CN108864289A

CN108864289A - The double target spot CAR-T therapy vectors and its construction method of gastric cancer and application

Info

Publication number: CN108864289A
Application number: CN201810384271.XA
Authority: CN
Inventors: 田晓丽
Original assignee: Shanghai Yi Hao Biotechnology Co Ltd
Current assignee: Shanghai Yi Hao Biotechnology Co Ltd
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2018-11-23

Abstract

The present invention relates to the double target spot CAR-T therapy vectors for providing gastric cancer and its construction method and applications.Double target spot CAR-T therapy vectors are made of Lentiviral pCDH-EF1 α-MCS- (PGK-Puro) and CAR structure two parts.The CAR structure is specially Igkappa-iRGD-EpCAMscFv-(G₄S)₅-Her-2scFv-CD8α-CD28-CD137-CD3ζ-2A-CCL19.The T cell containing the therapy vector, i.e. CAR-T cell (Chimeric Antigen Receptor T-Cell) are obtained in a manner of virus infection, it, can targets identification and killing stomach cancer cell by expressing CAR structure.

Description

The double target spot CAR-T therapy vectors and its construction method of gastric cancer and application

Technical field

The present invention relates to a kind of CAR-T therapy vectors, double target spot CAR-T therapy vectors more particularly, to gastric cancer and its Construction method and application.

Background technique

Gastric cancer is China's clinical relatively common one of malignant tumour at present, the spy with high incidence and high mortality Point, in China, the morbidity and mortality of gastric cancer account for the third position of malignant tumour.Currently, the therapeutic modality of gastric cancer mainly has hand Art treatment, radiotherapy, chemotherapy, targeted therapy, immunization therapy.Operation is the only possible mode for curing gastric cancer, but advanced gastric carcinoma Surgery alone treatment may exist naked eyes or mirror under remain stove, so that recurrence and metastatic rate is greatly increased；Toxic and side effects of chemoradiotherapy Greatly, curative effect can not reach promising result；There are drug resistance, the obvious benefits of nothing etc. for molecular targeted therapy such as Herceptin treatment Problem；Immunization therapy such as immunologic test point inhibitor PD-L1 antibody do not observe in gastric cancer and are relieved that this may It is related with tumour cell itself and its microenvironment.

CAR-T cell therapy is artificially to be overexpressed to identify specific tumour on T cell surface by technique for gene engineering The single-chain variable fragments of surface antigen, to make T cell identification specific antigen, kill the target for expressing the antigen Cell.Simultaneously as CAR-T cell is to identify antigen mode using antibody, therefore not by major histocompatibility complex The limitation of (main histocompatibility complex, MHC).In recent years, CAR-T cell is in neoplastic hematologic disorder treatment Achieve exciting effect, such as complete incidence graph to late recurrent refractory childhood acute lymphoblastic leukemia (ALL) treatment (CR) it can reach 90%, 50% or more reached to the CR of chronic lymphocytic leukemia (CLL) and partial B cell lymthoma.? In terms for the treatment of solid tumor, safety and validity have been confirmed.

Chinese patent CN107557388A discloses the slow virus carrier and its construction method for the preparation of CAR-T cell With in the method for preparing the application in antitumor cell product, while having also set up fluorescence quantitative PCR detection virus titer, it is Clinical application provides examination criteria.

Chinese patent CN107446938A discloses a kind of selectively targeted receptor protein MAGEA and its CAR-carrier T Construction method, the construction method include first extracting the RNA of the monoclonal hybridoma of anti-osteosarcoma, then using monoclonal The specific primer of antibody carries out RT-PCR, and the variable region DNA fragmentation of heavy chain of antibody and light chain is cloned into carrier T, surveys Sequence, obtains the receptor protein MAGEA of specific recognition tumour cell, and its expressed sequence is cloned on CAR-T carrier, obtains Obtain the novel C AR-T carrier of selectively targeted MAGEA.The invention can be the entity tumor of CAR-T cell therapy, especially Effective target is provided in the immunization therapy of osteosarcoma.

Chinese patent CN107325185A discloses a kind of bis- targeting chimeric antigens of the PSCA based on OCTS-CAR and PDL1 Receptor, encoding gene, OCTS-CAR-T recombinant expression carrier and its construction method and application.The bis- targetings of the anti-PSCA and PDL1 Chimeric antigen receptor includes the CD8leader membrane receptor signal peptide, double antigen binding domains, the CD8Hinge that are sequentially connected in series embedding Close receptor hinge, CD8Transmembrane Chimerical receptor transmembrane region, CD28 Chimerical receptor costimulating factor, OX40 it is chimeric by Body costimulating factor and TCR Chimerical receptor t cell activation domain, wherein double antigen binding domains include with series connection or corner connection Heavy chain VH and light chain VL, the antibody inner hinge Inner-Linker and list for PSCA the and PDL1 single-chain antibody that mode connects Hinge Inter-Linker between chain antibody.The bis- targeting Chimeric antigen receptors of the anti-PSCA and PDL1 are encoded in addition, additionally providing Gene, recombinant expression carrier and its construction method and application.

Above-mentioned patent is the correlative study about CAR-T cell therapy, but lacks carry out double targets for gastric cancer at present The carrier of point CAR-T treatment or correlative study.

Summary of the invention

It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide double target spot CAR- of gastric cancer T therapy vector and its construction method and application.

The purpose of the present invention can be achieved through the following technical solutions：

First purpose of the invention：

A kind of Chimeric antigen receptor, i.e. CAR be provided, CAR structure include EpCAM single-chain antibody, Her-2 single-chain antibody and Tri- specificity structures of Chemokines CC CL19.

Wherein, EpCAM single-chain antibody structure is for the special of stomach cancer cell surface tumours related antigen EpCAM design Property antigen binding domain, EpCAM single-chain antibody nucleotide sequence is as shown in SEQ ID NO.2.

Wherein, Her-2 single-chain antibody structure is for the special of stomach cancer cell surface tumours related antigen Her-2 design Property antigen binding domain, nucleotide sequence is as shown in SEQ ID NO.4.

Chemokines CC CL19 chemotactic T cell infiltrates tumor tissues, and mediate tumor is immune, while inhibiting that suppression is immunized in tumour The release of the factor processed promotes Immune Cell Antigens to offer to act on, and inhibits vascularization indirectly, the final growth for inhibiting tumour； Chemokines CC CL19 nucleotide sequence is as shown in SEQ ID NO.10.

In addition, Recent study is found, CCR7 receptor in expression in gastric carcinoma, CCL19 can directly with CCR7 receptor knot It closes, access inhibits tumor proliferation, migration and invasion after activated receptor.

Further, CAR structure composition is Igkappa-iRGD-EpCAM scFv- (G₄S)₅-Her-2scFv -CD8α- CD28-CD137-CD3ζ-T2A-CCL19。

Wherein iRGD structure can express iRGD peptide, and nucleotide sequence is as shown in SEQ ID NO.1, Kazuki Sugahara and its colleague once reported that the peptide of iRGD was a kind of peptide that can penetrate tumour, when iRGD is as a kind of individual When substance is injected together with anti-tumor drug, drug delivery and anti-tumor activity can be greatly enhanced.

EpCAM single-chain antibody is expressed as EpCAMscFv in structure.

(G₄S)₅The hinge Inner-Linker between single-chain antibody, nucleotide sequence is as shown in SEQ ID NO.3.

Her-2 single-chain antibody is expressed as Her-2scFv in the structure.

CD8 α is transmembrane region, connects extracellular antigen binding domain and intracellular signal domain, can be by CAR Structure anchor in T cell membrane On, nucleotide sequence is as shown in SEQ ID NO.5.

CD28-CD137 is costimulation structural domain, and the generation of transduction proliferation signal and inducing cytokine inhibits tumour raw Long, CD28 nucleotide sequence is as shown in SEQ ID NO.6, and CD137 nucleotide sequence is as shown in SEQ ID NO.7.

CD3 ζ is signal transduction domain, when extracellular region and target antigen combination, will be swashed to conduction TCR sample signal intracellular T cell living, plays the effect of targeting killing tumor cell, nucleotide sequence is as shown in SEQ ID NO.8.

T2A is that linker- is found in bright tetra- precursor virus of arteries and veins thosea siensis β (Thosea asigna), and effect is to make one Single fragment coding protein, its advantage is that two genes (ORF) can be connected to become an ORF by T2A polypeptide, mRNA is turned over It is translated into a fusion protein, which can be identified the proteolytic cleavage of T2A into two albumen, nucleotide sequence such as SEQ Shown in ID NO.9.

Second purpose of the invention：

A kind of double target spot CAR-T therapy vectors of gastric cancer are provided, the Chimeric antigen receptor including first goal of the invention, And PUC19 plasmid.The Chimeric antigen receptor of first goal of the invention is present on PUC19 plasmid vector.

Third purpose of the present invention：

A kind of double target spot CAR-T therapy vectors of gastric cancer, including Lentiviral pCDH-EF1 α-MCS- are provided (PGK-Puro) and CAR.

Wherein, Lentiviral pCDH-EF1 α-MCS- (PGK-Puro), structure is as shown in Figure 1.Pass through Snap Gene software analyzes the carrier and searches pertinent literature it is found that pCDH-EF1 α-MCS- (PGK-Puro) is bis- with EcoRI and NotI Digestion Insert Fragment.The expression vector includes：EF1 α promoter-is mammalian cell specificity promoter, driving capability compared with By force；Multiple cloning sites (MCS)-include multiple restriction enzyme sites (restriction site), are that foreign gene is inserted into Position；The polyA tailing efficiency of mRNA can be improved in WPRE element-, improves the expression efficiency of metastatic gene；SV40 polyA sequence It arrange-can effectively terminate transcription and add PolyA tail for the mRNA of transcription；Hybrid RSV/5 ' LTR- contains promoter and increasing The controlling elements such as hadron make its high-caliber expression overall length virus transcription object in 293T cell；Genetic element (cPPT, gag, Env, LTRs)-for packing, transduceing and steadily will be in the genomic DNA of expressing viral structural integrity to host；SV40 Origin- makes plasmid stablize proliferation in incasing cells.

The Lentiviral, which can be used as, makes target gene in nearly all mammalian cell include non-dividing cell With the most effective carrier expressed in dividing cell, can hold carry exogenous genetic fragment it is big, transfection efficiency is higher, also can to T cell Reach satisfied transfection.

4th purpose of the invention：A kind of construction method of double target spot CAR-T therapy vectors of gastric cancer is provided.

It by above-mentioned CAR structure according to gene order, is synthesized using standard biologic synthetic method, the CAR after synthesis is deposited It is on PUC19 plasmid vector；Lentiviral pCDH-EF1 α-MCS- (PGK-Puro) is purchased from SBI.By PUC19 matter Grain carrier and Lentiviral are all made of ECoR I, Not I double digestion, and digestion products are passed through agarose gel electrophoresis Separation, recycle purpose band, obtain the concentration of carrier and target fragment, by the two according to molar ratio be 1:5 be attached turn Change, plasmid extracts, final to obtain the recombinant plasmid for containing specific CAR structure.

5th purpose of the invention：The application of double target spot CAR-T therapy vectors of gastric cancer is provided, that is, a kind of CAR-T is provided Cell.

The therapy vector is subjected to slow virus packaging using three plasmid packaging systems, group becomes PSPAX2, pMD2G, gastric cancer Double target spot CAR-T therapy vectors, use 293T cell as the incasing cells of slow virus, after collecting culture 48h, 72h respectively Virus liquid, this virus liquid is concentrated, and survey virus titer, finally with MOI=20 infect T cell, it is thin to can be obtained CAR-T Born of the same parents.

This three kinds of plasmids content of (6cm ware system) in system is respectively 4 μ g, 0.5 μ g, 3 μ g.

In the present invention, CAR structure includes tri- EpCAM single-chain antibody, Her-2 single-chain antibody and Chemokines CC CL19 spies Anisotropic approach；EpCAM single-chain antibody, Her-2 single-chain antibody are to express tumor associated antigen according to stomach cancer cell surface The specificity structure that EpCAM, Her-2 are determined, two strain specific antibodies of Anti-EpCAM, Anti-Her-2 of the structure representation Be responsible for identification tumor associated antigen (tumor associated antigen, TAA), on the one hand can targeting killing gastric cancer it is thin Born of the same parents；On the other hand, it assigns T cell new antigentic specificity, can effectively avoid tumour cell MHC expression and lower this and be immunized and escape Ease mechanism.Chemokines CC CL19 can induce immunocyte infiltration and play immune function into tumor tissues.When T cell passes through disease After malicious mode of infection obtains the carrier and expresses the CAR structure, T cell can targeting identify and kill stomach cancer cell.Pass through CAR structure is expressed, it can targets identification and killing stomach cancer cell.

Compared with prior art, the present invention has the following advantages and beneficial effects：

Since the T cell can express the antibody of tumor associated antigen Her-2, EpCAM for stomach cancer cell surface simultaneously Anti-Her-2, Anti-EpCAM, so that its identification range greatly increases, it is wider for the hazard boundary of stomach cancer cell, it can obtain Obtain better therapeutic effect；Meanwhile the CAR-T cell of single antigen receptor can be fitted into avoid multiple infusion, not only alleviate Injury to patient can also mitigate its economic pressures；In addition, to impart T cell stronger for CAR structure mentioned in the present invention Proliferative, lasting vitality, and by Chemokines CC CL19 chemotactic T cell infiltrate tumor tissues, inhibit tumor in be immunized The release of the factor enables T cell to overcome tumor by local immunosupress microenvironment and break host immune tolerance status, fast run-up Vertical tumour immunity simultaneously eliminates small-sized or large-scale tumor load.

Detailed description of the invention

Fig. 1 is Lentiviral pCDH-EF1 α-MCS- (PGK-Puro) structural schematic diagram；

Fig. 2 is cytotoxicity analysis detection technique flow chart；

Fig. 3 is the Western blot testing result figure of targeting antigen EpCAM expression in ags cell system；

Fig. 4 is the Western blot testing result figure of targeting antigen Her-2 expression in ags cell system.

Fig. 5 is Ago-Gel electricity of pCDH-EF1 α-MCS- (PGK-Puro) plasmid after ECoR I, Not I digestion Swimming result.

Specific embodiment

Material source in following embodiment is described as follows：

1. slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro), slow virus packaging plasmid pMD2G, carrier matter Grain PSPAX2 is purchased from SBI；Slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro) structure is as shown in Figure 1.

2.CAR structure sequence is designed by Shanghai Yi Hao Biotechnology Co., Ltd, by raw work bioengineering (Shanghai) share Co., Ltd's synthesis, with the preservation of PUC19 plasmid form；

3. restriction endonuclease EcoR I, Not I are purchased from NEB；

4.T4DNA ligase、Free H₂O is purchased from precious biology；

5. competent cell is purchased from Trans；

6. plastic recovery kit, the small extraction reagent kit of plasmid is purchased from Tiangeng biochemical technology Co., Ltd；

7.293T cell, ags cell are purchased from Chinese Academy of Sciences's cell bank；

8.FBS, DMEM, 1640 culture mediums, PBS, Opti-MEM, lipofectamine 2000 are purchased from Gibco；

9. cell pyrolysis liquid RIPA, protease inhibitors is purchased from the green skies；

10.BCA protein quantification kit, the super quick colour reagent box of ECL chemiluminescence, Tween-20,5 × SDS-PAGE Albumen sample-loading buffer, HRP-labeled Goat Anti-Rabbit IgGHRP-labeled Goat Anti-Mouse IgG is holy purchased from assist；

11. electrophoresis liquid, fast-turn construction liquid, glue are purchased from Tanon；

12. skimmed milk power is purchased from BD；

13.PVDF film is purchased from Millipore；

14. membrane regeneration liquor is purchased from Suo Laibao Biotechnology Co., Ltd；

15.Anti-CD3zeta antiboby is purchased from abcam；

16.Beta Actin Antibody is purchased from Wuhan Sanying Bio-Technology Co., Ltd.；

17. albumen Marker is purchased from Thermo；

18. high-pressure steam sterilizing pan is pacified purchased from Shanghai Shen；

19. air dry oven is purchased from Shanghai Ba Jiu Industrial Co., Ltd.；

20.ProFlex PCR instrument is purchased from ABI；

21.Multiskan GO microplate reader+uDrop ultra micro template, flow cytometer are purchased from ThermoFisher；

22.ST16R tabletop refrigerated centrifuge, Fresco microcentrifuge are purchased from ThermoFisher；

23.HE120 Horizontal electrophoresis tank, Tanon gel imager are purchased from Tanon；

24. Biohazard Safety Equipment, CO₂4 DEG C of directly-heated type cell incubator, double door refrigerators are purchased from ThermoFisher；

25. pipettor, electronic suction assisting device are purchased from Eppendorf；

26. water isolation type constant incubator, constant-temperature shaking incubator are purchased from the permanent Science and Technology Ltd. in Shanghai one；

The 27.Bio-Rad small-sized electrophoresis system of Mini-PROTEAN Tetra Cell is purchased from Bio-Rad；

28. Olympus microscope is purchased from Olympus；

29. oese, spreading rod Jie Te biofiltration limited liability company；

30. syringe, the culture dish of 0.45 μm of filter membrane, each specification, culture bottle, porous culture plate, various specifications centrifuge tube Purchased from Corning；

A kind of Chimeric antigen receptor, i.e. CAR, CAR structure include EpCAM single-chain antibody, Her-2 single-chain antibody and chemotactic Tri- specificity structures of factor CCL19.

CAR structure composition is Igkappa-iRGD-EpCAM scFv- (G₄S)₅-Her-2scFv -CD8α-CD28- CD137-CD3ζ-T2A-CCL19。

Wherein iRGD structure can express iRGD peptide, and nucleotide sequence is as shown in SEQ ID NO.1, Kazuki Sugahara and its colleague once reported that the peptide of iRGD was a kind of peptide that can penetrate tumour, when iRGD is as a kind of individual When substance is injected together with anti-tumor drug, drug delivery and anti-tumor activity can be greatly enhanced.EpCAM single-chain antibody structure It is the specific antigen binding domain for stomach cancer cell surface tumours related antigen EpCAM design, EpCAM single-chain antibody nucleosides Acid sequence is as shown in SEQ ID NO.2.EpCAM single-chain antibody is expressed as EpCAMscFv in structure.(G₄S)₅For single-chain antibody Between hinge Inner-Linker, nucleotide sequence is as shown in SEQ ID NO.3.Her-2 single-chain antibody structure is for gastric cancer The specific antigen binding domain of cell surface tumor related antigen Her-2 design, nucleotide sequence is as shown in SEQ ID NO.4. Her-2 single-chain antibody is expressed as Her-2scFv in the structure.CD8 α is transmembrane region, connects extracellular antigen binding domain and letter intracellular Number domain, can be by CAR Structure anchor on T cell film, and nucleotide sequence is as shown in SEQ ID NO.5.CD28-CD137 is total Stimulus structure domain, the generation of transduction proliferation signal and inducing cytokine inhibit tumour growth, CD28 nucleotide sequence such as SEQ Shown in ID NO.6, CD137 nucleotide sequence is as shown in SEQ ID NO.7.CD3 ζ is signal transduction domain, when extracellular region and target When antigen binding, T cell will be activated to conduction TCR sample signal intracellular, play the effect of targeting killing tumor cell, core Nucleotide sequence is as shown in SEQ ID NO.8.T2A is that linker- is found in bright tetra- precursor virus (Thosea of arteries and veins thosea siensis β Asigna), effect is to make a single fragment coding protein, its advantage is that two genes (ORF) can pass through T2A polypeptide It is connected to become an ORF, mRNA translates into a fusion protein, which can be identified the proteolytic cleavage of T2A at two A albumen, nucleotide sequence is as shown in SEQ ID NO.9.Chemokines CC CL19 chemotactic T cell infiltrates tumor tissues, mediates Tumour immunity, while inhibiting the release of immunosuppressive factor in tumour, promote Immune Cell Antigens to offer to act on indirectly, and press down Vascularization processed, the final growth for inhibiting tumour；Chemokines CC CL19 nucleotide sequence is as shown in SEQ ID NO.10.In recent years Come the study found that CCR7 receptor is in expression in gastric carcinoma, CCL19 can be directly in conjunction with CCR7 receptor, access after activated receptor Inhibit tumor proliferation, migration and invasion.

Double target spot CAR-T therapy vectors of gastric cancer, including Lentiviral pCDH-EF1 α-MCS- (PGK-Puro) And CAR.

Wherein, Lentiviral pCDH-EF1 α-MCS- (PGK-Puro), structure is as shown in Figure 1.Pass through Snap Gene software analyzes the carrier and searches pertinent literature it is found that pCDH-EF1 α-MCS- (PGK-Puro) is bis- with EcoRI and NotI Digestion Insert Fragment.The expression vector includes：EF1 α promoter-is mammalian cell specificity promoter, driving capability compared with By force；Multiple cloning sites (MCS)-include multiple restriction enzyme sites (restriction site), are that foreign gene is inserted into Position；The polyA tailing efficiency of mRNA can be improved in WPRE element-, improves the expression efficiency of metastatic gene；SV40polyA sequence It arrange-can effectively terminate transcription and add PolyA tail for the mRNA of transcription；Hybrid RSV/5 ' LTR- contains promoter and increasing The controlling elements such as hadron make its high-caliber expression overall length virus transcription object in 293T cell；Genetic element (cPPT, gag, Env, LTRs)-for packing, transduceing and steadily will be in the genomic DNA of expressing viral structural integrity to host；SV40 Origin- makes plasmid stablize proliferation in incasing cells.The Lentiviral, which can be used as, makes target gene nearly all Mammalian cell includes the most effective carrier expressed in non-dividing cell and dividing cell, can hold load exogenous genetic fragment Greatly, transfection efficiency is higher, can also reach satisfied transfection to T cell.

The construction method of double target spot CAR-T therapy vectors of gastric cancer：By above-mentioned CAR structure according to gene order, using normal Rule biological synthesis method is synthesized, and the CAR after synthesis is present on PUC19 plasmid vector；Lentiviral pCDH- EF1 α-MCS- (PGK-Puro) is purchased from SBI.PUC19 plasmid vector and Lentiviral are all made of ECoR I, Not I couple Digestion products are separated by agarose gel electrophoresis, recycle purpose band, obtain the concentration of carrier and target fragment by digestion, By the two according to molar ratio be 1:5 are attached conversion, and plasmid extracts, final to obtain the recombination matter for containing specific CAR structure Grain.

The therapy vector is subjected to slow virus packaging using three plasmid packaging systems, group becomes PSPAX2, pMD2G, gastric cancer Double target spot CAR-T therapy vectors, content of this three kinds of plasmids in system (6cm ware system) is respectively 4 μ g, 0.5 μ g, 3 μ g.Use 293T cell as the incasing cells of slow virus, the virus liquid after collecting culture 48h, 72h respectively, by this virus liquid Concentration, and virus titer is surveyed, T cell is finally infected with MOI=20, can be obtained CAR-T cell.

The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.

Embodiment

(1) plasmid extracts

The preparation method of LB liquid medium：Electronic balance weighs 5g fluid nutrient medium dry powder in 500mL conical flask, adds 100mL ultrapure water sterilizes in high-pressure steam sterilizing pan after masking foil sealing, when being cooled to 40 DEG C~50 DEG C, with 1000:1, which is added 0.2% ampicillin, is transferred to spare, condition of storage in clean 500mL reagent bottle after careful mixing It is 4 DEG C.

The preparation method of LB solid medium：Electronic balance weighs 5g solid medium dry powder in 500mL conical flask, adds 100mL ultrapure water sterilizes in high-pressure steam sterilizing pan after masking foil sealing, when being cooled to 40 DEG C~50 DEG C, with 1000:1 is added 0.2% ampicillin, and after careful mixing, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after solidification, seals membrana oralis and is stored in 4 DEG C.

Glycerol stock is taken out respectively from -80 DEG C of refrigerators, and glycerol stock contains slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro) and PUC19 plasmid it, takes 5 μ L to be inoculated in 5mL LB liquid medium (AMP resistance) respectively, respectively takes 8 pipes, point It does not mark, in constant-temperature table 12~16h of shaken cultivation, condition is 37 DEG C, 250rmp.

The small extraction reagent kit of plasmid is purchased from Tiangeng biochemical technology Co., Ltd, carries out plasmid extraction by its specification.Specific behaviour Make as follows：Following CP3, P1, P2, P3, PW belong to the reagent inside mentioned reagent box, article No.： DP103-03

1) column equilibration：Adsorption column CP3,500 μ L equilibrium liquid BL, 12,000rpm, centrifugation 1min are outwelled useless in collecting pipe Liquid places back in adsorption column in collecting pipe.(processed pillar on the day of use)；

2) bacterium solution for taking 1mL to be incubated overnight, addition centrifuge tube, 12,000rpm, it is centrifuged 1min, absorption supernatant as far as possible is (more Bacterial sediment is collected into a centrifuge tube by secondary centrifugation)；

3) 250 μ L solution P1 are added into the centrifuge tube of bacterial sediment, suspends and mixes precipitating, if there is not mixing thoroughly Fungus block, will affect cracking, cause extracted amount and purity relatively low；

4) 250 μ L solution P2 are added, mild spins upside down 6-8 times, cracks thallus sufficiently.It (is gently blended, no It acutely to shake, in order to avoid interrupting genomic DNA, cause to be mixed with genomic DNA fragment in the plasmid extracted.Bacterium solution strains at this time Must be limpid sticky, 5min is not to be exceeded in the time used, in case plasmid is destroyed.It, may be due to thallus if not becoming limpid Excessively, cracking is not thorough, and should reduce biomass)；

5) 350 μ L solution P3 are added into centrifuge tube, leniently spins upside down 6-8 times, mixes well immediately, at this time will There is white flock precipitate.12,000rpm, it is centrifuged 10min, forms precipitating in centrifugation bottom of the tube at this time.(P3 should be stood after being added It mixes, avoids generating localized precipitation.If there are also minute whites to precipitate in supernatant, supernatant is taken after being centrifuged again)；

6) supernatant that previous step is collected is transferred in adsorption column CP3 (adsorption column is put into collecting pipe) with pipettor, Pay attention to trying not that precipitating is sucked out.12,000rpm, it is centrifuged 60s, the waste liquid in collecting pipe is outwelled, adsorption column CP3 is put into receipts In collector.

7) 600 μ L rinsing liquid PW are added into adsorption column CP3,12,000rpm, centrifugation 1min are outwelled useless in collecting pipe Adsorption column CP3 is put into collecting pipe by liquid.It is repeated once；

8) adsorption column CP3 is put into collecting pipe, 12,000rpm, 2min is centrifuged, by rinsing liquid remaining in adsorption column Removal, is placed in and is placed at room temperature for 2min, thoroughly to dry remnants；

9) adsorption column CP3 is placed in a clean centrifuge tube, 60 μ L Free H is added dropwise to adsorbed film centre₂O, room Temperature places 2min, 12000rpm, is centrifuged 2min, plasmid is collected into centrifuge tube, slow virus expression plasmid is respectively obtained PCDH-EF1 α-MCS- (PGK-Puro) and PUC19 plasmid.Microplate reader surveys production concentration, slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro) concentration is 358ng/ μ L；PUC19 plasmid be 254.3ng/ μ L, finish writing label, be stored in -20 DEG C it is standby With.

(2) digestion, connection, conversion

1. digestion

Slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro), PUC19 plasmid are carried out with following system simultaneously EcoRI, NotI double digestion, 37 DEG C of water-bath 1-2h；

pCDH-EF1α-MCS-(PGK-Puro)/PUC19

Sample-adding amount is determined according to plasmid concentration, and plasmid is 2 μ g in system

2. agarose gel electrophoresis

Slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro) digestion products need 0.8% Ago-Gel： Agarose 0.16g is weighed in 100mL conical flask, is added in 1 × TAE of 20mL, micro-wave oven dissolves by heating, and is cooled to 50 DEG C At~60 DEG C, the nucleic acid dye (glod view) of 2 μ L is added, shakes up, is poured into and has been plugged in comb gel slab, solidifies standby With.

PUC19 plasmid enzyme restriction product needs 1.0% Ago-Gel：Agarose 0.2g is weighed in 100mL conical flask In, it being added in 1 × TAE of 20mL, micro-wave oven dissolves by heating, when being cooled to 50 DEG C~60 DEG C, the nucleic acid dye of 2 μ L is added, It shakes up, is poured into and has been plugged in comb gel slab, solidify spare.

After glue cooled and solidified, 6 × Loading buffer is added according to final volume in plasmid enzyme restriction product, piping and druming is mixed It is even, it sequentially adds in loading hole.It is put into electrophoresis tank, 90V, 30min observe and record result with gel imager.Agarose Gel electrophoresis result pCDH-EF1 α-MCS- (PGK-Puro) is as shown in Figure 5.

3. glue recycles

Glue recycling is carried out with Tiangeng Ago-Gel QIAquick Gel Extraction Kit specification, concrete operation step is as follows：It adsorbs below It column CA2, solution PN, is reagent in mentioned reagent box, article No.：DP209-02

1) column equilibration step：Into adsorption column CA2, (adsorption column is put into collecting pipe) is added 500 μ L equilibrium liquid BL, and 12, 000rpm (~13,400g) is centrifuged 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.(it please make With the same day processed pillar)；

2) single target DNA band is cut from Ago-Gel (excision redundance as far as possible) be put into it is clean In centrifuge tube, weight is weighed；

3) be added into blob of viscose equimultiple bulk solution PN (if gel weight is 0.1g, volume can be considered 100 μ l, then plus Enter 100 μ l PN solution), 56 DEG C of water-baths are placed, and constantly centrifuge tube are leniently spun upside down therebetween, to ensure that blob of viscose is sufficiently molten Solution.If can continue to place a few minutes or add some sol solutions again there are also not molten blob of viscose, until blob of viscose be completely dissolved (if The volume of blob of viscose is excessive, blob of viscose can be cut into fragment in advance).

Pay attention to：For recycling<The small fragment of 300bp can add the different of 1/2 blob of viscose volume after the complete colloidal sol of PN is added Propyl alcohol is to improve the rate of recovery；Solution temperature is preferably down to room temperature upper prop again after being completely dissolved by blob of viscose, because adsorption column is in room temperature When in conjunction with DNA ability it is stronger；

4) previous step acquired solution is added in an adsorption column CA2 (adsorption column is put into collecting pipe), is placed at room temperature for 2min, 12,000rpm (~13,400g) are centrifuged 30-60s, outwell the waste liquid in collecting pipe, adsorption column CA2 is put into collecting pipe In.

Pay attention to：Absorption column volume is 800 μ L, if sample volume is greater than 800 μ L and can be added portionwise；

5) 600 μ L rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CA2, 12,000rpm (~13,400g) are centrifuged 30-60s, outwell the waste liquid in collecting pipe, adsorption column CA2 is put into collecting pipe.Note Meaning：If the DNA of recycling is the experiment for salt density value, such as blunt end cloning experiment or direct Sequencing, it is proposed that after PW is added 2-5min is stood to be centrifuged again；

6) repetitive operation step (5)；

7) adsorption column CA2 is put back in collecting pipe, 12,000rpm (~13,400g) are centrifuged 2min, eliminate rinsing as far as possible Liquid.Adsorption column CA2 is placed in and is placed at room temperature for several minutes, is thoroughly dried, to prevent remaining rinsing liquid from influencing the reality of next step It tests.Pay attention to：The residual of ethyl alcohol will affect subsequent enzyme reaction (digestion, PCR etc.) experiment in rinsing liquid；

4. adsorption column CA2 is put into a clean centrifuge tube, 30 μ L Free are vacantly added dropwise to adsorbed film middle position H₂O is placed at room temperature for 2min.12,000rpm (~13,400g) are centrifuged 2min and collect DNA solution, microplate reader detectable concentration： It is 2.3ng/ μ L that recycling concentration, which is 15.9ng/ μ L, CAR structure concentration, after pCDH-EF1 α-MCS- (PGK-Puro) digestion.Even It connects, convert

1) recycling segment is pressed into CAR structure:PCDH-EF1 α-MCS- (PGK-Puro)=5:1 molar ratio is attached, 16 DEG C, 4h is connected in ProFlex PCR instrument.

Linked system is as follows：

Subsequent operation carries out in superclean bench.

Wherein, CAR structure composition is Igkappa-iRGD-EpCAM scFv- (G₄S)₅-Her-2scFv -CD8α-CD28- CD137-CD3ζ-T2A-CCL19.Wherein iRGD structure can express iRGD peptide, nucleotide sequence as shown in SEQ ID NO.1, Kazuki Sugahara and its colleague once reported that the peptide of iRGD was a kind of peptide that can penetrate tumour, when iRGD is as a kind of When individual substance is injected together with anti-tumor drug, drug delivery and anti-tumor activity can be greatly enhanced.EpCAM is single-stranded anti- Body structure is the specific antigen binding domain for stomach cancer cell surface tumours related antigen EpCAM design, and EpCAM is single-stranded anti- Body nucleotide sequence is as shown in SEQ ID NO.2.EpCAM single-chain antibody is expressed as EpCAMscFv in structure.(G₄S)₅It is single-stranded Hinge Inner-Linker between antibody, nucleotide sequence is as shown in SEQ ID NO.3.Her-2 single-chain antibody structure is needle To the specific antigen binding domain of stomach cancer cell surface tumours related antigen Her-2 design, nucleotide sequence such as SEQ ID Shown in NO.4.Her-2 single-chain antibody is expressed as Her-2scFv in the structure.CD8 α is transmembrane region, connects extracellular antigen binding Domain and intracellular signal domain, can be by CAR Structure anchor on T cell film, and nucleotide sequence is as shown in SEQ ID NO.5.CD28- CD137 is costimulation structural domain, and the generation of transduction proliferation signal and inducing cytokine inhibits tumour growth, CD28 nucleotide Sequence is as shown in SEQ ID NO.6, and CD137 nucleotide sequence is as shown in SEQ ID NO.7.CD3 ζ is signal transduction domain, works as born of the same parents When outskirt and target antigen combine, T cell will be activated to conduction TCR sample signal intracellular, it is thin to play targeting killing tumour The effect of born of the same parents, nucleotide sequence is as shown in SEQ ID NO.8.T2A is that linker- is found in bright tetra- precursor virus of arteries and veins thosea siensis β (Thosea asigna), effect is to make a single fragment coding protein, its advantage is that two genes (ORF) can pass through T2A polypeptide is connected to become an ORF, and mRNA translates into a fusion protein, which can be identified the protease of T2A Two albumen are cut into, nucleotide sequence is as shown in SEQ ID NO.9.Chemokines CC CL19 chemotactic T cell infiltrates tumor group It knits, mediate tumor is immune, while inhibiting the release of immunosuppressive factor in tumour, promotes Immune Cell Antigens to offer to make indirectly With, and inhibit vascularization, the final growth for inhibiting tumour；Chemokines CC CL19 nucleotide sequence such as SEQ ID NO.10 institute Show.CAR structure sequence is designed by Shanghai Yi Hao Biotechnology Co., Ltd, by the raw limited public affairs of work bioengineering (Shanghai) share Department's synthesis, with the preservation of PUC19 plasmid form.

2) competent cell is taken out from -80 DEG C of refrigerators to melt on ice, be based on 37 DEG C of incubators from 4 DEG C of taking-up solid cultures Preheating；

3) connection product is transferred in 1.5mL centrifuge tube；

4) it is slowly added to 50 μ L competent cells thereto, agitates by adding, mixes the two；

5) after the centrifuge tube in 3 being put in 30min on ice, the heat shock 90s in 37 DEG C of water-baths；

6) it after heat shock, takes out be placed in 2min on ice immediately；

7) 950 μ L fluid nutrient mediums are added thereto, in constant-temperature table 40~60min of culture, condition is 37 DEG C, 200rmp；

8) 6000rpm is centrifuged 2min, discards most of supernatant, stay 40~60 μ L；

9) resuspended bacterium solution is blown and beaten with pipette tips, bacterium solution is dripped on the solid medium of preheating, is marked, 37 DEG C of incubators Overnight；

(3) upgrading grain, digestion verification, sequencing

1) for picking part bacterium colony in 5mL fluid nutrient medium, constant-temperature table carries out Zengjing Granule, and condition is 37 DEG C, 250rmp cultivates 12~16h；

2) plasmid extraction is carried out with the small extraction reagent kit of plasmid purchased from Tiangeng Biotechnology Co., Ltd, and measures concentration； Each 500 μ L that take out are used as fungi preservation, are stored in -80 DEG C in 1.5mLEp pipe before plasmid extraction；

3) gained plasmid is subjected to EcoRI, NotI double digestion, 37 DEG C of water-bath 1-2h.Digestion system is as follows：

Structure containing CAR pCDH-EF1 α-MCS- (PGK-Puro) plasmid

Sample-adding amount is determined according to plasmid concentration, and plasmid is 1 μ g in system

4) agarose gel electrophoresis.The Ago-Gel 50ml of method configuration 0.8% as above, and carry out electrophoresis, 90V, 20min observes and records result with gel imager；

5) take 1 μ g that Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced the correct plasmid of band, band is abnormal Plasmid and the bacterium solution of retention abandon.The correct plasmid of sequencing result is stripped, the plasmid of sequencing result mistake and Its bacterium solution abandons.

(4) preparation of concentrating virus liquid

1. virus liquid is collected

Slow virus packaging is carried out using three plasmid packaging systems.Three plasmids are respectively the slow virus expression containing CAR structure Plasmid pCDH-EF1 α-MCS- (PGK-Puro), slow virus packaging plasmid pMD2G, vector plasmid PSPAX2.Cell is that 293T is thin Born of the same parents.

Specific implementation step is as follows：

1) interior for 24 hours before transfection to carry out bed board：It is typically chosen cell of the passage number within 3 generations, is grown according to cell close Degree and state adjust cell density, and growth conditions are good in 10cm ware, and it is useless that the 293T cell that stand density reaches 80% inhales abandoning Liquid；

2) thereto slowly adherent addition 3mLPBS to wash cell；

3) it inhales after abandoning waste liquid, slow adherent addition 1mL pancreatin digests 2min in constant incubator；

4) the DMEM complete medium (10%FBS) of 3mL is added into the cell digested to terminate digestion, and is transferred to 800rpm is centrifuged 3min in 15mL centrifuge tube；

5) it discards supernatant, into cell precipitation plus cell is resuspended in 5mL DMEM complete medium (10%FBS), slowly blows It beats and mixes；

6) 6 6cm wares are taken, and add 5mL DMEM complete medium (10%FBS)；

7) 720 μ L cell suspensions are added into each 6cm ware, it is careful to mix, need cell completely adherent and adherent equal It is even；

8) for density to be grown up to 60~90%, cell state is good, can carry out viral packaging；

9) according to transfection cell quantity, by three plasmid mixed liquors needed for each 6cm ware of following proportional arrangement

3 μ g of recombinant plasmid

pMD2G 0.5μg

PSPAX2 4μg

The dosage for needing to be added is determined according to each plasmid concentration.The dosage that this need to be added is as follows：

Recombinant plasmid (124ng/ μ L) 24.2 μ L

pMD2G(436ng/μL) 1.15μL

PSPAX2(246ng/μL) 14.5μL

2mL Opti-MEM is added into plasmid mixed liquor, is stored at room temperature 5min；

10) lipofectamine 2000 (4 DEG C preservation) mixed liquor need to be separately configured, needed for each 6cm ware Lipofectamine 2000 is 2 μ L/ μ g, tri- plasmid mixed liquor, and 2mLOpti-MEM is slowly added dropwise thereto, is stored at room temperature 5min；

11) above (9) and (10) are mixed in a pipe, the static 20min of room temperature；

12) the 293T cell completed is taken out, inhales and abandons waste liquid, and careful adherent addition 1mLDMEM culture medium, prevented Cells float；

13) by the cell in the careful adherent addition (12) of the mixed liquor in (11), cells float is prevented, after mixing, after Continuous culture 72h；

14) culture supernatant after collection 72h, it is spare by 0.45 μm of membrane filtration；

2. virus liquid is concentrated

1) 5 × PEG8000NaCl is prepared：Weigh NaCl 8.766g；It is pure that PEG8000 50g is dissolved in 200mL Milli-Q In water；121 DEG C, moist heat sterilization 30min；It is stored in 4 DEG C；

2) 5 × PEG-8000NaCl mother liquor 7.5mL is added in the filtered initial liquid of virus of every 30mL；

3) every 20~30min mixing is primary, carries out 3-5 times altogether；

4) it stands overnight for 4 DEG C；

5) 4 DEG C, 4000g, it is centrifuged 20min；

6) it inhales and abandons supernatant, stand 1~2min of pipe, siphon away residual liquid；

7) 500 μ LPBS dissolution slow virus precipitating is added in the filtered initial liquid of virus of every 30mL；

8) viral suspension after collecting is distributed into 50 every part of μ L, is stored in production tube.With being stored after broken dry ice quick-frozen At -80 DEG C spare (avoiding concentrating virus liquid multigelation).

3. virus titer measures

1) the good 293T cell of growth conditions is taken, is counted after digestion, by 2 × 10⁵Cells/well cell is uniformly spread to 24 porocyte culture plates, 6~10h of culture are adherent to cell；

2) the concentrating virus liquid of various concentration is added in cells and supernatant into 24 orifice plates, gently pats 24 hole edges of boards Edge is uniformly mixed virus liquid with culture solution, and 48h is infected in incubator；

3) after infecting 48h, digest and collect cell, Flow cytometry positive cell percentage, and according to following public affairs Formula calculates virus titer, unit TU/mL：

T=(cell number × positive cell percentage × 1000 when bed board)/addition virus liquid volume (μ L)

(5) virus liquid infects T cell

1.PBMC separation

1) about 6mL people periphery new blood is collected with the vacuum blood collection tube containing heparin；

2) it dilutes：Isometric PBS is added at room temperature, gently piping and druming mixes；

3) it is loaded：50mL centrifuge tube is taken, draws 6mL Ficoll (lymphocyte separation medium) in centrifuge tube, (Ficoll It is 1 with the volume ratio for diluting preceding blood:1), pipe tilt 45 °, by the blood after dilution at Ficoll ullage about 1cm edge Tube wall is added slowly to above Ficoll；

4) it is centrifuged：18-20 DEG C, 2000rpm, 30min, points four layers from tube bottom to liquid level after centrifugation, be followed successively by red blood cell and Granulocyte layer, layering liquid layer, mononuclearcell layer, plasma layer；

5) it recycles：Pipette is inserted directly into mononuclearcell layer (or the blood plasma for first sucking upper layer), is gently sucked out single After a nucleus layer, it is put into new centrifuge tube；

6) it washs：The PBS of addition to 3 times of volume less than PBMC (peripheral blood mononuclear cells), 18-20 DEG C, 2000rpm, 10min, twice；

7) cell count：Supernatant is abandoned, adds 1mLRPMI-1640 culture medium (containing 10% fetal calf serum), piping and druming mixes, preparation At PBMC cell suspension.It is counted using blood cell counting plate：After taking a drop PBMC suspension and a 2% trypan blue dye liquor of drop to mix It is added in blood cell counting plate, counts 4 big lattice inner cell sums under the microscope.Cell number/mL=4 block plaid cell is total Number/4 × 10⁴× 2 (extension rates)

The activation and slow-virus infection of 2.T cell

1) cell density is adjusted to 1 × 10⁶Cell factor and antibody complex (final concentration 100U/mL is added in cell/mL IL-2,100ng/m L Anti-CD3 (OKT3), 250ng/mL Anti-CD28, continuously cultivate 48h；

2) according to MOI=20, virus quantity required for calculating.Calculation formula is as follows：Required virus quantity (mL)=(MOI × Cell quantity)/virus titer

3) after -80 DEG C of refrigerators taking-up virus, melt in 37 DEG C of water-baths rapidly.Above-mentioned meter is added in six orifice plates It calculates resulting virus quantity, adds the polybrene of final concentration of 8 μ g/m L, after mixing well, using ParafilmTM orifice plate, Orifice plate is placed in a centrifuge, 800g is centrifuged 60min；

4) orifice plate is taken out, orifice plate is placed in 37 DEG C, 5%CO₂Incubator in, continue culture for 24 hours；

5) 250g is centrifuged 10min, removes containing virulent culture medium supernatant, and cell precipitation is resuspended with fresh culture, will Cell is transferred in six new orifice plates, continue culture 3-6 days it is spare；

(6) Western blot verifies whether CAR structure is expressed

By the CD3 ζ chain in detection CAR structure, to detect the expression of CAR in cell.

1. the extraction and preparation of total protein of cell sample

1) cell, including untreated T cell and infected T cell are collected；

2) add cell pyrolysis liquid RIPA into cell according to cell concentration, mixing is carefully blown and beaten with pipette tips, is placed on ice 10min；

3) whether observation liquid is relatively limpid, if color is partially yellow, can mend appropriate RIPA, after the concussion that is vortexed mixes, continues at 10min is placed on ice；

4) step (3) are repeated；

5) after cell sufficiently cracks, 12000rmp, is centrifuged 10min by 4 DEG C；

6) supernatant is collected, protein concentration is surveyed using BCA method, BCA kit is holy purchased from assist；

It 7) is that 10 μ L determine protein sample sample-adding amount, while 2 μ L5 × loading Buffer are added with final applied sample amount, With Free H₂5min is boiled for 100 DEG C after 10 μ L of O polishing makes final total protein concentration 20ng seal membrana oralis；

8) residual protein sample saves after 5 × loading Buffer can be added.- 20 DEG C can be reserved for one week, and -80 DEG C can protect It deposits one month；

2.Western blot detection

1) glue is fixed with clip, electrophoresis liquid is added thereto, it is ensured that no leakage；

2) electrophoresis liquid is added into electrophoresis tank；

3) mixing of vortex oscillator and of short duration centrifugation are used before protein sample loading；

4) loading：Every 10 μ L of hole；

5) electrophoresis：80V, 30min；Turn 120V, until terminating；

6) transferring film：Appropriately sized pvdf membrane is cut, impregnates a few minutes activation in methanol liquid in advance.According to filter paper- Film-glue-filter paper sequence is put well, is carried out electricity and is turned, and condition is wet turn, constant current 400mA, 1h；

7) it closes：5% skim milk, room temperature close 1h (or 4 DEG C overnight)；

8) PBST washes film：5min/3 times；

9)Anti-CD3zeta antiboby(1:1000) it is incubated for, 4 DEG C overnight；

10) PBST washes film：5min/3 times；

11)HRP-labeled Goat Anti-Rabbit IgG(1:2000) it is incubated for, room temperature 1h；

12) PBST washes film：5min/3 times；

13) it develops the color and takes pictures；

14) PBST washes film：5min/3 times；Appropriate membrane regeneration liquor is added, washes film 1h；

15) closing is re-started, and is incubated for Beta Actin Antibody (1:4000),HRP-labeled Goat Anti-MouseIgG(1:2000) it, develops the color, observes internal reference β-actin；

3. cytotoxicity analysis detects

Techniqueflow chart is shown in Fig. 2, and concrete operations process is as follows：

1) detection plate is set, while setting 4 controls：Target cell maximum release group, volume correction control group, ground control Group and Spontaneous release group, experimental group are arranged by 20: 1,10: 1,5: 1,1: 1 (effector cell: target cell).Target cell selects AGS (Western blot detects its Her-2, EpCAM expression to cell line, and targeting antigen EpCAM expresses feelings in ags cell system Condition is as shown in figure 3, targeting antigen Her-2 expression is as shown in Figure 4 in ags cell system)；

A. effector cell hole is set up：+ 50 μ L culture medium of 50 μ L effector cell；

B. experimental group：Target cell is constant, changes effector cell：+ 50 μ L target cell of 50 μ L effector cell；

C. the spontaneous release group of target cell is set up：+ 50 μ L culture medium of 50 μ L target cell；

D. target cell maximum release group is set up：+ 10 μ L lysate (10 ×) of+50 μ L culture medium of 50 μ L target cell；

E. volume correction control group is set up：+ 10 μ L lysate (10 ×) of 100 μ L culture medium；

F. ground control group is set up：100 μ L culture mediums；The optimization of target cell inoculation number purpose

2) cell cracking and harvest supernatant：Every 100 μ L culture medium adds 10 37 DEG C of μ L cracked solutions (10 ×), 5%CO₂It is incubated for 45min, 250g are centrifuged 4min；

3) 50 μ L supernatants of transfer are protected from light the detection buffer for taking 12mL to thaw, by remaining rapid jelly to another orifice plate It deposits and (can be thawed, but can not be placed for a long time with 37 DEG C of water-baths).12mL detection buffer is added to one bottle of Substrate cocktail In (can be used for two 96 orifice plates), it is inverted and mixes；

4) diluted Substrate cocktail is added in 50 holes μ L/, and room temperature is protected from light incubation 30min, and (substrate is mixed after not used dilution Close thing liquid and be put in -20 DEG C, can be reserved for 6-8 weeks), add 50 μ L stop baths；

5) bubble removal that will contain in hole, 1h is interior to detect absorption value (490 or 492nm), at least detects and absorbs twice Value；

6) result counts

It is flat that all experimental group, the spontaneous release group of effector cell, the absorption value of the spontaneous release group of target cell should subtract background Equal absorption value；The absorption value of target cell maximum release group should subtract the mean absorbance of volume correction control group.After correction Value is for killing the statistics of rate：

Cell killing rate (%)=[(the spontaneous spontaneous release of release-target cell of experimental group release-effector cell)/(target cell The maximum spontaneous release of release-target cell)] × 100%.

As a result there are two types of manifestation modes：The intuitive of naked eyes is observed and reflects cell killing rate by measurement LDH；

Visually observe that (each group target is thin when bed board as it can be seen that experimental group target cell numbers are reduced compared with the spontaneous release group of target cell Born of the same parents' number is identical, uniform bed board), and with experimental group effector cell：The increase of target cell ratio, target cell numbers are reduced more Obviously, it is lethal to illustrate that effector cell has target cell.

In addition, can reflect cell killing rate by the measurement of LDH, by drawing effector cell：Target cell-cell kills Hurt rate line chart, it can be seen that the two is positively related relationship.

The above result shows that the CAR-T cell obtained through slow-virus infection, it can be thin for tumour by expressing and identifying The antibody of the specific antigen of cellular surface, the specific killing tumour cell.

Hair can be understood and used the above description of the embodiments is intended to facilitate those skilled in the art It is bright.Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein General Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to above-mentioned implementations Example, those skilled in the art's announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be Within protection scope of the present invention.

Sequence table

<110>Shanghai Yi Hao Biotechnology Co., Ltd

<120>The double target spot CAR-T therapy vectors and its construction method of gastric cancer and application

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

tgccgcggcg acaagggccc cgactgc 27

<210> 2

<211> 750

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

caggtcaagc tgcagcagtc aggggctgaa ctggtgaggc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc aactactgga taaactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggaaat atttatcctt cttatattta tactaactac 180

aatcaagagt tcaaggacaa ggtcacattg actgtagacg aatcctccag cacagcctac 240

atgcagctca gcagcccgac atctgaggac tctgcggtct attactgtac aagatcccct 300

tatggttacg acgagtatgg tctggactac tggggccaag gcaccacggt caccgtctcc 360

tcaggtggag gcggttcagg cggaggtggc tctggcggtg gcggatcgga catcgagctc 420

actcagtctc catcctccct gactgtgaca gcaggagaga aggtcactat gaactgcaag 480

tccagtcaga gtctgttaaa cagtagaaat caaaagaact acttgacctg gtaccagcag 540

aaaccagggc agcctcctaa actgttgata tactgggcat ccactaggga atctggggtc 600

cctgatcgct tcacaggcag tggatctgga acagatttca ctctcaccat cagcagtgtg 660

caggctgaag acctggcagt ttattactgt cagaatgatt atgtttatcc gctcacgttc 720

ggtgctggga ccaagctgga aataaaacgg 750

<210> 3

<211> 75

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcgggtgg aggcggttca 60

ggcggaggtg gctct 75

<210> 4

<211> 741

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

gacctccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca gagcattagc agctatgtcg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgtatagtgg ggtcccatca 180

aggttcagtg gcagtagatc tgggacagat tttactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag agttacagtc ccccattcac ttttggtcag 300

ggaaccaaag tcgagattaa gcgtggtggt ggtggttctg gtggtggtgg ttctggcggc 360

ggcggctccg gtggtggtgg atccgaagtg cagctggtgg agtctggggg aggcttggta 420

cgacctggcg gttccctgag actctcctgt gcagcctctg gcttcacctt cactgactac 480

tatatgcact gggtccggca agctccaggg aagggcctgg agtgggtcgc cagaattagt 540

cctggaggcg gatatatagg ctatgcggac tctgtgaagg gccgattcac catctccgcc 600

gacacctcta agaacacagc atatctgcaa atgaacagtc tgagagctga ggacacggcc 660

gtctattact gcgcaagaag ggactatggt gactacgggt ttgcttactg gggacagggt 720

actttggtta ccgtctcttc c 741

<210> 5

<211> 204

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttac 204

<210> 6

<211> 123

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 7

<211> 126

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 8

<211> 336

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

agagtgaagt tcagcaggag cgcagagccc 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> 9

<211> 75

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

ggaggcggag gcgggagagc agaagggaga ggaagcttgc taacctgtgg agacgttgag 60

gaaaatccag ggcca 75

<210> 10

<211> 297

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60

agtggcacca atgatgctga agactgctgc ctgtctgtga cccagaaacc catccctggg 120

tacatcgtga ggaacttcca ctaccttctc atcaaggatg gctgcagggt gcctgctgta 180

gtgttcacca cactgagggg ccgccagctc tgtgcacccc cagaccagcc ctgggtagaa 240

cgcatcatcc agagactgca gaggacctca gccaagatga agcgccgcag cagttaa 297

Claims

1. a kind of Chimeric antigen receptor, which is characterized in that including EpCAM single-chain antibody, Her-2 single-chain antibody and chemotactic factor (CF) Tri- specificity structures of CCL19,

Wherein, EpCAM single-chain antibody nucleotide sequence is as shown in SEQ ID NO.2,

Her-2 single-chain antibody nucleotide sequence is as shown in SEQ ID NO.4；

Chemokines CC CL19 nucleotide sequence is as shown in SEQ ID NO.10.

2. a kind of Chimeric antigen receptor according to claim 1, which is characterized in that Chimeric antigen receptor structure composition is Igkappa-iRGD-EpCAM scFv-(G₄S)₅-Her-2scFv-CD8α-CD28-CD137-CD3ζ-T2A-CCL19。

3. a kind of Chimeric antigen receptor according to claim 2, which is characterized in that

IRGD nucleotide sequence as shown in SEQ ID NO.1,

EpCAMscFv indicates EpCAM single-chain antibody,

(G₄S)₅The hinge Inner-Linker between single-chain antibody, nucleotide sequence as shown in SEQ ID NO.3,

Her-2scFv indicates Her-2 single-chain antibody,

CD8 α be transmembrane region, connect extracellular antigen binding domain and intracellular signal domain, nucleotide sequence as shown in SEQ ID NO.5,

CD28-CD137 is costimulation structural domain, and CD28 nucleotide sequence is as shown in SEQ ID NO.6, CD137 nucleotide sequence As shown in SEQ ID NO.7,

CD3 ζ be signal transduction domain, nucleotide sequence as shown in SEQ ID NO.8,

T2A is linker, and nucleotide sequence is as shown in SEQ ID NO.9.

4. a kind of double target spot CAR-T therapy vectors of gastric cancer, which is characterized in that comprising embedding described in any one of claim 1-3 Close antigen receptor and carrier.

5. double target spot CAR-T therapy vectors of gastric cancer according to claim 4, which is characterized in that the carrier includes PUC19 Plasmid, Lentiviral.

6. double target spot CAR-T therapy vectors of gastric cancer according to claim 5, which is characterized in that the slow virus expression carries Body is pCDH-EF1 α-MCS- (PGK-Puro).

7. a kind of construction method of double target spot CAR-T therapy vectors of gastric cancer as described in claim 5 or 6, which is characterized in that

By Chimeric antigen receptor structure according to gene order, is synthesized using standard biologic synthetic method, be present in after synthesis On PUC19 plasmid vector；PUC19 plasmid vector and Lentiviral are all made of ECoR I, I double digestion of Not, by digestion Product is separated by agarose gel electrophoresis, is recycled purpose band, is obtained the concentration of carrier and target fragment, by the two according to rubbing You are than being 1:5 are attached conversion, and plasmid extracts, final to obtain the recombinant plasmid containing Chimeric antigen receptor structure, i.e., described Double target spot CAR-T therapy vectors of gastric cancer.

8. a kind of application of double target spot CAR-T therapy vectors of gastric cancer as described in claim 5 or 6, which is characterized in that be used for structure CAR-T cell is built,

The therapy vector is subjected to slow virus packaging using three plasmid packaging systems, group becomes pair of PSPAX2, pMD2G, gastric cancer Target spot CAR-T therapy vector, uses 293T cell as the incasing cells of slow virus, and culture collects virus liquid, by this virus liquid After concentration, T cell is infected, can be obtained CAR-T cell.

9. the application of double target spot CAR-T therapy vectors of gastric cancer according to claim 8, which is characterized in that PSPAX2, PMD2G, gastric cancer double target spot CAR-T therapy vectors have constant concentration ratio in system.

10. a kind of CAR-T cell, which is characterized in that be prepared using the application method of claim 8.