CN106591363A - Preparation method of universal heterologous CAR-T cells and application - Google Patents
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Abstract
The invention provides a preparation method of universal heterologous CAR-T cells and an application. Constructed CD28-CD137-CD19-CD3 full-length gene is introduced into allogeneic T cells of healthy persons with CRISPR/Cas9 technology, in order to produce CAR-T cells with targeting cytotoxicity; after amplification in vitro, the cells are fed back to patients for carrying out antineoplastic treatment, and in the treatment process, a PNAs method is used for removing lymphocyte in order to avoid antigraft reaction of hosts. The chimeric antigen receptors can be prepared in large scale with T lymphocyte of healthy human, and antineoplastic treatment of heterologous cancer patients.
Description
Technical field
The present invention relates to medical science, immunology, Celluar and Molecular Biology field, and in particular to a kind of universal different
Body CAR-T cell preparation method and applications.
Background technology
Immune cell therapy is clinically obtained more and more as a kind of new cancer treatment method, its therapeutic effect
Confirm, CAR-T cells (Chimeric antigen receptor T cell) technology is to remove carcinoma cell immunization using the immunocyte of patient itself
Cell therapy, it is possible to cure acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin′ses and drench
The hematologic cancers such as bar tumor (NHL), treatment solid tumor also has a significant effect, because tumour immunotherapy is curing the same of tumor
When the treatment toxicity without conventional radiotheraphy, chemotherapy again, so immune cell therapy has broad prospects in the treatment of tumor.
Adoptive cellular treats (ACT) because it is expanded at short notice and activates the effector lymphocyte with anti-tumor activity, is facing
Receive much concern in bed research.TIL, CIK, NK, NKT and gamma delta T these cells for being used for ACT obtain one in clinical application research
Fixed curative effect, but the good immunocyte source of specific for tumour antigen, affinity is difficult, negligible amounts, kills tumor activity and in vivo
Persistent period is not enough, constrains the development of ACT.
(Kerkar SP. " Model T " cells in the entrance human body of T cell portability exogenous gene safety:a time-
tested vehicle for gene therapy Fronti Immunol,2012；4:304-304).T cell is readily available,
Can effectively be induced in vitro, and can be bred in a large number, can according to these features of T cell, using modern genetic transduction techniques,
Give T cell new characteristic, the T cell through modifying can recognize target antigen in the antigen expressed binding domain of its surface-stable
Meanwhile, without restricted (Ito F, the Chang AE.Cancer immunotherapy of MHC:current status and
future directions.Surg Oncol Clin N Am,2013；22(4):765-783.).
Then, researcher is attempted solving the problems, such as to restrict ACT development using genetic modification T cell, for modifying T cell
Gene have TCR, CAR, promote immune cell propagation cytokine (such as IL-2, IL-15).Wherein Chimeric antigen receptor T
Cell (CAR-T) is can encode the chimeric molecule of single-chain antibody-costimulatory moleculeses-immunity receptor tyrosine activation gene order
Fusion gene modification T cell.Because it has that tumor antigen identification specificity is strong, affinity is high, non-MHC is restricted and can be in body
The advantage of inside and outside a large amount of amplifications and receive more concern.
Recent studies have shown that, using T cells (TN) or Central memory T cell (TCM), these cells are than differentiation
T cell has more powerful internal killing activity.Concrete operations：T cell can be carried out by T cell stem cell labeling thing CD62L
Sorting.For example, by from the PBMC of cancer patient and allogeneic tumor cell and magnetic bead co-cultivation, the magnetic bead can pass through CD3
The combination for adding CD40 with CD28, CD40 or CD28 carrys out activating T cell.
CAR is the transmembrane molecule of artificial constructed fusion gene coding, has extracellular region, intracellular region and transmembrane region to constitute.Born of the same parents
The single-stranded variable region (scFv) of outskirt, is responsible for the identification of specific antigen；Intracellular region is responsible for the transduction of signal, when extracellular region and anti-
After original specifically binds, the signal needed for the activation of intracellular region active cell, so as to promoting T cell propagation, release cytokine, resisting
Apoptosis etc.；Transmembrane region links extracellular region and intracellular region, and to the different designs in each region CAR-T cell work(is directly affected
The performance of energy.
Bone-marrow-derived lymphocyte leukemia and malignant lymphoma are the evils for being primary in medulla hematopoietic system and lymph node and diffusing whole body
Property tumor.Although traditional chemicotherapy has certain curative effect, but without selectivity, normal tissue damages very big.In recent years, it is biological
Therapeutic Method is widely used in oncotherapy, especially monoclonal antibody, such as rituximabAnd it is special because of its
Targeting, high-affinity and have received good result.Monoclonal antibody is by Fc sections and effector lymphocyte surface activation receptor Fc γ RI/Fc
γ R III are combined, so as to mediate lethal effect, but the T cell with immunologic cytotoxicity effect because surface lacks above-mentioned receptor not
Can effectively be mediated, so as to weaken immunological effect of the body to tumor.The CAR-T cells modified with anti-CD19 can be recognized simultaneously
Various not synantigens, can be to solve exempting from for tumor with the tumor cell in the efficient specific killing bone-marrow-derived lymphocyte source of mediate T cell
Epidemic disease is escaped and brings dawn.
CD19 is ideal tumor associated antigen, and it is expressed in each of the development of the bone-marrow-derived lymphocyte in addition to stem cell
The individual stage, therefore the malignant cell in B cell source has CD19 to express.Show the CAR- that CD19 antibody scFvs build about research
T cell has the effect of high affinity and primary B cell acute lymphoblastic leukemia (B-ALL) oncocyte of cracking
(Cooper LJ,Topp MS,Serrano LM,et al.T-cell clones can be rendered specific
for CD19:toward the selective augmentation of the graft-versus-B-lineage
leukemia effect.Blood,2003；101(4):1637-1644.).Kalos et al. is in the second filial generation modified with anti-CD19
CAR-T cells carry out being found during therapeutic advance CLL that in vivo amplification rate is even up to soon more than 1000 times to CAR-T cells,
Persistent period was more than 6 months.Tumor cell is not only eliminated, a part of cell also persistently exists in vivo with Memorability CAR-T
(Kalos M,Levine BL,Porter DL,et al.T cells with chimeric antigen receptors
have potent antitumor effects and can establish memory in patients with
advanced leukemia.Sci Transl Med,2011；3(95):95ra73-95ra73.).B cell maturation antigen presentation
In mature B cell and plasma cell surface, both survivals can be promoted, B cell maturation antigen be tumor necrosis factor superfamily into
Member, can be a promising target mesh in treatment multiple myeloma with reference to B cell activation factor and proliferation-inducing ligand
Mark.
CD3 is T lymphocytic cell surface special moleculars, and function is that TCR is delivered to cell with the antigenic information of external combination
Interior, in active cell activation process, the early process for occurring to activate after T cell receives antigenic stimulus plays an important role.
4-1BB, also known as CD137, is T cell surfactant molecules, is expressed in the T cell of activation, and the participation of 4-1BB can
To amplify derivative immunne response.4-1BB can stimulate after contacting with the monoclonal antibody of anti-4-1BB and be swashed by antigen
CD8+T lymphopoiesis living, to have tumour-specific killing activity, stimulate interferon-γ (IFN-γ) and other Th1 types
Cytokine (interleukin II IL-2), the generation of tumor necrosis factor (TNF-α), release, and stimulate to anti-apoptotic
T cell protection.In addition 4-1BB has immunomodulatory effect.4-1BB parts (4-1BBL) can greatly enlarged CD8+T lymphs it is thin
The response of born of the same parents.Research shows that the CAR-T cells obtained after adding costimulatory moleculeses 4-1BB in the middle of CAR have secretion more thin
Intracellular cytokine and higher proliferation activity (Carpenito C, Milone MC, Hassan R, et al.Control of large,
established tumor xenografts with genetically retargeted human T cells
containing CD28and CD137domains.Proc Natl Acad Sci USA,2009；106(9):3360-
3365).Relevant research shows that the ability of the transmembrane region expression CAR of CD28 is most strong.CD28+4/1BB combinations provide costimulation letter
Number, after the first signal path of antigenic stimulus, the expression of anti-apoptotic gene can be raised, promote IL2 secretions.
Relevant research shows that CAR introduces costimulatory moleculeses signal (such as CD28, CD137, CD27, CD244), can improve T
The cytotoxicity of cell, proliferation activity, time-to-live, increase the release of cytokines of antigen induction, raise anti-apoptotic egg
In vain.These effects can strengthen T cell to the lethal effect of target tumor (Song DG, Ye Q, Poussin M,
etal.CD27costimulation augments the survival and antitumor activity of
Redirected human T cells in vivo.Blood, 2012；119(3):696-706)
Therefore can be by genetic modification, orientation builds scFv-CD28-CD137-CD19-CD3 genes, is then introduced into
CAR-T cells are fabricated to T cell, it is thin such that it is able to realize the tumor that mediate T cell specific killing bone-marrow-derived lymphocyte is originated
Born of the same parents, and costimulatory moleculeses signal can also improve the cytotoxicity and proliferation activity of CAR-T cells, strengthen T cell to target
The lethal effect of tumor cell.
In the preparation process of CAR-T cells, need the coding molecule of antibody sequence and φt cell receptor signal pathway is related
Sequence be directed in T cell, exogenous gene is imported and treated, and most research results show, this be it is a kind of relatively safely
Mode.At present substantially there are retrovirus, slow viruss, transposon in the U.S. for the mode that clinical trial imports exogenous gene
MRNA, electricity such as proceed at the mode.Early in nineteen ninety, Rosenberg groups propose using retrovirus that exogenous gene importing T is thin
It is safe and feasible that born of the same parents carry out tumor, and the same year U.S. FDA have approved gene therapy.The mainly division stage of retroviral infection
Cell, it is extremely weak for Unseparated Cell infection ability.The slow viruss for developing on this basis, then to division and Unseparated Cell
It is respectively provided with preferable infection ability.It is difficult to that exogenous gene is directed in T lymphocytes by the way of traditional direct transfection, because
When prepared by this most CAR-T cell, the slow viruss system of employing, because viral vector is related to striving in more patent
Discuss, therefore researchers attempt transduceing into exogenous gene to prepare CAR-T cells, preclinical study with reference to electricity using transposon again
Also confirm that the method is feasible, although it is considered as safety that retrovirus and slow viruss import exogenous gene to carry out treatment
, but due to exogenous gene be but random integration to host genome and long-term existence and stable expression in the cell, exist
Inappropriate insertion point mutation, may cause cell that the potential risk of conversion occurs, and the mode that transposon electricity turns there is also
Similar risk.Therefore, there is research and probe that encoding plasmids are directly transduceed into intracellular by electricity, but be because that plasmid is imported
Mode great majority be transient expression, can reach therapeutic effect need further experiment checking.Led as target by the use of RNA
To CRISPR/Cas9 gene editing technologies be considered as the newest achievement of gene editing technology, research find Cas9 can solve
Rotation DNA, gRNA and targeting DNA is matched after untwisting, and carries out cutting DNA using pairing the discharged energy of combination.
The T cell of expression CAR can be with Direct Recognition and with reference to the TAA of tumor cell surface, and CAR is by the incoming T cell of signal
Interior, activation T cell secrete cytokines include perforin, granzyme, INF- γ, TNF-α etc., so as to play killing tumor cell
Effect.Therefore, CAR-T cells are MHC nonrestrictive.CAR-T cells are by antibody-antigene specific binding capacity and cell
The killing ability of mediation is incorporated into one, makes simple, is widely used, and is the important method of immune antineoplaston.
CAR combines the killing mechanism of the high-affinity of antibodies on tumor antigen and T cell, and by gene T lymphs are transfected
Cell so as to the killing tumor cell of energy specificity.
Current immune cell therapy is mainly adoptive cellular treatment (ACT), and this therapy is to obtain T from patient itself to drench
Bar cell, is reinjected in patient's body in vitro after genetic modification and amplification, and this adoptive cellular therapy has MHC unrestricted
Property target cell identification function, obtains certain curative effect in clinical application research, but this kind of therapy needs production equipment and clinic
Accurately coordinating between use, and produce the CAR-T cells with therapeutic efficiency needs the long period, may be delayed patient
Golden hour.In order to solve this restricted problem, the present invention develop it is a kind of can be with the compatible allogeneic of adoptive transfer
CAR-T cells.
This method is extracted T cell, by industrial mass production goes out that great majority can be injected from a healthy volunteer
The CAR-T cells of patient, can largely increase the therapeutic domain of CAR-T on this theoretical method, when shortening treatment wait
Between, and cost substantially reduces.
When adoptive transfer treatment is carried out in allogeneic CAR-T cells input patient's body, it is necessary to avoid the anti-transplanting of host
Thing reacts and graft versus host disease, to prevent body from repelling the damage of graft or graft cell to host tissue of adopting
Evil, but also to ensure lethal effects of the CAR-T being input into host's tumor.Graft versus host disease can be by isolating lymph
Lymphocyte in knot or the target gene group by knockout φt cell receptor (TCR α β) in CAR-T cells, drench donor T
The TCR identification gene delection expression of bar cell, and suppress host-versus-graft reaction, can be carried out by lym phocytopheresis method
Avoid.PNAs is the reagent for removing lymphocyte, and under certain density PNAs, CAR-T cells can be expanded also can be controlled
More human cancer, and keep its multiplication capacity and targets neoplastic cells activity.
The content of the invention
The present invention provides a kind of universal allosome CAR-T cell preparation method and applications, by what is built
CD28-CD137-CD19-CD3 full-length genes are fabricated to targeting cell with CRISPR/Cas9 technological sourcings to T cell
The CAR-T cells of toxicity, then after amplification in vitro, feed back to and carry out in patient's body antineoplaston, in therapeutic process, use
PNAs removes the antigraft reaction that the method for lymphocyte avoids host.The purpose of the present invention is in tumor patient produced in vitro
Transgenic T lymphocytes, are input in patient's body for treating tumor disease after then expanding.
A kind of universal allosome CAR-T cell preparation methoies, including step is as follows：
A () extracts blood from healthy volunteer's body, isolate T cell；
B () builds scFv-CD28-CD137-CD19-CD3 full-length genes；
C () scFv-CD28-CD137-CD19-CD3 full-length genes import T cell；
(d) cultured and amplified in vitro CAR-T cell colonys；
E () removes patient lymphocytes with PNAs；
F () CAR-T cells are fed back in patient's body, carry out antineoplaston；
The concrete operations flow process of the step (a) is：1) anticoagulant blood vessel extracts healthy volunteer's peripheral blood；2) to Guan Zhongjia
Enter erythrocyte cracked liquid and equal-volume PBS, gently blow and beat into cell suspension；3) two centrifuge tubes are separately taken, LTS1077 lymphs are added
Cell is layered liquid；Cell suspension is drawn with suction pipe, at 0.5-3cm above lymphocytes separating solution that cell suspension is careful
And slowly add, cell suspension is overlapped with lymphocytes separating solution, 2000r/min is centrifuged 5-50min；4) take out from
Heart pipe, pipet sucks the blood plasma of the superiors, and the mononuclearcell that liquid-transfering gun is drawn under blood dress layer is inserted in centrifuge tube, added
PBS, gently blows and beats uniform rear recentrifuge, and 1500r/min is centrifuged 10min, removes supernatant, washs 2-5 time altogether；5) to going
To fall added in the centrifuge tube after supernatant the culture medium containing inactivated fetal bovine serum, mycillin and RPMI-1640 and be positioned over 37 DEG C thin
Amplification culture in born of the same parents' incubator；6) it is centrifuged, collects peripheral blood lymphocytes, is placed in liquid nitrogen container and saves backup.
The cumulative volume of the cell pyrolysis liquid and PBS equal-volumes, cell pyrolysis liquid and PBS and the peripheral blood volume ratio for extracting
For 2:1-1:2.
The volume ratio of lymphocytes separating solution and cell suspension is 2:1-1:2；Step 4) in add PBS volume be lymph
1/2-3 times of cell layering liquid；
Step 5) in add concentration be respectively 10% inactivated fetal bovine serum, 100U/ml mycillins, 100U/mlIL-2
RPMI-1640.
The step (b) synthesizes scFv-CD28-CD137-CD19-CD3 full-length genes using gene synthesis technology.
The step (c) is according to CRISPR/Cas9 shot design principles, using the design of crispr online tools, synthesis
The gRNA and primer of targeting scFv-CD28-CD137-CD19-CD3 full-length genes, and insert it into CRISPR/Cas9 plasmids
In skeleton carrier, recombiant plasmid is fabricated to, then by the recombiant plasmid in-vitro transfection T cell for building.
The detailed step of the step (d) is：It is (1-2) × 10 with OKM-100 cell culture fluids adjustment inoculum density6/
It is added to after ml containing allosome inactivation blood plasma in stimulating factor Tissue Culture Flask, is then added, cultivates to cell culture incubator,
4-5d is after cell is paved with bottom of bottle for culture, cell to be proceeded to continue in big Tissue Culture Flask and is cultivated, after 2～3d of culture, will be big
Tissue Culture Flask in cell go to the CO containing bigger OKM-200 cell culture fluids2Continue to cultivate in ventilative culture bag,
After culture 6-7d, harvesting.
It is an advantage of the current invention that：
1st, compared with autologous CAR-T Therapeutic Method, the method from patient without drawing blood, it is possible to decrease the pain of patient,
And the T cell activity of healthy volunteer is relatively strong, quality is easily unified.
2nd, compared with autologous CAR-T, allosome CAR-T is capable of achieving industrialization, large-scale production and prepares, and not only can significantly drop
Low cell production cost, is more beneficial for whole-course quality control.
3rd, allosome CAR-T cells can be prepared in advance, can be applicable to tumor patient within 1-3 days after the cell recovery for preparing,
The time that patient waits cell is thus greatlyd save, and traditional autologous CAR-T needs to wait for 3 weeks even for more time.
4th, using CRISPR/Cas9 gene editing technologies, simple operation, efficiently, targeting accuracy.
5th, CAR introduces costimulatory moleculeses signal (such as CD28, CD137, CD3, CD19), can improve the cell toxicant of T cell
Property, proliferation activity, the time-to-live, increase antigen induction release of cytokines, raise Anti-apoptotic proteins, these effects can
Strengthen lethal effect of the T cell to target tumor.
Specific embodiment
In order to illustrate in greater detail the present invention, be given and following prepare example.But the scope of the present invention is not limited thereto.
Embodiment 1
A () extracts blood from healthy volunteer's body, isolate T cell；
B () builds scFv-CD28-CD137-CD19-CD3 full-length genes；
C () scFv-CD28-CD137-CD19-CD3 full-length genes import T cell；
(d) cultured and amplified in vitro CAR-T cell colonys；
E () removes patient lymphocytes with PNAs；
F () CAR-T cells are fed back in patient's body, carry out antineoplaston；
The separation of (a), peripheral blood lymphocytes and activation
Choose hepatic and renal function normal；PBMC stimulates CD3/CD28 the healthy volunteer for having good response.
1st, anticoagulant blood vessel extracts healthy volunteer peripheral blood 10ml；
2nd, erythrocyte cracked liquid and equal-volume PBS are added in pipe, cell suspension 20ml is gently blown and beaten into；
3rd, two 50ml centrifuge tubes are separately taken, 10mlLTS1077 lymphocytes separating solutions are added.10ml cells are drawn with suction pipe
Suspension, cell suspension carefully and is slowly added at 1cm above lymphocytes separating solution, make cell suspension overlap with
On lymphocytes separating solution, 2000r/min is centrifuged 20min；
4th, centrifuge tube is taken out, pipet sucks the blood plasma of the superiors, and the mononuclearcell that liquid-transfering gun is drawn under blood dress layer is put
In entering centrifuge tube, 10mlPBS is added, gently blows and beats uniform rear recentrifuge, 1500r/min is centrifuged 10min, removes supernatant,
Wash 3 times altogether；
5th, add containing 10% inactivated fetal bovine serum, 100U/ml mycillins, 100U/ to removing in the centrifuge tube after supernatant
The RPMI-1640 culture medium of ml IL-2 is positioned over 37 DEG C, 5%CO2Cell culture incubator in amplification culture.
6th, it is centrifuged, collects peripheral blood lymphocytes, is placed in liquid nitrogen container and saves backup.
The design of (b) Hinge-TM-CD28-CD137-CD19-CD3 ξ
The relatively sequence (Hinge) and transmembrane region (TM) of CAR is from CD8a (aa135-205, GenBank：BC025715.1)、
CD28 functional areas (aa180-220, GenBank：BC025715.1)、CD137(aa214-255,GenBank:U03397.1)、
CD19 (aa422-433, Shenzhen Xin Bosheng biotechnologies) and CD3 ξ (aa52-163, Genbank：J04132.1, Hinge-
TM-CD28-CD137-CD19-CD3 ξ expression cassettes are completed by gene machine (Dr.Oligo192 synthesizers).
Hinge-TM-CD28-CD137-CD19-CD3 ξ are expanded
Hinge-TM-CD28-CD137-CD19-CD3 ξ amplimers：
F2:5-TGGCACCAAGCTGGAAATCAAAACCACGACGCCAGCGCCGCGA-3
R2:5-CGGGATCCTTAGCGAGGGGGCAGGGCCT-3, primer is synthesized by Shanghai life work biology company limited.
The genes of interest fragment Hinge-TM-CD28-CD137-CD19-CD3 ξ of acquisition, subpackage is frozen in -20 DEG C of refrigerators
Preserve.C () scFv-CD28-CD137-CD19-CD3 full-length genes import T cell；
1st, the structure of scFv-CD28-CD137-CD19-CD3-gRNA vector plasmids
According to CRISPR/Cas9 shot design principles, satisfactory gRNA is designed, effect of being screened to it and missed the target
Should assess, pick out the strong gRNA of characteristic.Then full-length genome comparison is carried out using bioinformatics software, it is to avoid select the wind that misses the target
The big target sequence in danger, carries out in vitro increasing success rate.
The Hinge-TM-CD28-CD137-CD19-CD3 ξ that above-mentioned steps are obtained are attached with vector plasmid, with even
Meet purpose fragment Hinge-TM-CD28-CD137-CD19-CD3 ξ of the enzyme by size for 2436bp to connect in vector gene.Instead
System is answered to be Solution I enzyme linked systems
Solution I enzyme linked systems
PCR reaction conditions：95 DEG C are reacted 3 minutes, and 95 DEG C slowly naturally cool to 25 DEG C, and 16 DEG C are reacted 5 minutes, then will
Return of goods product is connected in carrier：The μ l of pCAG-T7 1, the μ l of PCR product 2, are diluted with water to 10 μ l, after being sufficiently mixed, room
(25 DEG C) of temperature stands 5 minutes.Take the μ l of connection product 5 to be added in 50 μ l DH5 α competent cells of defrosting, flick mixing, ice
After bath 30 minutes, 42 DEG C of heat shocks 90 seconds stand 2 minutes on ice, are directly applied to flat board, second day, choose well-grown bacterium colony
In LB culture fluid, 37 DEG C of 200rpm shaking table cultures are overnight.Take 5ml bacterium solutions to be sequenced.
2nd, transfections of the pCAG-T7-CD28-CD137-CD19-CD3-gRNA to T cell
The recombiant plasmid for building is diluted to into equal-volume mixing after isoconcentration with TE buffer, well-grown T is thin
Born of the same parents are seeded to 12 orifice plates after digesting respectively, fusion carries out pCAG-T7-CD28-CD137-CD19-CD3- when reaching 60%-80%
GRNA is transfected.Take 2 μ l recombiant plasmid and add 2 μ l Easyfect, gently blow and beat, rock mixing, be incubated at room temperature 20 minutes.With sieve
Screening of Media is selected to go out the mixing clonal cell line of gene insertion, sequencing analysis detection gene transfection.
(d) CAR-T cell expansion ex vivo cultures
It is (1-2) × 10 with OKM-100 cell culture fluids adjustment inoculum density according to CAR-T cell counts6/ml
The 75cm containing stimulating factor is added to afterwards2In Tissue Culture Flask, 10% allosome inactivation blood plasma is then added, to 37 DEG C of CO2
Culture in cell culture incubator, 4-5d is after cell is paved with bottom of bottle for culture, and cell is proceeded to into 225cm2Continue to train in Tissue Culture Flask
Support, after culture 2-3d, by 225cm2Cell in Tissue Culture Flask goes to the CO containing 1000ml OKM-200 cell culture fluids2
Continue to cultivate in ventilative culture bag, after culture 6-7d, harvesting is counted.
E () removes lymphocyte in patient's body with PNAs, destroy the self immune system of patient；
F () CAR-T cells are fed back in patient's body, carry out antineoplaston
Cultured CAR-T cells carry out antibacterial, funguses, mycoplasma, adventitious viruseses and endotoxin detection it is no positive after, mix
In being suspended from 100ml normal saline, 1h is got over the slow intravenous infusion of transfusion device with filter membrane, can intramuscular injection diphenhydramine 20- before feedback
40mg.Returning step detects vital sign and side reaction.Phlebotomize before feeding back every time and do lymphocyte subpopulation detection.
Blood lymphocyte phenotypes application flow cytometer is determined, and CD3+, CD28+, CD19+, CD137+, CD3+CD28 are determined respectively
+, CD19+CD137+, CD3+CD28+CD19+CD137+, CD3+CD28+CD19+, CD28+CD19+CD137+ cell is in lymph
Percentage situation in cell, to obtain CAR-T cells more detailed data in vivo.Cultivated with mtt assay detection simultaneously
The activity of the killing tumor cell of CAR-T cells after amplification.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those of ordinary skill in the art the invention discloses technical scope in, the change or replacement that can be readily occurred in, all should
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be defined by scope of the claims.
Claims (8)
1. a kind of universal allosome CAR-T cell preparation methoies, it is characterised in that：It is as follows including step：
A () extracts blood from healthy volunteer's body, isolate T cell；
B () builds scFv-CD28-CD137-CD19-CD3 full-length genes；
C () builds the CRISPR/Cas9 gene plasmids of targeting scFv-CD28-CD137-CD19-CD3 full-length genes, and transfect T
Cell makes CAR-T cells.
(d) cultured and amplified in vitro CAR-T cell colonys；
E () removes patient lymphocytes with PNAs；
F () CAR-T cells are fed back in patient's body, carry out antineoplaston.
2. universal allosome CAR-T cell preparation methoies according to claim 1, it is characterised in that：The step (a)
Concrete operations flow process is：1) anticoagulant blood vessel extracts healthy volunteer's peripheral blood；2) erythrocyte cracked liquid is added in pipe and body is waited
Product PBS, gently blows and beats into cell suspension；3) two centrifuge tubes are separately taken, LTS1077 lymphocytes separating solutions are added；Inhaled with suction pipe
Obtained cell suspension, carefully and is slowly adding cell suspension at 0.5-3cm above lymphocytes separating solution, makes cell
Suspension is overlapped with lymphocytes separating solution, and 2000r/min is centrifuged 5-50min；4) centrifuge tube is taken out, pipet is sucked most to be gone up
The blood plasma of layer, the mononuclearcell that liquid-transfering gun is drawn under blood dress layer is inserted in centrifuge tube, adds PBS, after gently piping and druming is uniform again
Secondary centrifugation, 1500r/min is centrifuged 10min, removes supernatant, washs 2-5 time altogether；5) to removing in the centrifuge tube after supernatant plus
Enter the culture medium containing inactivated fetal bovine serum, mycillin and RPMI-1640 and be positioned over amplification culture in 37 DEG C of cell culture incubators；6)
Centrifugation, collects peripheral blood lymphocytes, is placed in liquid nitrogen container and saves backup.
3. universal allosome CAR-T cell preparation methoies according to claim 2, it is characterised in that：The cell lysis
The cumulative volume of liquid and PBS equal-volumes, cell pyrolysis liquid and PBS is 2 with the peripheral blood volume ratio for extracting:1-1:2.
4. universal allosome CAR-T cell preparation methoies according to claim 2, it is characterised in that：Lymphocyte is layered
The volume ratio of liquid and cell suspension is 2:1-1:2；Step 4) in add the volume of PBS to be 1/2-3 times of lymphocytes separating solution.
5. universal allosome CAR-T cell preparation methoies according to claim 2, it is characterised in that：Step 5) middle addition
Concentration be respectively 10% inactivated fetal bovine serum, 100U/ml mycillins, the RPMI-1640 of 100U/ml IL-2.
6. universal allosome CAR-T cell preparation methoies according to claim 1, it is characterised in that：Step (b) profit
Synthesize scFv-CD28-CD137-CD19-CD3 full-length genes with gene synthesis technology.
7. universal allosome CAR-T cell preparation methoies according to claim 1, it is characterised in that：Step (c) root
According to CRISPR/Cas9 shot design principles, using the design of Crispr online tools, synthesis targeting scFv-CD28-CD137-
The gRNA and primer of CD19-CD3 full-length genes, and insert it in CRISPR/Cas9 plasmid backbone carriers, it is fabricated to restructuring
Plasmid, then by the recombiant plasmid in-vitro transfection T lymphocytes for building.
8. CAR-T cells preparation method according to claim 4, it is characterised in that：The detailed step of the step (d)
For：It is (1-2) × 10 with OKM-100 cell culture fluids adjustment inoculum density6It is added to after/ml containing stimulating factor cell culture
In bottle；Then allosome inactivation blood plasma is added, is cultivated to cell culture incubator, 4-5d is after cell is paved with bottom of bottle for culture, will be thin
Dysuria with lower abdominal colic to enter continue in big Tissue Culture Flask cultivates, and after culture 2-3d, the cell in big Tissue Culture Flask is gone to containing more
The CO of big OKM-200 cell culture fluids2Continue to cultivate in ventilative culture bag, after culture 6-7d, harvesting.
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