CN108103105A - A kind of preparation method of CAR-T cells, CAR-T cells obtained and its application - Google Patents
A kind of preparation method of CAR-T cells, CAR-T cells obtained and its application Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of CAR T cells, CAR T cells obtained and its application, the preparation methods to comprise the following steps：Separation, activation and the amplification of S1, T lymphocyte：Amplification will be cultivated after separated CD3+CD8+T lymphocyte activators；S2, the recombinant slow virus for carrying CAR infect the step S1 treated T lymphocytes, the CAR T cells are made, wherein, the CAR genes that carry include CD28 intracellular regions and 4 1BB intracellular regions in the recombinant slow virus for carrying CAR, and the nucleotide sequences of the CD28 intracellular regions and 4 1BB intracellular regions is respectively as shown in SEQ ID7 and SEQ ID9.CAR T cells can be widely applied to the treatment of kinds cancer made from preparation method of the present invention.Compared with prior art, the CAR T cell proliferation rates that prepared by the method for the present invention are significantly improved compared to conventional method.
Description
Technical field
The present invention relates to field of immunology, and in particular to a kind of Chimeric antigen receptor T lymphocytes
Preparation method, CAR-T cells obtained and its application of (ChimericAntigen Receptor T-cell, CAR-T).
Background technology
In the past 10 years, immunotherapy has shown huge potentiality and prospect in treatment of cancer, and Chimeric antigen receptor T is thin
The appearance of born of the same parents' therapy is the quantum jump progress of immunotherapy field.1989, it is embedding that Gross etc. is put forward for the first time structure specificity
Close the T lymphocytes (CAR-T) of antigen receptor modification.CAR-T therapies are to utilize patient's autoimmunity cell killing tumour cell,
It is a kind of method of specific killing tumour cell, the therapy is by extracting the T lymphocytes in peripheral blood in patients, then general
The T lymphocytes of extraction are transformed by gene engineering method, and improved T lymphocytes possess powerful specific recognition
Improved T lymphocytes are fed back to patient's body by the ability of tumour antigen after amplification in vitro, have spy to tumour cell
Different in nature lethality.Compared with traditional immunization therapy, CAR-T therapies in vitro with better target has been shown in clinical test
Tropism and lethal, illustrates huge application potential and development prospect.
Nevertheless, there are still differences for effect of the CAR-T therapies in multinomial oncotherapy research, thus it is speculated that one of its reason
It is since the multiplication efficiency of the CAR-T cells prepared in the prior art is different with survival persistence.
The content of the invention
The technical problems to be solved by the invention are：A kind of system that can be ensured that and be proliferated efficient CAR-T cells is provided
Preparation Method, CAR-T cells obtained and its application.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：A kind of preparation method of CAR-T cells,
Comprise the following steps：
Separation, activation and the amplification of S1, T lymphocyte：Expand being cultivated after separated CD3+CD8+T lymphocyte activators
Increase；
S2, the recombinant slow virus for carrying CAR infect the step S1 treated T lymphocytes, and it is thin that the CAR-T is made
Born of the same parents, wherein, the CAR genes carried in the recombinant slow virus for carrying CAR include CD28 intracellular regions and 4-1BB intracellular regions, institute
The nucleotide sequence of CD28 intracellular regions and 4-1BB intracellular regions is stated respectively as shown in SEQ ID7 and SEQ ID9.
The beneficial effects of the present invention are：The CAR gene structures and sequence of optimization design are conducive to the expression and increasing in later stage
It grows, the multiplication of CAR-T cells is peomoted using two costimulatory molecules intracellular regions；CAR-T is prepared by the method for the present invention
Cell proliferation rate can promote more than 75% compared to customary preparation methods.
Present invention additionally comprises pass through CAR-T cells made from the above method.
Present invention additionally comprises the purposes by above-mentioned CAR-T cells in the drug for treating or preventing cancer is prepared.
Beneficial effects of the present invention also reside in：CAR-T cells application prospect of the present invention is extensive, will can be widely applied to various
It treats or prevents in the drug of cancer and with good treatment or prevention effect.
Specific embodiment
For the technology contents that the present invention will be described in detail, the objects and the effects, it is explained below in conjunction with embodiment.
The design of most critical of the present invention is：The CAR gene structures and sequence of optimization design are conducive to the translation in later stage, table
It reaches and is proliferated, wherein Leader sequences contribute to the translation in later stage, are peomoted using two costimulatory molecules intracellular regions
The multiplication of CAR-T cells.
A kind of preparation method of CAR-T cells, comprises the following steps：
Separation, activation and the amplification of S1, T lymphocyte：Expand being cultivated after separated CD3+CD8+T lymphocyte activators
Increase；
S2, the recombinant slow virus for carrying CAR infect the step S1 treated T lymphocytes, and it is thin that the CAR-T is made
Born of the same parents, wherein, the CAR genes carried in the recombinant slow virus for carrying CAR include CD28 intracellular regions and 4-1BB intracellular regions, institute
The nucleotide sequence of CD28 intracellular regions and 4-1BB intracellular regions is stated respectively as shown in SEQ ID7 and SEQ ID9.
As can be seen from the above description, the beneficial effects of the present invention are：The CAR gene structures and sequence of optimization design are conducive to
The expression in later stage and multiplication peomote the multiplication of CAR-T cells using two costimulatory molecules intracellular regions；Pass through the present invention
Method prepares CAR-T cell proliferation rates can promote more than 75% compared to customary preparation methods.
Further, in the step S2, when carrying the recombinant slow virus infection T lymphocytes of CAR, polybrene is added
(Polybrene), the addition concentration range of the Polybrene is 5~10 μ g/ml, is preferably 5 μ g/ml.
Further, in the step S2, the time of virus infection is 10~20 days, is preferably 14 days.
Further, the step S2 was carried out by the 3rd day after the T lymphocyte activators in the step S1.
Further, the CAR genes are CD19CAR genes, and the CD19CAR genes include Leader-CD19scfv-
Hinge-CD28TM-CD28 intracellular region-linker-4-1BB intracellular region-CD3zeta segments are made of above-mentioned segment, described
Leader-CD19scfv-hinge-CD28TM-CD28 intracellular region-linker-4-1BB intracellular region-CD3zeta segments by
Leader genetic fragments, CD19scfv genetic fragments, hinge genetic fragments, CD28TM genetic fragments, CD28 intracellular region gene pieces
Section, linker genetic fragments, 4-1BB intracellular regions genetic fragment and CD3zeta genetic fragments are spliced successively, the Leader
Genetic fragment, CD19scfv genetic fragments, hinge genetic fragments, CD28TM genetic fragments, CD28 intracellular regions genetic fragment,
The nucleotide sequence of linker genetic fragments, 4-1BB intracellular regions genetic fragment and CD3zeta genetic fragments successively as SEQ ID2,
Shown in SEQ ID3, SEQ ID5, SEQ ID6, SEQ ID7, SEQ ID8, SEQ ID9 and SEQ ID10.
As can be seen from the above description, the beneficial effects of the present invention are：Leader sequences are designed in CAR genes, are contributed to
The translation in later stage.
Further, the recombinant slow virus of CAR is carried in the step S2 to carry the recombinant slow virus of CD19CAR, institute
The preparation for stating the recombinant slow virus for carrying CD19CAR comprises the following steps：EF1 α-CD19CAR genes are obtained by gene chemical synthesis,
PMD18-T carriers are connected to, EF1 α-CD19CAR segments are obtained by PCR amplification, and genetic fragment both ends are respectively provided with
BstXI/BamHI restriction enzyme sites.
Further, it is described carry CD19CAR recombinant slow virus prepare it is further comprising the steps of：BstXI/BamHI
PGK promoters are replaced with EF1 α promoters, pass through In-Fusion by double digestion pRRLSIN.cPPT.PGK-GFP.WPRE carriers
EF1 α-CD19CAR segments are cloned into carrier by method；Screening positive clone and after expanding culture, using pMD2.G, pRSVRev,
PMDLg/pRRE helper plasmids with lipo2000 methods transfect 293FT cells, when 24 is small after collect virion.
Further, the gene for carrying CAR is EGFRvIII CAR genes, and the EGFRvIII CAR genes include
Leader-EGFRvIII scfv-hinge-CD28TM-CD28 intracellular region-linker-4-1BB intracellular region-CD3zeta segments or
It is made of above-mentioned segment, the Leader-EGFRvIII scfv-hinge-CD28TM-CD28 intracellular regions-linker-4-1BB
Intracellular region-CD3zeta segments are by Leader genetic fragments, EGFRvIII scfv genetic fragments, hinge genetic fragments, CD28TM
Genetic fragment, CD28 intracellular regions genetic fragment, linker genetic fragments, 4-1BB intracellular regions genetic fragment and CD3zeta gene pieces
Duan Yici is spliced, the Leader genetic fragments, EGFRvIII scfv genetic fragments, hinge genetic fragments, CD28TM
Genetic fragment, CD28 intracellular regions genetic fragment, linker genetic fragments, 4-1BB intracellular regions genetic fragment and CD3zeta gene pieces
Section nucleotide sequence successively as SEQ ID2, SEQ ID4, SEQ ID5, SEQ ID6, SEQ ID7, SEQ ID8, SEQ ID9 and
Shown in SEQ ID10.
Further, the recombinant slow virus of CAR is carried in the step S2 to carry the recombinant lentiviral of EGFRvIII CAR disease
Poison, the preparation of the recombinant slow virus for carrying EGFRvIII CAR comprise the following steps：Gene EF1 is obtained by gene chemical synthesis
α-EGFRvIII CAR, are connected to pMD18-T carriers, and EF1 α-EGFRvIII CAR segments, and gene are obtained by PCR amplification
Segment both ends are respectively provided with BstXI/BamHI restriction enzyme sites.
Further, the recombinant slow virus for carrying EGFRvIII CAR prepare it is further comprising the steps of：BstXI/
PGK promoters are replaced with EF1 α promoters, pass through In- by BamHI double digestion pRRLSIN.cPPT.PGK-GFP.WPRE carriers
EF1 α-EGFRvIII CAR segments are cloned into carrier by Fusion methods；Screening positive clone and after expanding culture, uses
PMD2.G, pRSVRev, pMDLg/pRRE helper plasmid with lipo2000 methods transfect 293FT cells, when 24 is small after collect virus
Particle.
Further, the CD3+CD8+T lymphocytes are isolated by following steps：From peripheral blood mononuclear cells
Middle separation T lymphocytes using anti-cd 3 antibodies, anti-CD8 antibody, sort to obtain CD3+CD8+T lymphs thin by flow cytometry
Born of the same parents.
Further, the activation operation in the step S1 is as follows：In isolated CD3+CD8+T lymphocyte suspensions
In, add CD3/CD28 antibody-coupled magnetic beads activated T lymphocytes, the CD3/CD28 antibody-coupled magnetic beads and T lymphocytes
Ratio be 1:1~3:1, it is preferably 3:1.
Further, the culture amplification operation in the step S1 is as follows：Divide above-mentioned from peripheral blood mononuclear cells
From the 2nd day after T lymphocytes, recombinant human il-2 that addition ultimate density is 300~500IU/ml, ultimate density be 5~
The recombined human IL-7 and ultimate density of 8ng/ml is the recombined human IL-15 of 5~8ng/ml.
Preferably, the culture operation in the step S1 is as follows：T lymphs are separated from peripheral blood mononuclear cells above-mentioned
From the 2nd day after cell, addition ultimate density be 300IU/ml recombinant human il-2, ultimate density be 6ng/ml recombined human
The IL-7 and recombined human IL-15 that ultimate density is 6ng/ml.
Further, the culture operation in the step S1 and S2 is that cell is placed in 37 DEG C, 5%CO2In incubator
Culture.
Present invention additionally comprises pass through CAR-T cells made from the above method.
Present invention additionally comprises the purposes by above-mentioned CAR-T cells in the drug for treating or preventing cancer is prepared.
As can be seen from the above description, beneficial effects of the present invention also reside in：CAR-T cells application prospect of the present invention is extensive, will
It can be widely applied in the drug of various treatment or prevention cancers and with good treatment or prevention effect.
The embodiment of the present invention one is：A kind of preparation method of CAR-T cells, comprises the following steps：
Separation, activation and the amplification of S1, T lymphocyte
Peripheral blood 50mL is gathered under aseptic condition, blood preparation is sent to laboratory, carries out peripheral blood mononuclear cells
Separation.Peripheral blood 2000rpm, l0min are centrifuged, collect upper strata autologous plasma.Remaining blood presses 1 with 0.01mol/L PBS liquid:
1 dilution piping and druming is uniform, separates mononuclearcell using lymphocyte separation medium, PBS washing cells use cell precipitation containing 10%
The GTT551 serum free mediums of autologous plasma are resuspended, by Pan T Cell Isolation Kit II (can directly city be purchased from U.S.
Its Ni company) operating instruction, from peripheral blood mononuclear cells separate T lymphocytes.Using anti-CD3, anti-CD8 antibody,
CD3+CD8+T lymphocytes are sub-elected by flow cytometry.
In isolated CD3+CD8+T lymphocyte suspensions, addition CD3/CD28 antibody-coupled magnetic beads activation T lymphs
Cell, the wherein ratio of CD3/CD28 antibody-coupled magnetic beads and T lymphocytes are 3:1, restructuring is added in above-mentioned cultivating system
Human IL-2 makes its final concentration of 300IU/mL, while adding recombined human IL-7 and recombined human IL-15 makes its final concentration be 6ng/
ml.Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
S2, the recombinant slow virus infection T lymphocytes for carrying CAR, prepare CAR-T cells
The 3rd day of timing since operating T lymphocyte activators, with the recombinant slow virus infection T leaching for carrying CD19-CAR
Bar cell, when infection, add Polybrene, make its final concentration of 5 μ g/mL, prepare CAR-T cells.10 days after virus infection, use
Limiting dilution assay screening positive clone cell line.By the K562 cells of CAR-T cells and the CD19 that transduceed by 1:1 ratio is trained altogether
It supports 5 days, cell proliferation rate is detected using CFSE decoration methods.
The recombinant slow virus preparation process of the carrying CD19CAR is as follows：
1st, CAR gene orders are built
CD19CAR gene structures：Leader-CD19scfv-hinge-CD28TM-CD28 intracellular regions-linker-4-1BB
Intracellular region-CD3zeta, sequence are shown in annex.
2nd, CD19CAR recombinant slow virus is carried to prepare
EF1 α-CD19CAR genes are obtained by gene chemical synthesis, pMD18-T carriers is connected to, EF1 is obtained by PCR amplification
α-CD19CAR segments, and genetic fragment both ends are respectively provided with BstXI/BamHI restriction enzyme sites.The nucleotide of the EF1 α genes
Sequence is as shown in SEQ ID1.BstXI/BamHI double digestion pRRLSIN.cPPT.PGK-GFP.WPRE carriers, by PGK promoters
EF1 α promoters are replaced with, EF1 α-CD19CAR segments are cloned into carrier by In-Fusion methods.Select positive colony survey
Sequence.Identify that correct plasmid expands culture.Turned using pMD2.G, pRSVRev, pMDLg/pRRE helper plasmid lipo2000 methods
Contaminate 293FT cells, when 24 is small after collect virion.
Control experiment one operates as follows：Except CD19CAR gene structures are in the recombinant slow virus for carrying CD19CAR
Outside Leader-CD19scfv-hinge-CD28TM-CD28 intracellular regions-CD3zeta, other steps are identical with embodiment one.
The CAR-T cell proliferative conditions of the embodiment of the present invention one and the preparation of control experiment one, knot are detected with CFSE decoration methods
Fruit is as follows：
CAR-T cell Proliferations efficiency prepared by control experiment one is 18%, CAR-T cells prepared by the embodiment of the present invention one
Proliferation rate 30%.
It can be seen from the results above that the cell proliferation rate of the CAR-T prepared using the method for the present invention is than control experiment one
The cell proliferation rate of the CAR-T prepared using conventional method is higher by 67%.
The embodiment of the present invention two is：A kind of preparation method of CAR-T cells, comprises the following steps：
Separation, activation and the amplification of S1, T lymphocyte
Peripheral blood 50mL is gathered under aseptic condition, blood preparation is sent to laboratory, carries out peripheral blood mononuclear cells
Separation.Peripheral blood 2000rpm, l0min are centrifuged, collect upper strata autologous plasma.Remaining blood presses 1 with 0.01mol/L PBS liquid:
1 dilution piping and druming is uniform, separates mononuclearcell using lymphocyte separation medium, PBS washing cells use cell precipitation containing 10%
The GTT551 serum free mediums of autologous plasma are resuspended, by Pan T Cell Isolation Kit II's (Mei Tian Ni companies)
Operating instruction separates T lymphocytes from peripheral blood mononuclear cells.Using anti-CD3, anti-CD8 antibody, pass through fluidic cell
Art sub-elects CD3+CD8+T lymphocytes.
In isolated CD3+CD8+T lymphocyte suspensions, addition CD3/CD28 antibody-coupled magnetic beads activation T lymphs
Cell, the wherein ratio of CD3/CD28 antibody-coupled magnetic beads and T lymphocytes are 1:1, restructuring is added in above-mentioned cultivating system
Human IL-2 makes its final concentration of 500IU/mL, while adding recombined human IL-7 and recombined human IL-15 makes its final concentration be 8ng/
mL.Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
S2, the recombinant slow virus infection T lymphocytes for carrying CAR, prepare CAR-T cells
The 3rd day of timing since operating T lymphocyte activators is infected with the recombinant slow virus for carrying EGFRvIII-CAR
T lymphocytes, when infection, add Polybrene, make its final concentration of 10 μ g/mL, prepare CAR-T cells.14 after virus infection
My god, limiting dilution assay screening positive clone cell line.By the U87 cells of CAR-T cells and the EGFRvIII that transduceed by 1:1 ratio
Example co-cultures 5 days, and cell proliferation rate is detected with CFSE decoration methods.
Prepared by the recombinant slow virus for carrying EGFRvIII CAR, comprise the following steps：
1st, CAR gene orders are built
EGFRvIII CAR gene structures：Leader-EGFRvIII scfv-hinge-CD28TM-CD28 intracellular regions-
Linker-4-1BB intracellular region-CD3zeta, sequence are shown in annex.
2nd, EGFRvIII CAR recombinant slow virus is carried to prepare
EF1 α-EGFRvIII CAR genes are obtained by gene chemical synthesis, pMD18-T carriers is connected to, is obtained by PCR amplification
To EF1 α-EGFRvIII CAR segments, genetic fragment both ends are respectively provided with BstXI/BamHI restriction enzyme sites.The EF1 α genes
Nucleotide sequence as shown in SEQ ID1.BstXI/BamHI double digestion pRRLSIN.cPPT.PGK-GFP.WPRE carriers, will
PGK promoters replace with EF1 α promoters, and EF1 α-EGFRvIII CAR segments are cloned into carrier by In-Fusion methods.
Select positive colony sequencing.Correct plasmid expands culture.It is used using pMD2.G, pRSVRev, pMDLg/pRRE helper plasmid
Lipo2000 methods transfect 293FT cells, when 24 is small after collect virion.
The control experiment operation of the embodiment of the present invention two is as follows：Except in the recombinant slow virus for carrying EGFRvIII CAR
EGFRvIII CAR gene structures for outside Leader-EGFR scfv-hinge-CD28TM-CD28 intracellular regions-CD3zeta, other
Step is identical with embodiment two.
The CAR-T cell proliferative conditions of the embodiment of the present invention two and the preparation of control experiment two, knot are detected with CFSE decoration methods
Fruit is as follows：
CAR-T cell Proliferations efficiency prepared by control experiment two is 20%, CAR-T cells prepared by the embodiment of the present invention two
Proliferation rate 36%.
It can be seen from the results above that it is adopted using CAR-T cell proliferation rates prepared by the method for the present invention than control experiment two
The CAR-T cell proliferation rates prepared with conventional method are higher by 80%.
The embodiment of the present invention three is：A kind of preparation method of CAR-T cells, comprises the following steps：
Separation, activation and the amplification of S1, T lymphocyte
Peripheral blood 50mL is gathered under aseptic condition, blood preparation is sent to laboratory, carries out peripheral blood mononuclear cells
Separation.Peripheral blood 2000rpm, l0min are centrifuged, collect upper strata autologous plasma.Remaining blood presses 1 with 0.01mol/L PBS liquid:
1 dilution piping and druming is uniform, separates mononuclearcell using lymphocyte separation medium, PBS washing cells use cell precipitation containing 10%
The GTT551 serum free mediums of autologous plasma are resuspended, by Pan T Cell Isolation Kit II (can directly city be purchased from U.S.
Its Ni company) operating instruction, from peripheral blood mononuclear cells separate T lymphocytes.Using anti-CD3, anti-CD8 antibody,
CD3+CD8+T lymphocytes are sub-elected by flow cytometry.
In isolated CD3+CD8+T lymphocyte suspensions, addition CD3/CD28 antibody-coupled magnetic beads activation T lymphs
Cell, the wherein ratio of CD3/CD28 antibody-coupled magnetic beads and T lymphocytes are 3:1, restructuring is added in above-mentioned cultivating system
Human IL-2 makes its final concentration of 400IU/mL, while adding recombined human IL-7 and recombined human IL-15 makes its final concentration be 5ng/
ml.Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
S2, the recombinant slow virus infection T lymphocytes for carrying CAR, prepare CAR-T cells
The 3rd day calculated since operating T lymphocyte activators, with the recombinant slow virus infection T leaching for carrying CD19-CAR
Bar cell, when infection, add Polybrene, make its final concentration of 5 μ g/mL, prepare CAR-T cells.After recombinant slow virus infection
20 days, with limiting dilution assay screening positive clone cell line.By the K562 cells of CAR-T cells and the CD19 that transduceed by 1:1
Ratio co-cultures 5 days, and cell proliferation rate is detected using CFSE decoration methods.
The recombinant slow virus preparation process of the carrying CD19CAR is as follows：
1st, CAR gene orders are built
CD19CAR gene structures：Leader-CD19scfv-hinge-CD28TM-CD28 intracellular regions-linker-4-1BB
Intracellular region-CD3zeta, sequence are shown in annex.
2nd, CD19CAR recombinant slow virus is carried to prepare
EF1 α-CD19CAR genes are obtained by gene chemical synthesis, pMD18-T carriers is connected to, EF1 is obtained by PCR amplification
α-CD19CAR segments, and genetic fragment both ends are respectively provided with BstXI/BamHI restriction enzyme sites.The nucleotide of the EF1 α genes
Sequence is as shown in SEQ ID1.BstXI/BamHI double digestion pRRLSIN.cPPT.PGK-GFP.WPRE carriers, by PGK promoters
EF1 α promoters are replaced with, EF1 α-CD19CAR segments are cloned into carrier by In-Fusion methods.Select positive colony survey
Sequence.Identify that correct plasmid expands culture.Turned using pMD2.G, pRSVRev, pMDLg/pRRE helper plasmid lipo2000 methods
Contaminate 293FT cells, when 24 is small after collect virion.
Control experiment three operates as follows：Except CD19CAR gene structures are in the recombinant slow virus for carrying CD19CAR
Outside Leader-CD19scfv-hinge-CD28TM-CD28 intracellular regions-CD3zeta, other steps are identical with embodiment three.
The CAR-T cell proliferative conditions of the embodiment of the present invention three and the preparation of control experiment three, knot are detected with CFSE decoration methods
Fruit is as follows：
CAR-T cell Proliferations efficiency prepared by control experiment three is 22%, CAR-T cells prepared by the embodiment of the present invention three
Proliferation rate 40%.
It can be seen from the results above that the cell proliferation rate of the CAR-T prepared using the method for the present invention is than control experiment three
The cell proliferation rate of the CAR-T prepared using conventional method is higher by 82%.
In conclusion a kind of preparation method of CAR-T cells provided by the invention, CAR-T cells obtained and its application,
CAR-T cell proliferation rates prepared by the method for the present invention are significantly improved compared to conventional method.
The foregoing is merely the embodiment of the present invention, are not intended to limit the scope of the invention, every to utilize this hair
The equivalent structure or equivalent flow shift that bright description is made directly or indirectly is used in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of CAR-T cells, it is characterised in that：Comprise the following steps：
Separation, activation and the amplification of S1, T lymphocyte：Amplification will be cultivated after separated CD3+CD8+T lymphocyte activators；
S2, the recombinant slow virus for carrying CAR infect the step S1 treated T lymphocytes, and the CAR-T cells are made,
Wherein, the CAR genes carried in the recombinant slow virus for carrying CAR include CD28 intracellular regions and 4-1BB intracellular regions, described
The nucleotide sequence of CD28 intracellular regions and 4-1BB intracellular regions is respectively as shown in SEQ ID7 and SEQ ID9.
2. the preparation method of CAR-T cells according to claim 1, it is characterised in that：In the step S2, CAR is carried
Recombinant slow virus infection T lymphocytes when, add Polybrene, the addition concentration range of the Polybrene is 5~10 μ
g/ml。
3. the preparation method of CAR-T cells according to claim 1, it is characterised in that：In the step S2, virus infection
Time be 10~20 days.
4. the preparation method of CAR-T cells according to claim 1, it is characterised in that：By the T lymphs in the step S1
Carry out the step S2 within the 3rd day after cell-stimulating.
5. the preparation method of CAR-T cells according to claim 1, it is characterised in that：What the recombinant slow virus carried
CAR genes are CD19CAR genes, and the CD19CAR genes include Leader-CD19scfv-hinge-CD28TM-CD28 intracellulars
- linker-4-1BB intracellular region-CD3zeta segments in area are made of, the Leader-CD19scfv-hinge- above-mentioned segment
CD28TM-CD28 intracellular region-linker-4-1BB intracellular region-CD3zeta segments are by Leader genetic fragments, CD19scfv genes
Segment, hinge genetic fragments, CD28TM genetic fragments, CD28 intracellular regions genetic fragment, linker genetic fragments, 4-1BB intracellulars
Area's genetic fragment and CD3zeta genetic fragments are spliced successively, the Leader genetic fragments, CD19scfv genetic fragments,
Hinge genetic fragments, CD28TM genetic fragments, CD28 intracellular regions genetic fragment, linker genetic fragments, 4-1BB intracellular region bases
Because the nucleotide sequence of segment and CD3zeta genetic fragments is successively such as SEQ ID2, SEQ ID3, SEQ ID5, SEQ ID6, SEQ
ID7, SEQ ID8, shown in SEQ ID9 and SEQ ID10.
6. the preparation method of CAR-T cells according to claim 1, it is characterised in that：What the recombinant slow virus carried
CAR genes are EGFRvIII CAR genes, and the EGFRvIII CAR genes include Leader-EGFRvIII scfv-hinge-
CD28TM-CD28 intracellular region-linker-4-1BB intracellular region-CD3zeta segments are made of, the Leader- above-mentioned segment
EGFRvIII scfv-hinge-CD28TM-CD28 intracellular region-linker-4-1BB intracellular region-CD3zeta segments are by Leader
Genetic fragment, EGFRvIII scfv genetic fragments, hinge genetic fragments, CD28TM genetic fragments, CD28 intracellular region gene pieces
Section, linker genetic fragments, 4-1BB intracellular regions genetic fragment and CD3zeta genetic fragments are spliced successively, the Leader
Genetic fragment, EGFRvIII scfv genetic fragments, hinge genetic fragments, CD28TM genetic fragments, CD28 intracellular region gene pieces
The nucleotide sequence of section, linker genetic fragments, 4-1BB intracellular regions genetic fragment and CD3zeta genetic fragments is successively such as SEQ
ID2, SEQ ID4, SEQ ID5, SEQ ID6, SEQ ID7, SEQ ID8, shown in SEQ ID9 and SEQ ID10.
7. the preparation method of CAR-T cells according to claim 1, it is characterised in that：Activation behaviour in the step S1
Make as follows：In isolated CD3+CD8+T lymphocyte suspensions, addition CD3/CD28 antibody-coupled magnetic beads activation T lymphs
The ratio of cell, the CD3/CD28 antibody-coupled magnetic beads and T lymphocytes is 1:1~3:1.
8. the preparation method of CAR-T cells according to claim 7, it is characterised in that：Culture in the step S1 is expanded
It is as follows to increase operation：The 2nd day after T lymphocytes is separated from peripheral blood mononuclear cells, addition ultimate density for 300~
The weight that the recombined human IL-7 and ultimate density that the recombinant human il-2 of 500IU/ml, ultimate density are 5~8ng/ml are 5~8ng/ml
Group human IL-15.
9. according to CAR-T cells made from claim 1~8 any one of them preparation method.
10. CAR-T cells according to claim 9 are applied to prepare in the drug for treating or preventing cancer.
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