CN105861531A - Chimeric antigen receptor T cell and preparation method thereof - Google Patents

Chimeric antigen receptor T cell and preparation method thereof Download PDF

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CN105861531A
CN105861531A CN201610251247.XA CN201610251247A CN105861531A CN 105861531 A CN105861531 A CN 105861531A CN 201610251247 A CN201610251247 A CN 201610251247A CN 105861531 A CN105861531 A CN 105861531A
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汪治宇
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Hebei Geltai Biotechnology Co.,Ltd.
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Abstract

The invention discloses a chimeric antigen receptor T cell and a preparation method thereof. The chimeric antigen receptor T cell has the specific esophagus cancer relevant functional activity, has important potential and practical values on the treatment of esophagus cancer, and provides a new technical direction for the treatment of the esophagus cancer.

Description

A kind of Chimeric antigen receptor T cell and preparation method thereof
Technical field
The present invention relates to a kind of gene engineering technology field, Chimeric antigen receptor T that a kind of cancer of the esophagus is relevant is thin Born of the same parents and preparation method thereof.
Background technology
Cancer always perplexs the huge difficult problem of the mankind.China newly sends out tumor cases and is about 3,120,000 examples, average every day 8550 people mean per minute just have 6 people to be just diagnosed as malignant tumour, these data all show, cancer is still that and poses a health risk One big killer.For many years, the method for the treatment of of cancer mainly operation, chemotherapy and radiation, effective to the clinical treatment of tumor patient Rate is the lowest, and according to statistics, the efficient of same antineoplastic is only 25%.For same tumour, with same medicine, The methods for the treatment of of standard dose, the curative effect and the toxic and side effect that are risen at different patients but have the biggest difference.Cause this Main reason is that of result, tumour is a kind of multi-pathogenesis, heterogeneous disease.Over the past decade, individual patients is depended on raw The tumour personalized treatment of thing mark is gradually replacing the Therapeutic mode according to Conventional wisdom, and shows superior curative effect With wide prospect, such as Imatinib and Herceptin.
Immunotherapy is a kind of novel therapies occurred after operation, chemotherapy, radiotherapy and targeted therapies, is referred to as treating cancer " the fifth-largest therapy " of disease.It is that the strength utilizing patient's self immune system is to resist cancer；Immunotherapy for cancer mainly wraps Include adoptive cell therapy, immunomodulator, tumor vaccine and immunity binding site blocking treatment etc..Although relevant immunization therapy Research the most carried out before 30 years, the international top periodical such as nearest 2 years " cell ", " science ", " naturally " publish multinomial should The breakthrough achievement in research in field, pharmacy giant the most constantly releases the heavy pound therapy of this type.On December 6th, 2014-No. 9 is at old gold In United States blood association annual meeting (ASH) that mountain is held, immunotherapy for cancer is to have earned enough eyeball, and CAR-T therapy is the most standby Concerned.
The concept of CART is that Israel doctor ZeligEshhar proposes the eighties in last century, 1989 develop first embedding Close antigen receptor T cell.CART therapy i.e. Chimeric antigen receptor T cell therapy, has walked around PD therapy and has started adoptive immunity response This difficulty, has started the medical history new page of great-leap-forward development.CART, for that the most reliable adoptive immunity, introduces complete New concept and method, successfully induce anticancer adoptive immunity response.First CART solves a difficult problem for antigen.Since Naturally under situation, immune system can not find antigen, that a kind of antigen of the most artificial appointment.Investigate acute lymphoblastic B cell leukemia CART therapy.There is a differentiation antigen CD19 on lymphocytic B cells surface, either normal B cells or canceration B cell has table Reach.Selecting CD19 is that target antigen may insure that do not have fish that has escape the net.It follows that how to guarantee that T cell can find CD19 target antigen？This Also it is the core technology of CART.Naturally T cell certainly will not be by CD19 as target antigen.CD19 is expressed in normal B cells, CD19 is tolerated by T cell.One antigen receptor is fitted to T cell surface by CART.This antigen receptor essence is a kind of antibody, right CD19 has the highest compatibility.Both encounter will be in conjunction with, does not abandons.Then, coupling location with antibody protein, T cell is just Peg cancer cell.So there being a metaphor, this antigen receptor, just as GPS, navigates to T cell, for finding target to provide guarantee. Antibody is again how to be inserted into T cell？This is realized by Gene transfer techniques.Here transfect is not antibody itself But the gene of encoding antibody, say, that transfection is DNA.Then the DNA of transfection can be expressed in T cell table by encoding antibody Face.
The T cell therapy being only fitted together to antibody is first generation CART, solves target antigen or the problem of the first signal.But Being that clinical effectiveness dispatches a person to anticipate, reason is the secondary signal i.e. disappearance of costimulatory signal.Costimulatory signal T cell is not had still not have Method activates, even if having pegged cancer cell does not has fighting capacity yet like that.Produce costimulatory signal and need T cell and antigen presenting cell Between interaction.Antigen presenting cell is also required to the first signal.So also same antibody chimeric certainly will be entered antigen Presenting cells such as DC is excessively complicated.Then, second generation CART is developed again for costimulatory signal.Second generation CART is at target On the basis of CD19, add CD28 or CD137, the common costimulatory receptor in T cell.The signal domain of T cell includes: CD3 ζ (T The constituent of cell antigen receptor signal transduction) CD28, or CD3 ζ CD137.The passing through sequence and can state of immune response For: CD19 activates antibody, and antibody activates CD28(CD137), CD28(CD137) activate CD3 ζ.It can be said that second generation CART is simultaneously Solve the first signal and secondary signal, eliminate the reliance on the most reliable tumor associated antigen, eliminate the reliance on antigen and offer carefully Born of the same parents, also there is no concern that the low expression of MHC.The feature of third generation CART is to add again costimulatory molecules albumen after CD28 OX40 or 4-1BB(4-1BB is exactly CD137).Both costimulatory molecules albumen can activating T cell further, extend life cycle. The evolution of three generations's CART technology reflects the important function of costimulatory molecules.
The CART therapy of acute lymphoblastic B cell leukemia obtains the responsiveness of surprising 90%-100%, permanently effective, no Few sufferer complete incidence graph i.e. clinical recovery.Still more, these sufferers entering to organize CART are all chemotherapy failure, bone-marrow transplantation Rear recurrence waits grave illness of being driven into a corner the soonest to suffer from.CART will become great milestone in human history: really cures the side of cancer Method.
Promote the major obstacle of CART therapy from effect of missing the target.It not the target spot of tumour as CD19, but B cell itself Target spot.While tumour cell is disposed, B cell is also disposed of.This is not the most to fight the enemy 1,000, feels sorrow for oneself 800.It is less Fight the enemy 1,000, feel sorrow for oneself 80.But mistake would rather kill 1,000, never let slip one.Fortunately patient does not has B cell can give birth in the same old way Deposit, if periodically input gamma immune globulin.As long as target spot is not very tumour-specific, just have effect of missing the target Should, this is the main source of CART side effect.It is reported, an example is controlled for the third generation CART of colon cancer with HER2 for target antigen Treating, there is respiratory distress after inputting CART 15 minutes in patient, dead after five days.High-caliber cell factor detected and find CART cellular infiltration lung tissue.One explanation is that target antigen has expression in normal lung tissue.Safety is treated in order to improve CART Property and validity, conservative dosage escalation method is to input dosage 2 days or above dispersion T cell, constantly monitors toxicity Sign, stoping may the extensive further T cell input reacted of induction.Alleviate with monoclonal antibody closing target antigen and miss the target Reaction is also a kind of selection if desired.Also proposed the scheme introducing suicide gene recently, start CART the most immediately certainly Kill, at present at III clinical trial phase.Another side effect of CART therapy is cytokine storm.It is reported when CART treats in early days The sufferer ICU to be lived of 60%.The generation of cytokine storm can be greatly reduced now by dosage escalation method.
In a word, Chimeric antigen receptor CAR can be combined with target antigen high-affinity and not affected by central tolerance, and this is special Property makes the immunization therapy of CAR can overcome most of defect of conventional immunization therapy.The most most of clinical CART Noticeable result both is from treating hematologic disease, and the CART clinic for solid tumor also begins to starting.The wind of CART therapy Danger is the non-specific effect of missing the target caused of antigen receptor.
Summary of the invention
The technical problem to be solved in the present invention is to provide the relevant Chimeric antigen receptor T cell of a kind of cancer of the esophagus and preparation thereof Method, the treatment for the cancer of the esophagus provides a new technique direction.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows.
A kind of Chimeric antigen receptor T cell, this Chimeric antigen receptor T cell is with T lymphocytes as host, thin at lymph T The Matrix attachment region of born of the same parents is closed by consideration convey dyeing and finishing and has the gene shown in SEQ ID NO:1.
Above-mentioned Chimeric antigen receptor T cell, with T lymphocytes as host, contains SEQ ID on the surface of T lymphocytes The antigen receptor of gene expression shown in NO:1.
The preparation method of above-mentioned Chimeric antigen receptor T cell, first proceeds to non-viral by the gene shown in SEQ ID NO:1 Carrier, is then incorporated into said gene in the Matrix attachment region of T lymphocytes by consideration convey dyeing technique so that said gene is being drenched Bar T cell surface expression, kills nonspecific lymphocyte directional transformation for being capable of identify that and tumour cell being carried out targeting The specific chimeric antigen receptor T cell of wound.
The preparation process of above-mentioned Chimeric antigen receptor T cell includes:
A, PBMC separate
A-1, transfer to new blood, in 50 mL centrifuge tubes, be diluted according to volume ratio 1:1 with physiological saline, blow up and down Beat 3-5 time mix, 6 mL Ficoll solution are joined in 15 mL centrifuge tubes, be vertically placed into stand on level table standby With；The blood 8 mL pipette diluted slowly is uniformly added in the above-mentioned centrifuge tube containing Ficoll solution；
A-2, the centrifuge tube having added blood and Ficoll solution is transferred in centrifuge gently, wants during transfer centrifuge tube Minimizing is rocked, and keeps centrifuge tube vertical, and to avoid breaking liquid level layering, room temperature 400g is centrifuged 30 minutes；
A-3, centrifugal terminate after, transfer centrifuge tube is in super-clean bench gently, with pipettor draw in the middle of tunica albuginea layer i.e. lymph thin Born of the same parents' layer, avoids being drawn to the cell on lower floor or upper strata, it is to avoid pollute in suction operation, be then transferred to 50 new mL from In heart pipe；
A-4, adding 30 mL physiological saline in the above-mentioned lymphocyte being transferred out, piping and druming mixing, 60-100g room temperature is centrifuged 10 minutes, repeated washing lymphocyte, remove supernatant, add complete medium re-suspended cell；
It is 5*10 that A-5, counting adjust density5Cell/ml, in 12 orifice plates anticipated, every hole adds 1ml cell；To cultivate Plate is positioned over 37 DEG C, 5% (volume ratio) CO2Incubator cultivate 48 hours；
B, slow-virus infection
B-1, collection step A gained PBMC, room temperature 300g is centrifuged 4min, and adjustment density is 5*105Cell/ml, 12 new orifice plates In every hole add 1ml cell, experimental group adds the slow virus of 7.5ul EGFR bis-generation CAR, and virus titer is 2*108/ mL, MOI= 3；Control group adds 7.5ul comparison virus, and virus titer is also 2*108/ mL, MOI=3, every hole adds TRANS B and infects reagent；
B-2, Tissue Culture Plate is placed in 37 DEG C, 5% (volume ratio) CO2Incubator is cultivated；
B-3, the 3rd day every hole of cultivation add the fresh culture medium of 1ml, continue to cultivate；.
B-4, the 7th day counting of cultivation calculate amplification times；
C, phenotype and parting detection
C-1, count each cell and take out 1.0 x106Individual, 1000rpm, centrifugal 3min, add 100ul FACS buffer weight Outstanding cell, adds 15ul APC anti-human CD8,15ul PE anti-human CD4,15ul PerCY5.5 anti-human CD3；
C-2,4 DEG C of lucifuges hatch 30min, 1000rpm, centrifugal 3min, remove supernatant addition FACS buffer and wash twice again；
C-3, addition 150ul FACS buffer re-suspended cell, FACS detects fluorescence；Determine the expression of prepared cell GFP, so After utilize BD Accure C6 software that data are carried out Treatment Analysis, i.e. can determine that the infection rate of T cell, complete to be fitted together to The preparation of antigen receptor T cell.
Use and have the beneficial effects that produced by technique scheme: Chimeric antigen receptor T prepared by the method for the present invention Cell has specific cancer of the esophagus correlation function activity, and the treatment to the cancer of the esophagus has important potential and real value, for food The treatment of pipe cancer provides a new technique direction.
Accompanying drawing explanation
Fig. 1 is the CAR structural representation of two generations and three generations.
Fig. 2 shows the preparation flow of CART cell (i.e. Chimeric antigen receptor T cell).
Fig. 3 shows the GFP expression of results of CART-EGFR and CART-control group.
Fig. 4 shows that CART-EGFR cell hives off.
Fig. 5 shows that CART-cellular control unit hives off.
Fig. 6 shows the result that flow cytomery cell EGFR expresses.
Fig. 7 shows that CART-EGFR and CART-cellular control unit secretes result.
Fig. 8 shows that CART-EGFR, CART-control group is to target cell and the lethal effect of non-target cell.
Fig. 9 shows CART-EGFR, CART-control group GFP cell detection results.
Detailed description of the invention
Following example are described in detail the present invention.Various raw material used in the present invention and items of equipment are conventional city Sell product, all can be bought by market and directly obtain.
Embodiment 1, the design of CAR-EGFR and structure
The specification of 1-1, CAR.Through statistics, current clinical trial use most CAR structures include two generations and three generations CAR (seeing accompanying drawing 1), in relevant clinical report, two generation CAR reports are more, the most especially dominate with the University of Pennsylvania CAR structure (NCT01626495, NCT01029366) evident in efficacy, therefore we have employed this two generations CAR mount structure.
The structure of 1-2, CAR and structure.CAR-EGFR sequence includes following several part: CD8-pre, EGFR-scFv, CD8 hinge+cross-film, 4-1BB(CD137), CD3 ζ.Its complete sequence sees SEQ ID NO:1.
The structure of 1-3, CAR control group and structure.CAR-control group sequential structure includes: CD8-pre, EGFR-scFv, CD8-ΔCD3ζ.Its complete sequence sees SEQ ID NO:2.
The preparation of the T cell (CART-EGFR) that embodiment 2, CAR-EGFR modify is verified with phenotype
The preparation of 2-1, CART-EGFR and CART-control group.CART-EGFR and CART-control group preparation flow such as accompanying drawing 2 institute Show.Specifically include following content:
2-1-1, experiment reagent and laboratory apparatus:
Experiment reagent includes:
Ficoll-Paque PLUS(GE, cat#17-1440-02)
Complete medium TexMACS (Miltenyi Biotechnolog, cat#170-076-309)+IL-2 (Miltenyi Biotechnolog, cat#130-097-748)
PerCP5.5 anti-human CD3 (BD, cat#552852)
PE anti-human CD4 (BD, cat#555347)
APC anti-human CD8 (BD, cat#555369)
FACS buffer:PBS+2.5%FBS
TRANS B。
Laboratory apparatus includes:
Centrifuge: Hunan instrument L550
Flow cytometer: BD C6
CO2 incubator: ESCO MCO-20AIC
Cell counter: Cellometer Auto 1000.
2-1-2, PBMC separate: transferred to by new blood in 50 mL centrifuge tubes.With physiological saline according to 1:1(volume Than) be diluted, 3-5 mixing of piping and druming the most up and down.6 mL Ficoll are joined in 15 mL centrifuge tubes, vertically places Stand for standby use on level table.Slowly it is even added to vertically place with pipette by the blood (cumulative volume 8 mL) diluted In the centrifuge tube containing Ficoll solution of table top.It is transferred to having added the blood centrifuge tube with Ficoll gently in centrifuge. Rock in transfer centrifuge tube process minimizing as far as possible, keep centrifuge tube vertical, to avoid breaking liquid level layering.Room temperature 400g is centrifuged 30 Minute.Centrifugal terminate after, transfer centrifuge tube is in super-clean bench gently.Middle tunica albuginea layer (i.e. lymphocyte is drawn with pipettor Layer) it is transferred in 50 new mL centrifuge tubes.Avoid being drawn to the cell on lower floor or upper strata, it is to avoid pollute as far as possible.Add 30 In the mL physiological saline extremely above-mentioned lymphocyte being transferred out, blowing and beating mixing gently, 60-100g room temperature is centrifuged 10 minutes. Repeated washing lymphocyte, removes supernatant, adds a certain amount of complete medium re-suspended cell.It is 5*10 that counting adjusts density5 Cell/ml, in 12 orifice plates anticipated, every hole adds 1ml.Culture plate is positioned over 37 ° of C, 5%CO2Incubator cultivates 48 Hour.
2-1-3, slow-virus infection: collect above-mentioned PBMC, room temperature 300g is centrifuged 4min, and adjustment density is 5*105Cell/ Ml, in 12 new orifice plates, every hole adds 1ml cell, and experimental group adds the slow virus (system of slow virus of 7.5ul EGFR bis-generation CAR Standby is to compare the technology routinized, and can prepare or provide the gene order of the present invention to transfer to the lucky triumphant chemical gene skill in Shanghai voluntarily Prepared by art Co., Ltd)；Virus titer is 2*108/ mL, MOI=3；Control group adds 7.5ul comparison virus, and virus titer is 2* 108/ mL, MOI=3, every hole adds certain TRANS B infection reagent, and (TRANS B infects reagent purchased from the lucky triumphant geneticization in Shanghai Learn a skill Co., Ltd, and its consumption determines according to product description).Tissue Culture Plate is placed in 37 ° of C, 5%CO2 incubator trainings Support.Within 3rd day, every hole adds the fresh culture medium of 1ml, continues to cultivate.Within 7th day, counting calculates amplification times.
This is prepared, and initial p BMC cell concentration is 1E+6, and when the 7th day, experimental group PBMC cell concentration is 2.50E+07, its In 97.9% be CD3+T cell, see that Fig. 2, cells expanded are about 25 times；Control group PBMC cell concentration is 1.77E+07, carefully Born of the same parents' amplification times is about 17 times, is shown in Table 1.
Table 1 CART-EGFR and CART-cellular control unit is bred
2-2, CART-EGFR Phenotypic examination.Experiment purpose: determine the efficiency of infection of CART-EGFR and CART-control group.Experiment Design: the expression of cell GFP prepared by flow cytomery.Count each cell and take out 1.0 x106Individual, 1000rpm, from Heart 3min, adds 100ul FACS buffer re-suspended cell, adds 15ul APC anti-human CD8,15ul PE anti-human CD4 、15ul PerCY5.5 anti-human CD3.4 DEG C of lucifuges hatch 30min, 1000rpm, centrifugal 3min, removes supernatant addition FACS buffer and washes twice again.Adding 150ul FACS buffer re-suspended cell, FACS detects Fluorescence.With BD Accure C6 software, data are carried out Treatment Analysis.Experimental result and analysis: CART-EGFR and CART-control group GFP expression of results such as Fig. 3.As it is shown on figure 3, CART-EGFR efficiency of infection is about 56.1%, CART-control group Efficiency of infection is about 19.3%.
The cell of 2-3, CART hives off detection.Experiment purpose: determine the subgroup distribution of T cell in the cell of CART-EGFR Feature.Experimental design: cell surface marker prepared by flow cytomery, examination target and meaning are as shown in table 2 below.
Table 2 T cell subgroup is distributed
Experimental result and analysis: CART-EGFR and CART-cellular control unit hive off testing result such as Fig. 4,5.Can by the above results Knowing, prepared CART-EGFR cell hives off such as table 3；Prepared CART-cellular control unit hives off such as table 4.
Table 3 CART-EGFR subsets distribution ratio
Table 4 CART-cellular control unit subgroup distribution proportion
T lymphocyte (CD3+) accounts for the ratio of total cell colony 99.6%
T effector cell (CD3+CD8+) accounts for the ratio of T cell 19.8%
T helper lymphocyte (CD3+CD4+) accounts for the ratio of T cell 74.4%
Embodiment 3, the checking of functional verification target cell of CART-EGFR
Experiment purpose: the expression of EGFR on detection target cell and non-target cell.Experimental design: use the streaming antibody of EGFR to target Cell (Eca109) and compared with control cells (MCF7) detect.Experimental result and analysis: flow cytomery Eca109 and MCF7 EGFR expression of results such as Fig. 6, in result display negative control MCF7, EGFR expresses relatively low, but in target cell Eca109 EGFR high expressed.
Embodiment 4, the external functional verification cytokine secretion experiment of CART-EGFR.
Experiment purpose: after jointly being hatched by detection CART-EGFR cell and target cell, on whether cytokine secretion has Rise and judge whether CART has function.Experimental design: contrast CART-EGFR, CART-control group and target cell and non-target cell respectively After jointly hatching, the secretion situation of cell factor in culture medium supernatant.Reagent includes: Human TH1/TH2 cytokine CBA Kit(BD, Cat#550749).Cell (Cell line) including: MCF7(Medium:DMEM+10%FBS)；ECA109 (Medium:DMEM+10%FBS).Instrument includes: centrifuge: Hunan instrument L550；Flow cytometer: BD C6.Experimental technique includes:
4-1, cytokine secretion: collect target cell, by dilution buffer, cell be washed once, 1000rpm 3min is centrifuged, Abandoning supernatant, with 1640 re-suspended cells containing 2% FBS, counting, cell is diluted to 1*10 the most at last6Individual/ml concentration；Collect CART Cell (effector cell), washed once cell by dilution buffer, and 1000rpm 3min is centrifuged, and abandons supernatant, 2% FBS's 1640 re-suspended cells, counting, cell is diluted to 1*10 the most at last6Individual/ml concentration；By thin to 100ul target cell and 100ul CART Born of the same parents 1:1 mixing adds in 96 orifice plates, 37 DEG C, 5%CO2Co-incubation 20-24 hour；1000rpm 5min is centrifuged, and collects supernatant inspection Survey cell factor.
4-2, cytokines measurement: prepare 15ml centrifuge tube, mark 1 on bottle cap, is transferred to by standard items in EP pipe, and Being added thereto to standard dilutions 2ml, gentle inversion mixes, and room temperature prevents 15min-20min from balancing.Take 9 1.5ml EP Pipe, and in often pipe, add 300ul standard dilutions, reference numerals 2-9 on bottle cap, mixed in 1 pipe takes 300ul to 2 pipe Even；Take out in 300ul to 3 pipe in 2 pipes, the like carry out 2 times of gradient dilutions, separately take 1.5ml EP and manage, addition 300ul standard dilutions is as negative control.Determine cell factor and the sample number needing detection.Take out to be detected Cell factor capture pearl bottle, firmly mixing.Every kind of pearl takes out (detected sample number+standard items number+negative control number) * The capture pearl amount of 10ul.And every kind of pearl is carried out turbine concussion mixing.Sample is diluted (dilute with standard dilutions Release multiple can decide in its sole discretion, such as 1:10,1:100 etc.) take N number of 1.5ml EP pipe (N=sample number+standard items number+comparison number) to Often pipe (standard items, sample, negative control) adds 50ul mixing pearl；And in each pipe, add corresponding test agent (standard Product, sample, comparison) 50ul and PE Detection Reagent 50ul, after being sufficiently mixed, lucifuge incubated at room 3 hours. In often pipe, add 1ml wash buffer, softly blow even rear 200g and be centrifuged 5 minutes.Carefully suck supernatant, and to every Guan Zhongjia Enter 300ul wash buffer, and resuspended precipitation.Sample is carried out flow cytomery.Soft with the FCAP Array v3 of BD Part carries out Treatment Analysis to data.
4-3, experimental result and analysis: this experiment have detected IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-respectively The secretion of γ, wherein IL-4 and TNF-α secretion level are relatively low, change inconspicuous, and result is as it is shown in fig. 7, CART-EGFR exists During target cell, the secretion of cell factor is higher than CART-control group, and result display CART-EGFR has certain functional activity.
Embodiment 5, the external functional verification cellkilling capacity detection of CART-EGFR.
Experiment purpose: the killing ability of detection CART-EGFR and CART-control group.Experimental design: real by LDH release Test, it is judged that the CART cell specific killing to target cell.Experimental result and analysis: CART-EGFR and CART-control group is thin to target Cracking effect such as Fig. 8 of born of the same parents and non-target cell.Compared with CART-control group, target cell is had and necessarily kills work by CART-EGFR Property, at CART-EGFR: target ration is after 20:1 co-cultures 4hr, target cell lysis rate is 40.42%.
Embodiment 6, the proliferation experiment of CART-EGFR when there is target cell
Experiment purpose: detection CART-EGFR and CART-control group proliferation function in the presence of target cell.Experimental design: CART-EGFR and CART-control group and target ration are 2:1, and co-incubation 48 hours, flow cytomery is with GFP The propagation of cell.Experimental result and analysis: CART-EGFR and CART-cellular control unit proliferation results such as Fig. 9, deposit target cell Under conditions, CART-EGFR and CART-control group propagation is inconspicuous.
From above-mentioned test example, utilize Chimeric antigen receptor T cell CART-prepared by the antigen receptor gene of the present invention EGFR compares CART-control group and has shown certain functional activity, and the gene of the present invention has weight for the treatment of the cancer of the esophagus Potential and the real value wanted.
Foregoing description is only used as the enforceable technical scheme of the present invention and proposes, single not as to its technical scheme itself Restrictive condition.
<110>Wang Zhiyu
<120>a kind of Chimeric antigen receptor T cell and preparation method thereof
<160> 2
<210> 1
<211> 1461
<212> DNA
<213>artificial sequence
<400> 1
ATGTACAGGA TGCAACTCCT GTCTTGCATT GCACTAAGTC TTGCACTTGT CACGAACTCG 60
GCCGAAGTGC AGCTGCAGCA GAGCGGCGCG GAACTGATGA AACCGGGCGC GAGCGTGAAA 120
ATTAGCTGCA AAGCGACCGG CTATACCTTT AGCAGCTATT GGATTGAATG GGTGAAACAG 180
CGTCCGGGCC ATGGCCTGGA ATGGATTGGC GAAATTCTGC CGGGCAGCAA GAAAACCAAC 240
TATAACGAAA AATTTAAAGG CAAAGCGACC TTTACCGCGG ATACCAGCAG CAACACCGCG 300
TATATGCAGT TTAGCAGCCT GACCAGCGAA GATAGCGCGG TGTACTATTG CGCGCGTTAT 360
TATTATCGTA ACGATGATTA TGGCATGGAT TATTGGGGCC AGGGCACCAC CGTGACCGTG 420
AGCAGCGGTG GTGGTGGTTC TGGCGGTGGT GGTAGCGGCG GTGGTGGCAG CGATATTGTG 480
ATGAGCCAGA GCCCGAAAAG CATGAGCATG AGCGTGGGCG AACGTGTGAC CCTGACCTGC 540
AAAGCGAGCG AAAACGTGGT GACCTATGTG AGCTGGTATC AGCAGAAACC GGAACAGAGC 600
CCGAAACTGC TGATTTATGG CGCGAGCAAC CGTTATACCG GCGTGCCGGA TCGTTTTACC 660
GGCAGCGGCA GCGCGACCGA TTTTACCCTG ACCATTAGCA GCGTGCAGGC GGAAGATCTG 720
GCGGATTATC ATTGCGGCCA GGGCTACAGC TATCCGTATA CCTTTGGCGG CGGCACCAAA 780
CTGGAAATTA AAACCACGAC GCCAGCGCCG CGACCACCAA CACCGGCGCC CACCATCGCG 840
TCGCAGCCCC TGTCCCTGCG CCCAGAGGCG TGCCGGCCAG CGGCGGGGGG CGCAGTGCAC 900
ACGAGGGGGC TGGACTTCGC CTGTGATATC TACATCTGGG CGCCCTTGGC CGGGACTTGT 960
GGGGTCCTTC TCCTGTCACT GGTTATCACC CTTTACTGCA AACGGGGCAG AAAGAAACTC 1020
CTGTATATAT TCAAACAACC ATTTATGAGA CCAGTACAAA CTACTCAAGA GGAAGATGGC 1080
TGTAGCTGCC GATTTCCAGA AGAAGAAGAA GGAGGATGTG AACTGAGAGT GAAGTTCAGC 1140
AGGAGCGCAG ACGCCCCCGC GTACAAGCAG GGCCAGAACC AGCTCTATAA CGAGCTCAAT 1200
CTAGGACGAA GAGAGGAGTA CGATGTTTTG GACAAGAGAC GTGGCCGGGA CCCTGAGATG 1260
GGGGGAAAGC CGAGAAGGAA GAACCCTCAG GAAGGCCTGT ACAATGAACT GCAGAAAGAT 1320
AAGATGGCGG AGGCCTACAG TGAGATTGGG ATGAAAGGCG AGCGCCGGAG GGGCAAGGGG 1380
CACGATGGCC TTTACCAGGG TCTCAGTACA GCCACCAAGG ACACCTACGA CGCCCTTCAC 1440
ATGCAGGCCC TGCCCCCTCG C 1461
<210> 2
<211> 1041
<212> DNA
<213>artificial sequence
<400> 2
ATGTACAGGA TGCAACTCCT GTCTTGCATT GCACTAAGTC TTGCACTTGT CACGAACTCG 60
GCCGAAGTGC AGCTGCAGCA GAGCGGCGCG GAACTGATGA AACCGGGCGC GAGCGTGAAA 120
ATTAGCTGCA AAGCGACCGG CTATACCTTT AGCAGCTATT GGATTGAATG GGTGAAACAG 180
CGTCCGGGCC ATGGCCTGGA ATGGATTGGC GAAATTCTGC CGGGCAGCAA GAAAACCAAC 240
TATAACGAAA AATTTAAAGG CAAAGCGACC TTTACCGCGG ATACCAGCAG CAACACCGCG 300
TATATGCAGT TTAGCAGCCT GACCAGCGAA GATAGCGCGG TGTACTATTG CGCGCGTTAT 360
TATTATCGTA ACGATGATTA TGGCATGGAT TATTGGGGCC AGGGCACCAC CGTGACCGTG 420
AGCAGCGGTG GTGGTGGTTC TGGCGGTGGT GGTAGCGGCG GTGGTGGCAG CGATATTGTG 480
ATGAGCCAGA GCCCGAAAAG CATGAGCATG AGCGTGGGCG AACGTGTGAC CCTGACCTGC 540
AAAGCGAGCG AAAACGTGGT GACCTATGTG AGCTGGTATC AGCAGAAACC GGAACAGAGC 600
CCGAAACTGC TGATTTATGG CGCGAGCAAC CGTTATACCG GCGTGCCGGA TCGTTTTACC 660
GGCAGCGGCA GCGCGACCGA TTTTACCCTG ACCATTAGCA GCGTGCAGGC GGAAGATCTG 720
GCGGATTATC ATTGCGGCCA GGGCTACAGC TATCCGTATA CCTTTGGCGG CGGCACCAAA 780
CTGGAAATTA AAACCACGAC GCCAGCGCCG CGACCACCAA CACCGGCGCC CACCATCGCG 840
TCGCAGCCCC TGTCCCTGCG CCCAGAGGCG TGCCGGCCAG CGGCGGGGGG CGCAGTGCAC 900
ACGAGGGGGC TGGACTTCGC CTGTGATATC TACATCTGGG CGCCCTTGGC CGGGACTTGT 960
GGGGTCCTTC TCCTGTCACT GGTTATCACC CTTTACTGCA GAGTGAAGTT CAGCAGGAGC 1020
GCAGACGCCC CCGCGTACTA A 1041

Claims (4)

1. a Chimeric antigen receptor T cell, it is characterised in that: this Chimeric antigen receptor T cell with T lymphocytes as host, The Matrix attachment region of T lymphocytes is closed by consideration convey dyeing and finishing and has the gene shown in SEQ ID NO:1.
2. according to the Chimeric antigen receptor T cell described in claim, it is characterised in that: described Chimeric antigen receptor T cell with T lymphocytes is host, contains the antigen receptor of gene expression shown in SEQ ID NO:1 on the surface of T lymphocytes.
3. the preparation method of Chimeric antigen receptor T cell described in claim 1, it is characterised in that: first by SEQ ID NO:1 institute The gene shown proceeds to non-virus carrier, and then said gene is incorporated into the Matrix attachment region of T lymphocytes by consideration convey dyeing technique In so that said gene is at T lymphocytes surface expression, by nonspecific lymphocyte directional transformation for being capable of identify that and right Tumour cell carries out the specific chimeric antigen receptor T cell of target killing.
Preparation method the most according to claim 3, it is characterised in that: its preparation process includes:
A, PBMC separate
A-1, transfer to new blood, in 50 mL centrifuge tubes, be diluted according to volume ratio 1:1 with physiological saline, blow up and down Beat 3-5 time mix, 6 mL Ficoll solution are joined in 15 mL centrifuge tubes, be vertically placed into stand on level table standby With；The blood 8 mL pipette diluted slowly is uniformly added in the above-mentioned centrifuge tube containing Ficoll solution；
A-2, the centrifuge tube having added blood and Ficoll solution is transferred in centrifuge gently, wants during transfer centrifuge tube Minimizing is rocked, and keeps centrifuge tube vertical, and to avoid breaking liquid level layering, room temperature 400g is centrifuged 30 minutes；
A-3, centrifugal terminate after, transfer centrifuge tube is in super-clean bench gently, with pipettor draw in the middle of tunica albuginea layer i.e. lymph thin Born of the same parents' layer, avoids being drawn to the cell on lower floor or upper strata, it is to avoid pollute in suction operation, be then transferred to 50 new mL from In heart pipe；
A-4, adding 30 mL physiological saline in the above-mentioned lymphocyte being transferred out, piping and druming mixing, 60-100g room temperature is centrifuged 10 minutes, repeated washing lymphocyte, remove supernatant, add complete medium re-suspended cell；
It is 5*10 that A-5, counting adjust density5Cell/ml, in 12 orifice plates, every hole adds 1ml cell；Culture plate is positioned over 37 DEG C, The CO of 5% volume ratio2Incubator cultivate 48 hours；
B, slow-virus infection
B-1, collection step A gained PBMC, room temperature 300g is centrifuged 4min, and adjustment density is 5*105Cell/ml, 12 new orifice plates In every hole add 1ml cell, experimental group adds the slow virus of 7.5ul EGFR bis-generation CAR, and virus titer is 2*108/ mL, MOI= 3；Control group adds 7.5ul comparison virus, and virus titer is also 2*108/ mL, MOI=3, every hole adds TRANS B and infects reagent；
B-2, Tissue Culture Plate is placed in 37 DEG C, the CO of 5% volume ratio2Incubator is cultivated；
B-3, the 3rd day every hole of cultivation add the fresh culture medium of 1ml, continue to cultivate；
B-4, the 7th day counting of cultivation calculate amplification times；
C, phenotype and parting detection
C-1, count each cell and take out 1.0 x106Individual, 1000rpm, centrifugal 3min, add 100ul FACS buffer resuspended Cell, adds 15ul APC anti-human CD8,15ul PE anti-human CD4,15ul PerCY5.5 anti- human CD3；
C-2,4 DEG C of lucifuges hatch 30min, 1000rpm, centrifugal 3min, remove supernatant addition FACS buffer and wash twice again；
C-3, addition 150ul FACS buffer re-suspended cell, FACS detects fluorescence；Determine the expression of prepared cell GFP, so After utilize BD Accure C6 software that data are carried out Treatment Analysis, i.e. can determine that the infection rate of T cell, complete to be fitted together to The preparation of antigen receptor T cell.
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Cited By (5)

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Publication number Priority date Publication date Assignee Title
CN106801067A (en) * 2016-12-26 2017-06-06 武汉波睿达生物科技有限公司 A kind of preparation method of Chimeric antigen receptor T cell
CN107557392A (en) * 2017-09-30 2018-01-09 山东兴瑞生物科技有限公司 A kind of preparation method and applications of the immunocyte of the safety-type Chimeric antigen receptor modifications of anti-EGFR
CN108085342A (en) * 2017-12-29 2018-05-29 深圳市茵冠生物科技有限公司 A kind of preparation method of the T lymphocytes of Chimeric antigen receptor genetic modification, CAR-T cells obtained and application thereof
CN108315305A (en) * 2017-12-26 2018-07-24 沣潮医药科技（上海）有限公司 Carry the preparation method and applications of the immunocyte excretion body of Chimeric antigen receptor
CN108728416A (en) * 2017-04-17 2018-11-02 沈阳美达博生物科技有限公司 A kind of CAR-T cell culture flow

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KAICHAO FENG ET AL.: "Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer", 《SCIENCE CHINA LIFE SCIENCES》 *
余晓玲等: "表达靶向癌胚抗原的嵌合受体的慢病毒载体构建及初步应用", 《第三军医大学学报》 *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106801067A (en) * 2016-12-26 2017-06-06 武汉波睿达生物科技有限公司 A kind of preparation method of Chimeric antigen receptor T cell
CN108728416A (en) * 2017-04-17 2018-11-02 沈阳美达博生物科技有限公司 A kind of CAR-T cell culture flow
CN107557392A (en) * 2017-09-30 2018-01-09 山东兴瑞生物科技有限公司 A kind of preparation method and applications of the immunocyte of the safety-type Chimeric antigen receptor modifications of anti-EGFR
CN107557392B (en) * 2017-09-30 2020-06-30 山东兴瑞生物科技有限公司 Preparation method and application of anti-EGFR safe chimeric antigen receptor modified immune cells
CN108315305A (en) * 2017-12-26 2018-07-24 沣潮医药科技（上海）有限公司 Carry the preparation method and applications of the immunocyte excretion body of Chimeric antigen receptor
CN108315305B (en) * 2017-12-26 2020-11-06 沣潮医药科技（上海）有限公司 Preparation method and application of immune cell exosome carrying chimeric antigen receptor
CN108085342A (en) * 2017-12-29 2018-05-29 深圳市茵冠生物科技有限公司 A kind of preparation method of the T lymphocytes of Chimeric antigen receptor genetic modification, CAR-T cells obtained and application thereof

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