by hinge area, the hinge area packet Hinge area containing IgG1 and/or CD8 α hinge area.
According to the present invention, the Chimeric antigen receptor further includes signal peptide, and the signal peptide is that can instruct chimeric antigen Receptor transmembrane transfer signal peptide be all it is feasible, those skilled in the art can according to need selection this field routine signal Peptide, the Chimeric antigen receptor further include signal peptide, preferably CD8 alpha signal peptide or Secretory signal peptide.
Chimeric antigen receptor of the invention further includes promoter, and the promoter can be the high expression of EF α or any Promoter, those skilled in the art can select according to the actual situation, not do particular determination, the presence of promoter herein The performance of Chimeric antigen receptor of the invention will not be had an impact.
According to the present invention, the Chimeric antigen receptor includes signal peptide, the extracellular domain in conjunction with BCMA antigen, hinge Area, transmembrane domain, costimulatory signal conducting structure domain and CD3 ζ signal transduction structural domain are connected in series.
According to the present invention, the Chimeric antigen receptor is CD8 alpha signal peptide, in conjunction with the extracellular domain of BCMA antigen, CD8 α Hinge area, CD8 α transmembrane domain, 4-1BB signal transduction structural domain and CD3 ζ signal transduction structural domain are connected in series.
According to the present invention, the amino acid sequence of the Chimeric antigen receptor includes the sequence as shown in SEQ ID NO.3, institute It is specific as follows to state amino acid sequence shown in SEQ ID NO.3: MALPVTALLLPLALLLHAARPQVQLVESGGGLVQAGGS LRLSCAASGRTFSDHTLGWFRQAPGKEREFVGAISWSGGSTYYADSVSGRFTISRDKAKNTGYLQMNSLKPEDTAV YYCAAADDRYSDYRYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR.
According to the present invention, the amino acid sequence of the Chimeric antigen receptor has extremely with sequence shown in SEQ ID NO.3 Few 90% identity, for example, can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, preferably 95%, the more preferably variant of 98% identity.
Second aspect encodes the nucleic acid of Chimeric antigen receptor as described in relation to the first aspect.
The nucleic acid is specific as shown in SEQ ID NO.4, specific as follows: atggcactgccagtgacagccctgctgct gccactggccctgctgctgcacgcagcacgccctcaggtgcagctggtagagtctgggggaggattggtgcaggct gggggctctctgagactctcctgtgcagcctctggacgtaccttcagtgaccataccctgggctggttccgccagg ctcccgggaaggagcgtgagtttgtaggagctatttcctggagtggtggtagcacatactatgcagactccgtgag cggccgattcaccatctctcgagacaaggccaagaacacgggctatctgcaaatgaacagcctgaaacctgaggac acggccgtttattactgtgcagcagccgacgatcgctatagtgactatcgctactggggccaggggacccaggtca ccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccct gcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctac atctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcagggtgaagt tttctcggagcgccgatgcaccagcatatcagcagggacagaatcagctgtacaacgagctgaatctgggcaggcg cgaggagtacgacgtgctggataagcggagaggcagagatcccgagatgggaggcaagccaaggaggaagaaccct caggagggcctgtataatgagctgcagaaggacaagatggccgaggcctactctgagatcggcatgaagggagagc ggagaaggggcaagggacacgatggcctgtatcagggcctgagcacagccaccaaggacacctacgatgcactgca catgcaggccctgccacctagg.
The third aspect, the present invention provide a kind of viral vectors, including nucleic acid described in second aspect.
According to the present invention, the viral vectors is slow virus carrier and/or retroviral vector, and preferably slow virus carries Body.
Fourth aspect, the present invention provide a kind of T cell, are compiled using Chimeric antigen receptor as described in relation to the first aspect by it The nucleic acid sequence of code, which is transfected into T cell, to be expressed.
According to the present invention, the mode of the transfection be by viral vectors and/or eukaryotic expression plasmids to T cell, T cell is transfected into preferably by viral vectors.
In the present invention, the T cell has good targeting killing effect, while can discharge low dosage immune factor, Has the high immunologic cytotoxicity reaction property of hypotoxicity.
5th aspect, the present invention provide a kind of recombinant slow virus, will include the viral vectors and packet as described in the third aspect The recombinant slow virus that dress helper plasmid gag/pol, Rev and VSV-G cotransfection mammalian cell obtains.
According to the present invention, the mammalian cell be 293 cells, 293T cell or 293F cell in any one or at least Two kinds of combination.
6th aspect, the present invention provide a kind of composition, and the composition includes chimeric antigen as described in relation to the first aspect Receptor and/or the recombinant slow virus as described in terms of the 5th.
7th aspect, the present invention provide Chimeric antigen receptor as described in relation to the first aspect, the nucleic acid as described in second aspect, Viral vectors as described in the third aspect, the T cell as described in fourth aspect, the recombinant slow virus as described in terms of the 5th or such as Composition described in 6th aspect is in preparation Chimeric antigen receptor T cell, immunocyte or the application in tumor therapeutic agent.
According to the present invention, the tumour is disease relevant to the expression of B cell maturation antigen protein, such as multiple bone Myeloma, Hodgkin lymphoma, leukaemia or glioblastoma.
Compared with prior art, the invention has the following beneficial effects:
(1) present invention is by screening a large amount of BCMA antibody, and having found a new anti-BCMA antibody can be with BCMA antigen appropriate combination, reaches better therapeutic effect, provides a more effective therapeutic choice for B cell related disease；
(2) special construction of the single domain antibody (VHH) of the alpaca in Chimeric antigen receptor of the present invention, be currently known can The minimum unit (only 15KDa) of combining target antigen, other than it can identify some conventional epitopes, very due to its molecular weight Small, CDR3 area head, cyclization extends outwardly, and can also identify conventional antibody without identification epitope, and it is good that in addition there are stability, The advantages such as weak to the immunogenicity of people；
(3) Chimeric antigen receptor the positive expression rate of the invention is 32% or more, has specificity to BCMA positive cell Lethal effect, and there is high specificity and good therapeutic effect almost without killing to BCMA negative cells.
Detailed description of the invention
Fig. 1 is the sequence diagram of Chimeric antigen receptor of the invention；
Fig. 2 is Lentiviral pLVX-EF1 α-G8 CAR-Km element schematic；
Fig. 3 is Lentiviral pLVX-EF1 α-G8 CAR-Km plasmid map；
Fig. 4 is the expression result figure of flow cytometer detection CAR-T, wherein Fig. 4 (a) is the G8 CAR of the T cell of untransfected Expression ratio, Fig. 4 (b) are that the G8 CAR of Chimeric antigen receptor of the present invention expresses ratio, and Fig. 4 (c) is the T cell of untransfected FMC63 CAR expresses ratio, and the FMC63 CAR that Fig. 4 (d) is FMC63CAR-T expresses ratio；
Fig. 5 is that CART kills target cell situation；
Fig. 6 is CART and detects the secretion result figure of IFN-gama in supernatant after cell co-cultures.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art, Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from The conventional products of acquisition.
Embodiment 1: the design of Chimeric antigen receptor
The present embodiment constructs the Chimeric antigen receptor (G8 CAR) of anti-BCMA, and as shown in sequence diagram Fig. 1, this is chimeric Antigen receptor includes the signal peptide sequence (Leader) of one section of CD8 α, the single domain antibody sequence in conjunction with BCMA antigentic specificity (Anti-BCMA VHH), the hinge area (Hinge) and transmembrane domain (Transmembrane) of CD8 α, 4-1BB costimulation domain Sequence and CD3 ζ signal transduction domain sequence, specific each section sequence are as follows:
The amino acid sequence (SEQ ID NO.5) of CD8 alpha signal peptide (leader): MALPVTALLLPLALLLHAARP；
The nucleotide sequence (SEQ ID NO.6) of CD8 alpha signal peptide (leader): ATGGCACTGCCAGTGACAGCCCTG CTGCTGCCACTGGCCCTGCTGCTGCA CGCAGCACGCCCT；
The amino acid sequence (SEQ ID NO.7) of CD8 α hinge area (hinge): TTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACD；
The nucleotide sequence (SEQ ID NO.8) of CD8 α hinge area (hinge): ACCACGACGCCAGCGCCGCGACCAC CAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGC AGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT；
The amino acid sequence (SEQ ID NO.9) of CD8 α transmembrane region (TM): IYIWAPLAGTCGVLLLSLVITLYC；
The nucleotide sequence (SEQ ID NO.10) of CD8 α transmembrane region (TM): ATCTACATCTGGGCGCCCTTGGCCGGG ACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC；
The amino acid sequence (SEQ ID NO.11) of 4-1BB costimulation domain (ICD) intracellular: KRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCEL；
The nucleotide sequence (SEQ ID NO.12) of 4-1BB costimulation domain (ICD) intracellular: AAGAGAGGCAGGAAGAAGC TGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTT CCCAGAGGAGGAGGAGGGAGGATGTGAGCTG；
The amino acid sequence (SEQ ID NO.13) in CD3 ζ signal transduction domain: RVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR；
The nucleotide sequence (SEQ ID NO.14) in CD3 ζ signal transduction domain: AGGGTGAAGTTTTCTCGGAGCGCCGAT GCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGC TGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAA TGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGA CACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCAC CTAGG.
Embodiment 2: the Chimeric antigen receptor expression vector of anti-BCMA is constructed
(1) full genome synthesizes G8 CAR sequence, the G8 CAR synthesized with EcoRI and BamHI double digestion full genome and zero load Body in 37 DEG C of water-baths after digestion 30min, carries out DNA electrophoresis using 1.5% Ago-Gel, then uses the fine jade of Tiangeng The recovery processing of sepharose kits；
(2) connection of pCDH-EF1-MCS carrier and G8 CAR genetic fragment:
Linked system is as follows:
Component Additive amount (μ l)
PCDH-EF1-MCS carrier 2(50ng)
G8 CAR gene 10(150ng)
T4 DNA connection buffer 2
T4 DNA ligase (NEB) 1
dd H2O 5
In total 20
In 22 DEG C of connection 1h, connection product directly converts Stbl3 competent escherichia coli cell, takes 200 μ l converted products It is coated with the LB plate of ammonia benzyl resistance, LB plate is inverted overnight incubation in 37 DEG C of incubator.Select 3 lists the next morning at random Clone carries out bacterium colony PCR identification, and positive colony sample presentation is sequenced.
The element of the Chimeric antigen receptor Lentiviral pLVX-EF1 α-G8 CAR-Km of anti-BCMA is shown in Fig. 2, carrier Map is shown in Fig. 3.
Embodiment 3: slow virus packaging
Slow virus packaging is carried out to the Lentiviral in embodiment respectively, using four pUC pUCs, specific steps It is as follows:
Gag/pol, Rev, VSV-G and work of the present invention needed for (1) four pUC pUC expresses slow virus carrier packaging respectively Artificial chimeric's antigen receptor that the single-chain antibody of Cheng Wending is constituted: four plasmids are carried out to transiently transfect 293T cell, gross mass is 10μg；
(2) above-mentioned plasmid is added into the DMEM of the serum-free of certain volume, is placed 15 minutes after mixing, it will be above-mentioned mixed It closes liquid to be added into the T75 culture bottle for the cell for being covered with 293T cell, mix gently, in 37 DEG C, 5%CO2Cell incubator training Support 6h；
(3) fresh culture is replaced after 6h, continues to cultivate, and the butyric acid sodium solution of 10mM is added, after 72 hours The culture supernatant for collecting slow virus carries out purification assays.
The amplification of embodiment 4:CAR-T cell
Every volunteer adopts the whole blood of 30ml, and peripheral blood and physiological saline 1:1 are diluted, are added in centrifuge tube Ficoll, the peripheral blood after being slowly added into dilution, 1500rpm are centrifuged PBMC layers of 30min gentle aspiration and move into another centrifuge tube；
With brine PBMC more times, it is transferred to the X-VIVO culture medium (IL- of the OKT3 containing 50ng/mL, 300IU/mL 2) it is cultivated in, after PBMC separation, needs to be activated with the OKT3 containing 50ng/mL, the X-VIVO of the IL-2 of 300IU/ml, Culture medium is replaced with the X-VIVO containing 300IU/mL after 2 days to expand culture, carries out within then every two days primary count and more The X-VIVO containing 300IU/mL is changed, and cell concentration is maintained 0.5 × 106-1×106/ mL is observed continuously 10 days.
Embodiment 5: slow-virus infection T cell
It improves slow virus using RetroNectin the RetroNectin of 30 μ g is coated in the efficiency of infection of T cell In 6 orifice plates, it is put in 37 DEG C of cell incubator 2h；RetroNectin is drawn, closes packet using Hank ' the s solution containing 2.5%BSA 6 orifice plates by after are put in 37 DEG C of cell incubator 0.5h；Confining liquid is drawn, Hank ' the s solution washing 6 containing 2%Hepes is utilized X-VIVO culture medium is added in orifice plate, and suitable slow virus solution is added, and 2000g is centrifuged 2h；Supernatant is abandoned, is added 1 × 106T Cell (the CD3 positive > 90%), 1000g is centrifuged 10min, in 37 DEG C, 5%CO2It is cultivated in the cell incubator of certain humidity, Second day repeats the above process.
Infection 5 days after measure G8 CAR expression, using FITC-Labeled Human BCMA (article No. BCA-HF2H1, Acrobiosystems) and the combination of G8 VHH, by the expression of flow cytomery G8 CAR, as a result such as Fig. 4 (a)-Fig. 4 (b) shown in；From Fig. 4 (a)-Fig. 4's (b) the results show that the positive expression rate of G8 CAR is 32.95%.
Also with FITC-Labeled Human CD19 (20-291) Protein, Fc Tag (article No. CD9-HF251, Acrobiosystems) and the combination of FMC63scFv, by the expression of flow cytomery FMC63 CAR, as a result such as Fig. 4 (c) shown in-Fig. 4 (d)；From Fig. 4 (c)-Fig. 4's (d) the results show that the positive expression rate of FMC63 CAR is 55.55%.
Embodiment 6: cytotoxicity detection
Cytotoxicity test experience is carried out using G8 CAR, and (clones) building from FMC63 with anti-CD19 scFv CAR and blank control NC (T cell of untransfected) is as control, the specific steps are as follows:
Using LDH detection CAR-T cell to K562, K562-CD19 (the K562 cell for stablizing expression CD19), K562- The toxicity of BCMA (the K562 cell for stablizing expression BCMA) and 8226 cell of RPMI；
After the centrifugation of various cells, with serum-free without phenol red, RPMI1640 culture medium counts after repeatedly washing, take 1 × 106Each 50 μ L of 8226 cell of K562, K562-CD19, K562-BCMA and RPMI, be plated in 96 orifice plates, as target cell, By target cell: effector cell=1:1 be separately added into untransfected T cell and each CAR-T cell, in 37 DEG C, 5%CO2With it is certain 12h is cultivated in the cell incubator of humidity；Lysate is added as positive control, then 250g is centrifuged 5min, and every hole takes 100 μ L Culture supernatant is added in 96 new orifice plates, and 20 μ L reaction solutions are added, is put in darkroom and reacts 20-30min.
Microplate reader 590nm measurement calculates dissolution percentage according to formula:
Cytotoxicity (%)=[(experimental port-culture medium background hole)-(spontaneous LDH relief hole-culture medium of effector cell Background hole)-(the spontaneous LDH relief hole of target cell-culture medium background hole)]/[(target cell maximum LDH relief hole-volume correction Hole)-(the spontaneous LDH relief hole of target cell-culture medium background hole)] × 100%.
Specific step referring to application No. is 201510362935.5 patent " CD33 specific chimeric antigen receptor and its Using ".
As a result show such as Fig. 5, G8 CAR-T cell can specificity the killing BCMA positive cell (K562-BCMA with RPMI 8226), and to BCMA negative cells (K562 and K562-CD19) almost without killing ability；In addition, FMC63 CAR-T The K562-CD19 cell of the killing CD19 positive of cell energy specificity, and it is several to CD19 negative cells (K562 and RPMI 8226) There is no killing ability.
Embodiment 7:ELISA detects cell line and CAR-T cell co-cultures the level of IFN-γ in supernatant
Respectively by K562, K562-CD19 (the K562 cell for stablizing expression CD19), K562-BCMA (stablizes expression BCMA's K562 cell) and 8226 cell of RPMI according to 5 × 105Cells/well is inoculated with 24 orifice plates.By every hole 5 × 105Cell is separately added into T cell (T mock) cell of CAR-T, untransfected, supplement culture solution to 1.5mL, after being co-cultured 12 hours in incubator；Using Human IL-2, IFN-γ ELISA detection kit (Xin Bosheng biology) are detected that (specific steps are shown in ELISA to supernatant is co-cultured Detection kit specification), as a result as shown in Figure 6.
From fig. 6, it can be seen that G8 CAR-T cell and the cell (K562-BCMA and RPMI 8226) of the BCMA positive are trained altogether Supporting IFN-γ cytokine levels in supernatant has conspicuousness raising compared with BCMA negative cells (K562 and K562-CD19) group.
In conclusion CART of the invention has better effect compared to other Chimeric antigen receptors and other tumour antigens Fruit, the present invention, which has found a new anti-BCMA antibody, to be reached better therapeutic effect with BCMA antigen appropriate combination, be B cell related disease provides a more effective therapeutic choice.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
SEQUENCE LISTING
<110>hundred caip gene Science and Technology Ltd. of Guangzhou
<120>a kind of Chimeric antigen receptor and its application for targeting BCMA
<130> 2018
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 119
<212> PRT
<213>artificial synthesized sequence
<400> 1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His
20 25 30
Thr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Ser Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Asp Arg Tyr Ser Asp Tyr Arg Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 2
<211> 357
<212> DNA
<213>artificial synthesized sequence
<400> 2
caggtgcagc tggtagagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg taccttcagt gaccataccc tgggctggtt ccgccaggct 120
cccgggaagg agcgtgagtt tgtaggagct atttcctgga gtggtggtag cacatactat 180
gcagactccg tgagcggccg attcaccatc tctcgagaca aggccaagaa cacgggctat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagccgac 300
gatcgctata gtgactatcg ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 3
<211> 363
<212> PRT
<213>artificial synthesized sequence
<400> 3
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 Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg
35 40 45
Thr Phe Ser Asp His Thr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Phe Val Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr
65 70 75 80
Tyr Ala Asp Ser Val Ser Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala
85 90 95
Lys Asn Thr Gly Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Ala Ala Asp Asp Arg Tyr Ser Asp Tyr Arg
115 120 125
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro
130 135 140
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
145 150 155 160
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
165 170 175
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
180 185 190
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
195 200 205
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
210 215 220
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
225 230 235 240
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
245 250 255
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
260 265 270
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
275 280 285
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
290 295 300
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
305 310 315 320
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
325 330 335
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
340 345 350
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 4
<211> 963
<212> DNA
<213>artificial synthesized sequence
<400> 4
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc agctggtaga gtctggggga ggattggtgc aggctggggg ctctctgaga 120
ctctcctgtg cagcctctgg acgtaccttc agtgaccata ccctgggctg gttccgccag 180
gctcccggga aggagcgtga gtttgtagga gctatttcct ggagtggtgg tagcacatac 240
tatgcagact ccgtgagcgg ccgattcacc atctctcgag acaaggccaa gaacacgggc 300
tatctgcaaa tgaacagcct gaaacctgag gacacggccg tttattactg tgcagcagcc 360
gacgatcgct atagtgacta tcgctactgg ggccagggga cccaggtcac cgtctcctca 420
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 480
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 540
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 600
ctgtcactgg ttatcaccct ttactgcagg gtgaagtttt ctcggagcgc cgatgcacca 660
gcatatcagc agggacagaa tcagctgtac aacgagctga atctgggcag gcgcgaggag 720
tacgacgtgc tggataagcg gagaggcaga gatcccgaga tgggaggcaa gccaaggagg 780
aagaaccctc aggagggcct gtataatgag ctgcagaagg acaagatggc cgaggcctac 840
tctgagatcg gcatgaaggg agagcggaga aggggcaagg gacacgatgg cctgtatcag 900
ggcctgagca cagccaccaa ggacacctac gatgcactgc acatgcaggc cctgccacct 960
agg 963
<210> 5
<211> 21
<212> PRT
<213>artificial synthesized sequence
<400> 5
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 6
<211> 63
<212> DNA
<213>artificial synthesized sequence
<400> 6
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cct 63
<210> 7
<211> 45
<212> PRT
<213>artificial synthesized sequence
<400> 7
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> 8
<211> 135
<212> DNA
<213>artificial synthesized sequence
<400> 8
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 9
<211> 23
<212> PRT
<213>artificial synthesized sequence
<400> 9
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
1 5 10 15
Leu Val Ile Thr Leu Tyr Cys
20
<210> 10
<211> 72
<212> DNA
<213>artificial synthesized sequence
<400> 10
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 11
<211> 42
<212> PRT
<213>artificial synthesized sequence
<400> 11
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> 12
<211> 126
<212> DNA
<213>artificial synthesized sequence
<400> 12
aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60
acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120
gagctg 126
<210> 13
<211> 112
<212> PRT
<213>artificial synthesized sequence
<400> 13
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 Glu Glu 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> 14
<211> 336
<212> DNA
<213>artificial synthesized sequence
<400> 14
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 tgcacatgca ggccctgcca cctagg 336

Claims (10)

1. a kind of Chimeric antigen receptor for targeting BCMA, which is characterized in that the Chimeric antigen receptor includes that can combine antigen Extracellular domain, transmembrane domain and at least one intracellular domain, wherein the extracellular domain be anti-BCMA single domain resist Body；
Wherein, the amino acid sequence of the anti-BCMA single domain antibody is selected from:
(a) amino acid sequence as shown in SEQ ID NO.1；Alternatively,
(b) amino acid sequence as shown in SEQ ID NO.1 by one or more replacing, adding or deleting for amino acid shape At, can specific bond in Chimeric antigen receptor, have in conjunction with BCMA and inducing T cell signal transduction function variant.
2. Chimeric antigen receptor according to claim 1, which is characterized in that the amino as shown in SEQ ID NO.1 The acid sequence amino acid sequence formed and SEQ ID NO.1 institute by one or more replacing, adding or deleting for amino acid The amino acid sequence shown has at least 90% identity, preferably 95%, the more preferably variant of 98% identity；
Preferably, the nucleotide sequence of the anti-BCMA single domain antibody include have the sequence as shown in SEQ ID NO.2, or with It has at least 85%, preferably 90%, the variant of further preferred 95% identity.
3. Chimeric antigen receptor according to claim 1 or 2, which is characterized in that the transmembrane domain is CD28 cross-film Structural domain and/or CD8 α transmembrane domain；
Preferably, the intracellular domain further includes costimulatory signal transduction domain and/or CD3 ζ signal transduction domain intracellular；
Preferably, costimulatory signal conducting structure domain behaviour 4-1BB intracellular region, people CD28 intracellular region, people CD27 intracellular region, Any one in people OX40 intracellular region, people CD30 intracellular region, people CD40 intracellular region or people's OX40 intracellular region or at least two Combination, is preferably people's 4-1BB intracellular region；
Preferably, the extracellular domain with transmembrane domain is connected by hinge area；
Preferably, the hinge area includes IgG1 hinge area and/or CD8 α hinge area；
Preferably, the Chimeric antigen receptor further includes signal peptide, preferably CD8 alpha signal peptide or Secretory signal peptide.
4. Chimeric antigen receptor according to any one of claim 1-3, which is characterized in that the Chimeric antigen receptor packet Include signal peptide, the extracellular domain in conjunction with BCMA antigen, hinge area, transmembrane domain, costimulatory signal conducting structure domain and CD3 ζ signal transduction structural domain is connected in series；
Preferably, the Chimeric antigen receptor is CD8 alpha signal peptide, in conjunction with the extracellular domain of BCMA antigen, CD8 α hinge area, CD8 α transmembrane domain, 4-1BB signal transduction structural domain and CD3 ζ signal transduction structural domain are connected in series；
Preferably, the amino acid sequence of the Chimeric antigen receptor includes the sequence as shown in SEQ ID NO.3 or has with it At least 90% identity, preferably 95%, the more preferably variant of 98% identity.
5. encoding the nucleic acid of Chimeric antigen receptor according to any one of claims 1-4.
6. a kind of viral vectors, which is characterized in that including nucleic acid as claimed in claim 5；
Preferably, the viral vectors is slow virus carrier and/or retroviral vector, preferably slow virus carrier.
7. a kind of T cell, which is characterized in that pass through it using such as Chimeric antigen receptor of any of claims 1-4 The nucleic acid sequence of coding, which is transfected into T cell, to be expressed；
Preferably, the mode of the transfection is by viral vectors and/or eukaryotic expression plasmids to T cell, preferably logical It crosses viral vectors and is transfected into T cell.
8. a kind of recombinant slow virus, which is characterized in that viral vectors as claimed in claim 6 and packaging helper plasmid will be included The recombinant slow virus that cotransfection mammalian cell obtains；
Preferably, the mammalian cell be 293 cells, 293T cell or 293F cell in any one or at least two group It closes.
9. a kind of composition, which is characterized in that the composition includes such as chimeric antigen of any of claims 1-4 Receptor and/or recombinant slow virus as claimed in claim 8.
10. as Chimeric antigen receptor of any of claims 1-4, recombinant slow virus as claimed in claim 8 or Composition as claimed in claim 9 is in preparation Chimeric antigen receptor T cell, immunocyte or answering in tumor therapeutic agent With；
Preferably, the tumour is B cell malignant tumour.
CN201811598951.8A 2018-12-26 2018-12-26 BCMA (brain cell activating antigen) targeted chimeric antigen receptor and application thereof Active CN109651511B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811598951.8A CN109651511B (en) 2018-12-26 2018-12-26 BCMA (brain cell activating antigen) targeted chimeric antigen receptor and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811598951.8A CN109651511B (en) 2018-12-26 2018-12-26 BCMA (brain cell activating antigen) targeted chimeric antigen receptor and application thereof

Publications (2)

Publication Number Publication Date
CN109651511A true CN109651511A (en) 2019-04-19
CN109651511B CN109651511B (en) 2020-06-09

Family

ID=66116737

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811598951.8A Active CN109651511B (en) 2018-12-26 2018-12-26 BCMA (brain cell activating antigen) targeted chimeric antigen receptor and application thereof

Country Status (1)

Country Link
CN (1) CN109651511B (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109942709A (en) * 2019-04-22 2019-06-28 广州百暨基因科技有限公司 The single domain antibody of anti-BCMA a kind of and its application
CN110041433A (en) * 2019-04-26 2019-07-23 上海科棋药业科技有限公司 A kind of Chimeric antigen receptor and its application targeting BCMA
CN111850013A (en) * 2019-06-25 2020-10-30 浙江康佰裕生物科技有限公司 Chimeric antigen receptor with synergistic co-stimulation receptor and application thereof
CN111848820A (en) * 2020-07-31 2020-10-30 广东昭泰体内生物医药科技有限公司 CD19 and BCMA double-target chimeric antigen receptor and application thereof
CN111909271A (en) * 2020-08-12 2020-11-10 深圳市茵冠生物科技有限公司 BCMA chimeric antigen receptor based on single domain antibody and application thereof
CN112028996A (en) * 2020-10-30 2020-12-04 南京北恒生物科技有限公司 Single domain antibodies targeting BCMA and uses thereof
CN112251412A (en) * 2020-10-12 2021-01-22 广东昭泰体内生物医药科技有限公司 BCMA (brain cell activating antigen) targeted chimeric antigen receptor T cell and application thereof
CN114075287A (en) * 2020-08-18 2022-02-22 湖南远泰生物技术有限公司 Humanized BCMA antibodies and BCMA-CAR-T cells
CN114195896A (en) * 2021-12-06 2022-03-18 东莞清实生物科技有限公司 BCMA chimeric antigen receptor based on single domain antibody and application thereof
WO2022199590A1 (en) * 2021-03-22 2022-09-29 浙江纳米抗体技术中心有限公司 Nanobody targeting bcma and application thereof
WO2023191526A1 (en) * 2022-03-30 2023-10-05 바이젠셀 주식회사 Chimeric antigen receptor including cd30-derived intracellular signaling domain, immune cell expressing same, and use thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104877032A (en) * 2015-06-26 2015-09-02 中国医学科学院血液病医院（血液学研究所） CD33 specificity chimeric antigen receptor and application thereof
WO2017025038A1 (en) * 2015-08-11 2017-02-16 Nanjing Legend Biotech Co., Ltd. Chimeric antigen receptors based on single-domain antibodies and methods of use thereof
CN106795217A (en) * 2014-07-24 2017-05-31 蓝鸟生物公司 BCMA chimeric antigen receptors
CN107207598A (en) * 2014-12-12 2017-09-26 蓝鸟生物公司 Bcma chimeric antigen receptors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106795217A (en) * 2014-07-24 2017-05-31 蓝鸟生物公司 BCMA chimeric antigen receptors
CN107207598A (en) * 2014-12-12 2017-09-26 蓝鸟生物公司 Bcma chimeric antigen receptors
CN104877032A (en) * 2015-06-26 2015-09-02 中国医学科学院血液病医院（血液学研究所） CD33 specificity chimeric antigen receptor and application thereof
WO2017025038A1 (en) * 2015-08-11 2017-02-16 Nanjing Legend Biotech Co., Ltd. Chimeric antigen receptors based on single-domain antibodies and methods of use thereof

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN109942709A (en) * 2019-04-22 2019-06-28 广州百暨基因科技有限公司 The single domain antibody of anti-BCMA a kind of and its application
CN110041433A (en) * 2019-04-26 2019-07-23 上海科棋药业科技有限公司 A kind of Chimeric antigen receptor and its application targeting BCMA
CN111850013A (en) * 2019-06-25 2020-10-30 浙江康佰裕生物科技有限公司 Chimeric antigen receptor with synergistic co-stimulation receptor 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
CN111848820A (en) * 2020-07-31 2020-10-30 广东昭泰体内生物医药科技有限公司 CD19 and BCMA double-target chimeric antigen receptor and application thereof
WO2022033057A1 (en) * 2020-08-12 2022-02-17 深圳市茵冠生物科技有限公司 Single-domain antibody-based bcma chimeric antigen receptor, and application thereof
CN111909271A (en) * 2020-08-12 2020-11-10 深圳市茵冠生物科技有限公司 BCMA chimeric antigen receptor based on single domain antibody and application thereof
CN114075287B (en) * 2020-08-18 2023-07-21 湖南远泰生物技术有限公司 Humanized BCMA antibodies and BCMA-CAR-T cells
CN114075287A (en) * 2020-08-18 2022-02-22 湖南远泰生物技术有限公司 Humanized BCMA antibodies and BCMA-CAR-T cells
CN112251412A (en) * 2020-10-12 2021-01-22 广东昭泰体内生物医药科技有限公司 BCMA (brain cell activating antigen) targeted chimeric antigen receptor T cell and application thereof
CN112251412B (en) * 2020-10-12 2023-03-28 汤朝阳 BCMA (brain cell activating antigen) targeted chimeric antigen receptor T cell 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
CN112028996A (en) * 2020-10-30 2020-12-04 南京北恒生物科技有限公司 Single domain antibodies targeting BCMA and uses thereof
WO2022199590A1 (en) * 2021-03-22 2022-09-29 浙江纳米抗体技术中心有限公司 Nanobody targeting bcma and application thereof
CN114195896A (en) * 2021-12-06 2022-03-18 东莞清实生物科技有限公司 BCMA chimeric antigen receptor based on single domain antibody and application thereof
CN114195896B (en) * 2021-12-06 2022-07-05 东莞清实生物科技有限公司 BCMA chimeric antigen receptor based on single domain antibody and application thereof
WO2023191526A1 (en) * 2022-03-30 2023-10-05 바이젠셀 주식회사 Chimeric antigen receptor including cd30-derived intracellular signaling domain, immune cell expressing same, and use thereof

Also Published As

Publication number Publication date
CN109651511B (en) 2020-06-09

Similar Documents

Publication Publication Date Title
CN109651511A (en) A kind of Chimeric antigen receptor and its application targeting BCMA
CN109485734A (en) It is a kind of target BCMA and CD19 bispecific chimeric antigen receptor and its application
CN108395482B (en) Construction of targeting CD20 antigen chimeric antigen receptor and activity identification of engineered T cell thereof
KR102663253B1 (en) Chimeric antigen receptor for solid tumors and T cells expressing chimeric antigen receptor
CN109485733A (en) A kind of anti-BCMA Chimeric antigen receptor and its application of full source of people
CN110144326A (en) A kind of antitumor T cell of targeting and its preparation method and application
CN109836496A (en) It is a kind of to target the single-chain antibody of CD317, Chimeric antigen receptor T cell and its preparation method and application
CN110144328A (en) A kind of antitumor T cell of targeting and its preparation method and application
CN109266667A (en) Target the Chimeric antigen receptor and its application of CD5
CN109836497A (en) A kind of single-chain antibody of targeting EGFR, Chimeric antigen receptor T cell and its preparation method and application
CN109836495A (en) It is a kind of to target the single-chain antibody of CD22, Chimeric antigen receptor T cell and its preparation method and application
CN110157679B (en) Targeting T lymphocyte and preparation method and application thereof
CN109836493A (en) It is a kind of to target the single-chain antibody of CD19, Chimeric antigen receptor T cell and its preparation method and application
CN109957018A (en) It is a kind of to target the single-chain antibody of CD22, Chimeric antigen receptor T cell and its preparation method and application
CN113527514B (en) Application of Gstp1 in preparation of synergistic CAR-T
US20210046113A1 (en) Dual-activating costimulatory molecule receptor and use thereof
WO2019129056A1 (en) Cd137 bidirectional activation costimulatory molecule receptor and use thereof
CN109957025A (en) It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application
WO2021057932A1 (en) Modified immune cell and use thereof
CN109957020A (en) It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application
CN110157675A (en) A kind of targeting T lymphocyte and its preparation method and application
CN110526979A (en) Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP
CN109957546A (en) A kind of Chimeric antigen receptor T cell and its preparation method and application targeting CD317
CN110157677A (en) A kind of targeting T lymphocyte and its preparation method and application
CN109957016A (en) It is a kind of to target the single-chain antibody of CD38, Chimeric antigen receptor T cell and its preparation method and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant