CN107236762A - A kind of method that minicircle dna transfecting T cells prepare clinical grade CAR T cell preparations - Google Patents
A kind of method that minicircle dna transfecting T cells prepare clinical grade CAR T cell preparations Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses a kind of method that minicircle dna transfecting T cells prepare clinical grade CAR T cell preparations, methods described includes applying non-viral minicircle dna（minicircle DNA,mcDNA）Carrier high-efficiency rate transfecting T cells prepare the technology of clinical grade CAR T cell preparations, and the mcDNA rotaring dyeing technologies include carrying the Chimeric antigen receptor of target gene（CAR）Design and synthesis, the structure of target gene CAR parent vector plasmids, the extraction of target gene CAR mcDNA vector plasmids, target gene CAR mcDNA vector plasmids transfecting T cells and the clinical grade CAR T cell preparations prepared by application mcDNA rotaring dyeing technologies, the purposes of the mcDNA rotaring dyeing technologies is further disclosed, it is found that this mcDNA rotaring dyeing technologies have, expression stabilization, function-stable high on mammalian T cell transfection efficiency, do not influence the feature of cytogene characteristic；Prepared CAR T cells preparation has the features such as targeting strong, killing-efficiency is high, clinical safety, and the mcDNA rotaring dyeing technologies can be used for the preparation for treating the clinical grade CAR T cell preparations of various entity tumors and blood and lymphsystem tumor.
Description
Technical field
The invention belongs to biological technical field, and in particular to apply minicircle dna rotaring dyeing technology, prepare clinical grade CAR-T thin
The method of born of the same parents' preparation.
Background technology
Tumor biotherapy has been acknowledged as the fourth-largest hand after operation, radiotherapy, the great tradition treatment means of chemotherapy three
Section.Biological therapy mainly includes cellular immunotherapy and the major class of antibody target medicine two.Cellular immunotherapy development in recent years is fast
Suddenly, on the basis of CTL/TIL obtains good clinical efficacy, immune detection point blocks CTL/TIL, TCR-T and CAR-T etc. new
Type cell preparation largely enters clinical test, and targetting CAR-T using CD-19/CD20 treats clinic of the B cell lymthoma as representative
Experiment, obtain more than 90% killing-efficiency and as milestone formula break through.Thus CAR-T cellular immunotherapies are likely to become
The critical treatment means of tumour.
Chimeric antigen receptor（CAR）Typical structure include by identification antigen single-chain antibody light chain and weight chain variable district
Hinge area and transmembrane region and intracellular that the scFv connected into constitutes extracellular binding area, single-chain antibody can be made to have flexibility
Signal peptide area.Extracellular binding area scFv need not recognize antigen in the form of MHC/ antigenic peptide complexes, but Direct Recognition is anti-
Original, and no matter the essence of antigen is protide or glycolipid class.The size of hinge area, flexibility ratio and extending range are determined
The ability that scFv is combined with tumor cell surface antigen.Transmembrane region plays important signal transduction, Chang Xuan in T cell activation
Built with CD3, CD8, CD28 transmembrane region.Intracellular signal area is by costimulatory molecules and tyrosine activation motifs ITAM groups
Into different its acknowledgement type of costimulatory molecules, secrete cytokines level maintains each not phase in terms of the cell survival time
Together.CAR structures are transferred to by genetic engineering by the CAR-T prepared by T cell, if specific recognition and killing ability are strong, table
Up to whether whether stable, function is permanent, the structure with CAR structures is closely related.The improvement that CAR design experienced three generations is gone through
Journey.First generation CAR only includes CD3 ζ or Fc ε RI γ chains；Second generation CAR includes a CD3 ζ and costimulatory molecules such as CD28/
4-1BB/CD27/ICOS/OX40, these costimulatory moleculeses can strengthen CD3 ζ signal transduction；Third generation CAR contain CD3 ζ and
Two costimulatory moleculeses, make CAR function more powerful.It is another have scholar in CAR genetic modification except Chimeric antigen receptor
Outside gene, one or more coding CAR and its carrier of promoter are also add, and carrier can be by some cell factors such as
IL-12 release further activates CAR signal path, referred to as forth generation CAR.CAR with Direct Recognition and can combine tumour
The tumor associated antigen of cell surface（TAA）, and by the incoming T cell of antigen signals, make T cell activation and secrete and discharge and be a large amount of
The cell factors such as IL-2, TNF-α, INF- γ, perforin and granzyme, killing ability is played to tumour cell.
CAR-T cell preparations can be prepared by transfecting above-mentioned CAR gene orders into T cell using suitable carrier.At present
Conventional carrier is mainly viral vector and the major class of non-virus carrier two.Viral vector based on slow virus and adenovirus vector,
Wherein adenovirus system infection cell when mechanism of action be that viral DNA can not be incorporated into chromosome, be only free in nucleus,
Therefore the expression of gene for a long time can not be maintained, and triggers immune response after application repeatedly.And slow virus carrier system can infect
Division stage and non-division cells, and external source target gene can be incorporated on chromosome, realize that target gene has for a long time
Effect expression, thus it is approved by the fda in the United States for clinic.But application viral vector prepares clinical grade CAR-T cell preparations, still has
Transfection efficiency is low（40%）, there is potential safety hazard（Genetic mutation and to knurl）The problems such as.Viral vector import foreign gene be
Random integration has insertion point mutation and conversion even intoxicating, the risk of tumorigenesis occurs for inducing cell to chromosome.Non- disease
What poisonous carrier was commonly used has liposome, polymer and Molecular Conjugates etc., emerging recently sleeping beauty's carrier and Transposon System
For the modification of CAR T cells, but it is low with transfection efficiency（20%-40%）, the shortcoming such as complex process, time length, cost be higher.
Minicircle dna is a kind of in recent years emerging non-virus carrier, be by traditional plasmid in Escherichia coli body by enzyme
The effect of cutting produces a kind of small ring supercoil expression cassette that locus specificity restructuring is obtained.There is research to carry purpose using minicircle dna
The cardiac muscle cell of gene Successful transfection mouse and Skeletal Muscle Cell, make it stable expression people vascular epidermal growth factor, separately have
Scholar has successfully transfected the versatile stem cell of people using minicircle dna.Research shows that non-virus carrier minicircle dna is gene
The best selection for the treatment of, because it lacks resistant maker gene and bacterium replication sequence, significantly improves minicircle dna and clinically should
Security.Can minicircle dna carry the transfection and modification that target gene realizes human T-cell, there is no experiment to confirm so far.
We take the lead in having made intensive studies this technology, and we devise the third generation CAR sequences for carrying target gene PSCA first
Row, by the target gene-CAR sequence constructs into micro-loop parent vector plasmid, and are successfully extracted comprising the micro- of target gene
The various means of minicircle dna Successful transfection of the target gene of carrying, are difficult to by circular DNA vector plasmid by means of electroporation method
The human T-cell of transfection, and more than 58% high transfection efficiency is obtained, far above existing virus or non-viral transfection means, Er Qiezhuan
Then 8 days, target gene-CAR expression rate was up to 89%, and is expressed more than 2 weeks as the amplification passage of T cell is stable.Electricity turns
Dye can make part T cell film destruction and cause cell death, we have discovered that, T cell survival rate is 60% or so after electrotransfection
And it is slow-growing, but culture can recover normal amplification and passage completely after 3 days, and T cell phenotype is identical with normal T-cell.
Had shown that through in vitro and in vivo killing experiments researchs, prepared target gene-CAR-T cell preparations can be efficient
The corresponding tumour antigen of rate, specific recognition, and produce high efficiency, specific tumour killing.It is of the invention to prepare skill with existing CAR-T
Art is compared, with following prominent characteristics：（1）The non-viral minicircle dna carrier transfection human T-cell's success of first Application；（2）Prove
Minicircle dna carrier is up to 58% to people T transfection efficiency, higher than existing any virus or non-viral transfection technology；（3）
Target gene-CAR is up to 90% in T cell expression rate, and with the stable expression of amplification passage of T cell；（4）It is of the invention prepared
Target gene-CAR-T cell preparations, to target gene height expression tumour cell there is powerful specific killing function；
（5）Target gene-CAR-T cell preparations prepared by the present invention are replicated in the absence of virus replication, bacterium, long term is potential to knurl etc.
Risk, thus clinical safety.
The content of the invention
It is an object of the invention to provide the new skill that a kind of minicircle dna transfecting T cells prepare clinical grade CAR-T cell preparations
Art.
To realize above target, the present invention provides a kind of minicircle dna transfecting T cells and prepares clinical grade CAR-T cell preparations
Method, methods described comprises the following steps：
（1）Target gene-CAR design and synthesis；
（2）The structure of target gene-CAR parent vector plasmids and preservation；
（3）The extraction of target gene-CAR-mcDNA vector plasmids；
（4）Target gene-CAR-mcDNA transfecting T cells prepare CAR-T cells；
（5）The preparation and application of clinical grade, target gene-CAR-T cell preparations.
It is preferred that, the method for the invention step is as follows：
（1）Target gene-CAR design, its method is as follows：1. filters out anti-purpose base using DNAstar8.0 software analysis
The Fab sequences and VH and VL sequences of cause, for designing the scFv sequences in CAR；2. modelled signals peptide sequence IgGkappa, puts
In target gene-scFv front ends to guide CAR structures to penetrate target cell membrane；3. is according to Genebank（NCBI）Middle CD8, CD28,
CD137 and CD3 ζ gene order purpose of design gene-CAR integral frame structure, and set respectively in the front and back end of the sequence
Put EcoR1 and BamH1 restriction enzyme sites；
（2）Target gene-CAR synthesis, its method is as follows：1. is carried out target gene-CAR full genome sequence by said structure
Row synthesis；2. the target gene-CAR complete genome sequences after synthesis are subcloned into pUC57 carriers be prepared into target gene-
CAR-pUC57 plasmids；
（3）The structure of target gene-CAR parent vector plasmids, its method is as follows：1. applications plasmid extraction kit is right respectively
Above-mentioned purpose gene-CAR-pUC57 plasmids and minicircle dna support agent box（MC-EasyTM Minicircle DNA
Production kit；SBI）The minicircle dna parent vector plasmid of offer（pMC.CMV-MCS-EF1-GFP-SV40PolyA）Enter
Row plasmid extraction, obtains plasmid extraction thing；2. is to step 1. obtained target gene-CAR-pUC57 and pMC.CMV-MCS-EF1-
GFP-SV40PolyA plasmid extractions thing carries out EcoR1 and BamH1 endonuclease reactions and obtains digestion products；3. is by step 2. obtained mesh
Gene-CAR-pUC57 and pMC.CMV-MCS-EF1-GFP-SV40PolyA digestion products be attached reaction obtain connection production
Thing；4. by step, 3. obtained connection product is transformed into the special competence bacterium solution that minicircle dna support agent box is provided to
（ZYCY10P3S2TE.coli）In, 30 DEG C, 250rpm shake bacterium culture 90min, obtain the parent vector containing target gene-CAR
Plasmid bacterial solution；5. by step, 4. obtained bacterium solution 50-200 μ l are laid in the LB agarose culture plates containing 50 μ g/ml kanamycins to
On, 37 DEG C of overnight incubations, selected clone, low dose extracts plasmid, digestion, electrophoresis, sequencing identification；Such as gene order correctly, then
4. step is obtained to parent vector -80 DEG C of glycerin storages of the plasmid bacterial solution of-CAR containing target gene, in case follow-up target gene -
The extraction of CAR-mcDNA vector plasmids；
（4）The extraction of target gene-CAR-mcDNA vector plasmids, its method is as follows：1. is by step（2）The purpose base of acquisition
Because-CAR parent vectors plasmid bacterial solution is from -80 DEG C of removals, the LB agaroses training of the 2ml containing 50 μ g/ml kanamycins is inoculated in
Support on base, 30 DEG C, 250rpm, shake bacterium 1-2h, survey OD values in case condition of culture regulates and controls；2. is by target gene-CAR parent vectors
Plasmid bacterial solution is inoculated in the sterile culture flask of the growth medium containing 200ml, 30 DEG C, 250rpm shakes bacterium and stay overnight；3. takes a small amount of training
Base detection pH value and OD values are supported, adjustment pH value is between 6.9-7.4, OD600Value is between 4-6, and 30 DEG C of continuation, 250rpm shake bacterium 5-
5.5hr；4. extracts target gene-CAR- by QIAGEN EndoFree Plasmid Maxi Kit operating procedures heavy dose
McDNA vector plasmids, detect that qualified rear -20 DEG C of refrigerators are saved backup；
It is preferred that, the bacterium solution by target gene-CAR parent vector plasmids is inoculated in the sterile of the growth medium containing 200ml
30 DEG C in blake bottle, 250rpm shake bacterium and stay overnight, it is characterized in that：1. shakes bacterium and stays overnight the time within 16hr；2. ensures growth training
Base pH value is supported between 6.9-7.4, most preferably 7.0；3. ensures growth medium OD600Value is between 4-6.
（5）Target gene-CAR-T cell preparations are prepared, its method is as follows：1. is autologous or allogeneic PBMC separation is obtained
Take T cell, after 1000U/ml IFN-γ is stimulated, 37 DEG C, overnight incubation in 5% CO2 incubators, next day day 1 are separately added into
1 μ g/ml, CD28 monoclonal antibody of CD3 monoclonal antibodies 1 μ g/ml and IL-2 500U/ml is cultivated, and day 2 adds IL-15 10U/ml cultures, day
3 addition 500U/ml containing IL-2 complete medium culture amplification T cell, culture medium was changed per 2-3 days；2. presses 4D-
Nucleofector electroporations（LONZA）Operating instruction prepares electricity and turns liquid, and above-mentioned T cell is resuspended, and adds target gene-CAR-
McDNA vector plasmids move to electric revolving cup after softly mixing, and are put into electroporation, set electric carryover sequence, and electricity turns 3s and completes CAR-T systems
It is standby；3. above-mentioned purposes gene-CAR-T is moved to cultivate in the IL-2 containing 500U/ml of preheating AIM-V serum free mediums and expanded
Increase the clinical order of magnitude target gene-CAR-T cells of 10-15 days harvests；4. target gene-CAR-T cell physiologicals salt solution is centrifuged
Wash 3-4 times and dispel cell fragment, move into 200ml feedback normal saline bag/bottles of the 2.0g containing human serum albumin, be prepared into clinic
Level target gene-CAR-T cell preparations.
The method of the invention, wherein the design and synthesis of the target gene-CAR, wherein designed target gene
Fab sequences, are selected from：PSCA、PSMA、CEA、EGFR、EGFRvIII、HER2、MSLN、GD2、IL13Rα2、GPC3、CAIX、L1-
CAM、CA125、CD133、FAP、CTAG1B、MUC1、FR-α、GUCY2C；
Wherein designed CAR sequences are the third generation containing scFv-CD3 ζ, CD28 and 4-1BB/OX40/ICOS gene orders
CAR sequences, and the second generation CAR sequences only containing scFv-CD3 ζ and CD28 gene orders, and only contain scFv-CD3 ζ
The first generation CAR sequences of gene order, and the TRUCK cell CAR sequences only containing forth generation；
The extraction of the target gene-CAR-mcDNA vector plasmids, wherein, the target gene-CAR-mcDNA carriers addressed
Plasmid, is any target gene-CAR-mcDNA vector plasmids；
The electricity turns T cell and prepares target gene-CAR-T cell preparations, wherein, transfection T cell used is selected from：Autologous T is thin
Born of the same parents, allogeneic T cells and each T cell subgroup.
Present invention additionally comprises the clinical grade target gene-CAR-T cell preparations prepared with method of the present invention.With
And application of the described target gene-CAR-T cell preparations in antineoplastic is prepared.
It is the detailed description of the technology of the present invention content below：
1. target gene-CAR design and synthesis：
1.1 go out Fab, VH and VL sequence of anti-target gene using biological software DNAstar8.0 Analysis and Screening, for designing
Chimeric antigen receptor（CAR）ScFv sequences；Modelled signal peptide sequence IgGkappa, is placed in target gene-scFv sequence leading portions
To guide CAR structure penetration cell films；According to Genebank（NCBI）Middle CD8, CD28, CD137 and CD3 ζ gene orders synthesis
Third generation CAR frame structure, and EcoR1 and BamH1 restriction enzyme sites are set respectively in CAR front and back end.The full base more than
Because of sequent synthesis target gene-CAR（Accompanying drawing 1）.
1.2 will contain EcoR1, and the target gene-CAR of BamH1 restriction enzyme sites is subcloned into pUC57 vector plasmids, life
Entitled target gene-CAR-pUC57 vector plasmids（Accompanying drawing 2）.
2. structure and the preservation of target gene-CAR parent vector plasmids：
2.1 plasmid extraction：Using plasmid extraction kit（TIANGEN）Respectively to above-mentioned purpose gene-CAR-pUC57 plasmids
And minicircle dna support agent box（MC-EasyTMMinicircle DNA Production kit；SBI）The micro-loop provided
DNA parent vector plasmids（pMC.CMV-MCS- EF1-GFP-SV40PolyA）Plasmid extraction is carried out, plasmid extraction thing is obtained.
2.2 digestion：To above-mentioned purpose gene-CAR-pUC57 and pMC.CMV-MCS-EF1-GFP- SV40PolyA plasmids
Extract carries out EcoR1 and BamH1 endonuclease reactions, and reaction system is as follows：
EcoRI 2μl
BamHI 2μl
10×K buffer 10μl
PSCA-CAR-pUC57 or
pMC.CMV-MCS-EF1-GFP-SV40PolyA 15μl
Plus endonuclease reaction pipe is put into 37 DEG C of water-bath 1h by ultra-pure water to 100 μ l, adds buffer solution and terminate endonuclease reaction；1% fine jade
Purpose fragment is cut under sepharose electrophoresis, ultraviolet light, using DNA gel QIAquick Gel Extraction Kit（TIANGEN）Purpose is separately recovered
Fragment, obtains target gene-CAR and pMC.CMV-MCS-EF1-GFP-SV40PolyA digestion products.
2.3 coupled reaction：The digestion products of above-mentioned purpose gene and carrier are attached instead by following reaction system
Should：
The μ l of target gene digestion products 5
The μ l of carrier digestion products 1
The μ l of 10 × T4 DNA ligases buffer solution 1
The μ l of T4 DNA ligases 0.6
Plus ultra-pure water is to 10 μ l, coupled reaction pipe is placed in 4 DEG C of connections overnight and obtains connection product, its structural representation is shown in accompanying drawing
3。
2.4 conversion：Above-mentioned connection product is added into the special competence bacterium solution that minicircle dna support agent box is provided
（ZYCY10P3S2TE.coli）In, incubated cell 30 minutes, 42 DEG C of water bath 30 seconds, are placed 2 minutes on ice on ice；Add
Room temperature S.O.C. culture mediums are placed in bacteriological incubator into the bottle equipped with competent cell, 30 DEG C, 250rpm shake bacterium culture
90min, obtains target gene-CAR parent vector plasmid bacterial solutions.
2.5 clone identification：Above-mentioned bacterium solution is tiled and contains the LB agarose culture plates of 50 μ g/ml kanamycins, 37 DEG C of cultures
Overnight, next day selects bacterial clone, and low dose extracts plasmid, EcoR1 and BamH1 double digestions, electrophoresis, sequencing identification；Sequencing knot
Fruit shows：Gene order is correct, shows the success of target gene-CAR parent vectors plasmid construction（Accompanying drawing 3）.
2.6 by above-mentioned purpose gene-CAR parent vectors -80 DEG C of glycerin storages of plasmid bacterial solution, in case follow-up target gene -
The extraction of CAR-mcDNA vector plasmids.
3. the extraction of target gene-CAR-mcDNA vector plasmids：
3.1 by 5 times of growth medium（5X Minicircle Growth medium, SBI）It is diluted to 1 times.
3.2 remove target gene-CAR parent vectors plasmid from -80 DEG C, are inoculated into 1 times of growth medium 200ml,
Move to aseptic bottle（Aseptic bottle volume/culture volume=5:1）30 DEG C, 250rpm, shake bacterium and stay overnight（<16hr）.
3.3 next day, a small amount of culture medium is taken to survey pH value and OD600Value, it is ensured that pH value is in 7 or so, OD600Value is between 4-6.
Need to adjust pH value and OD respectively with NaOH and fresh 1 times of growth medium if necessary600Value reaches above-mentioned standard.
3.4 continue 30 DEG C, 250rpm, shake bacterium 5-5.5hr（<5.5hr）, low dose extraction plasmid, EcoR1 and BamH1 enzymes
Cut, gel electrophoresis, examine target gene-CAR-mcDNA vector plasmids quality.
3.5 is up-to-standard, and purpose base is extracted by QIAGEN EndoFree Plasmid Maxi Kit steps heavy dose
Because of-CAR-mcDNA vector plasmids, EndoFree buffer TE weight solute grains simultaneously quantify plasmid concentration, obtain target gene-
CAR-mcDNA vector plasmids（Accompanying drawing 4-5）, -20 DEG C save backup.
4. target gene-CAR-mcDNA transfecting T cells prepare CAR-T cells：
4.1 PBMC acquisition：Tumour patient autologous peripheral blood, gene is taken to be harmonious entirely or half-matched healthy human peripheral blood 50-
100ml, liquaemin anti-freezing is transported to GMP like cells culturing room；50ml centrifuge tubes are taken to add Ficoll separating liquid 20ml, along wall
Above-mentioned blood 30ml is slowly added in separating liquid upper strata, 20 DEG C, 2500rpm, gradient centrifugation 20min；Carefully draw autologous plasma
Separately deposit standby, carefully draw tunica albuginea layer（PBMC）New centrifuge tube is placed in, physiological saline 1500rpm centrifugations 5min, which is washed 3 times, to be abandoned
Cell count is resuspended in supernatant, AIM-V serum free mediums.
4.2 T cell cultures and amplification：Above-mentioned T cell suspension moves into suspension blake bottle, adds 1000U/ml IFN-
γ, is placed in 37 DEG C, 5%CO2 incubator overnight incubations；Next day（day 1）Add 1 μ g/mlCD3 monoclonal antibodies, 1 μ g/ml CD28 monoclonal antibodies,
500U/ml IL-2 are cultivated；Day 2 adds 10U/ml IL-15 cultures 48hr；The addition 500U/ml of Day 3 IL-2 complete training
Base is supported to continue to expand passage, it is every to change liquid within 2-3 days.
4.3 minicircle dna electricity turn T cell and prepare CAR-T：
4.3.1 blake bottle coating and preheating：Blake bottle adds the CD3 monoclonal antibodies and CD28 monoclonal antibodies that concentration is 1 μ g/ml, is placed in 4 DEG C
Refrigerator coating is stayed overnight, and next day abandons supernatant, the PBS washings containing 0.5% BSA, adds the IL-2 cell culture mediums containing 500U/ml, 37
Pre- stand-by heat in DEG C incubator.
4.3.2 electricity turns liquid configuration：The electricity of 100 microlitres of electric revolving cups turns liquid configuration：82μl nucleofector solution
+ 18 μ l supplement, room temperature is placed standby；
4.3.3 minicircle dna electricity turns T cell preparation CAR-T：Above-mentioned T cell is taken, often pipe 5 × 106It is individual, room temperature 200g centrifugations
10min, dispels supernatant completely, and electricity turns the μ l of liquid 100 and the electric revolving cup of cell addition is resuspended；Pipette tips are placed in glass bottom and softly add purpose base
Because of-CAR-mcDNA vector plasmids（Control group adds the pmaxGFP plasmids that kit is provided）, it is to avoid produce bubble；Setting
The electric carryover sequence of LONZA Nucleofector TM4D electroporations, 3s completes electricity and turned, prepared by target gene-CAR-T cells
Into.
4.3.4 the culture and amplification of target gene-CAR-T cells：Standby target gene-the CAR-T of the above-mentioned electric transformation of ownership is thin
Born of the same parents, move into the IL-2 containing 500U/ml of preheating AIM-V serum free mediums, and culture amplification 10-15 days changes liquid in every 3 days, periodically
Count cell quantity.
4.4 target gene-CAR-T identification：
4.4.1 electricity turns the influence to T cell survival rate and increment activity：Electricity turns to produce damage to T cell, and breeds work to it
Property causes of short duration influence.FCM analysis electricity turns the discovery of the T cell after 24h, its survival rate about 60%；T is thin in electrotransfection 3 days
Born of the same parents' proliferation activity is reduced, and proliferation activity substantially recovers after 3 days.
4.4.2 electricity turns the influence to T cell phenotype：24 hours after transfection, flow cytometer detection T cell surface C D3, CD4 and
CD8 expression and normal T-cell indifference, show that electricity turns not influenceing the phenotypic characteristic of T cell.
4.4.3 minicircle dna transfects the transfection efficiency of human T-cell：The a small amount of cells of 6hr after fluorescence microscopy Microscopic observation, transfection
There is fluorescence, 24hr about 60% cell, 48hr about more than 90% cell expression fluorescence（Accompanying drawing 6）.24hr streamings are examined after transfection
Survey and find, GFP luciferase expression rates are up to more than 58% in prepared CAR-T（Accompanying drawing 7）.
4.4.4 expression of the flow cytometer detection target gene on CAR-T surfaces：Biotin-protein L combination CAR-T surfaces
Flow cytometer detection is found after light chain VL, PE-SA mark in single-chain antibody, expression of the target gene-scFv-CAR on T cell surface
Rate is up to more than 80%（Accompanying drawing 8）.
4.4.5 the expression of Western testing goals gene-CAR albumen：Molecular weight is 57kDa target gene-CAR eggs
It is white clear in the expression of CAR-T cells, and control cell is without expression（Accompanying drawing 9）.
5. the preparation and application of clinical grade target gene-CAR-T cell preparations：
The preparation of 5.1 clinical grade target gene-CAR-T cell preparations：Take 4.3.4 obtain target gene-CAR-T cells 1-2 ×
109Individual, physiological saline centrifugation washes 3-4 times and dispels cell fragment, and routinely cell is moved into 200ml feedbacks and given birth to by galenic pharmacy standard
Manage bag of saline/bottle（Containing 1% 200,000 unit of human serum albumin 2.0g, IL-2）, it is prepared into clinical grade target gene-CAR-T cells
Preparation.5.2 cells freeze：4.3.4 is taken to obtain target gene-CAR-T cells, changing medium culture 24hr makes at cell
In exponential phase；A centrifuge tube is taken to add culture medium, 5% autoserum, 20% DMSO configuration frozen stock solutions；It is collected by centrifugation thin
Born of the same parents, fresh culture is resuspended, and adds equivalent frozen stock solution（DMSO concentration is maintained at 5-10%）, cell count and vitality assessment, packing
In cryopreservation tube（2-3×109Individual cell/pipe）Sealing mark, program cooling is stored in -80 DEG C of refrigerators or liquid nitrogen container.Taken when using
Go out cryopreservation tube rapid fluid resuscitation, cell preparation is prepared by 5.1 methods describeds.
The transport of 5.3 cells：Take 5.1 obtain or cryopreservation resuscitation after by 5.1 methods prepare target gene-CAR-T cells
Preparation, bottle sealing, mark prepares date, time and lot number, is placed in 4 DEG C of environment incubators（Dry ice or refrigerator）, it is anti-to jolt
Land route or aviation are delivered to ward, and 12 hr internal jugular veins infusion is used.
The application of 5.4 target gene-CAR-T cell preparations：Before venous re-transfusion treatment, preparation time and transfusion need to be checked
The tightness of bag；The soft cell preparation that mixes is to reduce cell mass, using defeated after the blood transfusion apparatus physiological saline washing pipe with filter screen
Enter cell, input finishes physiological saline washing pipe again；By the various adverse reactions of clinical procedure anti symptom treatment.
Brief description of the drawings：
Third generation target gene-CAR the structure charts of accompanying drawing 1.（PSCAscFv-CD28-CD137-CD3ζ）.
The PSCA-CAR-pUC57 vector plasmids of the restriction enzyme site containing EcoR1 and BamH1 of accompanying drawing 2..
The minicircle dna parent vector plasmid of accompanying drawing 3.（pMC.CMV-MCS-EF1-GFP-SV40PolyA）Structure chart.
The PSCA-CAR-mcDNA vector plasmids of accompanying drawing 4. extract product schematic diagram.
The gel electrophoresis of the PSCA-CAR-mcDNA vector plasmids of accompanying drawing 5..L1：Parent vector plasmid (8651bp)；L2：
PSCA-CAR-mcDNA vector plasmids (4575bp).
The transfection efficiency of the fluorescence microscopy Microscopic observation T cell of accompanying drawing 6.（100×）.
The PSCA-CAR-mcDNA vector plasmids of accompanying drawing 7.（B）It is up to 58.66% to the transfection efficiency of human T-cell, it is significantly high
In control plasmid（A）Transfection efficiency.
Expression rates of the 24hr flow cytometer detections PSCA-CAR on T cell surface is up to more than 80% after accompanying drawing 8. is transfected
（Triplicate）.
The Western blot of accompanying drawing 9. detect 57kDa CAR protein expressions（C）, and untransfected T（A）And mock
cell（B）15kDa CD3 ζ are only expressed, CAR albumen is not expressed.
The in vitro killing-efficiencies of accompanying drawing 10..PSCA-CAR-T has powerful kill to the RT4 cells of PSCA height expression
Hinder efficiency（p<0.05）, and the PC-3M cells of PSCA low expressions are killed without obvious.
After accompanying drawing 11. and high expression PSCA AT4 cells are co-cultured, PSCA-CAR-T INF- γ and IL-2 secretory volumes
Significantly rise（p<0.05, respectively）.
The human immunity of accompanying drawing 12. rebuilds NOD/SCID mouse（B）Peripheral blood people CD3（T cell）And CD19（B cell）Table
It is respectively 67.29% and 21.5% up to rate, and does not rebuild mouse（A）Expression is negative.
The In vivo killing-efficiencies of accompanying drawing 13..PSCA-CAR-T is notable to the killing-efficiency of the AT4 tumours of PSCA height expression
Higher than mock T（p<0.05）.
The PSCA-CAR-T of accompanying drawing 14. can significantly inhibit the AT4 tumour growths of PSCA height expression.
Embodiment：
The present invention is further illustrated below by way of specific embodiment, but not as the limitation of the present invention.
Minicircle dna transfecting T cells prepare the technology of clinical grade PSCA-CAR-T cell preparations, and step is as follows：
1. the design and synthesis of PSCA-CAR gene orders：
1.1 applied biology software DNAstar8.0 design anti-PSCA Fab, VH and VL sequence, the scFv for designing CAR
Sequence；Modelled signal peptide sequence IgGkappa is placed in PSCA-scFv sequence leading portions；By Genebank（NCBI）Middle CD8, CD28,
CD137 and CD3 ζ gene orders synthesis third generation CAR frame structure, and in CAR front and back end set respectively EcoR1 and
BamH1 restriction enzyme sites.Designed more than, synthesize PSCA-CAR complete genome sequence（Accompanying drawing 1）.
PSCA-CAR gene orders are subcloned into pUC57 vector plasmids by 1.2, are named as PSCA-CAR-pUC57（Accompanying drawing
2）.
The structure of 2.PSCA-CAR parent vector plasmids：
2.1 plasmid extraction：Using the small extraction reagent kit of TIANGEN plasmids, respectively to above-mentioned PSCA-CAR-pUC57 plasmids and micro-loop
The minicircle dna parent vector plasmid that DNA vector kit is provided（pMC.CMV-MCS- EF1-GFP-SV40PolyA）Carry out
Plasmid extraction, obtains plasmid extraction thing.
2.2 digestions and coupled reaction：By PSCA-CAR-pUC57 and pMC.CMV-MCS-EF1-GFP- SV40PolyA matter
Grain extract carries out EcoR1 and BamH1 digestions, recovery and coupled reaction, obtains connection product.
2.3 competence bacterium solutions are converted and clone：By connection product add minicircle dna support agent box provided it is special
Competence bacterium solution（ZYCY10P3S2TE.coli）, be placed in bacteriological incubator, 30 DEG C, 250rpm shake bacterium culture, obtain PSCA-CAR
Parent vector plasmid bacterial solution；Plasmid bacterial solution is tiled and contains the LB agarose culture plates of 50 μ g/ml kanamycins, 37 DEG C were cultivated
Night, next day selects bacterial clone；Low dose extracts plasmid, EcoR1 and BamH1 double digestions, electrophoresis, sequencing are correct, PSCA-CAR
The success of parent vector plasmid construction（Accompanying drawing 3）.
The preservation of 2.4 parent vector plasmids：By -80 DEG C of glycerin storages of above-mentioned PSCA-CAR parent vectors plasmid bacterial solution, with
The extraction of standby follow-up PSCA-CAR-mcDNA vector plasmids.
The extraction of 3.PSCA-CAR-mcDNA vector plasmids：
3.1 remove PSCA-CAR parent vectors plasmid from -80 DEG C, are inoculated into 1 × Minicircle Growth medium
In 200ml, aseptic bottle is moved to（Aseptic bottle volume/culture volume=5:1）30 DEG C, 250rpm, shake bacterium and stay overnight（<16hr）.
3.2 next day, a small amount of culture medium is taken to survey pH value and OD600Value, adjustment pH value is in 7 or so, OD600Value is between 4-6；
Continue to shake bacterium culture 5-5.5hr（<5.5hr）；Low dose extracts plasmid, EcoR1 and BamH1 digestions, gel electrophoresis, inspection
PSCA-CAR-mcDNA vector plasmids it is up-to-standard, it is heavy dose of by QIAGEN EndoFree Plasmid Maxi Kit steps
PSCA-CAR-mcDNA vector plasmids are extracted, EndoFree buffer TE weight solute grains are simultaneously quantitative, obtain PSCA-CAR-
McDNA vector plasmids（Accompanying drawing 4-5）, -20 DEG C save backup.
4.PSCA-CAR-mcDNA transfections T prepares PSCA-CAR-T cells：
4.1 PBMC acquisition：Tumour patient autologous peripheral blood 50-100ml is taken, takes 50ml centrifuge tubes to add Ficoll separation
Liquid 20ml, above-mentioned blood 30ml is slowly added in separating liquid upper strata along wall, 20 DEG C, 2500rpm, gradient centrifugation 20min；Carefully inhale
Take autologous plasma separately to deposit standby, carefully draw tunica albuginea layer（PBMC）Be placed in new centrifuge tube, physiological saline 1500rpm, 5min from
The heart is washed 3 times, abandons supernatant, and cell count is resuspended in AIM-V serum free mediums.
4.2 T cell cultures and amplification：Above-mentioned T cell suspension moves into suspension blake bottle, adds 1000U/ml IFN-
γ, is placed in 37 DEG C, 5%CO2 incubator overnight incubations；Next day day 1 adds 1 μ g/ml CD3 monoclonal antibodies, 1 μ g/ml CD28 monoclonal antibodies
And 500U/ml IL-2 cultures；Day 2 adds 10U/ml IL-15 cultures 48hr；The additions of Day 3 500U/ml IL-2's is complete
Full culture medium continues to expand passage, every to change liquid within 2-3 days.
4.3 minicircle dna electricity turn T and prepare CAR-T：
4.3.1 blake bottle coating and preheating：Blake bottle adds the CD3 monoclonal antibodies and CD28 monoclonal antibodies that concentration is 1 μ g/ml, is placed in 4 DEG C
Refrigerator coating is stayed overnight, and next day abandons supernatant, the PBS washings containing 0.5% BSA, adds the AIM-V serum-frees of the IL-2 containing 500U/ml
Pre- stand-by heat in culture medium, 37 DEG C of incubators.
4.3.2 electrotransfection：82 μ l nucleofector solution are mixed with 18 μ l supplement, configuration 100
Electricity needed for microlitre electric revolving cup turns liquid；Take above-mentioned T cell 5 × 106Individual, room temperature 200g centrifugation 10min dispel supernatant, electricity completely
Turn the μ l of liquid 100 and the electric revolving cups of 100 microlitres of cell addition are resuspended；Pipette tips are placed in glass bottom and softly add PSCA-CAR-mcDNA carrier matter
Grain, notes that bubble can not be produced；The electric carryover sequence of LONZA Nucleofector TM4D electroporations is set, starts electricity and turns,
Prepared by PSCA-CAR-T cells completes.
4.3.3 PSCA-CAR-T culture and amplification：PSCA-CAR-T cells are moved into the culture medium of above-mentioned preheating, training
Amplification 10-15 days is supported, liquid, regular count cell quantity is changed within every 3 days.
4.4 PSCA-CAR-T identification：
4.4.1 electricity turns the influence to T cell：FCM analysis finds that electricity turns T cell survival rate about 60%, loss percentage after 24h
About 40%.Electricity turns T cell proliferation activity reduction in 3 days, and proliferation activity substantially recovers after 3 days.T cell CD3, CD4 and CD8 expression
With normal T-cell indifference.
4.4.2 transfection efficiency of the minicircle dna to human T-cell：Fluorescence microscopy Microscopic observation, a small amount of cells of the rear 6hr of electricity turn,
The cells of 24hr about 60%, the cells of 48hr about more than 90% expression fluorescence（Accompanying drawing 6）.Flow cytometer detection finds that 24hr is intracellular after electricity turns
Luciferase expression rate is up to 58%（Accompanying drawing 7）.
4.4.3 flow cytometer detection T surfaces PSCAscFv-CAR expression rate：Biotin-protein L combination CAR-T surfaces
Flow cytometer detection is found after light chain VL in single-chain antibody, row PE-SA mark, expression rates of the PSCAscFv-CAR on T cell surface
Up to more than 80%（Accompanying drawing 8）.
4.4.4 Western detects PSCA-CAR protein expressions：Molecular weight is 57kDa PSCA-CAR albumen in CAR-T
Cell expression is clear, and control cell is without expression（Accompanying drawing 9）.
5. the preparation and application of clinical grade PSCA-CAR-T cell preparations：
The preparation of 5.1 clinical grade PSCA-CAR-T cell preparations：4.3.3 is taken to obtain the PSCA-CAR-T cells 1-2 of amplification cultivation
×109Individual, physiological saline centrifugation is washed 3-4 times, moves into 200ml normal saline bags/bottle（Containing 1% human serum albumin 2.0g, IL-2 20
Ten thousand units）Clinical grade PSCA-CAR-T cell preparations are made.
5.2 PSCA-CAR-T's freezes：Take 4.3.3 to obtain exponential phase PSCA-CAR-T, add equivalent frozen stock solution
（DMSO containing 5% autoserum and 5-10%）, it is sub-packed in cryopreservation tube（2-3×109Individual cell/pipe）Sealing mark, program drop
Temperature is stored in -80 DEG C of refrigerators or liquid nitrogen container.
5.3 PSCA-CAR-T transport：5.1 are taken to obtain clinical grade PSCA-CAR-T cell preparations, 4 DEG C of environment incubators
（Dry ice or refrigerator）, anti-to jolt land route or aviation is delivered to ward, 12 hr internal jugular veins infusion is used.
5.4 PSCA-CAR-T Cytotoxicity in vitro efficient studies（in vitro）：
5.4.1 target cell：Prostatic cancer cell lines RT4（Experimental group）And PC-3M（Control group）As target cell；Flow cytometer detection
Determine RT4 cells height expression PSCA（82%）, PC-3M cells do not express PSCA（0.01%）.
5.4.2 packet：General T（nomal T）, GFP-mcDNA idle running T（mock T）, PSCA-CAR-mcDNA transfection T
（PSCA-CAR-T）.
5.4.3 CCK-8 assay：96 orifice plates are separately added into experimental group and control group target cell（Every group of 4 multiple holes）, 4
DEG C, adhere-wall culture 2hr under the conditions of 5%CO2, respectively by E:T = 2:1 / 5:1 / 10:1 / 20:1 adds effector cell, 37
DEG C, co-culture 12hr under the conditions of 5%CO2；Washing is changed after ordinary culture medium, and CCK-8 liquid 10 μ l, 37 DEG C, 5%CO2 are added per hole
Under the conditions of cultivate 4hr, ELIASA determines OD values；Killing-efficiency（%）={ 1-E+T groups OD values/T group OD values } x100%.
As a result：PSCA-CAR-T energy specific recognitions and the positive RT4 cells of killing psca expression, killing-efficiency are up to
85.16%, it is significantly higher than normal T and the mock T of control group（p<0.05;Accompanying drawing 10）；And it is thin to the negative PC-3M of expression
Born of the same parents are several without killing.
5.4.4 ELISA detects cytokine secretion amount：24 orifice plates are separately added into target cell（RT4 and PC-3M）, 37 DEG C,
Adhere-wall culture 2hr under the conditions of 5%CO2；Normal T, mock T and PSCA-CAR-T effector cell are separately added into by packet, is continued
Cultivate 24hr；Take each group supernatant 500 μ l, 4000rpm, centrifugation 10min to remove particulate matter and polymer, reagent is then pressed respectively
Box specification detects IFN-γ and IL-2 secretory volume.As a result：RT4 cells positive PSCA can be with obvious stimulation PSCA-CAR T
Cell secretes a large amount of IFN-γs and IL-2, and normal T and mock T secretory volumes are very low（p<0.05;Accompanying drawing 11）.
5.5 PSCA-CAR-T internal killing-efficiency research（in vivo）：
5.5.1 NOD/SCID mouse human immunity is rebuild：Take Healthy People PBMC 4 × 107Individual/0.5ml mouse tail vein injections, are raised
Support and obtain within 4 weeks mouse peripheral blood, flow cytometer detection is shown in that the expression rate of CD3+T lymphocytes is 67.29%, CD19+B lymphocytes
Expression rate is 21.5%, and human immunity is rebuild successfully（Accompanying drawing 12）.
5.5.2 humanization NOD/SCID mouse modeling：Take humanization NOD/SCID mouse, respectively dorsal sc inoculation 3 ×
106Individual AT4 cells or PC-3M cell modeling, tumor formation rate 100%；3 days visible subcutaneous nodules, it is 10-15 days long to 250-300mm3,
Modeling success.
5.5.3 in vivo fragmentation tests：Model mouse is randomly divided into 3 groups, every group 8；Respectively at the 1st, 8 days tail vein notes
Penetrate 1 × 107Individual normal T, mock T and PSCA-CAR-T, observe mouse animation, adverse reaction, tumour growth etc. and refer to
Mark.As a result：Change of each group mouse without life habit and behavior, the adverse reaction such as no allergy, fash, diarrhoea, depilation；
PSCA-CAR-T group gross tumor volumes are reduced significantly（p<0.05;Accompanying drawing 13-14）.
5.6 PSCA-CAR-T clinical practice：Before venous re-transfusion treatment, the sealing of preparation time and infusion bag need to be checked
Degree；Softly cell preparation is rocked to reduce cell mass, it is defeated using cell is inputted after the blood transfusion apparatus physiological saline washing pipe with filter screen
Enter and finish physiological saline washing pipe again；By adverse reactions such as clinical procedure anti symptom treatment fever, fash, allergic reactions.
Claims (6)
1. a kind of method that minicircle dna transfecting T cells prepare clinical grade CAR-T cell preparations, it is characterised in that methods described bag
Include following steps：
（1）Target gene-CAR design and synthesis；
（2）The structure of target gene-CAR parent vector plasmids and preservation；
（3）The extraction of target gene-CAR-mcDNA vector plasmids；
（4）Target gene-CAR-mcDNA transfecting T cells prepare CAR-T cells；
（5）The preparation and application of clinical grade, target gene-CAR-T cell preparations.
2. according to the method described in claim 1, it is characterised in that methods described step is as follows：
Target gene-CAR design, its method is as follows：1. filters out anti-target gene using DNAstar8.0 software analysis
Fab sequences and VH and VL sequences, for designing the scFv sequences in CAR；2. modelled signals peptide sequence IgGkappa, is placed in mesh
Gene-scFv front ends to guide CAR structures to penetrate target cell membrane；3. is according to Genebank（NCBI）Middle CD8, CD28,
CD137 and CD3 ζ gene order purpose of design gene-CAR integral frame structure, and set respectively in the front and back end of the sequence
Put EcoR1 and BamH1 restriction enzyme sites；
Target gene-CAR synthesis, its method is as follows：1. the complete genome sequence that carries out target gene-CAR by said structure is closed
Into；2. target gene-CAR the complete genome sequences after synthesis are subcloned into pUC57 carriers and are prepared into target gene-CAR- by
PUC57 plasmids；
The structure of target gene-CAR parent vector plasmids, its method is as follows：1. applications plasmid extraction kit is respectively to above-mentioned
Target gene-CAR-pUC57 plasmids and minicircle dna support agent box（MC-EasyTM Minicircle DNA Production
kit；SBI）The minicircle dna parent vector plasmid of offer（pMC.CMV-MCS-EF1-GFP-SV40PolyA）Carry out plasmid extraction,
Obtain plasmid extraction thing；2. is to step 1. obtained target gene-CAR-pUC57 and pMC.CMV-MCS-EF1-GFP-
SV40PolyA plasmid extractions thing carries out EcoR1 and BamH1 endonuclease reactions and obtains digestion products；3. is by step 2. obtained purpose base
Connection product is obtained because-CAR-pUC57 and pMC.CMV-MCS-EF1-GFP-SV40PolyA digestion products are attached reaction；
4. by step, 3. obtained connection product is transformed into the special competence bacterium solution that minicircle dna support agent box is provided to
（ZYCY10P3S2TE.coli）In, 30 DEG C, 250rpm shake bacterium culture 90min, obtain the parent vector containing target gene-CAR
Plasmid bacterial solution；5. by step, 4. obtained bacterium solution 50-200 μ l are laid in the LB agarose culture plates containing 50 μ g/ml kanamycins to
On, 37 DEG C of overnight incubations, selected clone, low dose extracts plasmid, digestion, electrophoresis, sequencing identification；Such as gene order correctly, then
4. step is obtained to parent vector -80 DEG C of glycerin storages of the plasmid bacterial solution of-CAR containing target gene, in case follow-up target gene -
The extraction of CAR-mcDNA vector plasmids；
The extraction of target gene-CAR-mcDNA vector plasmids, its method is as follows：1. is by step（2）The target gene of acquisition-
CAR parent vectors plasmid bacterial solution is inoculated in the LB agarose medias of the 2ml containing 50 μ g/ml kanamycins from -80 DEG C of removals
On, 30 DEG C, 250rpm shakes bacterium 1-2h, surveys OD values in case condition of culture regulates and controls；2. is by target gene-CAR parent vector plasmids
Bacterium solution is inoculated in the sterile culture flask of the growth medium containing 200ml, 30 DEG C, 250rpm shakes bacterium and stay overnight；3. takes a small amount of culture medium
PH value and OD values are detected, adjustment pH value is between 6.9-7.4, OD600Value is between 4-6, and 30 DEG C of continuation, 250rpm shake bacterium 5-5.5hr；
4. extracts target gene-CAR-mcDNA carriers by QIAGEN EndoFree Plasmid Maxi Kit operating procedures heavy dose
Plasmid, detects that qualified rear -20 DEG C of refrigerators are saved backup；
Target gene-CAR-T cell preparations are prepared, its method is as follows：1. is autologous or allogeneic PBMC separation obtains T cell,
After 1000U/ml IFN-γ is stimulated, 37 DEG C, overnight incubation in 5% CO2 incubators, next day day 1 are separately added into CD3 monoclonal antibodies 1
μ g/ml, CD28 monoclonal antibody 1 μ g/ml and IL-2 500U/ml is cultivated, and day 2 adds IL-15 10U/ml cultures, 3 additions of day
The complete medium culture amplification T cell of the 500U/ml containing IL-2, culture medium was changed per 2-3 days；2. presses 4D-
Nucleofector electroporations（LONZA）Operating instruction prepares electricity and turns liquid, and above-mentioned T cell is resuspended, and adds target gene-CAR-
McDNA vector plasmids move to electric revolving cup after softly mixing, and are put into electroporation, set electric carryover sequence, and electricity turns 3s and completes CAR-T systems
It is standby；3. above-mentioned purposes gene-CAR-T is moved to cultivate in the IL-2 containing 500U/ml of preheating AIM-V serum free mediums and expanded
Increase the clinical order of magnitude target gene-CAR-T cells of 10-15 days harvests；4. target gene-CAR-T cell physiologicals salt solution is centrifuged
Wash 3-4 times and dispel cell fragment, move into 200ml feedback normal saline bag/bottles of the 2.0g containing human serum albumin, be prepared into clinic
Level target gene-CAR-T cell preparations.
3. according to the method described in claim 1, it is characterised in that methods described step is as follows：
The design and synthesis of the target gene-CAR, it is characterized in that：Designed target gene Fab sequences, are selected from：PSCA、
PSMA、CEA、EGFR、EGFRvIII、HER2、MSLN、GD2、IL13Rα2、GPC3、CAIX、L1-CAM、CA125、CD133、
FAP、CTAG1B、MUC1、FR-α、GUCY2C；
The design and synthesis of the target gene-CAR, it is characterized in that：Designed CAR sequences are to contain scFv-CD3 ζ, CD28
And the third generation CAR sequences of 4-1BB/OX40/ICOS gene orders, and only containing scFv-CD3 ζ and CD28 gene orders
Second generation CAR sequences, and the first generation CAR sequences only containing scFv-CD3 ζ gene orders, and only containing forth generation
TRUCK cell CAR sequences；
The extraction of the target gene-CAR-mcDNA vector plasmids, it is characterized in that：Target gene-the CAR-mcDNA addressed
Vector plasmid, is any target gene-CAR-mcDNA vector plasmids；
The electricity turns T cell and prepares target gene-CAR-T cell preparations, it is characterized in that：Transfection T cell used, is selected from：
Autologous T cells, allogeneic T cells and each T cell subgroup.
4. the method according to claim 2 method, it is characterised in that wherein, step（4）It is described that target gene-CAR is close
The bacterium solution of this vector plasmid be inoculated in the sterile culture flask of the growth medium containing 200ml 30 DEG C, 250rpm shake bacterium and stay overnight, it is special
Levying is：1. shakes bacterium and stays overnight the time within 16hr；2. ensures growth medium pH value between 6.9-7.4, is most preferably
7.0；3. ensures growth medium OD600Value is between 4-6.
5. the clinical grade target gene-CAR-T cell preparations prepared with the method described in claim 1.
6. application of the target gene-CAR-T cell preparations in antineoplastic is prepared described in claim 5.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561069A (en) * | 2013-10-23 | 2015-04-29 | 深圳先进技术研究院 | Micro-annullus DNA recombinant plasmid containing recombinant chimeric antigen receptor gene expression box, micro-annullus DNA containing expression box and application |
| CN104774270A (en) * | 2015-05-06 | 2015-07-15 | 河北利同康生物科技有限公司 | Adenocarcinoma specificity EpCAM-GM-CSF genetic recombinant fusion protein and preparation method thereof |
| CN105452287A (en) * | 2013-04-17 | 2016-03-30 | 贝勒医学院 | Immunosuppressive tgf-[beta] signal converter |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978226B (en) * | 2011-09-05 | 2015-03-04 | 中国科学院深圳先进技术研究院 | Micro-ring DNA gene vector composition, and preparation method and application thereof |
| JP6817069B2 (en) * | 2014-04-25 | 2021-01-20 | ブルーバード バイオ， インコーポレイテッド | An improved way to manufacture adoptive cell therapies |
| CN111394317A (en) * | 2014-06-06 | 2020-07-10 | 蓝鸟生物公司 | Improved T cell compositions |
| CN105316314B (en) * | 2014-07-29 | 2018-07-27 | 深圳新诺微环生物科技有限公司 | A kind of minicircle dna of high-purity and its preparation method and application |
| EP3352798A1 (en) * | 2015-09-22 | 2018-08-01 | Julius-Maximilians-Universität Würzburg | A method for high level and stable gene transfer in lymphocytes |
| CN107236762A (en) * | 2017-06-19 | 2017-10-10 | 河北浓孚雨生物科技有限公司 | A kind of method that minicircle dna transfecting T cells prepare clinical grade CAR T cell preparations |
-
2017
- 2017-06-19 CN CN201710462493.4A patent/CN107236762A/en active Pending
-
2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105452287A (en) * | 2013-04-17 | 2016-03-30 | 贝勒医学院 | Immunosuppressive tgf-[beta] signal converter |
| CN104561069A (en) * | 2013-10-23 | 2015-04-29 | 深圳先进技术研究院 | Micro-annullus DNA recombinant plasmid containing recombinant chimeric antigen receptor gene expression box, micro-annullus DNA containing expression box and application |
| CN104774270A (en) * | 2015-05-06 | 2015-07-15 | 河北利同康生物科技有限公司 | Adenocarcinoma specificity EpCAM-GM-CSF genetic recombinant fusion protein and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 唐晓义 等: "嵌合抗原受体修饰T细胞免疫治疗的机遇与挑战", 《解放军医药杂志》 * |
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| WO2018233589A1 (en) * | 2017-06-19 | 2018-12-27 | 蔡子琪 | Method for preparing clinical-grade car-t cell preparation by transfecting t cell with minicircle dna |
| CN110129268A (en) * | 2018-02-08 | 2019-08-16 | 上海细胞治疗集团有限公司 | A kind of electricity turns the cultural method of rear PBMC |
| CN112004552A (en) * | 2018-03-16 | 2020-11-27 | 托马斯杰斐逊大学 | anti-GUCY 2C chimeric antigen receptor compositions and methods |
| CN109439632A (en) * | 2018-11-16 | 2019-03-08 | 中国科学院合肥肿瘤医院 | A method of improving CAR-T cell transfecting efficiency |
| CN112689679A (en) * | 2019-07-23 | 2021-04-20 | 株式会社东芝 | Methods, nucleic acid introduction vectors, and kits for generating CAR-T cells |
| CN112481284A (en) * | 2020-12-07 | 2021-03-12 | 深圳市瑞吉生物科技有限公司 | mRNA (messenger ribonucleic acid) for coding CAR (cancer cell receptor) gene, combined mRNA, construction method, CAR-T cell and application |
| CN112481284B (en) * | 2020-12-07 | 2023-07-25 | 深圳瑞吉生物科技有限公司 | mRNA (messenger ribonucleic acid) encoding CAR (CAR-T) gene, combined mRNA, construction method, CAR-T cell and application |
| CN112662631A (en) * | 2021-03-16 | 2021-04-16 | 合源生物科技(天津)有限公司 | CAR-T cell perfusion culture method |
| CN112662631B (en) * | 2021-03-16 | 2021-06-29 | 合源生物科技(天津)有限公司 | CAR-T cell perfusion culture method |
| WO2022194118A1 (en) * | 2021-03-16 | 2022-09-22 | 合源生物科技(天津)有限公司 | Perfusion culture method for car-t cells |
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| WO2018233589A1 (en) | 2018-12-27 |
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