CN105907790A - Preparation method of CD70-contained chimeric antigen receptor modified T cell specifically recognizing EGFRvIII - Google Patents

Preparation method of CD70-contained chimeric antigen receptor modified T cell specifically recognizing EGFRvIII Download PDF

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CN105907790A
CN105907790A CN201610451776.4A CN201610451776A CN105907790A CN 105907790 A CN105907790 A CN 105907790A CN 201610451776 A CN201610451776 A CN 201610451776A CN 105907790 A CN105907790 A CN 105907790A
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cell
plasmid
pegfp
cd70car
slow virus
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林志国
黄建平
暴洪博
金林春
葛海涛
慕璐岩
殷博
殷一博
龙宇
杨昌霖
夏松松
王佳斌
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Priority to PCT/CN2016/100208 priority patent/WO2017219528A1/en
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Abstract

The invention discloses a preparation method of a CD70-contained chimeric antigen receptor modified T cell specifically recognizing EGFRvIII. The method comprises the following steps of A, plasmid construction, amplification and purification; B, slow virus packaging, wherein 1, a packaging cell is cultured, 2, a DNA-EndoFectin slow virus mixture is prepared, 3, the packaging cell is transfected, and 4, slow virus particles are harvested; C, T cell transfection, wherein 1, a culture plate is preprocessed, 2, viruses are centrifuged, and 3, the T cell is transfected. The CAR-T technology is subjected to three-generation development, and the third-generation dual-costimulatory-molecule-contained CAR is mainly applied to a clinical experiment at present. On this basis, the novel costimulatory molecule CD70 is added. In the CAR-T cell, the CD70-CD27 mutual effect can induce the CD8+T cell to volatilize the stronger tumor killing and virus fusion effects. Accordingly, the CD70-contained CAR-T has the stronger tumor resistance, and the life time is prolonged.

Description

The preparation method modifying T cell containing CD70 Chimeric antigen receptor of specific recognition EGFRvIII
Technical field
The invention belongs to gene engineering technology field, relate to a kind of Chimeric antigen receptor modification T thin The preparation method of born of the same parents, is specifically related to a kind of specific recognition EGF-R ELISA type III sudden change The preparation method modifying T cell containing CD70 Chimeric antigen receptor of body (EGFRvIII).
Background technology
Chimeric antigen receptor modification T cell (Chimeric Antigen Receptors T cell, CAR-T) technology is to identify single-chain antibody and the activation sequences of T cell of tumor associated antigen It is combined as a whole, will the killing machine of high-affinity and T lymphocyte of antibodies on tumor antigen System combines, by gene transfer method transfecting T cells so that it is have specific recognition and kill Hinder the ability of tumor cell, owing to this method is compared with other immunotherapy of tumors method, effect Fruit is definite, is therefore considered as the new skill of a target biology treatment in immunotherapy of tumors field Art.Within 1989, proposed first by Eshhar etc., Rosenberg and Restifo doctor in 2006 Late etc. first Application tumor surface receptor (TCR) transfecting T cells technology targeted therapy 17 example Phase metastatic melanoma patient, the completed tumor regression of 2 example patients, 15 example conditions of patients are long-term Stable.Within 2010, lymphoma is entered by doctor's Rosenberg first Application CAR-T treatment concept Row treatment, result shows, after treatment, patient's not only tumor disappearance, and there is Bone Marrow of Patients In cancerous cell precursor be also selectively cleared.Next year, June et al. is again at " NEW ENGL J MED " and " SCI TRANSL MED " on delivered application CAR-T success Curing the Clinical Report of chronic lymphocytic leukemia, therefore, this method is quilt at present Think a treatment means the most potential in immunotherapy of tumors.CAR-T technology warp Go through three generations's development, be applied to now the mainly third generation of clinical experiment containing double costimulatory moleculeses CAR。
CD70 is a kind of transmembrane protein, and molecular weight is about 50000, combines including extracellular protein District, cross-film district and three constituents of intracellular region.After CD70 with CD27 is combined, can To play the effect of secondary signal, make T cell activate further, cell proliferation, cell because of The ability of the aspects such as sub-secretion significantly improves.And at the end of immunne response, it is expressed the most therewith Lower.Having research to confirm, interleukin-22,12 and tumor necrosis factor α etc. can promote CD70 Expression, contrary, IL-4 and IL-10 suppression CD70 expression.In CAR-T cell, The interaction of CD70-CD27 can induce CD8+T cell plays higher tumor-killing and melts Virus function, the CAR-T introducing CD70 has more powerful anti-tumor capacity.But mesh Before containing CD70 Chimeric antigen receptor modify T cell preparation method.
Summary of the invention
For the vacancy of prior art, the present invention adds new costimulatory molecules CD70, it is provided that one The preparation side modifying T cell containing CD70 Chimeric antigen receptor of species specificity identification EGFRvIII Method.
It is an object of the invention to be achieved through the following technical solutions:
A kind of specific recognition EGFRvIII modify T cell containing CD70 Chimeric antigen receptor Preparation method, comprises the following steps:
A, plasmid construction: build slow virus packaging plasmid pEGFP-CD70CAR plasmid；
Plasmid amplification and purification: ultra-vioket radiation Bacteriology Room super-clean bench 30min, cut in super-clean bench Carry 1/3 being placed in centrifuge tube of filter paper circle of pEGFP-CD70CAR plasmid, add aseptic Distilled water soaks 30min, centrifuging and taking supernatant after stirring, is placed in-20 DEG C and saves backup.
Join in EP pipe for expanding the competence bacillus coli DH 5 alpha of plasmid, add PEGFP-CD70CAR plasmid 1 μ L/ manages, and stands 30min after mixing on ice, if the most right According to: the competent cell that 1. 2 μ L pcDNA3.1 convert is as positive control；It is not added with the most completely The competent cell of plasmid DNA is as negative control.42 DEG C of heat shocks of low temperature 1.5～2min, Be placed in ice 5 minutes, add Luria-Bertani culture medium, shaking table 150rpm shaking 45～ 60min, makes bacteria resuscitation and expresses pEGFP-EFGRvIII and pEGFP-CD70CAR matter Grain ampicillin AMP Drug resistance, transfers to ammonia by the competent cell that 200 μ L have converted On the solid medium flat board of benzylpcnicillin (Amp+) (flat board needs 37 DEG C of incubator preheatings), Coating uniformly, after drying, is cultivated 12-16 hour.
After picking conversion pEGFP-EFGRvIII and pEGFP-CD70CAR on Amp+ flat board The single bacterium colony grown is put in test tube added with 3mL Amp+LB, and 180rpm shakes 12-16 hour, according to plasmid extraction kit plasmid extraction.
B, slow virus packaging
1) incasing cells is cultivated
Before transfecting, carry a few days ago by 1.3～1.5 × 106GeneCopoeia 293T thin Born of the same parents move into 10 cm cell plates and cultivate, and add complete medium in plate (90%DMEM+10% hyclone), fusion rate transfects when fitting 70～80%.
2) DNA-EndoFectin slow virus mixture is prepared
PEGFP-CD70CAR plasmid and 5.0ul LentiPac HIV is added toward aseptic EP pipe Mix reagent, is diluted to 200 μ l with Opti-MEM I culture medium.In another aseptic EP manages, 15ul EndoFectin transfection reagent is diluted with Opti-MEM I.Limit softly carries out vortex, limit EndoFectin is diluted more than the mixed solution and dripping of DNA and LentiPac HIV reagent Transfection reagent.This solution 10 of incubated at room temperature～25 minutes, make DNA-EndoFectin mixture Produce.
3) Transfection of packaging cells
DNA-EndoFectin slow virus complex is directly added into cell plates, and soft vortex is put down Plate makes complex disperse.Incubated overnight 6～8 hours, to add 2～5% heat inactivation tire Sanguis Bovis seu Bubali Clearly, the fresh culture (90%DMEM+10% hyclone) of penicillin and streptomycin is more Change old culture medium, culture medium adds the TiterBoost reagent of 1/500 volume.
4) results lentiviral particle
Culture medium is collected in transfection two days later, is centrifuged 10 minutes with 500g, can obtain containing slow virus The culture medium of granule.After Li Xin, filtering with the low protein binding PES filter membrane of 0.45 μm should Culture medium.
C, T cell transfect
1) culture plate pretreatment
The restructuring laminins Retro Nectin of 20～100 μ g/ml is coated on untreated 24 On orifice plate, 4 DEG C overnight.Half an hour is closed by 2% bovine serum albumin BSA.Rush with buffer Wash three times.
2) virus is centrifugal
By viral supernatants, i.e. by step B obtain containing Virus culture base with 125～250 μ l/cm2 Concentration be added on 24 orifice plates of pretreatment, 4～6h, remove supernatant with PBS rinse once.
3) T cell transfection
Pipe adds 5ml human lymphocyte separation liquid in advance, takes anticoagulation 5ml, with normal saline After 1:1 mixing, it is slowly dropped into, is centrifuged 20 minutes with 1500 revs/min, thin film seen from naked eyes, Draw mononuclear cell herein, put in the test tube containing normal saline 10 milliliters, fully mix After, it is centrifuged 10 minutes with 500g.Abandoning liquid, precipitation is repeatedly washed 2 times through aseptic PBS and is i.e. obtained institute Need cell.Stimulate the T of antibody OKT3 and IL-2 activation thin by having shifted to an earlier date three days with CD3 Born of the same parents are with 0.2～1 × 105The concentration of individual/ml adds, with the centrifugal force 10min of 2000g, Hatching 72h, obtain specific recognition EGFRvIII modifies T containing CD70 Chimeric antigen receptor Cell.
CAR-T technology experienced by three generations's development, is applied to now the mainly the 3rd of clinical experiment the Dai Hanshuan costimulatory molecules CAR.On this basis, present invention adds new costimulatory molecules CD70.In CAR-T cell, the interaction of CD70-CD27 can induce CD8+T thin Born of the same parents play higher tumor-killing and melt virus function.Therefore, the CAR-T of CD70 is introduced There is more powerful anti-tumor capacity, life cycle will be extended.
Detailed description of the invention
Below technical scheme is further described, but is not limited thereto, all It is technical solution of the present invention to be modified or equivalent, without deviating from the technology of the present invention side The spirit and scope of case, all should contain in protection scope of the present invention.
The chimeric antigen containing CD70 that the invention provides a kind of specific recognition EGFRvIII is subject to Body modifies the preparation method of T cell, specifically includes following steps:
A, plasmid construction: build slow virus packaging plasmid pEGFP-CD70CAR plasmid, CD70CAR by homing sequence, variable region of heavy chain, hinge region, variable region of light chain, CD8 across Film district, CD28 domain, CD137 domain, CD70 domain and CD3zeta chain structure Become, according to sequence chemistry synthetic gene fragment；
Plasmid amplification and purification: ultra-vioket radiation Bacteriology Room super-clean bench 30min, built-in tweezers, height The pressure instruments box (pincet, shears) of sterilizing, 1.5mL sterile centrifugation tube, 200 μ L move liquid Device and sterile pipette tip.The filter paper circle carrying pEGFP-CD70CAR plasmid is cut in super-clean bench 1/3 be placed in centrifuge tube, add appropriate aseptic double-distilled water and soak 30min, centrifugal after stirring Take supernatant, be placed in-20 DEG C and save backup.Remaining 2/3 carries pEGFP-CD70CAR matter Preservation (-80 DEG C of refrigerators) sealed by the filter paper of grain.
Join in EP pipe for expanding the competence bacillus coli DH 5 alpha of plasmid, add PEGFP-CD70CAR plasmid 1 μ L/ manages, and stands 30min after mixing on ice, if the most right According to: the competent cell that 1. 2 μ L pcDNA3.1 convert is as positive control；It is not added with the most completely The competent cell of plasmid DNA is as negative control.42 DEG C of heat shocks of low temperature 1.5～2min, Be placed in ice 5 minutes, add Luria-Bertani culture medium, shaking table 150rpm shaking 45～ 60min, makes bacteria resuscitation and expresses pEGFP-EFGRvIII and pEGFP-CD70CAR matter Grain ampicillin AMP Drug resistance, transfers to ammonia by the competent cell that 200 μ L have converted On the solid medium flat board of benzylpcnicillin (Amp+) (flat board needs 37 DEG C of incubator preheatings), Coating uniformly, after drying, is cultivated 12-16 hour.
After picking conversion pEGFP-EFGRvIII and pEGFP-CD70CAR on Amp+ flat board The single bacterium colony grown put in test tube added with 3mL Amp+LB (in each test tube only Add a bacterium colony), 180rpm shakes 12-16 hour, according to plasmid extraction kit (sky root Company, DP117) plasmid extraction: in adsorption column CP6 (plasmid extraction kit offer) Adding the balance liquid BL (plasmid extraction kit offer) of 2.5ml, 8,000rpm are centrifuged 2 Min, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column.Take 100ml The bacterium solution of incubated overnight adds centrifuge tube, room temperature 8, and 000rpm is centrifuged 3min and collects antibacterial, Absorb supernatant as far as possible.8ml solution P1 (plasmid is added in the centrifuge tube leave bacterial sediment Extract test kit to provide), use pipettor or turbula shaker thorough suspended bacterial cell precipitation. 8ml solution P2 (plasmid extraction kit offer) is added, the most leniently in centrifuge tube Spinning upside down 6～8 times, room temperature places 5min.8ml solution P4 (matter is added in centrifuge tube Grain extracts test kit and provides), the most leniently spin upside down 6～8 times, fully mix, extremely There is white dispersion flocculent deposit in solution.Then room temperature places 10min.8,000rpm be centrifuged 5～ 10min, makes white precipitate at the bottom of pipe, complete soln is carefully poured into filter CS1 (matter Grain extracts test kit and provides) in, slowly promote push handle to filter, filtrate collection is at clean 50ml Pipe in.In filtrate, add the isopropanol of 0.3 times of filtrate volume, turn after mixing of turning upside down Move on in adsorption column CP6.Room temperature 8,000rpm (～8,228 × g) centrifugal 2min, outwells receipts Waste liquid in collector, places back in adsorption column CP6 in collecting pipe.In adsorption column CP6 Adding 10ml rinsing liquid PW (plasmid extraction kit offer), 8,000rpm are centrifuged 2min, Discard the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.To adsorption column CP6 Middle addition 3ml dehydrated alcohol, room temperature 8,000rpm is centrifuged 2min, outwells waste liquid.To inhale Attached column CP6 places back in collecting pipe, and 8,000rpm are centrifuged 5min, it is therefore an objective to will absorption Rinsing liquid remaining in post is removed.Adsorption column CP6 is placed in a clean 50ml collecting pipe In, to the unsettled dropping in the middle part of adsorbed film 1～2ml elution buffer TB, (plasmid carries Take test kit to provide), room temperature places 5min, then room temperature 8, and 000rpm is centrifuged 2min. Eluent in 50ml centrifuge tube is all moved into a clean 1.5ml centrifuge tube ,-20 DEG C Preserve.Extracted plasmid is detected with nucleic acid-protein detector (NanoDrop 2000) The concentration of pEGFP-CD70CAR and purity.
B, slow virus packaging and titer determination
Use slow virus package kit (purchased from GeneCopoeia, No.HPK-LvTR-20) Packaging virus.
1) incasing cells is cultivated
Before transfecting, carry a few days ago by 1.3～1.5 × 106GeneCopoeia 293T thin Born of the same parents move into 10 cm cell plates and cultivate, and add complete medium in plate (90%DMEM+10% hyclone), fusion rate transfects when fitting 70～80%.
2) DNA-EndoFectin slow virus mixture is prepared
PEGFP-CD70CAR plasmid and 5.0ul LentiPac HIV is added toward aseptic EP pipe Mix reagent (is provided by slow virus package kit), (purchases with Opti-MEM I culture medium From Gibco company) it is diluted to 200 μ l.In another aseptic EP manages, with Opti-MEM I Dilution 15ul EndoFectin transfection reagent (being provided by slow virus package kit).Limit is soft Carry out vortex, while toward more than the mixed solution and dripping of DNA and LentiPac HIV reagent dilute Release EndoFectin transfection reagent.Order of addition can not be overturned, in order to avoid affecting transfection efficiency.As System is relatively big, and round bottom centrifuge tube (such as Falcom 5ml or 15ml centrifuge tube) can be used to contain molten Liquid.This solution 10 of incubated at room temperature～25 minutes, make DNA-EndoFectin mixture produce.
3) Transfection of packaging cells
DNA-EndoFectin slow virus complex is directly added into cell plates, and soft vortex is put down Plate makes complex disperse.Incubated overnight 6～8 hours, to add 2～5% heat inactivation tire Sanguis Bovis seu Bubali Clearly, the fresh culture (90%DMEM+10% hyclone) of penicillin and streptomycin is more Change old culture medium, culture medium adds the TiterBoost reagent of 1/500 volume (by slow virus Package kit provides).
4) results lentiviral particle
Culture medium is collected in transfection two days later, is centrifuged 10 minutes with 500g, can obtain containing slow virus The culture medium of granule.After Li Xin, filtering with the low protein binding PES filter membrane of 0.45 μm should Culture medium.
5) slow virus titer determination
According to lentiviral particle titre detection kit (purchased from GeneCopoeia, No. HPR-LTK-050) qRT-PCR detection (ABI 7300 real-time PCR system) is carried out. Draw standard curve, calculate titre.
C, T cell transfect
1) culture plate pretreatment
By the restructuring laminins Retro Nectin of 20～100 μ g/ml (purchased from Takara company, T100a) being coated on untreated 24 orifice plates, 4 DEG C overnight.Use 2% bovine serum albumin BSA Close half an hour.With wash buffer three times.Sealed membrane seals, and can preserve one under the conditions of 4 DEG C Week.
2) virus is centrifugal
By viral supernatants viral supernatants, i.e. by step B obtain containing Virus culture base with 125～ 250μl/cm2Concentration be added on the culture plate of pretreatment, 4～6h, remove supernatant PBS Rinse once.
3) T cell transfection
Pipe adds 5ml human lymphocyte separation liquid (purchased from Hao ocean, Tianjin company) in advance, takes anti- Blood coagulation 5ml, after mixing with normal saline 1:1, is slowly dropped into, with 1500 revs/min, rises slowly Centrifugal 20 minutes of slow fall, thin film seen from naked eyes, draw mononuclear cell herein, put into containing raw In the test tube of reason saline 10 milliliters, fully after mixing, it is centrifuged 10 minutes with 500g.Abandon liquid, Precipitation is repeatedly washed 2 times through aseptic PBS and is i.e. obtained required cell.CD3 is used by having shifted to an earlier date three days Stimulate the T that antibody OKT3 (purchased from Mei Tian Ni company, 130-093-387) and IL-2 activates Cell is with 0.2～1 × 105The concentration of individual/ml adds, with the centrifugal force 10min of 2000g, Hatch 72h.
D, build specific recognition EGFRvIII containing CD70CAR-T cell
T cell after transfection is observed under fluorescence microscope, it is seen that have green fluorescence table Reach, it was demonstrated that specific recognition EGFRvIII containing CD70CAR-T cell construction success.

Claims (3)

1. the modification T containing CD70 Chimeric antigen receptor of specific recognition EGFRvIII is thin The preparation method of born of the same parents, it is characterised in that described method step is as follows:
A, plasmid construction: build slow virus packaging plasmid pEGFP-CD70CAR plasmid；
Plasmid amplification and purification: cut in the super-clean bench after irradiating through ultraviolet and carry The 1/3 of the filter paper circle of pEGFP-CD70CAR plasmid is placed in centrifuge tube, adds aseptic double steaming Water soaking 30min, centrifuging and taking supernatant after stirring, it is placed in-20 DEG C and saves backup；
The competence bacillus coli DH 5 alpha being used for expanding plasmid is joined in EP pipe, adds PEGFP-CD70CAR plasmid 1 μ L/ manages, and stands 30min after mixing on ice；Low temperature 42 DEG C Heat shock 1.5～2min, is placed in ice 5 minutes, adds Luria-Bertani culture medium, shaking table 150rpm shaking 45～60min, make bacteria resuscitation and express pEGFP-EFGRvIII and 200 μ L have been converted by pEGFP-CD70CAR plasmid ampicillin AMP Drug resistance Competent cell is transferred on the solid medium flat board of ampicillin, and coating uniformly, is dried After, cultivate 12-16 hour；
After picking conversion pEGFP-EFGRvIII and pEGFP-CD70CAR on Amp+ flat board The single bacterium colony grown is put in test tube added with 3mL Amp+LB, and 180rpm shakes 12-16 hour, according to plasmid extraction kit plasmid extraction；
B, slow virus packaging
1) incasing cells is cultivated
Before transfecting, carry a few days ago by 1.3～1.5 × 106GeneCopoeia 293T thin Born of the same parents move into 10 cm cell plates and cultivate, and add complete medium in plate, fusion rate be 70～ Transfect when 80%；
2) DNA-EndoFectin slow virus mixture is prepared
PEGFP-CD70CAR plasmid and 5.0ul LentiPac HIV is added toward aseptic EP pipe Mix reagent, is diluted to 200 μ l with Opti-MEM I culture medium；In another aseptic EP manages, 15ul EndoFectin transfection reagent is diluted with Opti-MEM I；While carry out vortex, while toward DNA Try with more than the mixed solution and dripping of LentiPac HIV reagent diluting EndoFectin transfection Agent；This solution 10 of incubated at room temperature～25 minutes, make DNA-EndoFectin mixture produce；
3) Transfection of packaging cells
DNA-EndoFectin slow virus complex is directly added cell plates, dispersed complex； Incubated overnight 6～8 hours, to add 2～5% heat-inactivated fetal bovine serum, penicillin and strepto- The fresh culture of element changes old culture medium, adds the TiterBoost of 1/500 volume in culture medium Reagent；
4) results lentiviral particle
Culture medium is collected in transfection two days later, is centrifuged 10 minutes with 500g, can obtain containing slow virus The culture medium of granule；After Li Xin, filtering with the low protein binding PES filter membrane of 0.45 μm should Culture medium；
C, T cell transfect
1) culture plate pretreatment
The restructuring laminins Retro Nectin of 20～100 μ g/ml is coated on untreated On 24 orifice plates, 4 DEG C overnight；Half an hour is closed by 2% bovine serum albumin BSA；Use buffer Rinse three times；
2) virus is centrifugal
By viral supernatants, i.e. by step B obtain containing Virus culture base with 125～250 μ l/cm2 Concentration be added on 24 orifice plates of pretreatment, 4～6h, remove supernatant with PBS rinse once；
3) T cell transfection
Pipe adds 5ml human lymphocyte separation liquid in advance, takes anticoagulation 5ml, with normal saline After 1:1 mixing, it is slowly dropped into, is centrifuged 20 minutes with 1500 revs/min, thin film seen from naked eyes, Draw mononuclear cell herein, put in the test tube containing normal saline 10 milliliters, fully mix After, it is centrifuged 10 minutes with 500g；Abandoning liquid, precipitation is repeatedly washed 2 times through aseptic PBS and is i.e. obtained institute Need cell；T cell that antibody OKT3 and IL-2 activate will be stimulated with 0.2～1 × 10 with CD35 The concentration of individual/ml adds, and with the centrifugal force 10min of 2000g, hatches 72h, obtains special The opposite sex identifies that EGFRvIII's modifies T cell containing CD70 Chimeric antigen receptor.
Being fitted together to containing CD70 of specific recognition EGFRvIII the most according to claim 1 Antigen receptor modifies the preparation method of T cell, it is characterised in that described solid medium flat board makes Used time needs 37 DEG C of incubator preheatings.
Being fitted together to containing CD70 of specific recognition EGFRvIII the most according to claim 1 Antigen receptor modifies the preparation method of T cell, it is characterised in that the composition of described complete medium For: 90%DMEM+10% hyclone.
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