Bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its building sideMethod and applicationTechnical field
The invention belongs to field of biotechnology, are related to antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method, toolBody is related to bispecific chimeric antigen receptor c-Met/PD-1 scFv-CAR-T and its construction method and application.The present invention utilizesTechnique for gene engineering obtains the fusion of the recombination Chimeric antigen receptor containing bispecific, makes T cell expression should after infecting T cellBispecific chimeric antigen receptor.The bispecific chimeric antigen receptor T cell be capable of tumor cell surface c-Met andThe Inhibitory receptor PD-1 for blocking activation T cell surface avoids activation T cell from being induced apoptosis, keeps sustained activation ability, fromAnd the tumour cell of the specific killing c-Met positive, it can be applied to the immunization therapy of entity tumor.
Background technique
Prior art discloses in normal immunoreaction process, non-sensitized T cell at least needs the stimulation of two kinds of signalsSpecific immune response can be activated to generate；Wherein first signal is that T cell is identified by T cell receptor (TCR)Antigen in conjunction with MHC-I or II class molecule transmits antigen recognizing signal by TCR/CD3 complex；Second signal be withThe costimulatory molecules of T cell surface receptor molecule identification antigen presenting cell (APC) or other cell surfaces based on CD28, passPass costimulatory signal.Studies have shown that tumor specific T cells play mainly in the immune response that body removes tumour cellEffect, while tumour cell is by inhibiting to participate in T cell identification and antigen reactive developed by molecule, reducing the approach such as immunogenicityGenerate immunologic escape, thus prevent the immune system of body from play remove tumour cell effect.
Studies have found that replacing α, β chain of TCR variable with the single-stranded variable region (scFv) of tumor specific monoclonal antibodiesArea, and scFv is directly connected with costimulatory molecules (such as CD28, CD137) and T cell signal transduction region CD3 ζ, it is formedChimeric antigen receptor (CAR) is expressed in T cell surface, and tumour specific antigen can be identified by scFv, and directly generation T cell is livingThe first, second signal changed promotes T cell activation, proliferation, thus specific killing tumour cell.The process relies primarily onThe scFv of CAR-T cell surface is to the specific recognition of tumour antigen, the specificity of immune response and lethal relatively strong.
Currently, CAR-T cell obtains significant curative effect in Malignancy treatment, but answered in the clinic of solid tumorWith being but faced with lot of challenges.Major obstacle first is that due to entity tumor immunosupress microenvironment, i.e., by up-regulation suppressionProperty receptor processed inhibits tumor infiltrating lymphocyte (TIL) and the genetic engineering to modify the function of T cell, especially immunosupressThe up-regulation of property receptor PD-1.PD-1 is the most inhibitive ability of immunity receptors studied at present, is expressed in T cell and precursorB cell surface, a member of contactin.PD-L1 is the PD-1 major ligand having now been found that, research is foundPD-L1 is increased in the expression of kinds of tumor cells film surface, and tumour cell dexterously goes to combine T cell table using PD-L1The PD-1 receptor in face transmits inhibition signal, so as to cause immunologic escape.
There are preclinical study and clinical test to point out, enhances cancer patient's using PD-L1 or PD-1 blocking antibodyAnti-tumor immune response can be used as a kind of novel immunotherapy of tumors means, and can obtain the clinical response of duration.In addition, there is research to prompt, CAR-T cell therapy, which combines anti-PD-1 antibody, can greatly enhance the anti-tumor effect of CAR-T cell,PD-1-scFv-CAR fusion receptors so are expressed in CAR-T cell membrane surface, i.e., CAR-T cell table are closed using PD-1 scFvDoes the PD-1 receptor in face enhance the effectiveness of CAR-T cell therapy to avoid the inhibition signal of PD-L/PD-1? utilize anti-c-MetThe bispecific chimeric antigen receptor hc-Met scFv-Linker-PD-1 scFv-CD8 α-of scFv and anti-PD-1 scFv buildingCD28-CD137-CD3 ζ is expressed in T cell surface, can make the tumour cell of the T cell specific killing c-Met positive, seal simultaneouslyThe Inhibitory receptor PD-1 for closing T cell surface makes T cell sustained activation, therefore the T of bispecific chimeric antigen receptor modification is thinBorn of the same parents, which are expected to break to adopt, transmits the immunosupress signal shaft of the PD-L/PD-1 in T cell treatment, leads in entity tumor immunization therapyDomain has broad application prospects.
Status based on the prior art, present inventor is quasi- to provide bispecific chimeric antigen receptor c-Met/PD-1ScFv-CAR-T and its construction method and for the purposes in the immunization therapy of entity tumor.
Summary of the invention
The invention aims to solve technical problem of the existing technology, provide a kind of bispecific chimeric antigen byBody c-Met/PD-1 scFv-CAR-T and its construction method and for the purposes in the immunization therapy of entity tumor.
The present invention constructs the anti-receptor of chimeric antigen (the hc-Met scFv- of anti-hc-Met scFv-PD-1scFv a kind ofLinker-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ), the T cell for expressing the Chimeric antigen receptor has its uniqueness,It is verified by experiments to tumor cell specific killing ability, with the application prospect in clinical treatment.
The present invention is obtained a kind of containing bispecific recombination Chimeric antigen receptor hc-Met scFv- using technique for gene engineeringThe genetic fragment is inserted into Lentiviral system by the fusion of PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ,The supernatant containing virus is obtained by carrying out packaging to slow virus, T cell is infected, T cell is made to express the bispecific chimeric antigenReceptor.This bispecific chimeric antigen receptor T cell (CAR-T), which is capable of the c-Met on tumor cell surface and blocks, swashsThe Inhibitory receptor PD-1 on T cell surface living avoids activation T cell from being induced apoptosis, sustained activation ability is kept, thus specificallyProperty killing the c-Met positive tumour cell, can be applied to the immunization therapy of entity tumor.
More specifically, the technical solution adopted by the present invention is that: bispecific chimeric antigen receptor hc-Met scFv-Linker-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ, the Chimeric antigen receptor is by anti-human c-Met and PD-1 monoclonalAntibody light chain and heavy chain variable region hc-Met scFv, PD-1 scFv, people CD8 α hinge area, people CD28 transmembrane region and intracellular region,People's CD137 and CD3 ζ intracellular region structures in series is constituted；The amino acid sequence such as sequence table of the bispecific chimeric antigen receptorShown in middle SEQ ID NO.1；
The nucleic acid sequence of the bispecific chimeric antigen receptor is as shown in SQE ID NO.2；
The bispecific chimeric antigen receptor contains the single-chain antibody hc-Met scFv for people's c-Met antigen, ammoniaBase acid sequence is connected between the heavy chain and light chain of single-chain antibody with the connection small peptide of 15 amino acid as shown in SEQ ID NO.3；
The Chimeric antigen receptor contains the single-chain antibody hc-MetscFv for people's c-Met antigen, and nucleic acid sequence is such asShown in SEQ ID NO.4；
The Chimeric antigen receptor contains the extracellular combined area receptor PD-1 for human PD-L 1, amino acid sequence such as SEQShown in ID NO.5；
The Chimeric antigen receptor contains the extracellular combined area receptor PD-1 for human PD-L 1, nucleic acid sequence such as SEQShown in ID NO.6；
The amino terminal of the Chimeric antigen receptor contains a CD8 alpha signal peptide, in amino acid sequence such as sequence tableShown in SEQ ID NO.7；
The amino terminal of the Chimeric antigen receptor contains a CD8 alpha signal peptide, in nucleic acid sequence such as sequence tableShown in SEQ ID NO.8.
Invention further provides the bispecific antigen receptor hc-Met scFv-Linker-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ is in preparation Chimeric antigen receptor T cell and its application in immunotherapy of tumors.
The present invention uses anti-human c-Met scFv and PD-1 scFv amino acid sequence, is carried out codon optimization, fromPeople's CD8 alpha signal peptide gene, people's CD8 α hinge area gene, people CD28 transmembrane region and born of the same parents are searched in NCBI GenBank databaseInner region gene, CD137 and CD3 ζ intracellular region gene sequence information, full genome synthesize CD8 alpha signal peptide, CD8 α-CD28-Then CD137-CD3 ζ genetic fragment connects into bispecific chimeric antigen receptor hc-Met scfv-PD-1 by recombinaseScFv-CD8 α-CD28-CD137-CD3 ζ is inserted into Lentiviral pKC-EF1 α-DS-Red (pKC- empty carrier),Construct anti-human c-Met/PD-1 scFv-CAR expression vector (as shown in Figure 1)；It sends sequencing outside and complies fully with design (such as Fig. 2 instituteShow)；The packaging virus in 293T cell using the plasmid and the packaging plasmid VSVG and r-8.91 of slow virus, infection Jurkat are thinBorn of the same parents (as shown in Figure 3)；Jurkat cell is set to express the Chimeric antigen receptor.The CAR-Jurkat cell of acquisition in vitro with c-The Met and tumour cell MKN45 of the PD-L1 positive is co-cultured, and ELISA method detection co-cultures the cytokine levels in supernatant(as shown in Figure 4)；Radioactivity cytotoxicity method detects CAR-Jurkat cell to the killing activity of MKN45 cell(as shown in Figure 5)；Experiment in vivo detects its influence to tumour growth, it was confirmed that the T cell pair of Chimeric antigen receptor modificationThe specific killing action of tumour cell.Meanwhile it is single that targeting c-Met scFv-CAR and PD-1-CAR are constructed in the present inventionThe Chimeric antigen receptor of target spot, as comparing superiority of the bispecific chimeric antigen receptor in terms of inhibiting tumour function.
Chimeric antigen receptor of the invention is for modifying T lymphocyte, and the T cell (CAR-T cell) after modification is for makingStandby entity tumor immunity therapeutic preparation, can especially be used for the oncotherapy preparation of surface c-Met and the PD-L1 positive；DescribedBispecific chimeric antigen receptor hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ.Immune in tumour is controlledIt has broad application prospects in treatment,
Detailed description of the invention
Fig. 1 is pKC-CAR Lentiviral schematic diagram.
Fig. 2 is that peak value figure is sequenced in pKC-CAR Lentiviral part.
The red fluorescence (Red) of Jurkat T cell expresses feelings after Fig. 3 is fluorescence microscopy microscopic observation slow-virus infection 48hCondition.
Fig. 4 is CAR-T experimental group, IL-2 concentration compares in cellular control unit supernatant.
Fig. 5 is under different effect target ratio situations, and CAR-T experimental group is compared with cellular control unit toxicity.
Fig. 6 is the influence that CAR-T cell grows transplanted tumor in nude mice.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and detailed description:
The determination of embodiment 1:hc-MetscFv-PD-1scFv-CD8 α-CD28-CD137-CD3 ζ gene order
People's PD-1 extracellular fragment gene, people's CD8 alpha signal peptidyl are searched from U.S. national library of medicine site databasesCause, people's CD8 α hinge area gene, people CD28 transmembrane region and intracellular region gene, CD137 and CD3 ζ intracellular region gene order；Anti- c-The monoclonal antibody sequence that Met and PD-1 single-chain antibody (anti-c-Met scFv and PD-1 scFv) gene order constructs in fact from thisColumn, are carried out codon optimization, to guarantee to be more suitable for the expression of T cell in the case where encoding amino acid sequence is constant, respectivelyShown in gene sequence information such as SEQUENCE LISTING (sequence table SEQ ID NO.1-8)；
Said gene sequence is successively pressed into people's CD8 alpha signal peptide gene, anti-human c-Met scFv, anti-human PD-1 scFv, CAR(including people's CD8 α hinge area gene, people CD28 transmembrane region and intracellular region gene, people's CD137 and CD3 ζ intracellular region gene order) intoRow connection, introduces different restriction enzyme sites in each sequence junction, ultimately forms complete hc-Met scFv-PD-1 scFv-CAR gene sequence information.
Embodiment 2
Construct hc-Met scFv-PD-1 scFv-CD8 α-CD28-CD137-CD3 ζ expression plasmid
It overlaps PCR and synthesizes genetic fragment:
C-Met scFv, PD-1 scFv, CAR sequence are synthesized using overlapping PCR, is introduced in each sequence junction differentRestriction enzyme site is linked to be complete c-Met scFv-PD-1 scFv-CAR sequence using recombinase, is cloned into pKC-EF1 α-DS-In RED Lentiviral (pKC- empty carrier), anti-human c-Met scfv-PD-1scFv-CAR expression plasmid (pKC- is obtainedCAR plasmid) and DH5 α bacterium solution containing the plasmid, plasmid information it is as shown in Figure 1；
Recombinant plasmid sequencing:
Send Science and Technology Ltd., Shanghai Major Biological Medical Technology Co., Ltd. to be sequenced the recombinant plasmid, by sequencing result be fitted toHc-Met scFv-PD-1 scFv-CAR sequence alignment is to confirm that sequence is correct, on sequencing primer pKC-EF1 α-DS-Red carrierUniversal sequencing primer object:
Sense:5 '-GACTCAGCCGGCTCT-3 '
Anti-sense:5 '-CTCGCCCTCGATCTC-3 '
Part sequencing result is as shown in Figure 2.
Embodiment 3: a large amount of extractings of purpose plasmid and packaging plasmid
By the bacterial strain of pKC-CAR plasmid, pKC- empty carrier plasmid and VSVG and r-8.91 packaging plasmid in LB culture solutionMiddle mass propgation, with alkaline lysis large quantity extracting plasmid, (Beijing Tiangeng biochemical technology Co., Ltd endotoxin-free plasmid extracts examinationAgent box), in case transfection.
1) bacterium solution is added in 100ml LB culture solution with ampicillin in 1: 1000 ratio, 37 DEG C of constant temperature incubators220rpm shakes 12-16h；
2) a small amount of bacterium solution is taken to survey A600Absorbance stops shaking up to 0.4 or so；
3) the equilibrium liquid BL, 8000rpm that 2.5ml is added into adsorption column CP6 are centrifuged 2min, outwell useless in collecting pipeLiquid places back in adsorption column in collecting pipe；
4) bacterium solution is centrifuged 3min in 4 DEG C of 8000rpm, as far as possible absorption supernatant, collects bacterial precipitation；
5) 8ml solution P1 (RNaseA has been added in determination), thorough suspended bacterial cell precipitation is added；
6) 8ml solution P2 is added, leniently turns upside down 6-8 times immediately, cracks thallus sufficiently, be placed at room temperature for 5min；
7) 8ml solution P4 is added, leniently turns upside down 6-8 times, mixes well immediately, until white dispersion wadding occurs in solutionShape precipitating, is placed at room temperature for 10min, and 8000rpm is centrifuged 10min；Make white precipitate to tube bottom, complete soln is carefully poured intoIn filter CS1, push handle filtering is slowly pushed, filtrate is collected in the centrifuge tube of clean 50ml；
8) isopropanol of 0.3 times of filtrate volume is added into filtrate, is transferred to after mixing of turning upside down in absorption note CP6；
9) room temperature 8000rpm is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column CP6 is placed back in collecting pipe；
10) 10ml rinsing liquid PW (please first check whether and dehydrated alcohol has been added) is added into adsorption column CP6,8000rpmIt is centrifuged 2min, the waste liquid in collecting pipe is discarded, adsorption column is placed back in collecting pipe；
11) repetitive operation step 10；
12) 3ml dehydrated alcohol is added into adsorption column CP6, room temperature 8000rpm is centrifuged 2min, outwells waste liquid；
13) adsorption column CP6 is placed back in collecting pipe, 8000rpm is centrifuged 5min, by rinsing liquid remaining in adsorption columnRemoval；
14) it is sterile it is ultra-clean it is interior adsorption column CP6 is placed in a new 50ml collecting pipe, phase adsorbed film middle part position is outstanding2ml elution buffer TB is added dropwise in sky, is placed at room temperature for 5min, and then 8000rpm is centrifuged 2min, by the eluent in 50ml centrifuge tube2 clean 1.5ml centrifuge tubes all are distributed into, -20 DEG C save backup；
15) 2 μ l DNA solution ultraviolet spectrophotometries will be taken to survey plasmid DNA concentration and A260/A280, and carry out fine jade simultaneouslyThe quality of sepharose electrophoretic analysis plasmid.
Embodiment 4: the packaging of slow virus
Cell processing: being digested for 24 hours with pancreatin before transfection, collect the 4-12 band 293T cell in logarithmic growth phase, willCell is transferred in 10cm culture dish, and cell is grown in the DMEM culture medium that 10ml contains 10%FBS, 1%PS, sets 37 DEG C5%CO2Incubator is cultivated for 24 hours, is transfected when converging rate up to 60-70%；
Rotaring redyeing system configuration:
1) in 50 μ l serum-free Opti-MEM culture mediums are added in 1.5ml sterilizing EP pipe, 10 μ g plasmid (pKC- are addedThe plasmid or pKC- empty plasmid of CAR mesh: VSVG: r-8.91=4: 3: 3), mixing well, be stored at room temperature 5min；Another 1.550 μ l serum-free Opti-MEM culture mediums and 30 μ l transfection reagent lipofectamine 2000 are added in sterile centrifugation tube(Lipo2000: total DNA (μ g)=2: 1), mixes well, is stored at room temperature 5min, then mixes solution in two pipes, is stored at room temperature20min；
2) culture solution in former culture dish is carefully sucked out, 8ml serum-free Opti-MEM culture medium is added；
3) above-mentioned plasmid-Lipo2000 mixed liquor is added in 10cm culture dish, it is careful to mix；
4) culture dish is put into 37 DEG C of 5%CO212-16h is cultivated, 2ml FBS is added, continues to cultivate；
5) transfection for 24 hours after after fluorescence microscopy microscopic observation 293T cell transfecting Red luciferase expression situation, respectively at transfection48h, 2h collect cells and supernatant afterwards, and room temperature 3000rpm is centrifuged 15min, collect supernatant；
6) with 0.45 μm of filter filter virus supernatant, pKC- zero load and pKC-CAR virus stock solution used are obtained respectively.
Culture, infection and the amplification of embodiment 5Jurkat T cell
1) the JurkatT cell in the T cell source that this room of Soviet Union freezes is incubated, 4 generations of biography are seeded to 24 orifice plates later, to cultureThe polybrene pretreatment 4h of final concentration of 4 μ g/ml is added in every hole in Jurkat cell for 24 hours, and pKC- empty carrier is then addedOr 200 μ l of pKC-CAR viral concentration liquid, it is placed in incubator culture, it is interior for 24 hours not change liquid；
2) infection carries out infecting for second afterwards for 24 hours, and cell 1000rpm is centrifuged 10min, carefully sucks supernatant, is added newThe fresh RPMI 1640 culture medium containing 10%FBS adjusts cell concentration to 1 × 10 after being resuspended6/ ml pre-processes 4h with polybreneAfter 200 μ l pKC-CAR viral concentration liquid be added continue to cultivate；
3) continue after cultivating 96h, with the RED red fluorescence expression of fluorescence microscope JurkatT cell and shinePhase.Jurkat infection rate is up to 50% or so (as shown in Figure 3) as the result is shown.
Embodiment 6
Co culture system in vitro measures CAR-Jurkat T cell secrete cytokines and tests to tumor cell killing potential
ELISA method measures cytokine levels:
ELISA kit (is purchased from R&D company), operates by kit specification.
1) it will be taken out from the supernatant of C, D, E group culture 36h from -20 DEG C, room temperature is melted；
2) concentrated cleaning solution, dilution, standard items, sealing lath are taken out into room temperature pre-temperature from refrigerator；
3) concentrated cleaning solution and dilution are diluted to working concentration with distilled water respectively；
4) standard items are prepared: 500ml dilution is added into freeze-drying standard items, dissolves at least 20min naturally, mixes, it is diluteIt is interpreted into 2000,1000,500,250,125,62.5,31.25,15.6 and 0pg/ml；
5) standard items of sample and various concentration being added in corresponding aperture, 100 holes μ l/ seal reacting hole with sealing plate gummed paper,37 DEG C of incubation 2h；
6) board-washing 5 times dry liquid in plate；
7) corresponding antibodies are balanced to room temperature, 100 holes μ l/ is added, seal reacting hole, 37 DEG C of incubation 1h with sealing plate gummed paper；
8) board-washing 5 times dry liquid in plate；
9) chromogenic substrate is balanced to room temperature, 100 μ l are added in every hole, and 37 DEG C are protected from light incubation 30min；
10) terminate liquid is balanced to room temperature, 50 hole μ l/ of terminate liquid is added, microplate reader 450nm (detection in 30min after mixingWavelength) and 570nm (tuning wavelength) wavelength measurement absorbance value；
11) 4 parameters (4-PL) linear standard curve is drawn, its concentration is found by the OD value of sample.Double spies as the result is shownThe Jurkat cell of anisotropic Chimeric antigen receptor modification can be obviously promoted the secretion (as shown in Figure 4) of IL-2 and IFN-γ.
It is living to the killing of MKN45 cell that 96 non-radioactive cell toxicity method of CytoTox detects CAR-Jurkat T cellProperty:
Kit is purchased from Promega company, and by specification requires operation.
1) prepared by target cell: 800rpm is centrifuged 5min and collects logarithmic growth phase MKN45 cell, with the RPMI containing 5%FBS1640 adjust cell concentrations to 2 × 106/ml；
2) prepared by effector cell: 1000rpm is centrifuged 10min and collects the CAR-Jurkat for infecting latter week, with containing 5%FBSRPMI 1640 adjust cell concentration to 2 × 106/ml。
3) following control group and experimental group (three wells) are set in 96 orifice plate of the bottom U:
4) in different effects: target ratio (2: 1,1: 2,1: 4,1: 8 and 1: 16) adding effector cell and target cell, with containing 5%It is 200 holes μ l/ that the RPMI 1640 of FBS, which supplies volume,；
5) 200g is centrifuged 96 hole plate 4min, sets 37 DEG C, 5%CO2, saturated humidity incubator culture 7h, 45min exists in advanceTarget cell maximum relief hole adds 10 μ l cell pyrolysis liquids (10X), and 200g is centrifuged plate 4min；
6) LDH activity measures；
7) CTL activity calculates: calculating each group (3 multiple holes) mean light absorbency (A490), calculates CTL by operating procedure and crack targetThe percentage of cell, the Jurkat cell killing target cell activity of bispecific chimeric antigen receptor modification is obvious strong as the result is shownIn control group (as shown in Figure 5).
CAR-T cell inhibits the capacity experimental of tumour growth in 7 body of embodiment
Using CD3+T cell in immuno magnetic cell separation human peripheral
1) healthy volunteer is chosen, 20mL periphery whole blood is extracted from ulnar vein, isolates PBMC first；
2) RPMI 1640 culture medium of the isolated PBMC containing 10%FBS is resuspended, and counts cell concentration；
3) to wash PBMC with MACS buffer primary, and 1000rpm is centrifuged 10min sedimentation cell, abandons supernatant；
4) suitable anti-human-CD3 immunomagnetic beads (20uL/10 is added7A PBMC)) it mixes, 4 DEG C of incubation 15min；
5) appropriate MACS buffer is added, 1000rpm is centrifuged 10min, and sedimentation cell is resuspended with MACS buffer；
6) MiniMACS separator is taken out, MS splitter is put into separator, and wash MS with appropriate MACS bufferSplitter；
7) cell suspension in step (5) is slowly added in MS splitter, carries out cell sorting, is not to be immunized by CD3The negative cells of marked by magnetic bead flow out first；
8) splitter is washed three times with appropriate MACS buffer, every time flow to end the buffer in pillar；
9) prepare a new 15mL centrifuge tube on, MS splitter is removed from MiniMACS separator, place it is new fromOn heart pipe；
10) 1mL MACS buffer is added on splitter, the cell of delay is eluted completely quickly, repeats threeSecondary, the positive cell of CD3 immunomagnetic beads label finally all elutes；
11) supernatant is removed after the cell centrifugation collected, cell, cytometer is resuspended with 1640 culture medium of RPMI containing 10%FBSNumber is used for subsequent experimental.
Activation, amplification and the infection experiment of T lymphocyte
CD3+T lymphocyte is needed to activate and be expanded before being infected, by CD3/CD28 immune activation magnetic bead specificationIt carries out, specific steps are as follows:
1) by above-mentioned isolated CD3+T cell count, adjusting concentration is 2 × 106/ mL, every hole total volume are 500 μ LIt plants in 24 orifice plates；
2) 25 μ L immunomagnetic beads are added in every hole, to obtain magnetic bead and cell ratio as 1: 1；
3) recombinant human il-2 is added, makes its final concentration of 30U/mL；
4) cell checks daily, observes the upgrowth situation of cell.Every 2-3 days replacement culture mediums, and rejoin recombined humanIL-2；
5) when finding that cell density is larger, cell centrifugation is collected, count after resuspension and density is adjusted to 1 ×106/ mL is passaged to and continues to cultivate in 6 orifice plates；
6) cell is collected after cultivating 1 week, 1640 culture medium of RPMI that serum-free is added is resuspended, and is added in 6 orifice plates, overallProduct is 2mL, with 8 μ g/mL polybrene pretreatment cell 4h；
7) appropriate concentration slow virus suspension is slowly homogeneously added into 6 orifice plates after 4h, replaces culture medium afterwards for 24 hours；
8) fluorescence microscope T cell state is used daily, is taken pictures under the same visual field with red fluorescence and white light, is unitedCell infection efficiency is counted, is infecting the 5th day collection each group T cell in centrifuge tube, 1000rpm is centrifuged 5min；
9) it is resuspended, is cleaned one time with sterile PBS after abandoning supernatant, cell activity identification is placed under microscope through Trypan BlueObservation counts；
10) 1 × 10 finally is made with PBS8The suspension of/mL is for being transfused nude mice.
Zoopery:
Establish Nude Mouse Model
1) Male nude mice of 6-8 week old, which is raised, at least shifts to an earlier date 1 week before University Animal room SPF receptacle, transplantation tumorIt is purchased from Shanghai Slac Experimental Animal Co., Ltd.；
2) MKN45 cell DMEM in high glucose culture medium (10%FBS, 1%PS), 37 DEG C, 5%CO2It is cultivated in incubator；
3) 0.25% pancreatin of MKN45 cell in logarithmic growth phase is digested, neutralized, be centrifuged, abandoned on centrifuge tubeClearly；
4) cell is resuspended with sterile PBS, 1000rpm is centrifuged 5min, abandons supernatant, sterile PBS gravity treatment cell is added and countsNumber, adjustment concentration are 5 × 107The suspension of/mL；
5) with skin of back on the right side of iodophor disinfection nude mice, then with 75% ethanol back, hanging on allows alcoholVolatilization, the cell suspension of 100 μ L of dorsal sc injection on the right side of every nude mice, gently oppressing pin hole prevents cell from overflowing, thenIt puts back to and continues to cultivate in cage；
6) it observes mouse 2 times weekly, weighs, surveys lump length and width.Calculating gross tumor volume V=1/2 × length × wideSquare.
The infusion of CAR-T cell:
1) CAR-T cell is calculated and prepared in advance, measures and calculate transplanted tumor in nude mice volume, when growth reaches 50mm3It is leftWhen right (1 week after about injecting), nude mouse is randomly divided into 3 groups, every group 5；
2) CAR-T cell is resuspended in sterile PBS, is transfused in Mice Body by tail vein.First group is control group input 1×107T cell；Second group is the input of experimental group 11 × 107PKC-c-Met-CAR-T cell；Third group be experimental group 2 input 1 ×107PKC-c-Met/PD-1-CAR-T cell；
3) it then proceedes to observe 2 nude mice diet, defecation and the state of mind weekly, weigh, survey length of tumor and width,Totally 6 weeks；
4) chloral hydrate anesthesia nude mice removes tumor tissues and dissects nude mice, and statistics each group of data is analyzed, and as a result showsShow that the T cell of bispecific chimeric antigen receptor modification can significantly press down the growth (as shown in Figure 6) of in-vivo tumour.
In conclusion the contents of the present invention are not limited in the above embodiments, the knowledgeable people of same area can be withIt is proposed other embodiments easily within technological guidance's thought of the invention, but this embodiment is included in model of the inventionWithin enclosing.