CN110272493A - Target specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinical application - Google Patents

Target specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinical application Download PDF

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CN110272493A
CN110272493A CN201910484370.XA CN201910484370A CN110272493A CN 110272493 A CN110272493 A CN 110272493A CN 201910484370 A CN201910484370 A CN 201910484370A CN 110272493 A CN110272493 A CN 110272493A
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cell
people
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targeting
sequence
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代红久
李艳云
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Nanjing Kaidi Biotechnology Co Ltd
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Nanjing Kaidi Biotechnology Co Ltd
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Abstract

The present invention relates to a kind of specific chimeric antigen receptor T cell for targeting CD19 and preparation method and clinical applications.The present invention is based on targeting people CD19 single chain antibody sequence building targeting CD19 specific chimeric antigen receptor and the immune response cells of its modification, the immune response cell of the novel modification can be effectively targeted to attack kinds of tumor cells, CD19 positive tumor cell is especially expressed, can be used to prepare the preparation for treating tumour.The immune response cell preparation method step of the modification of targeting CD19 of the invention is simple, the immune response cell of the modification of the targeting CD19 of acquisition is high to the killing rate of tumour cell, according to clinical verification after million grades of low dosages are fed back, it recurs refractory advanced stage CD19 positive lymphomas patient and obtains clinical symptoms after two weeks and be relieved, obtained in the 77th day close to complete incidence graph curative effect, fever caused by cell-free factor release syndrome and any neurotoxicity side reaction occur.

Description

Target specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinic Using
Technical field
The invention belongs to immunotherapy of tumors biomedicine technical fields, are related to specific chimeric antigen receptor T cell.
Background technique
With the rapid development of biotechnology, immune cell therapy has become the fourth-largest therapy of field of cancer treatment.
Immunotherapy for cancer mainly includes adoptive cell therapy, immunomodulator, tumor vaccine and immunity inspection point Blocking treatment etc..Wherein, in cell therapy field, immunocyte (the especially Chimeric of Chimeric antigen receptor modification Antigen Receptor T-Cell, CAR-T) therapy undoubtedly has become research institution and drugmaker falls over each other the bright of " pursuing " Star.
CAR-T (Chimeric Antigen Receptor T-Cell, the T cell of Chimeric antigen receptor modification) is representative Immunotherapy, principle is mainly the T cell progress Chimeric antigen receptor for passing through genetic engineering means and extracting to patient itself Modification form CAR-T cell, which can specifically identify tumor surface related antigen (tumour cell marker), from And target killing tumour.Relative to routine immunization cell, the targeting of CAR-T cell, killing activity and persistence are all higher, and And tumor by local immunosupress microenvironment can be overcome and break host immune tolerance status.This is using CAR-T cell as representative Modification immune cell therapy has significant curative effect in the treatment of acute leukemia and non-Hodgkin lymphoma, it is considered to be most One of promising oncotherapy mode.
CAR-T immunotherapy is most to overturn one of the emerging technology of begetting power at present, and American-European and countries in Asia are all in product The clinical test of the therapy is carried out in pole, wherein China's development clinical test project is most, up to 204, occupies global CAR-T and treats The 50% of method clinical test.Hospital, the University of Pennsylvania, The Children's Hospital of Philadelphia, american cancer research institute (NCI), Fred Hutchinson Cancer Research Center commemorates that Si Long Caitlin Cancer center and Seattle children's hospital etc. are earliest development CAR- The research institution of T cell therapy.Emerging world biopharmaceutical company such as Juno, Kite, Celgene, Cellectis, Bluebird, tradition world pharmacy giant such as Novartis, Bristol Myers Squibb, Pfizer, Johnson & Johnson, lucky moral etc. have put into a large amount of moneys Gold and manpower are into the research and development of CAR-T therapy, and wherein KITE and Novartis Co., Ltd research and develop targeting CD19CART new drug product K TE- C19 and CTL-019 is because prominent curative effect successively obtains FDA approval.
However up to the present, target CD19 CAR-T product it is generally existing because of cytokine release syndrome caused by Fever or neurotoxicity side reaction, bring biggish challenge for the extensive clinical use and clinical treatment of the product.Synthesising biological The rise and application of technology provide for the CAR-T immunization therapy of tumour and more develop the selection of safe and efficient CAR-T, be The exploitation for developing safer CAR-T new treatment and drug provides enlightenment.
Summary of the invention
In view of the above problem and/or other problems of the relevant technologies, the purpose of the invention is to overcome existing CAR-T to face Faced in bed technique because caused by cytokine release syndrome (CRS) fever and neurotoxicity side reaction to patient with arriving Bed Therapeutic safety problem and low dosage CAR-T treat the unconspicuous challenge of drug effect, provide targeting people CD19 chimeric antigen by Body and its gene and recombinant expression carrier, engineering the modification of CD19 targeting Chimeric antigen receptor immune response cell and Its clinical application.The immune response cell of the CD19 targeting of engineering of the invention is capable of the killing effect of ground raising tumour cell Rate and safety clinical treatment, validity, to provide a kind of safe and reliable, the efficient oncotherapy with application prospect New tool.
In a first aspect, this application provides single chain antibody protein or its variant that a kind of targeting combines people CD19, it includes Sequence selected from the group below:
(1) SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7, shown in anti human CD 19 list The variant that amino acid modification generates is passed through in the amino acid sequence of chain antibody albumen, or (2) (1).
Inventor passes through creative work, constantly carries out the permutation and combination and sieve of amino acid sequence design and sequence Choosing, to nearly tens of CAR molecular sequences carry out random screening tests and targeting functional verification (such as building viral vectors, with And T cell is further infected, the test such as cytotoxicity of T cell for obtaining the T cell of modification, and detecting gained modification), It is compared later according to the result of multiple random combines, then carries out sequence adjustment, finishing screen selects the best sequence of effect, obtains The anti human CD 19 single-chain antibody Chimeric antigen receptor sequence of high-titer targeting people CD19 of the invention and its functional variant thereof.One In a little embodiments, the amino acid modification includes but is not limited to substitution, missing and the addition of amino acid, the functional variant thereof Including but not limited to pass through the substitution of one or more amino acid, the derived peptides or peptide analogues of missing and addition generation.
In some embodiments, the targeting is SEQ ID in conjunction with the single chain antibody protein of people CD19 or its variant There are 70~99% homologys shown in No.4 or with amino acid sequence shown in SEQ ID No.4, preferably 80~99%, more preferably 90~99%, the most preferably polypeptide of 95~99% homologys.
In some embodiments, the targeting is SEQ ID in conjunction with the single chain antibody protein of people CD19 or its variant There are 70~99% homologys shown in No.5 or with amino acid sequence shown in SEQ ID No.5, preferably 80~99%, more preferably 90~99%, the most preferably polypeptide of 95~99% homologys.
In some embodiments, the targeting is SEQ ID in conjunction with the single chain antibody protein of people CD19 or its variant There are 70~99% homologys shown in No.6 or with amino acid sequence shown in SEQ ID No.6, preferably 80~99%, more preferably 90~99%, the most preferably polypeptide of 95~99% homologys.
In some embodiments, the targeting is SEQ ID in conjunction with the single chain antibody protein of people CD19 or its variant There are 70~99% homologys shown in No.7 or with amino acid sequence shown in SEQ ID No.7, preferably 80~99%, more preferably 90~99%, the most preferably polypeptide of 95~99% homologys.
In some non-limiting embodiments, it is described with SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or It is by SEQ ID No.4, SEQ ID No.5, SEQ ID that amino acid sequence shown in SEQ ID No.7, which has the polypeptide of homology, Any shown amino acid sequence passes through 1~10 amino acid residue in No.6 or SEQ ID No.7, preferable through 1~5 Amino acid residue or the derived peptides more preferably formed by 1~3 replacing, missing or adding for amino acid residue.
Other than full-length polypeptide, theme disclosed in the application first aspect also provides the polypeptide of theme disclosed in the present application Or either segment segment in peptide domain.In some embodiments, the segment can be at least 5~15 amino acid.One In a little embodiments, the segment is at least 20 continuous amino acids, at least 30 continuous amino acids or at least 50 Continuance ammines Base acid.In some embodiments, the segment is at least 60 to 80,100,200,300 or more continuous amino acids.
Second aspect, this application provides it is a kind of targeting combine people CD19 specific chimeric antigen receptor, it includes from Aminoterminal combines single-chain antibody, transmembrane domain and the letter intracellular of people CD19 to the sequentially connected boot sequence of c-terminus, targeting Number structural domain, the targeting include targeting combination people CD19 described in the application first aspect single-stranded in conjunction with people's CD19 single-chain antibody Antibody protein or its variant, the targeting combine the preferred people CD19 of anti human CD 19 single chain antibody protein of people CD19, the targeting The immune response cell modified in conjunction with the specific chimeric antigen receptor of people CD19 kills tumour cell when imitating target ratio and being 10:1 Hurt efficiency up to 40%~60%.It is 6x10^4/ kilograms feeding back targeting CD19 dosage in clinical treatment lymthoma clinical test CAR-T cell after, clinic be displayed without it is any because caused by cytokine release syndrome fever and neurotoxicity side reaction, Safety clinical treatment and significant curative effect, patient's the 77th day curative effect obtained close to clinically complete remission after feeding back CAR-T cell.
In some embodiments, the targeting further includes hinge area in conjunction with the specific chimeric antigen receptor of people CD19.
In some embodiments, the transmembrane domain includes transmembrane region.
In some embodiments, the intracellular signal structural domain includes immunoreceptor tyrosine activating motif and costimulation Signal domain；
In some embodiments, the transmembrane region includes the transmembrane structure CD8 area, the transmembrane structure CD28 area, CD3 ζ cross-film Any one of structural area, the transmembrane structure CD4 area, the transmembrane structure 4-1BB area, the transmembrane structure OX40 area, the transmembrane structure ICOS area.
In some embodiments, the immunoreceptor tyrosine activating motif includes the intracellular signal structural domain of CD3 ζ chain Or Fc ε RI γ intracellular signal structure,
In some embodiments, the costimulatory signal domain includes CD28 intracellular signal structural domain, CD137/4-1BB born of the same parents At least one of interior signal domain, CD134/OX40 intracellular signal structural domain, ICOS intracellular signal structural domain.
In one embodiment, the hinge area is selected from CD8 α hinge area, IgG hinge area, or comprising all or in part Variant of the hinge area or hinge area of immunoglobulin by one or more amino acid modifications.
In one embodiment, the boot sequence is selected from amino acid sequence shown in SEQ ID No.3.
In some embodiments, the transmembrane region behaviour CD8 polypeptide.In some embodiments, the hinge area is People CD8 polypeptide or its variant.
In some embodiments, people's CD8 polypeptide of the hinge area is selected from polypeptide shown in SEQ ID No.8.
In this embodiment, the people CD8's of the transmembrane region is selected from polypeptide shown in SEQ ID No.9.
In some embodiments, the people 4-1BB intracellular domain is selected from amino acid sequence shown in SEQ ID NO.10 Polypeptide.
In some embodiments, the people CD28 intracellular domain is selected from amino acid sequence shown in SEQ ID No.11 Polypeptide.
In some embodiments, the CD3 ζ intracellular domain is selected from the more of amino acid sequence shown in SEQ ID No.12 Peptide.
In some embodiments, the CD3 ζ intracellular domain is selected from the more of amino acid sequence shown in SEQ ID No.13 Peptide.
In some embodiments, it is described targeting in conjunction with people CD19 specific chimeric antigen receptor be recombinant expression or by Carrier expression.
In one embodiment, the amino acid sequence of the Chimeric antigen receptor is from aminoterminal to c-terminus by guidance sequence Targeting described in column, the application first aspect combines the amino acid sequence of people CD19 single-chain antibody amino acid sequence, people's CD8 hinge area Column, the amino acid sequence of people's CD8 transmembrane region, the amino acid sequence of people's 4-1BB intracellular domain and people's CD3 ζ intracellular domain Amino acid sequence is sequentially connected in series；
In one embodiment, the amino acid sequence of the Chimeric antigen receptor is from aminoterminal to c-terminus by guidance peptide Targeting described in amino acid sequence, the application first aspect combines the single-chain antibody amino acid sequence of people CD19, people's CD8 hinge area sequence The amino acid sequence of column, the amino acid sequence of people's CD8 transmembrane region, the amino acid sequence of people's CD28 intracellular domain and CD3 ζ are intracellular The amino acid sequence of structural domain is sequentially connected in series.
In certain non-limiting embodiments, the CAR of theme disclosed in the application second aspect includes by antigen binding Structural domain is connected to the spacer region (spacer) of transmembrane domain, also known as hinge area.One spacer region can be sufficiently flexible , to allow antigen-binding domains to orient in different directions, in favor of antigen recognizing.Trivial can be of spacer comes from The part in the hinge area of IgG1 or the area CH2CH3 of immunoglobulin and CD3.Some spacer regions include immunoglobulin CH3 The some spacer regions in domain or both the domain CH3 and the domain CH2 include all or in part immunoglobulin (for example, IgG1, IgG2, IgG3, IgG4) hinge area, that is, the sequence between the domain CH1 and CH2 of immunoglobulin is fallen in, for example, IgG4Fc hinge or CD8 Hinge.The sequence in immunoglobulin source may include one or more amino acid modifications, for example, 1,2,3,4 or 5 substitution. In some non-limiting embodiments, the spacer region is hinge area.
In certain non-limiting embodiments, the CAR of theme disclosed in the application second aspect includes more containing CD28 The transmembrane domain of peptide and costimulatory signal conducting region containing CD28 polypeptide.
In some embodiments, hinge area includes people CD8 polypeptide.Hinge area CD8 polypeptide is selected from SEQ ID No.8 institute Show the polypeptide of amino acid sequence.
In some embodiments, transmembrane region includes people CD8 polypeptide.Transmembrane region CD8 polypeptide is selected from SEQ ID No.9 institute Show the polypeptide of amino acid sequence.
In some embodiments, the amino acid sequence of the people 4-1BB intracellular domain is selected from SEQ ID NO:10 institute Show the polypeptide of amino acid sequence.
In some embodiments, the people CD28 ζ intracellular domain is selected from amino acid sequence shown in SEQ ID No.11 Polypeptide.
In certain non-limiting embodiments, the intracellular domain of CAR may include that can activate or stimulate cell (example Such as, the cell of lymphoid, such as T cell) CD3 ζ polypeptide.CD3 ζ includes 3 immunity receptor Tyrosine Activating Motifs (Immunoreceptor tyrosine-based activation motif, ITAM), and will activation after combining antigen Signal is transmitted to cell (for example, the cell of lymphoid, such as T cell).
In some embodiments, the CD3 ζ intracellular domain is selected from the more of amino acid sequence shown in SEQ ID No.12 Peptide.In some non-limiting embodiments, the amino acid sequence of the people CD3 ζ intracellular domain is selected from SEQ ID No.13 Shown amino acid sequence.
In an illustrative embodiments, the boot sequence is as shown in SEQ ID No.3.
In an illustrative embodiments, the anti human CD 19 single-chain antibody amino acid sequence is as shown in SEQ ID No.4. In an illustrative embodiments, the anti human CD 19 single-chain antibody amino acid sequence is as shown in SEQ ID No.5.In an example In property embodiment, the anti human CD 19 single-chain antibody amino acid sequence is as shown in SEQ ID No.6.In an exemplary embodiment party In formula, the anti human CD 19 single-chain antibody amino acid sequence is as shown in SEQ ID No.7.In an illustrative embodiments embodiment In, the amino acid sequence of the people CD8 hinge area is as shown in SEQ ID No.8；
In an illustrative embodiments embodiment, the amino acid sequence of the people CD8 transmembrane region such as SEQ ID No.9 institute Show；
In an illustrative embodiments embodiment, the amino acid sequence of the people 4-1BB intracellular domain such as SEQ ID Shown in No.10；
In an illustrative embodiments embodiment, the amino acid sequence of the CD28 structural domain such as SEQ ID No.11 institute Show；
In an illustrative embodiments, the amino acid sequence of the CD3 ζ structural domain is as shown in SEQ ID No.12.
In an illustrative embodiments, the amino acid sequence of the CD3 ζ structural domain is as shown in SEQ ID No.13.
In a non-limiting embodiment, targeting is recombination in conjunction with the specific chimeric antigen receptor (CAR) of people CD19 Expression is expressed by carrier.
In certain unrestricted embodiments, the targeting of the application combines the intracellular domain of the CAR of people CD19 It include also at least one costimulatory signal conducting region, it includes at least one costimulations that can provide best lymphocyte activation to match Body molecule.The effector cell function of itself and CAR combined signal induction CAR+T cell.
Wherein, the costimulation ligand includes but is not limited to CD80, CD86, CD70, OX40L, 4-1BBL, ICOSL.
In some non-limiting embodiments, targeting includes costimulation in conjunction with the intracellular domain of the CAR of people CD19 Signal transduction area, the costimulatory signal conducting region, include two kinds of costimulatory molecules: CD28 and 4-1BB or CD28 and OX40 or 4-1BB and ICOS.
In a non-limiting embodiment, the targeting of the application includes in conjunction with the CAR intracellular domain of people CD19 4-1BB polypeptide.4-1BB can be used as tumor necrosis factor (TNF) ligand and have stimulating activity.4-1BBL can be covalently attached 5 ' ends of the extracellular antigen binding structural domain arrived.Alternatively, the 3 ' of the intracellular domain that 4-1BBL can be covalently attached to End.
In in the unrestricted embodiment of the application, 4-1BB polypeptide is selected from the amino acid sequence of SEQ ID NO:10.
In certain non-limiting embodiments, CAR also may include that antigen-binding domains are connected to transmembrane domain Spacer region (spacer).Spacer region can be it is sufficiently flexible, to allow antigen-binding domains to orient in different directions, In favor of antigen recognizing.Spacer region can be the area CH2CH3 of hinge area or immunoglobulin from IgG1 and the portion of CD3 Point.
In certain non-limiting embodiments, the intracellular domain of CAR may include that can activate or stimulate cell (example Such as, the cell of lymphoid, such as T cell) people's CD3 ζ polypeptide.CD3 ζ includes 3 ITAM, and will swash after combining antigen Signal living is transmitted to cell (for example, the cell of lymphoid, such as T cell).
In certain unrestricted embodiments, the intracellular domain of CAR also includes at least one costimulatory signal Conducting region, it includes the costimulatory molecules that at least one can provide best lymphocyte activation." costimulation point used herein Son " refers to the cell surface molecule needed for effective response of the lymphocyte to antigen in addition to antigen receptor or its ligand.Extremely A few costimulatory signal conducting region may include CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, or combinations thereof.Altogether Stimulation molecule can be in conjunction with costimulation ligand, and costimulation ligand is the protein expressed on cell surface, combine its by Costimulation response is generated when body, that is, realizes that the intracellular of provided stimulation is answered when its CAR molecule of antigen binding.Costimulation is matched Body includes but is not limited to CD80, CD86, CD70, OX40L, 4-1BBL, ICOSL.As an example, 4-1BB ligand (i.e. 4- 1BBL) in combination with 4-1BB (also referred to as " CD137 ") to provide Intracellular signals, CAR+T cell is induced with CAR combined signal Effector cell function.
In some embodiments, the costimulatory signal conducting region of the intracellular domain of CAR includes two kinds of costimulations point Son: CD28 and 4-1BB.
4-1BB can be used as tumor necrosis factor (TNF) ligand and have stimulating activity.It is bad that 4-1BB can be used as tumour Necrosis factor (TNF) ligand simultaneously has stimulating activity.In unrestricted embodiment, 4-1BB polypeptide has SEQ ID NO: The amino acid sequence of 10 continuous part, the length is at least 20 or at least 30 or at least 40 or at least 50, and at most 255 amino acid.Alternatively or in addition, the amino acid sequence of 4-1BB polypeptide is SEQ in unrestricted various embodiments The amino acid of ID NO:10.In one embodiment, the CAR intracellular domain of theme disclosed by the invention includes 4-1BB Polypeptide.
The third aspect, this application provides the specific chimerics that targeting described in a kind of coding second aspect combines people CD19 The nucleic acid molecules of antigen receptor, the nucleic acid molecules include from 5 ' to the 3 ' nucleotide for the encoding leader sequence being sequentially connected in series Sequence, the nucleotide sequence of the anti-CD19 single-chain antibody of coding, the nucleotide sequence of encoding transmembrane domain and coding intracellular signal The nucleotide sequence of structural domain.
In some embodiments, the intracellular signal structural domain includes immunoreceptor tyrosine activating motif and costimulation Signal domain.
In some illustrative embodiments, the nucleic acid molecules include that ' to 3, ' what is be sequentially connected in series is separately encoded from 5 Anti human CD 19 single chain antibody sequence described in boot sequence, the application first aspect, people CD8 hinge legion sequence, people's CD8 transmembrane region The nucleotide sequence of sequence, people's 4-1BB intracellular domain sequence and people's CD3 ζ intracellular domain.
In some illustrative embodiments, the nucleic acid molecules include that ' to 3, ' what is be sequentially connected in series is separately encoded from 5 The nucleosides of the nucleotide sequence of the single-chain antibody of anti human CD 19 described in boot sequence, the application first aspect, people's CD8 hinge area The nucleotide of acid, the nucleotide of people's CD8 transmembrane region, the nucleotide sequence of people's CD28 intracellular domain and people's CD3 ζ intracellular domain Sequence.
In some embodiments, the nucleic acid molecules of specific chimeric antigen receptor of the targeting in conjunction with people CD19 also wrap Include the nucleotide sequence of coding hinge area.
In some embodiments, the transmembrane domain includes transmembrane region.
In some embodiments, the transmembrane region includes the transmembrane structure CD8 area, the transmembrane structure CD28 area, CD3 ζ cross-film Any one of structural area, the transmembrane structure CD4 area, the transmembrane structure 4-1BB area, the transmembrane structure OX40 area, the transmembrane structure ICOS area,
In some embodiments, the immunoreceptor tyrosine activating motif includes the intracellular signal structural domain of CD3 ζ chain Or Fc ε RI γ intracellular signal structure.
In some embodiments, the costimulatory signal domain includes CD28 intracellular signal structural domain, CD137/4-1BB born of the same parents At least one of interior signal domain, CD134/OX40 intracellular signal structural domain, ICOS intracellular signal structural domain.
In some embodiments, the hinge area is selected from CD8 α hinge area, IgG hinge area, or comprising all or in part Immunoglobulin variant by one or more amino acid modifications of hinge area or hinge area.
In some embodiments, the nucleotide of the encoding leader sequence as shown in SEQ ID No.14 or with SEQ ID Nucleotide sequences homologous shown in No.14.
In some embodiments, the coding anti human CD 19 single-chain antibody nucleotide sequence is as shown in SEQ ID No.15 Or with nucleotide sequences homologous shown in SEQ ID No.15.
In one embodiment, the coding anti human CD 19 single-chain antibody nucleotide sequence as shown in SEQ ID No.16 or with Nucleotide sequences homologous shown in SEQ ID No.16.
In one embodiment, the coding anti human CD 19 single-chain antibody nucleotide sequence as shown in SEQ ID No.17 or with Nucleotide sequences homologous shown in SEQ ID No.17.
In one embodiment, the coding anti human CD 19 single-chain antibody nucleotide sequence as shown in SEQ ID No.18 or with Nucleotide sequences homologous shown in SEQ ID No.18.
In some embodiments, the nucleotide sequence of the people CD8 of the coding hinge area is as shown in SEQ ID No.19 Or with nucleotide sequences homologous shown in SEQ ID No.19.
In some embodiments, the nucleotide sequence of the people CD8 of the coding transmembrane region is as shown in SEQ ID No.20 Or with nucleotide sequences homologous shown in SEQ ID No.20,
In some embodiments, the nucleotide sequence of the encoding human 4-1BB intracellular domain such as SEQ ID No.21 It is shown or with nucleotide sequences homologous shown in SEQ ID No.21.
In some embodiments, the nucleotide sequence of the encoding human CD28 intracellular domain such as SEQ ID No.22 institute Show or with nucleotide sequences homologous shown in SEQ ID No.22.
In some embodiments, the nucleotide sequence of the encoding human CD3 ζ intracellular domain such as SEQ ID No.23 institute Show or with nucleotide sequences homologous shown in SEQ ID No.23.
In some embodiments, the nucleotide sequence of the encoding human CD3 ζ intracellular domain such as SEQ ID No.24 institute Show or with nucleotide sequences homologous shown in SEQ ID No.24.
Fourth aspect, this application provides a kind of Chimeric antigen receptor comprising the application second aspect or the application thirds The recombinant vector or expression plasmid of the nucleic acid of aspect.
The gene modification of immune response cell (for example, T cell, CTL cell, NK cell) can by with recombinant DNA or RNA construct transduces substantially homologous cell composition to realize.In one embodiment, carrier is retrovirus vector DNA or RNA construct can be imported host cell gene group by body (for example, γ retrovirus or slow virus).For example, compiling The polynucleotides of the single-chain antibody of code anti human CD 19, targeting people CD19 specific C AR can be cloned into retroviral vector, And expression can be driven from its endogenesis promoter, retrovirus long terminal repeats or from the internal promoter of substitution.
Non-virus carrier or RNA can also be used.Random chromosomal integration or targeted integration can be used (such as using nucleic acid Enzyme, transcriptional activation increment effector nuclease (TALEN), the short palindrome of Zinc finger nuclease (ZFN) and/or regular cluster interval repeat (CRISPR) or transgene expression (such as using natural or chemical modification RNA).
In some embodiments, the carrier be selected from γ-retroviral vector, slow virus carrier, adenovirus vector, Adeno-associated virus vectors.In an illustrative embodiments, the carrier is γ-retroviral vector.
5th aspect, this application provides a kind of promoters, for constructing recombinant vector described in the application fourth aspect With expression the application second aspect described in targeting combine people CD19 specific chimeric antigen receptor, the promoter include but It is not limited to nucleotide sequence EF1alpha promoter as shown in SEQ No.25 and/or the starting of the EFS as shown in SEQ No.26 Son.
In an exemplary preferred embodiment, it is described for for construct recombinant vector described in the application fourth aspect and It is such as SEQ that targeting described in the application second aspect, which is expressed, in conjunction with the promoter of the specific chimeric antigen receptor of people CD19 EFS promoter shown in No.26.
In an exemplary preferred embodiment, it is described for for construct recombinant vector described in the application fourth aspect and It is such as such as SEQ that targeting described in the application second aspect, which is expressed, in conjunction with the promoter of the specific chimeric antigen receptor of people CD19 EF1alpha promoter shown in No.25.
6th aspect, is that can express target described in second aspect of the present invention this application provides a kind of recombinant virus To CD19 specific chimeric antigen receptor and the virus of immune response cell can be infected.
In some embodiments, the immune response cell be cytotoxic T lymphocyte, NK cell, NKT cell or Helper T lymphocyte etc..
In the exemplary embodiment, the immune response cell is cytotoxic T lymphocyte.
In some embodiments, the virus is slow virus, adenovirus, adeno-associated virus or retrovirus etc..
In an illustrative embodiments, the virus is slow virus.
In an illustrative embodiments, the virus is retrovirus.
7th aspect, this application provides a kind of immune response cells of isolated modification, and it includes the application second party Targeting described in face combines the Chimeric antigen receptor of people CD19, the recombinant vector as described in fourth aspect present invention or expression matter Grain conversion gained.
For the initial gene modification of cell to provide targeting people CD19 specific immune response cell, usually using reverse Record viral vectors is transduceed, however any other suitable viral vectors or non-viral delivery systems can be used.For thin The subsequent gene of born of the same parents is modified to provide the cell comprising the antigen submission compound containing at least two costimulation ligands, reverse transcription Viral gene transfer (transduction) is also demonstrated that it is effective.Retroviral vector and combining for suitable assembly line are also suitable , wherein capsid protein is functional for infection people's cell.
In some embodiments, the immune response cell includes cytotoxic T lymphocyte, NK cell, NKT cell Or helper T lymphocyte.
In some embodiments, the immune response cell also includes the costimulation ligand of at least one external source.
Possible transduction method further includes by the Co-culture of cell and production cell.Transduction viral vectors can be used for Costimulation ligand (such as 4-1BBL and IL-12) is expressed in immune response cell.Preferably, the carrier of selection shows high sense Contaminate efficiency and stable integration and expression.
In some embodiments, it is preferred ground, at least one costimulation ligand be selected from 4-1BBL, CD80, CD86, CD70, OX40L, ICOSL and combinations thereof, or it is highly preferred that the costimulation ligand is 4-1BBL.
In some embodiments, the immune response cell is selected from T cell, natural kill (NK) cell, cytotoxic T Lymphocyte (CTL), regulatory T cells, human embryo stem cell and the multipotential stem cell that lymphoid cell can be divided into, preferably T Cell and natural kill (NK) cell, more preferably T cell.
It can use the multiple T cell subgroups separated for the carrier transduction of CAR expression from patient.
The disclosed modification immune response cell of 7th aspect can be other with the extracellular mark of expression specificity combination people CD19 Structural domain, for treating or preventing tumor formation, immunity disease or anti-aging.
In one exemplary embodiment, wherein the immune response cell of the modification is CAR-T cell.
It can use the central memory-type T of the genetic modification of targeting people CD19 single-chain antibody Chimeric antigen receptor preparation modification Cell, it is then stored refrigerated.
Eighth aspect, this application provides immune the answering of the isolated Chimeric antigen receptor modification of the 7th aspect of the application Answer the preparation method of cell, comprising the following steps:
Firstly, nucleic acid molecules described in the third aspect are connected in expression vector by way of molecular cloning, obtain Target the expression vector of the specific chimeric antigen receptor of CD19；
Then the CAR expression vector of the specificity of the targeting CD19 of acquisition is transfected into 293T cell, obtains virus liquid；
Finally with the virus liquid infection immunity responsive cell, the target of expression second aspect is obtained from metainfective cell The immune response cell of modification in terms of the 7th of the specific chimeric antigen receptor of CD19.
In some non-limiting embodiments, the immune response cell that the present invention modifies can be the thin of lymphoid Born of the same parents.The cell of the lymphoid is selected from B, T and natural kill (NK) cell, provides the generation of antibody, cell immune system Adjust, the detection of foreign substance in blood, to functions such as the detections of host's foreign cell.The cell of lymphoid it is non-limiting Example includes T cell, natural kill (NK) cell, cytotoxic T lymphocyte (CTL), regulatory T cells, embryonic stem cell With multipotential stem cell (for example, multipotential stem cell that lymphoid cell can be divided into).
In some embodiments, the immune response cell is selected from T cell, natural kill (NK) cell, cytotoxic T Lymphocyte (CTL), regulatory T cells, human embryo stem cell and the multipotential stem cell that lymphoid cell can be divided into, preferably T Cell or natural kill (NK) cell.
In some exemplary embodiments, T cell is mature lymphocyte in thymus gland, and is mainly responsible for cell What is mediated is immune.T cell participates in acquired immune system.
In some non-limiting embodiments, T cell includes but is not limited to t helper cell, cytotoxic T cell, note Recall T cell (including Central memory T cell, stem-like cell memory T cell (or dry sample memory T cell) and two kinds of effect Memory T cell (for example, TEM cell and TEMRA cell), regulatory T cells (also referred to as suppressor T lymphocyte), natural killer T are thin Born of the same parents, mucous membrane associated constant T cell and gamma delta T cells.In some embodiments, the T cell expression Foxp3 of CAR is expressed with reality Now phenotype is adjusted with maintenance T.
The immune response cell of the modification of the application can further include at least one exogenous costimulatory ligand, make Immune response cell is obtained to co-express or be induced to co-express the single-chain antibody target based on anti human CD 19 exogenously exogenously To the specific chimeric antibody and at least one exogenous costimulatory ligand of people CD19.Target people CD19 specific C AR and Interaction between at least one costimulation ligand provides the complete activation for immune response cell (for example, T cell) For important non-antigen specific signals.In some embodiments, at least one costimulation ligand be selected from 4-1BBL, CD80, CD86, CD70, OX40L, ICOSL, and combinations thereof.In one embodiment, costimulatory ligand is 4-1BBL.
In a preferred embodiment, the immune response cell of the isolated modification is T cell.
In a preferred embodiment, the immune response cell of the isolated modification is natural kill (NK) cell.
In some non-limiting embodiments, the immune response cell (such as T cell) of the isolated modification can be with It is self, non-self (such as allogeneic) or progenitor cells or stem cell in vitro derived from engineering.
9th aspect, this application provides a kind of pharmaceutical compositions, and it includes a effective amount of as described in one aspect of the present invention Targeting combination people CD19 single chain antibody protein or its functional variant thereof, second aspect described in targeting in conjunction with the chimeric of people CD19 Nucleic acid described in antigen receptor, third aspect present invention or the 7th aspect the isolated modification immune response cell and Pharmaceutically acceptable excipient.
Tenth aspect, this application provides a kind of for treating or preventing the subject's of disease, discomfort or health disorders Tumor formation, pathogenic infection, autoimmune disease, allograft or graft rejection or anti-aging kit, packet Containing targeting combination people CD19 single chain antibody protein described in first aspect present invention and its functional variant thereof, second can be described Target separation described in nucleic acid described in the Chimeric antigen receptor in conjunction with people CD19, third aspect present invention or the 7th aspect The immune response cell of modification.
In some embodiments, the kit also includes to treat or prevent disease, discomfort using immune response cell Or the printed instructions of the subject of health disorders.
Tenth on the one hand, and this application provides targeting combination people's CD19 single chain antibody proteins described in first aspect present invention And its it recombinates and carries described in the specific chimeric antigen receptor of targeting combination people CD19, fourth aspect described in variant, second aspect Body or expression plasmid, the immune response cell of isolated modification described in the 6th aspect recombinant virus, the 7th aspect, the 9th The composition of aspect or the kit of the tenth aspect are treated in preparation, or in the product of prevention disease, discomfort or health disorders Using, wherein described treat or prevent includes step to the specific phase with CD19 and anti human CD 19 single chain antibody protein Patient's application of disease caused by interaction is described in a effective amount of the 7th aspect of the application comprising targeting to treating or preventing The Chimeric antigen receptor T cell (CAR-T cell) of CD19 single-chain antibody.Described, uncomfortable or health disorders and CD19 and anti-human The specificity interaction of CD19 single-chain antibody ligandin is related.
In some embodiments, the disease, discomfort or health disorders include that tumor is formed, infected, autoimmune disease Disease, allograft, graft rejection, aging.
The present invention is based on the specific chimeric antigen receptors (CAR) that anti human CD 19 single-chain antibody molecules construct targeting people CD19 And the T cell (CAR-T cell) of CAR modification, novel C AR-T cell can be effectively targeted to attack kinds of tumor cells, It can be used to prepare the preparation for treating tumour, the especially pharmaceutical preparation of the tumour of positive expression CD19.
The present inventor has been surprisingly found that under study for action, is divided using what the present invention constructed comprising specific recognition people CD19 From the single-chain antibody based on anti human CD 19 Chimeric antigen receptor modification T cell, preparation method step is simple, effect target ratio When for 10:1, with up to 40%~60% tumor cell destruction, immunocyte depositing in patient's body can be obviously prolonged Live time, enhancing immunocyte target recognition of tumor cell are especially expressed the ability of the tumour of CD19, are reinforced thin to lymthoma The specific killing activity of born of the same parents, the refractory Lymphoma of late recurrent is through feeding back latter two month follow-up CT scan of KD-019CAR-T Show that Lymphoma tumor load reduces nearly 71%~98%, the discovery of whole blood CD19+B cell is nearly no detectable tumour Cell.The immune response cell of the Chimeric antigen receptor of targeting CD19 based on anti human CD 19 single-chain antibody of the invention is treatment Tumour provides a kind of new Scheme Choice, has good industrial application prospect.
This application provides the methods of the disease for the immune response for using such cell therapy for example to need to enhance.
Detailed description of the invention
The slow virus carrier structural schematic diagram that Fig. 1 is used as the exemplary present invention.
Fig. 2 is the schematic diagram of the order of connection of Chimeric antigen receptor each section in Examples 1 and 2.
1 is the amino acid sequence of people CD3 ζ structural domain, and 2 be the amino acid sequence of people 4-1BB intracellular domain, and 3 be people CD8 The amino acid sequence of transmembrane region, 4 be CD8 hinge area amino acid sequence, 5 be anti human CD 19 single-chain antibody amino acid sequence, 6 It is EF1alpha promoter for boot sequence, 7,8 be EFS promoter.
Fig. 3 is Healthy People donor peripheral blood T cell purity flow cell detection results in embodiment 2.
Fig. 4 is the FCM analysis result in embodiment 5 to CAR-T cell activity.
A. blank control group: without the T cell of virus infection liquid；B.KD-022 control group: the spy of targeting people CLDN18.2 Anisotropic CAR-T cell；C.KD-019: the specific C AR-T cell of targeting people CD19.
Fig. 5 is the FCM analysis result in embodiment 5 to KD-019CAR developed by molecule.
A. blank control group: without the T cell of virus infection liquid；B.KD-022 control group: the spy of targeting people CLDN18.2 Anisotropic CAR-T cell；C.KD-019: the specific C AR-T cell of targeting people CD19.
Fig. 6 is the specific KD-019CAR-T that the targeting CD19 of the application is measured by target cell of lymphoma cell Raji Cells against tumor cells killing rate test result.
Fig. 7 is the specific KD-019CAR-T that the targeting CD19 of the application is measured by target cell of lymphoma cell Raji The animal model for tumour of cell kills test result.
Fig. 8 is TNF α (Fig. 8 A) when KD-019CAR-T targets Raji tumour cell, IFN-γ (Fig. 8 B), IL-10 (figure 8C), IL-2 (Fig. 8 D) cytokine release testing result.
Fig. 9 is the test result that KD-019CAR toxicity and safe dose experiment are carried out with B-NSG animal.
A. the test result of toxicological experiment；B. the test result of safe dose experiment.
Figure 10 is that the test result that experiment is retained in KD-019CAR body is carried out with B-NSG animal.
Figure 11 is the FCM analysis result of Lymphoma T cell purity in embodiment 2.
Figure 12 is the FCM analysis result in embodiment 12 to KD-019CAR developed by molecule.
Blank control group: without the T cell of virus infection liquid；KD-019 group: the specific C AR-T cell of CD19 is targeted.
CAR-T groups of testing results of Lymphoma peripheral blood after Figure 13 KD-019CAR-T is fed back.
Lymphoma whole blood hs-CRP testing result after Figure 14 KD-019CAR-T is fed back.
Lymphoma whole blood levels of iron Protein Detection result after Figure 15 KD-019CAR-T is fed back.
Lymphoma CT scan inspection result (one) after Figure 16 KD-019CAR-T is fed back.
Lymphoma CT scan inspection result (two) after Figure 17 KD-019CAR-T is fed back.
Specific embodiment
Unless otherwise defined, all technical and scientific terms used herein is logical with technician of the art The meaning understood.
Material used in following example, reagent etc., are commercially available unless otherwise specified.
Embodiment 1
The expression plasmid of the specific chimeric antigen receptor of preparation targeting CD19
Step 1) targets the determination of the amino acid sequence of the specific chimeric antigen receptor of CD19
Firstly, searching FMC63-28Z clone's (target from the Genbank database of U.S. national library of medicine NCBI To the Chimerical receptor sequence of CD19 antigen protein) full length amino acid sequence (as shown in SEQ ID No.1) and full length nucleotide Sequence (as shown in SEQ ID No.2).
Secondly, the specific chimeric antigen receptor of building targeting CD19.
It is specific as follows:
Target the amino acid sequence of CD19 specific C AR molecule: from aminoterminal to c-terminus, by guidance peptide amino acid sequence (as shown in SEQ ID No.3), anti human CD 19 single-chain antibody amino acid sequence (such as SEQ ID No.4, shown), people's CD8 hinge Region amino acid sequence (as shown in SEQ ID No.8), the amino acid sequence (as shown in SEQ ID No.9) of people's CD8 transmembrane region, people 4-1BB intracellular domain amino acid sequence (as shown in SEQ ID No.10) and people's CD3 ζ domain amino acid sequence (such as SEQ Shown in ID No.12) it is sequentially connected in series.
Target the nucleotide sequence of CD19 specific C AR molecule: from 5 ' ends to 3 ' ends, by the nucleotide of encoding leader sequence Sequence (as shown in SEQ ID No.14), encode anti human CD 19 single chain antibody sequence nucleotide sequence (such as SEQ ID No.15, It is shown), the nucleotide sequence (as shown in SEQ ID No.19) of encoding human CD8 hinge area, encoding human CD8 transmembrane region nucleotide Sequence (as shown in SEQ ID No.20), encoding human 4-1BB intracellular domain nucleotide sequence (such as SEQ ID No.21 institute Show) and coding CD3 ζ structural domain nucleotide sequence (as shown in SEQ ID No.23) be sequentially connected in series.
The building and identification of the plasmid of the specific C AR molecule of step 2) expression targeting CD19
The nucleotide sequence of the specific C AR molecule of full genome synthesis targeting CD19, is connected by way of molecular cloning To slow virus carrier lentiGuide-Puro (Addgene), the overall length CAR sequence expression cassette of single encoder block is constructed, is utilized EF1alpha promoter (shown in sequence table SEQ ID No.25) is expressed.Specific steps are as follows:
Full genome synthesis contains promoter, boot sequence, anti human CD 19 single-chain antibody, CD8 hinge area, CD8 transmembrane region, 4- Nucleotide sequence (the life together with Nanjing of 1BB intracellular domain and the specific C AR molecule of the targeting CD19 of CD3 ζ structural domain Object), it utilizes
Primer
5’-cactttggcgccggctcgagggggcccgggtgcaaagatggataaagttttaaacagagagga-3’ (sequence table SEQ ID No.27) or
5 '-cactttggcgccggctcgagggggcccgggtaggtcttgaaaggagtgggaattgg ctcc-3 ' (sequences List SEQ ID No.28) and
Primer 5'-tccagaggttgattgtcgacttaacgcgtttagcgagggggcagggcctgcat gtgaag-3'
The artificial synthesized CAR molecular sequences of (sequence table SEQ ID No.29) PCR amplification, through Axygen plastic recovery kit (Hangzhou pool weighing apparatus) recycling, with the carrier lentiGuide-Puro (Addgene) digested by Restriction enzyme Sma I and MluI Carry out homologous recombination connection.The system and condition of specific recombination connection reaction are as follows:
Recombinate linked system:
The 5 μ l of PCR product of glue recycling, SmaI the and MluI digestion lentiGuide-Puro plasmid (Addgene) of glue recycling 3μl；4X 1402QuickCloning Kit (Nanjing Jino beauty) 5 μ l；7 μ l of deionized water；20 μ l of coupled reaction system volume；
Recombination condition of contact: above-mentioned reaction system is placed in 50 DEG C of water-baths, reacts 15min postposition 1min. on ice.
10ul recombination connection product is taken to convert through competence Stbl3, steps are as follows for specific conversion operation.
5 μ l connection products are added to the competent cell of 50 μ l (Stbl3 is bought from Invitrogen company, the U.S.) In, ice bath 30min, 42 DEG C of 45s, ice bath 2min, after 500 μ l nonreactive LB liquid mediums are then added, 37 degrees Celsius, 200rpm Shake culture 40min is coated with the LB solid plate of ammonia benzyl resistance, in 37 degrees Celsius of incubators overnight.Single colonie is waited to occur Afterwards, 5 bacterium colonies being of moderate size of picking extract plasmid, and are sent to the sequencing of business sequencing company (Nanjing is biological together), will be sequenced As a result comparing with the anti human CD 19 single-chain antibody specific C AR molecular sequences being fitted to confirms that sequence is completely correct, it was demonstrated that obtains The plasmid (abbreviation KD-019CAR slow virus carrier, EFS promoter) of the specific C AR molecule of expression targeting CD19.
Step 3) targets the extraction and purifying of the specific C AR expression plasmid of CD19
By the Stbl3 bacterial strain of the specific C AR developed by molecule plasmid of the targeting CD19 constructed in step 2) in LB culture medium Middle mass propgation carries out the extracting of high-purity endotoxin-free using Qiagen Plasmid Midi Kit (German Qiagen company), Dye is fully felt.Specific steps are as follows:
1. the bacterium solution for taking 150ml to be incubated overnight is added in centrifuge tube, 6000 × g is centrifuged 15 minutes, as far as possible absorption supernatant (bacterium solution precipitating can be collected into a centrifuge tube by being repeatedly centrifuged when bacterium solution is excessive).
2. 4ml solution P1 (please first check whether and RNase A has been added) is added into the centrifuge tube there are bacterial sediment, make With pipettor or the thorough suspended bacterial precipitating of turbula shaker.
3. 4ml solution P2 is added into centrifuge tube, leniently spinning upside down 4-6 times cracks thallus sufficiently, room temperature (15- 25 DEG C) it is incubated for 5 minutes.
4. the solution P3 of 4ml pre-cooling is added into centrifuge tube, leniently spins upside down 4-6 times, mix well immediately, on ice It is incubated for 5 minutes.
5. centrifuge tube is put into 4 DEG C of ultracentrifuges, 20000 × g is centrifuged 10 minutes.
6. column equilibration: 4ml equilibrium liquid QBT is added to adsorption column, stands, until liquid flows completely out.
7. the supernatant collected in step 5 is transferred in adsorption column, stand, until liquid is completely into resin medium.
8. the rinsing liquid QC of 10ml is added into adsorption column, stand, until liquid flows completely out, repetitive operation 1 time.
9. the eluent QF of 5ml is added into adsorption column, it is collected into 15ml centrifuge tube.
10. the isopropanol of 3.5ml is added into centrifuge tube, mix, 4 DEG C of >=15000 × g are centrifuged 30 minutes, discard Clearly.
11. the ethanol solution of 2ml 70% is added into the centrifuge tube precipitated there are DNA, >=15000 × g is centrifuged 10 points Clock discards supernatant.
12. suggesting being placed at room temperature for there are the centrifuge tubes that DNA is precipitated to uncap 5-10 minutes, the solution of suitable volumes being added DNA is re-dissolved, the solution containing DNA is transferred completely into new 1.5ml centrifuge tube, -20 DEG C of preservations.
The expression plasmid of the specific chimeric antigen receptor of the preparation targeting of embodiment 2 CD19
In addition to targeting anti human CD 19 single chain antibody sequence in the amino acid sequence of CD19 specific C AR molecule in step 1 (as shown in SEQ ID No.5), the amino acid sequence of people's CD3 ζ structural domain is as shown in SEQ ID No.13, targeting CD19 specificity The nucleotide sequence of anti human CD 19 single chain antibody sequence is encoded in the nucleotide sequence of CAR molecule (such as SEQ ID No.16 institute Show),
Building and the identification part of the plasmid of the specific C AR molecule of targeting CD19 are expressed with step 2)
The nucleotide sequence of the specific C AR molecule of full genome synthesis targeting CD19, is connected by way of molecular cloning To slow virus carrier lentiGuide-Puro (Addgene, Fig. 1), the overall length CAR sequence expression cassette rank of single encoder block is constructed Section, using EFS promoter (sequence table SEQ ID No.26), remaining is same as Example 1.
The expression plasmid of the specific chimeric antigen receptor of the preparation targeting of embodiment 3 CD19
In addition to targeting anti human CD 19 single chain antibody sequence in the amino acid sequence of CD19 specific C AR molecule in step 1 (as shown in SEQ ID No.6) is targeted and is encoded anti human CD 19 single-chain antibody sequence in the nucleotide sequence of CD19 specific C AR molecule (as shown in SEQ ID No.17, remaining is same as Example 1 for the nucleotide sequence of column.
The expression plasmid of the specific chimeric antigen receptor of the preparation targeting of embodiment 4 CD19
In addition to targeting anti human CD 19 single chain antibody sequence in the amino acid sequence of CD19 specific C AR molecule in step 1 (as shown in SEQ ID No.7), the amino acid sequence of people's CD3 ζ structural domain is as shown in SEQ ID No.13, targeting CD19 specificity In the nucleotide sequence of CAR molecule encode anti human CD 19 single chain antibody sequence nucleotide sequence (as shown in SEQ ID No.18, Remaining is same as Example 1.
Embodiment 5
Healthy human peripheral blood T cell is separately cultured
The fresh peripheral blood for taking health donors separates fresh peripheral blood mononuclear cells by density gradient centrifugation；It is sharp again It (is bought from Invitrogen company, the U.S., product information is with the paramagnetic beads for being coupled anti-CD 3 antibodies and anti-CD28 antibodyHuman T-Activator CD3/CD28, article No.: 11161D) it is enriched with CD3+T cell specifically will It is (10~30) × 10 that peripheral blood mononuclear cells, which is diluted to concentration,6A individual cells/ml, then by magnetic bead and cell with the ratio of 3:1 Example mixes in culture dish, is incubated for 2~3 hours altogether at room temperature, utilizes magnetic particle collector (Magnetic particles Concentrator, abbreviation MPC, article No.: 12301D is bought from Invitrogen company, the U.S.) enrichment CD3+T cell.Finally By the CD3 of enrichment+T cell is resuspended in culture solution, and (from Life Technologies company, the U.S., product information is for purchase OpTmizerTMT-Cell Expansion SFM, A1048503), being adjusted to cell solubility is 1 × 106A/ml, finally 37 DEG C, 5%CO2It is cultivated 2 days in incubator.T cell purity utilizes anti-PE anti-human CD3 antibody by flow cytometry (from BioLegend company, the U.S., article No.: 300408) being detected, as the result is shown the Healthy People T after enrichment with magnetic bead for purchase Cell purity is more than 99% (Fig. 3 periphery blood T cell, control group are Raji cell).
The preparation of 6 virus liquid of embodiment
The specific C AR expression plasmid and packaging plasmid psPAX2 of the targeting CD19 that the step 3) of embodiment 1 obtains and VSVG, according to ratio polyethyleneimine transfection reagent (408727, the Sigma) transfection reagent of 10:8:5, cotransfection 293T is thin Born of the same parents are (for ATCC product, product number are as follows: CRL-3216TM)；The preparation method of specific packaging plasmid is referring to Lenti-X Packaging Single Shots (Takara) specification；Specific transfection procedure process transfects specification referring to Sigma.
Be changed within 6 hours after transfection complete medium (purchase from Life Technologies company, product article No.: 11995-065), after cultivating 48 hours and 72 hours, vial supernatant is collected respectively, and in 4 DEG C, 3000rpm is centrifuged 10~15 points 0.45 μm of membrane filtration of Zhong Houjing, finally in 4 DEG C, 25000rpm ultracentrifugation 2~3 hours, concentrating virus is dense by the virus of collection Contracting liquid is transferred to -80 DEG C of preservations.
Embodiment 7
Target the preparation of the specific C AR-T cell of CD19
The CD3 that Example 2 obtains+T cell, is inoculated into 24 orifice plates, and inoculum density is 1 × 105A cell/ml, 37 DEG C, 5%CO2(in general incubation time, guarantees to infect in virus liquid according to depending on concrete practice within about 24 hours for environment culture When cell confluency rate between 50-70%).The viral concentration liquid of Example 3, second day value according to MOI=1~10 will After Tissue Culture Flask is added in virus liquid, mouth is sealed, after being put into straight angle centrifuge, low speed (500g-1000g/min) centrifugation 30~60 Minute, it is then placed in 37 DEG C of cultures in incubator.Expression anti human CD 19 single-chain antibody CAR molecule is obtained after infection after 48 hours T cell KD-019CAR-T cell, i.e. novel C AR-T cell, CAR molecular structure such as Fig. 2, the function that can carry out next step are real It tests.
Wherein, A is containing EF1a long promoter CAR molecular structure；B is containing the short promoter CAR molecular structure of EFS.
Embodiment 8 is identified
It (is bought later from Biovision company, article No.: K315- using 7-AAD/CFSE cytotoxicity test kit 100), and according to the operational manual of the kit to the CAR-T cell prepared cell activity test is carried out.Fluidic cell knot Fruit shows that KD-019CAR-T cell activity is all larger than 98% (Fig. 4).
Then using the expression of CAR molecule after flow cytometry detection infection, concrete operations are as follows.
Blank control group: without the T cell of virus infection liquid.
KD-022 control group: the specific C AR-T cell of targeting people CLDN18.2.
KD-019 group: the specific C AR-T cell of targeting people CD19.
Collected cell to be detected in the preparation process of cell.
By above-mentioned experimental group and control group, PBS cleaning is resuspended in FACS liquid (containing 0.1% sodium azide and 0.4% afterwards twice The PBS of BSA) in.
MFab antibody (the Alexa for marking APC according to antibody specification647-AffiniPure F(ab') 2Fragment Goat Anti-Mouse IgG, 115-606-006, Jackson) it is added to groups of cells to be detected and control group Cell suspension in, 4 DEG C be incubated for 60 minutes.Staining cell is obtained using flow cytometer (BD FacsCanto II), and is used FlowJo software analyzes result.(another note: in Fig. 5, abscissa PE-A indicates to emit after APC is stimulated streaming result as shown in Figure 5 The fluorescence intensity of fluorescence (part received by PMT) out；Ordinate SSC-A represents the granularity of cell), control group In be nearly no detectable the expression of CAR molecule, CAR developed by molecule rate reaches 25.3% in KD-019 group.
The cell to be detected that can be seen that the collection of embodiment 4 from the streaming result of Fig. 5 expresses the special of targeting CD19 Sex-mosaicism antigen receptor.
Application examples is with its effect data
For trying tumor cell line (also referred to as target cell system): lymphoma cell Raji is (limited purchased from one hundred biotechnology of Nanjing section Company).
Next it (is bought from Biovision company, article No.: K315- using 7-AAD/CFSE cytotoxicity test kit 100) the specific C AR-T cell pair of the resulting targeting CD19 of embodiment 4, and according to the operational manual of the kit is assessed The killing situation of above-mentioned target cell system.
Specifically, each target cell system is after CSFE fluorescent staining, with every hole 2 × 104The inoculum density of a/ml It is laid in culture plate.
Each target cell system is arranged in correspondence with an experimental group and two control groups, wherein experimental group addition is real Apply the cell suspension of the specific C AR-T cell of the resulting targeting CD19 of example 4；Blank control group addition is no virus infection T cell (i.e. embodiment 2 obtain CD3+T cell)；The addition of KD-022 control group is the unrelated CAR-T for targeting CLDN18.2 Cell.
Above-mentioned experimental group, according to three different effect target ratios (10:1,5:1 and 1:1), by the targeting CD19 of embodiment 4 Specific C AR-T cell mixed with target cell.Herein, term " effect target ratio " refers to effector cell's (specificity of targeting CD19 CAR-T cell) with the quantity ratio of target cell (tumour cell).
Likewise, blank control group and KD-022 control group, also respectively according to three different effect target ratios by T cell and target Mixing with cells.
Supernatant is removed in centrifugation after culture 20 hours, is dyed after cell precipitate is cleaned with 7AAD, using flow cytometer (BD FacsCanto II) obtains staining cell, and analyzes result using FlowJo software.
Fig. 6 show the tumor cell destruction test result using lymphoma cell Raji as target cell.It can from Fig. 6 Out, the specific C AR-T cell KD-019 of the targeting CD19 of embodiment 4 has apparent lethal effect to lymphoma cell Raji (being apparently higher than two control groups), and imitating the corresponding tumor cell destruction of target ratio 10:1 is more than 50%.
Fig. 7 show CAR-T cell to the tumor suppression test result of lymphoma Raji transplantable tumor nude mice.It can be with from Fig. 7 Find out, the specific C AR-T cell KD-019 of the targeting CD19 of embodiment 4 has apparent inhibit to lymphoma Raji transplantable tumor Effect, in inoculation 30 days or so, two nude mice of control group were all dead, and survivorship curve statistical result showed KD-019CAR-T is thin Born of the same parents feedback group nude mice life span is obviously (Fig. 7) longer than control group.
Cytokine release detection: CAR-T cell and target cell co-culture, and step is the same, difference: effect target ratio be 5:1 and 5:0, and be inoculated with when killing and co-cultured CAR-T cell and target cell 12~16 hours with the T cell culture medium without IL-2, it uses Labelled amount ddH2O dissolves cytokine standards product (depending on labelled amount is because of cell factor), is placed at room temperature for 15~20min and guarantees sufficiently Dissolution extracts sample cell supernatant by standard items by gradient doubling dilution is recommended, and 10 times is diluted with ddH2O, respectively by standard items It is added in respective reaction hole, every 100 μ L of hole, is incubated at room temperature 1~3 hour with laboratory sample, configure 1 × cleaning solution, every hole is used 360ul cleaning solution cleans 4 times, and liquid in hole is patted dry, and every hole is added 200 μ L enzyme marks and detects antibody, and incubation at room temperature 1~3 is small When, 200 μ L chromogenic substrates are added in repetitive operation step 6), every hole, and room temperature is protected from light incubation 30~60 minutes, and it is whole that 50ul is added in every hole Only liquid, 450nm measure light absorption value, and Fig. 8 shows that compared with no addition target cell and corresponding control group, KD-019 group is adding After entering target cell Raji, TNF α (Fig. 8 A), IFN-γ (Fig. 8 B), IL-10 (Fig. 8 C), IL-2 (Fig. 8 D) expression exist more Apparent up-regulation.
Fig. 9 show influence of the KD-019CAR-T cell of various dose to mouse main organs and life cycle.From figure 9 as can be seen that the specific C AR-T cell KD-019 of the targeting CD19 of embodiment 4 will not cause mouse core, liver, lung, kidney etc. main Internal organs are wanted to be inflamed, oedema and necrosis (Fig. 9 A), and to not having any negative effect (Fig. 9 B) life cycle of mouse.
Figure 10 show KD-019CAR-T in the intracorporal retention time of mouse.From fig. 10 it can be seen that control group and KD- Amount retained drops to about 50% to 019CAR-T cell in vivo after injection mouse 10 days, falls to approximately 25% after 17 days, and 31 days After be substantially not detectable remaining CAR-T cell (Figure 10).
Embodiment 9
Lymphoma T cell is separately cultured
RosetteSep Human T Cell is added into blood for the fresh peripheral blood for taking lymthoma donor Enrichment Cocktail (it buys from STEMCELL Technologies, article No.: 15061), and incubation at room temperature 20~ Then 30min isolates and purifies fresh T cell by density gradient centrifugation；Recycling has been coupled anti-CD 3 antibodies and anti-CD28 (from thermofisher Scientific company, the U.S., product information is Dynabeads CD3/ to the paramagnetic beads of antibody for purchase CD28CTS, article No.: 40203D) activate CD3+T cell.Specifically, first magnetic bead is cultivated with 1mL 1 × DPBS or AIM-V Base is washed 2 times, and magnetic bead is collected (Magnetic particles concentrator, abbreviation MPC, goods using magnetic particle collector Number: 12301D is bought from Invitrogen company, the U.S.).Then by the T cell isolated and purified AIM-V complete medium (purchase Buy from thermofisher Scientific company, the U.S., product information AIM-V, A3021002) be diluted to concentration be (3~ 5)×106/ mL, then magnetic bead and cell are uniformly mixed in centrifuge tube with the ratio of 2~4:1.Finally by total cell density tune Whole is 1 × 106/ mL is placed in 37 DEG C, cultivates 2 days in 5%CO2 incubator point to 24 porocyte culture plates.T cell purity passes through Flow cytometry, using anti-PE anti-human CD3 antibody (purchase from BioLegend company, the U.S., article No.: 300408) into Row detection, the Lymphoma T cell purity after enrichment with magnetic bead is more than that 97% (Figure 11 peripheral blood in patients T is thin as the result is shown Born of the same parents, control group are Raji cell).
Embodiment 10
Virus needed for reference implementation example 6 prepares the isolated T cell of infection embodiment 9 under the conditions of GMP.
Embodiment 11 is identified
The isolated lymthoma of the virus infection embodiment 9 that reference implementation example 6 is obtained under the conditions of GMP using embodiment 10 is suffered from Person's T cell come prepare feed back Lymphoma needed for CAR-T cell.
The cell to be detected that can be seen that the collection of embodiment 4 from the streaming result of Figure 12 expresses 26.6% targeting The specific chimeric antigen receptor of CD19, and the expression of CAR molecule is nearly no detectable in control group.
Shown in Figure 13, CAR-T groups of reference implementation examples 8 of Lymphoma peripheral blood use mFab after KD-019CAR-T is fed back Antibody test result shows that peak value is arrived in amplification in the 7th day after KD-019CAR-T is fed back, and is fallen after rise within the 14th day, is returned again within the 28th day To the 7th day early period of position level, it is reduced within the 77th day 8% level.
The refractory Lymphoma peripheral blood of late recurrent completes T cell separation, virus infection, CAR-T under gmp conditions After preparation and related quality inspection, in the form of 67ml normal saline solution in 30 minutes through venous re-transfusion to patient's body, KD- 019CAR-T feeds back Whole blood analysis (golden domain medical test) and CT scan detection (Kunming Yan'an hospital) CAR-T feedback preceding the 26 days and feed back after the 28th day, the 77th day the results show that KD-019CAR-T feed back after the 7th day CD19+B tumour cell obviously subtract Few to disappear up to basic, CT scan tumor regression 71~98% is clinical close to complete incidence graph.
Lymphoma whole blood hs-CRP testing result is shown after KD-019CAR-T shown in Figure 14 is fed back, super quick The apparent increase in the 14th day after feedback of c reactive protein level, the 77th day super quick C in whole blood compared with feeding back first 26th day after feedback Reactive protein is horizontal not to have significant change substantially.
Lymphoma whole blood levels of iron Protein Detection is the results show that ferritin levels after KD-019CAR-T shown in Figure 15 is fed back The apparent increase in the 15th day after feedback, after feedback the 77th day with feed back the preceding smaller decline of ferritin levels in 26th day.
Lymphoma CT scan result (one) after KD-019CAR-T shown in Figure 16 is fed back, CAR-T are treated first 26 days, right Enlargement of lymph nodes (Figure 16 A) by lung, after treatment 28 days, lymph node reduces by right lung, and maximum perpendicular diameter product reduces 53% (figure 16B), after treating 77 days, maximum perpendicular diameter product reduces 71% (Figure 17 C) before relatively treating.
Lymphoma CT scan result (two) after KD-019CAR-T shown in Figure 17 is fed back, before CAR-T treatment, CAR-T is controlled It treats first 26 days, there are high-visible lesser tubercle (Figure 17 A) by right lung, and after treatment 28 days, lesser tubercle reduces, and maximum perpendicular diameter multiplies Product reduces 85% (Figure 17 B), and after 77 days, lesser tubercle is continued to zoom out, and maximum perpendicular diameter product reduces 98% or more.
It can be seen that the specificity of the targeting CD19 of the embodiment of the present invention 4 from above-mentioned Fig. 6-7 in vitro and in vivo studies result CAR-T cell is capable of the tumour cell of the specific recognition CD19 positive, has specifical and efficient target killing power.
It can be seen that the specific C AR- of the targeting CD19 of the embodiment of the present invention 4 from above-mentioned Figure 16-17 clinical study results T cell is capable of the lympha tumour of the specific recognition CD19 positive, and patient is through feeding back the targeting of synthesising biological technical optimization of the present invention 6x10^4/ kilograms of the KD-019CAR-T cell of CD19, after amounting to 3,000,000 cells, acquisition clinical symptoms are relieved within the 28th day, It obtains within 77th day extraordinary clinical part to alleviate, close to the complete incidence graph on clinical meaning.
Therefore, the present invention constructed by targeting CD19 specific chimeric antigen receptor and through its viral vector infection The specific C AR-T cell of targeting CD19 can be applied to treatment leukaemia, lymthoma and combinations thereof.
It should be appreciated that although this specification is described in terms of embodiments, but not each embodiment only includes one A independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should will say As a whole, the technical solution in each embodiment may also be suitably combined to form those skilled in the art can for bright book With the other embodiments of understanding.
The series of detailed descriptions listed above only for feasible embodiment of the invention specifically Protection scope bright, that they are not intended to limit the invention, it is all without departing from equivalent implementations made by technical spirit of the present invention Or change should all be included in the protection scope of the present invention.
Sequence table
<110>Nanjing Kai Di Biotechnology Co., Ltd
<120>specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinical application are targeted
<141> 2019-06-05
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Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
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Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
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Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
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Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
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Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
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Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Ile Glu
260 265 270
Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr
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Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro
290 295 300
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu
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Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
325 330 335
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
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Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
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Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
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Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
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Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
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Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
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Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
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Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
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Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
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Leu His Met Gln Ala Leu Pro Pro Arg
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atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120
gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180
aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240
ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300
gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360
ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420
ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480
ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540
gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600
agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660
tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720
tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780
acctcagtca ccgtctcctc agcggccgca attgaagtta tgtatcctcc tccttaccta 840
gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 900
cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 960
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc ccctcgc 1467
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<212> PRT
<213> Artificial Sequence
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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> 4
<211> 242
<212> PRT
<213> Artificial Sequence
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Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser
130 135 140
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
165 170 175
Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
195 200 205
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
210 215 220
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Thr
<210> 5
<211> 245
<212> PRT
<213> Artificial Sequence
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Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys
115 120 125
Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln
130 135 140
Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
145 150 155 160
Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln
165 170 175
Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu
180 185 190
His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
195 200 205
Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr
210 215 220
Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr
225 230 235 240
Lys Leu Glu Ile Thr
245
<210> 6
<211> 245
<212> PRT
<213> Artificial Sequence
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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser
130 135 140
Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu
180 185 190
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
225 230 235 240
Val Thr Val Ser Ser
245
<210> 7
<211> 242
<212> PRT
<213> Artificial Sequence
<400> 7
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 8
<211> 45
<212> PRT
<213> Artificial Sequence
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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> 9
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 9
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> 10
<211> 42
<212> PRT
<213> Artificial Sequence
<400> 10
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> 11
<211> 107
<212> PRT
<213> Artificial Sequence
<400> 11
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
35 40 45
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
50 55 60
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
65 70 75 80
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
85 90 95
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
100 105
<210> 12
<211> 112
<212> PRT
<213> Artificial Sequence
<400> 12
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> 13
<211> 112
<212> PRT
<213> Artificial Sequence
<400> 13
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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> 63
<212> DNA
<213> Artificial Sequence
<400> 14
atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccg 63
<210> 15
<211> 726
<212> DNA
<213> Artificial Sequence
<400> 15
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300
tacggtggta gctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360
ggtggaggag gcagcggcgg tggagggtct ggtggaggtg gttctgacat ccagatgaca 420
cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 480
agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 660
tacttttgcc aacagggtaa tacgcttccg tacacgttcg gaggggggac taagttggaa 720
ataaca 726
<210> 16
<211> 735
<212> DNA
<213> Artificial Sequence
<400> 16
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300
tacggtggta gctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360
ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggcgacatc 420
cagatgacac agactacatc ctccctgtct gcctctctgg gagacagagt caccatcagt 480
tgcagggcaa gtcaggacat tagtaaatat ttaaattggt atcagcagaa accagatgga 540
actgttaaac tcctgatcta ccatacatca agattacact caggagtccc atcaaggttc 600
agtggcagtg ggtctggaac agattattct ctcaccatta gcaacctgga gcaagaagat 660
attgccactt acttttgcca acagggtaat acgcttccgt acacgttcgg aggggggact 720
aagttggaaa taaca 735
<210> 17
<211> 735
<212> DNA
<213> Artificial Sequence
<400> 17
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggactaagt tggaaataac aggctccacc tctggatccg gcaagcccgg atctggcgag 360
ggatccacca agggcgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca 420
cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc 480
tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa 540
accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag 600
agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt 660
gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca 720
gtcaccgtct cctca 735
<210> 18
<211> 726
<212> DNA
<213> Artificial Sequence
<400> 18
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggactaagt tggaaataac aggtggagga ggcagcggcg gtggagggtc tggtggaggt 360
ggttctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggtc aaggaacctc agtcaccgtc 720
tcctca 726
<210> 19
<211> 135
<212> DNA
<213> Artificial Sequence
<400> 19
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 20
<211> 72
<212> DNA
<213> Artificial Sequence
<400> 20
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 21
<211> 126
<212> DNA
<213> Artificial Sequence
<400> 21
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 22
<211> 321
<212> DNA
<213> Artificial Sequence
<400> 22
attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60
catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120
tgggtgctgg tggtggttgg gggagtcctg gcttgctata gcttgctagt aacagtggcc 180
tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240
atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300
gacttcgcag cctatcgctc c 321
<210> 23
<211> 336
<212> DNA
<213> Artificial Sequence
<400> 23
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 24
<211> 336
<212> DNA
<213> Artificial Sequence
<400> 24
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 25
<211> 1259
<212> DNA
<213> Artificial Sequence
<400> 25
tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg 60
tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 120
cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc 180
gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 240
gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 300
ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 360
gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc 420
ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc 480
gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc 540
ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg 600
ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg 660
gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt 720
cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg 780
gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa 840
ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg 900
cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac 960
aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg 1020
cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg 1080
gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc 1140
cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt 1200
tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1259
<210> 26
<211> 256
<212> DNA
<213> Artificial Sequence
<400> 26
taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg 60
cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc ctagagaagg 120
tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt 180
gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt 240
gccgccagaa cacagg 256
<210> 27
<211> 63
<212> DNA
<213> Artificial Sequence
<400> 27
cactttggcg ccggctcgag ggggcccggg tgcaaagatg gataaagttt taaacagaga 60
gga 63
<210> 28
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 28
cactttggcg ccggctcgag ggggcccggg taggtcttga aaggagtggg aattggctcc 60
<210> 29
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 29
tccagaggtt gattgtcgac ttaacgcgtt tagcgagggg gcagggcctg catgtgaag 59

Claims (22)

1. a kind of targeting combines the single chain antibody protein or its variant of people CD19, include the following group:
(1) SEQ ID No.4, SEQ ID No.5, anti human CD 19 single-chain antibody shown in SEQ ID No.6 or SEQ ID No.7 The variant that amino acid modification generates is passed through in the amino acid sequence of albumen, or (2) (1).
2. single chain antibody protein or its variant that targeting as described in claim 1 combines people CD19, which is characterized in that with
SEQ ID No.4, SEQ ID No.5, amino acid sequence shown in SEQ ID No.6 or SEQ ID No.7 have 70~ 99%
Homology.
3. the specific chimeric antigen receptor that a kind of targeting combines people CD19, which is characterized in that comprising from aminoterminal to c-terminus
Sequentially connected boot sequence, targeting combine single-chain antibody, transmembrane domain and the intracellular signal structural domain of people CD19,
The targeting people CD19 single-chain antibody includes the targeting of claims 1 or 2 in conjunction with the single-stranded anti-of people CD19
Body protein or its variant, the targeting combine the preferred people CD19 of single chain antibody protein of people CD19.
4. targeting as claimed in claim 3 combines the specific chimeric antigen receptor of people CD19,
Wherein the boot sequence is selected from the group:
(1) there is amino acid sequence shown in SEQ ID No.3；
The transmembrane domain behaviour CD8 polypeptide or its variant are selected from polypeptide shown in SEQ ID No.8；And/or it is selected from SEQ Polypeptide shown in ID No.9.
5. the specific chimeric antigen receptor that a kind of targeting combines people CD19, which is characterized in that by drawing from aminoterminal to c-terminus Lead sequence, anti human CD 19 single chain antibody protein of any of claims 1 or 2, people CD8 hinge legion sequence, people's CD8 transmembrane region sequence Column, people's 4-1BB intracellular domain sequence and CD3 ζ intracellular domain sequence are sequentially connected in series；It or is amino acid sequence from amino Hold c-terminus by boot sequence, anti human CD 19 single chain antibody protein of any of claims 1 or 2, people CD8 hinge legion sequence, The transmembrane domain people CD8, people's CD28 intracellular domain sequence and CD3 ζ intracellular domain sequence are sequentially connected in series.
6. targeting as claimed in claim 5 combines the specific chimeric antigen receptor of people CD19, wherein
The boot sequence is as shown in SEQ ID No.3；And/or
The anti human CD 19 single chain antibody protein such as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7, in shown in any one, and/or
The people CD8 hinge legion sequence is as shown in SEQ ID No.8；And/or
The sequence of the people CD8 transmembrane region is as shown in SEQ ID No.9；And/or
The people 4-1BB intracellular domain sequence is as shown in SEQ ID No.10；And/or
The CD28 domain sequence is as shown in SEQ ID No.11；And/or
The CD3 ζ domain sequence is as shown in SEQ ID No.12 or SEQ ID No.13.
It is recombination table 7. the described in any item targetings of claim 3-6 combine the specific chimeric antigen receptor of anti human CD 19 It reaches or is expressed by carrier.
8. targeting as claimed in claim 7 combines the specific chimeric antigen receptor of people CD19, wherein the carrier is selected from γ- Retroviral vector, slow virus load, adenovirus, adeno-associated virus or preferred γ-retroviral vector.
9. encoding the nucleic acid point of specific chimeric antigen receptor of the described in any item targetings of claim 3-6 in conjunction with people CD19 Son, characterized in that comprising ' ' nucleotide sequence for the encoding leader sequence being sequentially connected in series, coding targeting combine people to 3 from 5 The nucleotide sequence of the single chain antibody protein of CD19, the nucleotide sequence of encoding transmembrane domain and coding intracellular signal structural domain Nucleotide sequence, wherein the transmembrane domain includes transmembrane region, and the intracellular signal structural domain includes immunity receptor junket ammonia Acid activation motif and costimulatory signal domain；Optionally, the nucleic acid molecules further include the nucleotide sequence for encoding hinge area.
10. nucleic acid molecules according to claim 9, it is characterised in that: include from 5 ' to the 3 ' difference being sequentially connected in series The targeting as claimed in claim 1 or 2 of encoding leader sequence, coding combines the single chain antibody protein of people CD19, people's CD8 hinge area sequence Column, the transmembrane domain people CD8, people's 4-1BB intracellular domain sequence and CD3 ζ intracellular domain nucleotide sequence；Or comprising from What 5 ' to 3 ' were sequentially connected in series be separately encoded boot sequence, targeting as claimed in claim 1 or 2 combines the single-chain antibody of people CD19 Albumen, CD8 hinge area, CD8 transmembrane region, CD28 and CD3 ζ sequence nucleotide sequence.
11. nucleic acid molecules according to claim 10, wherein
The nucleotide sequences of the boot sequence are encoded as shown in SEQ ID No.14,
Or nucleotide sequence such as the SEQ ID No.15, SEQ ID of single-chain antibody of the coding targeting in conjunction with people CD19 Shown in No.16, SEQ ID No.17 or SEQ ID No.18,
Or the people CD8 hinge legion sequence is encoded as shown in SEQ ID No.19,
Or the transmembrane domain the people CD8 is encoded as shown in SEQ ID No.20,
Or the people 4-1BB intracellular domain sequence is encoded as shown in SEQ ID No.21,
Or the CD28 intracellular domain sequence is encoded as shown in SEQ ID No.22,
Or the coding CD3 ζ intracellular domain sequence is as shown in SEQ ID No.23 or SEQ ID No.24.
12. a kind of recombinant vector or expression plasmid, it includes the nucleic acid molecules described in any one of claim 9-11.
13. a kind of promoter, for constructing described in recombinant vector described in claim 12 and expression any one of claim 3-6 Targeting combine people CD19 specific chimeric antigen receptor.
14. promoter according to claim 13, which is characterized in that the promoter is selected from EF1 shown in SEQ No.25 α long promoter and EFS promoter or the promoter as shown in SEQ No.26 are the short starting of EFS shown in SEQ No.26 Son.
15. a kind of recombinant virus, in which:
The recombinant virus is that can express the described in any item targetings of claim 3-6 to resist in conjunction with the specific chimeric of people CD19 Original receptor, and the virus of immune response cell can be infected；The virus includes slow virus, adenovirus, adeno-associated virus or reverse Record virus；
The immune effector cell includes cytotoxic T lymphocyte, NK cell, NKT cell or helper T lymphocyte.
16. a kind of immune response cell of isolated modification, it includes the targeting knots described in any one of preceding claims 3-6 Close the specific chimeric antigen receptor of people CD19, the recombinant vector as described in claim 12 or expression plasmid conversion gained.
17. the immune response cell of isolated modification according to claim 16, wherein the immune response cell is selected from T Cell, natural killer cells, cytotoxic T lymphocyte, regulatory T cells, human embryo stem cell and lymph sample can be divided into The multipotential stem cell of cell, or it preferably is selected from T cell and natural killer cells or preferably T cell.
18. a kind of preparation method of the immune response cell of isolated modification, it is characterised in that:
The following steps are included:
Firstly, nucleic acid molecules described in any one of claim 9-11 are connected to initial table by way of molecular cloning Up in carrier, the expression vector of the specific chimeric antigen receptor of the expression targeting CD19 is obtained；
Then, the expression vector of the specific chimeric antigen receptor of the targeting CD19 of acquisition is transfected into 293T cell, obtained Virus liquid；
Finally with the virus liquid infection immunity responsive cell, the specificity of expression targeting CD19 is obtained from metainfective cell The immune response cell of Chimeric antigen receptor；
Wherein the immune response cell be selected from T cell, natural killer cells, cytotoxic T lymphocyte, regulatory T cells, Human embryo stem cell and the multipotential stem cell that can be divided into lymphoid cell or the immune response cell are selected from T cell or certainly Natural killer cell.
19. a kind of pharmaceutical composition for treating or preventing tumor disease, discomfort or health disorders, characterized in that comprising a effective amount of The immune response cell and pharmaceutically acceptable excipient of isolated modification as described in claim 16 or 17, the disease Disease, uncomfortable or health disorders are related with the specificity interaction of people CD19 target spot and its anti human CD 19 single chain antibody protein, institute State disease, discomfort or health disorders include, pathogenic infection, autoimmune disease, inflammatory disease, allograft, shifting Plant repulsion and aging.
20. a kind of kit for treating or preventing tumor disease, discomfort or health disorders or aging is formed, characterized in that packet Immune response cell or such as claim 9-11 containing the isolated modification as described in any one of claim 16 or 17 are any Nucleic acid described in.
21. promoter described in recombinant vector or expression plasmid described in claim 12 or claim 19 or 20 or right are wanted Ask the immune response cell or right of 21 recombinant viruses or the described in any item isolated modifications of claim 22~24 It is required that nucleic acid described in 14~17 or the described in any item targeting CD19 single chain antibody proteins of claims 1 to 3 or its functionality The specific chimeric antibody of variant or the described in any item targeting CD19 of claim 4~14 are treating or preventing disease, health Application in imbalance or uncomfortable or aging product, the disease, health disorders, discomfort or aging be with people CD19 target spot and its The specificity interaction of anti human CD 19 single chain antibody protein is related, and the disease, health disorders, discomfort include CD19 in lesion The highly expressed tumour formation of position cell surface, pathogenic infection, autoimmune disease, inflammatory disease, allograft, Graft rejection；Wherein, the CD19 antigen protein of the tumour expression people.
22. application as claimed in claim 21, wherein the treatment or prevention tumor forms negative including reducing tumour in subject Lotus, increase extend the survival of the subject formed with tumor or respond cancer cell or the pathogen of subject and increase immune sharp The living cells factor.
CN201910484370.XA 2019-06-05 2019-06-05 Target specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinical application Pending CN110272493A (en)

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