CN107254447A - Anti AFP CAR T cells and its preparation method and application - Google Patents

Anti AFP CAR T cells and its preparation method and application Download PDF

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CN107254447A
CN107254447A CN201710406296.0A CN201710406296A CN107254447A CN 107254447 A CN107254447 A CN 107254447A CN 201710406296 A CN201710406296 A CN 201710406296A CN 107254447 A CN107254447 A CN 107254447A
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刘未斌
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Abstract

The present invention provides a kind of Anti AFP CAR T cells and its preparation method and application, and the Anti AFP CAR T cells express SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins in T cell；In the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins, the SCFV is used for reference to the AFP albumen, and the amino acid sequence of the SCFV is First ray.The cell passes through to be obtained to T cell modification and transformation, and being capable of specific recognition and killing liver cancer, it is adaptable to the prevention and treatment of corresponding tumor disease.

Description

Anti AFP CAR-T cells and its preparation method and application
Technical field
The present invention relates to a kind of Anti AFP CAR-T cells and its preparation method and application, belong to genetically modified cell and Oncotherapy technical field.
Background technology
Primary carcinoma of liver (hepatocellular carcinoma, referred to as：HCC the 5th of Cancer Mortality) is occupied Position, the annual death toll about 1,000,000 in the whole world.At present, most of Patients with Primary lacks effective treatment means, total life Deposit rate only 3%-5%.Though liver transfer operation turns into the important means for the treatment of primary carcinoma of liver, donor shortage, surgical injury, exempt from The problems such as epidemic disease repulsion and high cost, constitutes the major obstacle of liver organ implantation technique development.Therefore, it is necessary to explore A kind of new, effective therapy approach.With molecular biology, technique for gene engineering and it is immunologic develop rapidly, controlled with immune Biological therapy based on treatment has shown that good application prospect.
Chimeric antigen receptor T cell (Chimeric antigen receptor T cell, referred to as：CAR-T cell) be By the means of genetic modification, single-stranded variable region (the single chain of the monoclonal antibody of specific recognition target antigen are enabled Fv domain, referred to as：SCFV) expression is in cell surface, while being increased by the activation of transmembrane region and the cell intracellular of engineer Signal domain is grown mutually to couple.The T cell for expressing CAR can be anti-by the tumour correlation of SCFV Direct Recognitions and combination tumor cell surface Original, CAR is by the incoming T cell of signal, and activation T cell secrete cytokines include perforin, granzyme, INF- γ, TNF-α Deng so as to play the effect of killing tumor cell.Therefore, CAR-T cells are major histocompatibility complex (major Histocompatibility complex, referred to as：MHC) nonrestrictive, its key applied is to determine that a kind of tumour is related Antigen, is expressed in tumor cell surface height, and in the normal tissue without expression or low expression.
Alpha-fetoprotein (alpha-feto protein, referred to as：AFP) it is a kind of interior sugared egg synthesized of fetal period liver In vain, level is very low in serum after adult, and the liver cell for occurring canceration can then recover the ability of its synthesis again, and it is in 70%-80% Primary carcinoma of liver (HCC) in have higher expression.Research confirms the murine hepatocarcinoma cell strain by mAFP genes are expressed Hepa1-6 is inoculated with C57BL/6J mouse, then with the Plasmid DNA immunization mouse of insertion AFP genes, as a result finds this curative Immune AFP DNAs can induce stronger specific CTL immunity reaction.The immunodominance polypeptide that also research application AFP originates 5 display AFP polypeptides have immunogenicity in vivo in vaccine therapy IV phase HCC patients, 6 patients, can be in serum afp Higher patient's moderate stimulation T cells with antigenic specificity.Therefore, AFP can be used as CAR-T as the specific antigen of liver cancer cells One promising target of cell anti-tumor treatment.
The content of the invention
The present invention provides a kind of Anti AFP CAR-T cells and its preparation method and application, and the cell is repaiied by T cell Decorations and transformation are obtained, and are capable of the liver cancer cells of specific recognition and killing AFP height expression, it is adaptable to corresponding tumor disease Prevention and treatment.
The present invention provides a kind of Anti AFP CAR-T cells, and the Anti AFP CAR-T cells are expressed in T cell SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins；It is described in the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins SCFV is used for reference to the AFP albumen, and the amino acid sequence of the SCFV is First ray, and the First ray is：
EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYYTTYNPSFQGHV TISIDKSTNTAYLHWNSLAASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPAKVSGS PGQKITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGAKSGNTASLTISGLQAEDEADYYC SSYTTGSRSVFGGGTKLTVLG
The Anti AFP CAR-T cells are using AFP as target spot, specifically, the inosculating antibody of the Anti AFP CAR-T cells Original receptor CAR includes after birth exoantigen land, hinge area and intracellular signal transduction area, and the after birth exoantigen land is SCFV, the hinge area is IgD, and the intracellular signal transduction area is CD28-OX40-CD3 ζ.Fig. 1 is Anti AFP of the invention CAR design diagram, refer to Fig. 1.
Further, in the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins, the amino acid sequence of the IgD is Second sequence.
Specifically, the second sequence is：
TFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLAHPS LPPQRLMALREPAAQAPVKLSTNLLASSDPPEAA
Further, in the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins, the amino acid sequence of the CD28 is 3rd sequence.
Specifically, the 3rd sequence is：
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSALLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
Further, in the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins, the amino acid sequence of the OX40 is 4th sequence, the amino acid sequence of the CD3 ζ is the 5th sequence.
Specifically, the 4th sequence is：
RRDQRLPPDSHKPPGGGSFRTPIQEEQADAHSTLAKI
Specifically, the 5th sequence is：
RVKFSRSAEPPAYQQGQNQLYNELNLGRSEEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
Further, in the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins,
The amino acid sequence of the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins is the 6th sequence.
Specifically, the 6th sequence is：
EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYTTYNPSFQGHVT ISIDKSTNTAYLHWNSLKASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSP GQSITISCTGTSSKVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYKS SYTTGSRAVFGGGTKLTVLGTFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRALWNA GTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAAFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVK FSRSAEPPAKQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR
Further, the Anti AFP CAR-T cells are obtained by slow-virus infection T lymphocytes, the slow virus Obtained by the Lentiviral infection 293T cells for carrying the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins Arrive.
The present invention also provides a kind of preparation method of any of the above-described described Anti AFP CAR-T cells, including following step Suddenly：
1) Lentiviral is built, the Lentiviral has described
SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins；
2) using slow virus packaging plasmid and Lentiviral infection 293T cells, slow virus is obtained, it is described Slow virus is packed by the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins；
3) T lymphocytes are separated, and with T lymphocytes described in the slow-virus infection, obtain the Anti AFP CAR- Expression SCFV-IgD-CD28-OX40-CD3 ζ the fusion proteins in T cell, the T cell of the Anti AFP CAR-T cells.
In step 1) in, by genetic engineering means, SCFV-IgD-CD28-OX40-CD3 ζ antigen-4 fusion protein genes are cloned Onto Lentiviral, so as to complete the structure to Lentiviral.In step 2) in, utilize step 1) obtained by Lentiviral obtains the slow disease packed by SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins to 293T cell infections Poison.In step 3) in, by step
2) slow virus in is expressed in the T lymphocytes that separate and collect in human peripheral to obtain CAR-T cells.
Further, in the step 1) before, in addition to synthesize and expand the SCFV-IgD-CD28-OX40-CD3 ζ Antigen-4 fusion protein gene.Specific synthesis and amplification method can be obtained according to this area conventional technical means.
Further, in addition to the slow virus purify, and drenched with T described in the slow-virus infection after purification Bar cell.General, the means of purification can use this area conventional meanses to carry out, such as filtering, ultrafiltration.
Present invention also offers any of the above-described described Anti AFP CAR-T cells in treatment liver-cancer medicine is prepared Using, can be used particularly for AFP molecules height expression liver cancer treatment.
The present invention program has advantages below：
1st, AFP antibody is used for the structure of CAR-T cells by Anti AFP CAR-T cells of the invention, can be to AFP eggs Effectively combined in vain.
2nd, the Anti AFP CAR-T cells preparation method that the present invention is provided, which simple, effective can be prepared, has specificity knowledge Not with the Anti AFP CAR-T cells of killing tumour.
3rd, Anti AFP CAR-T cells of the invention have efficient anti-tumor activity, can be used in preparing treatment liver In the medicine of cancer, enable in particular to using AFP molecules as target antigen, the treatment for the liver cancer of AFP molecules height expression.
Brief description of the drawings
Fig. 1 is the Anti AFP CAR of present invention design diagram；
Fig. 2 is the Anti AFP CAR-T cells of the embodiment of the present invention and the T cell being uninfected by target cell HepG-2 The fragmentation effect schematic diagram of (liver cancer)；
Fig. 3 is the Anti AFP CAR-T cells of the embodiment of the present invention and the T cell being uninfected by target cell Hep3B The fragmentation effect schematic diagram of (liver cancer)；
Fig. 4 is the Anti AFP CAR-T cells of the embodiment of the present invention, reference examples and blank example are to treatment liver cancer (HepG-2) the survival time of mice schematic diagram after Transplanted tumor model mouse.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiments of the invention, to this Technical scheme in inventive embodiments is clearly and completely described, it is clear that described embodiment is that a part of the invention is real Apply example, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creation Property work under the premise of the every other embodiment that is obtained, belong to the scope of protection of the invention.
Embodiment 1
The preparation of Anti AFP CAR-T cells of the present invention
1st, Lentiviral is built
By conventional genetic engineering means, SCFV-IgD-CD28-OX40-CD3 ζ antigen-4 fusion protein gene sequences are synthesized, will SCFV-IgD-CD28-OX40-CD3 ζ antigen-4 fusion protein gene sequences are cloned on Lentiviral, and acquisition SCFV-IgD- CD28-OX40-CD3 ζ Lentiviral pGreen puro-CAR.Wherein, SCFV-IgD-CD28-OX40-CD3 ζ are merged Each sequence of albumen is as previously described.
2nd, slow virus is packed
1) it is 10wt% hyclones (fetal bovine serum, abbreviation with mass fraction：FBS 1640 culture mediums) Cultivate 293T cells；
2) by 293T cells with 3x 105/cm2Density reach in diameter 15cm culture dish and cultivate 20h, it is ensured that during transfection Cell confluency degree is 80-90%；
Liquid is changed with 1640 culture mediums without serum, it is standby；
3) two EP pipes are taken, 1ml 1640 are added in two EP pipes respectively, by slow virus carrier pGreen puro-CAR With pMDLg PRRE, pRSV-Rev and pMD2.G plasmid according to mol ratio 1：1：1：1 mixes in one of EP pipes；Another 150ul Lipo2000 are added in individual EP pipes, mixes and places 5min；The 1640 of lipo2000 will be mixed with and add the EP containing plasmid Guan Zhong, mixes to obtain mixed liquor, and room temperature places 20min, and mixed liquor then is added dropwise into above-mentioned 2) the middle Tissue Culture Dish prepared In, continue to cultivate after 4h, nutrient solution is changed with 1640 culture mediums containing 10%FBS, continue to collect cell conditioned medium after cultivating 48h Liquid, obtains the slow virus packed by SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins.
3rd, slow virus purifies
The packaged slow virus obtained in 2 is filtered into membrane filtration with 0.22 μm, filtrate is collected in 50ml super filter tubes, with 3000g/min centrifugation 45min；Remaining upper strata viral concentration liquid is transferred to EP pipes, -80 DEG C is put and saves backup.
4th, the separation of CD8+ T cells
1) whole blood pours into 50ml centrifuge tubes, and room temperature is centrifuged 20 minutes, and it is 700g (common centrifugation) to control centrifugal force ref；
2) above-mentioned centrifugation bottom cell component, plus Du Shi phosphate buffers DPBS to 50ml are taken；
3) take 25ml aforesaid liquids to be added to 20ml human lymphocyte separating liquids respectively, centrifuge tube room temperature centrifuged 15 minutes, It is 800g to control centrifugal force ref；
5) tunica albuginea confluent monolayer cells are taken, DPBS is added, complements to 50ml；
6) centrifuge 10 minutes, it is 600g to control centrifugal force ref, abandons supernatant, obtains peripheral mononuclear blood cell (peripheral Blood mononuclear cell, referred to as：PBMC)；
7) using CD8+T cell magnetic bead sortings kit sorting CD8+T cells.
5th, slow virus carrier infection T lymphocytes
The complete medium culture CD8+T cells of the RPMI for being 10wt%FBS with mass fraction 1640, are added anti-for first day CD3 monoclonal antibody acs；Adding within 3rd day in the 3 of 150MOI will training after slow virus (Anti AFP CAR) after purification, 16h Foster base is replaced by the complete mediums of RPMI 1640 containing 50IU/ml recombinant human il-2s, continues to cultivate 10-20 days, obtains Anti AFP CAR-T cells.
Embodiment 2
Anti AFP CAR-T cells in vitro antitumous effects prepared by embodiment 1 are evaluated, comprised the following steps：
Using the stable hepatoma carcinoma cells HepG-2 and Hep3B for expressing AFP as target cell, slow virus carrier is used respectively The T cell (that is, Anti AFP CAR-T cells are made in embodiment 1) and the T cell system being uninfected by of pGreen puro-CAR infection Effector cell is obtained, by target cell according to density 1x 105Individual/ml is inoculated with 96 orifice plates, per the μ l of hole 100, according to 5:1,10:1,20:1 Effector cell is added target cell by effect target ratio, is placed in 5%CO2, 37 DEG C of incubator culture 4h, using WST-1 detection cell viabilities, Calculate killing-efficiency.
Fig. 2 is the Anti AFP CAR-T cells of the embodiment of the present invention and the T cell being uninfected by target cell HepG-2 The fragmentation effect schematic diagram of (liver cancer)；Fig. 3 is the Anti AFP CAR-T cells and the T cell being uninfected by of the embodiment of the present invention To target cell Hep3B (liver cancer) fragmentation effect schematic diagram.
Fig. 2 and Fig. 3 results show, expression AFP CAR T lymphocytes have very strong kill to AFP masculine liver cancers cell Wound is acted on.
Embodiment 3
Antitumous effect is evaluated in the Anti AFP CAR-T cell bodies prepared to embodiment 1, is comprised the following steps：
The female nude mice of 6 week old 15 is taken, 5x 10 is subcutaneously injected in right oxter6HepG-2 (liver cancer) cell, treats that tumour grows to 60mm3 Size, tumor model is randomly divided into 3 groups：Control group, Anti AFP CAR-T groups of cells, T cell group；Control group tail vein injection Physiological saline 200ul/ times, 2 times a week；Anti AFP CAR-T groups of cells tail vein injection Anti AFP CAR-T cells 1 × 107Individual/time, 2 times a week；T cell group tail vein injection T cell 1 × 107Individual/time, 2 times a week；Count mouse survival in 100 days State, does survival rate curve.Fig. 4 is the Anti AFP CAR-T cells of the embodiment of the present invention, reference examples and blank example are to controlling Treat the survival time of mice schematic diagram after liver cancer (HepG-2) Transplanted tumor model mouse.
Fig. 4 results show that Anti AFP CAR-T cells can postpone AFP relative to T cell and the blank example being uninfected by The life cycle of the transplanted human hepatocellular carcinoma mouse model of height expression.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology The scope of scheme.

Claims (10)

1. a kind of Anti AFP CAR-T cells, it is characterised in that the Anti AFP CAR-T cells are expressed in T cell SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins；
In the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins, the SCFV is used for reference to the AFP albumen, and institute The amino acid sequence for stating SCFV is First ray, and the First ray is：
EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYYTTYNPSFQGHVTISI DKSTNTAYLHWNSLAASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPAKVSGSPGQK ITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGAKSGNTASLTISGLQAEDEADYYCSSYT TGSRSVFGGGTKLTVLG。
2. Anti AFP CAR-T cells according to claim 1, it is characterised in that the SCFV-IgD-CD28- In OX40-CD3 ζ fusion proteins, the amino acid sequence of the IgD is the second sequence, and second sequence is：
TFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLAHPSLPPQ RLMALREPAAQAPVKLSTNLLASSDPPEAA。
3. Anti AFP CAR-T cells according to claim 2, it is characterised in that the SCFV-IgD-CD28- In OX40-CD3 ζ fusion proteins, the amino acid sequence of the CD28 is the 3rd sequence, and the 3rd sequence is：
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSALLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS。
4. Anti AFP CAR-T cells according to claim 3, it is characterised in that the SCFV-IgD-CD28- In OX40-CD3 ζ fusion proteins, the amino acid sequence of the OX40 is the 4th sequence；The amino acid sequence of the CD3 ζ is the 5th Sequence；
4th sequence is：
RRDQRLPPDSHKPPGGGSFRTPIQEEQADAHSTLAKI；
5th sequence is：
RVKFSRSAEPPAYQQGQNQLYNELNLGRSEEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
5. Anti AFP CAR-T cells according to claim 4, it is characterised in that the SCFV-IgD-CD28- In OX40-CD3 ζ fusion proteins,
The amino acid sequence of the SCFV-IgD-CD28-OX40-CD3 ζ is the 6th sequence, and the 6th sequence is：
EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYTTYNPSFQGHVTISID KSTNTAYLHWNSLKASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSI TISCTGTSSKVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYKSSYTT GSRAVFGGGTKLTVLGTFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRALWNAGTSV TCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAAFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRS AEPPAKQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR。
6. according to any described Anti AFP CAR-T cells of claim 1-5, it is characterised in that the Anti AFP CAR-T cells by slow-virus infection T lymphocytes obtain, the slow virus by carry the SCFV-IgD-CD28- The Lentiviral infection 293T cells of OX40-CD3 ζ fusion proteins are obtained.
7. the preparation method of any described Anti AFP CAR-T cells of claim 1-6, it is characterised in that including following step Suddenly：
1) Lentiviral is built, there is the Lentiviral SCFV-IgD-CD28-OX40-CD3 ζ to melt Hop protein；
2) using slow virus packaging plasmid and Lentiviral infection 293T cells, slow virus, the slow disease are obtained Poison is packed by the SCFV-IgD-CD28-OX40-CD3 ζ fusion proteins；
3) T lymphocytes are separated, and with T lymphocytes described in the slow-virus infection, obtain the AntiAFP CAR-T thin Expression SCFV-IgD-CD28-OX40-CD3 ζ the fusion proteins in born of the same parents, the T cell of the Anti AFP CAR-T cells.
8. the preparation method of Anti AFP CAR-T cells according to claim 7, it is characterised in that in the step 1) Before, in addition to synthesize and expand the SCFV-IgD-CD28-OX40-CD3 ζ antigen-4 fusion protein genes.
9. application of any described Anti AFP CAR-T cells of claim 1 to 6 in treatment liver-cancer medicine is prepared.
10. application according to claim 9, it is characterised in that the liver cancer is the tumour of AFP molecules height expression.
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CN111511383A (en) * 2017-11-14 2020-08-07 阿奇利克斯股份有限公司 Multifunctional immune cell therapy
JP2021503006A (en) * 2017-11-14 2021-02-04 アーセルクス インコーポレイテッド Multifunctional immune cell therapy
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US11377482B2 (en) 2017-11-14 2022-07-05 Arcellx, Inc. D-domain containing polypeptides and uses thereof
US11464803B2 (en) 2017-11-14 2022-10-11 Arcellx, Inc. D-domain containing polypeptides and uses thereof
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