CN109722420A - A kind of preparation and its application improveing Chimeric antigen receptor T cell - Google Patents
A kind of preparation and its application improveing Chimeric antigen receptor T cell Download PDFInfo
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- CN109722420A CN109722420A CN201910196890.0A CN201910196890A CN109722420A CN 109722420 A CN109722420 A CN 109722420A CN 201910196890 A CN201910196890 A CN 201910196890A CN 109722420 A CN109722420 A CN 109722420A
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Abstract
The present invention relates to cellular immunotherapy technical field, in particular to a kind of preparation and its application for improveing Chimeric antigen receptor T cell；A kind of improvement Chimeric antigen receptor T cell, it is characterised in that the T cell is transduceed into T cell simultaneously during viral transduction, by three kinds of slow virus including CAR；Existing CAR-T cell is transformed, while three kinds of slow virus corotation are imported into T cell, CAR on the one hand can have both been played and the special target of tumour is acted on, and tumour is killed；On the other hand, IL-7 the and IL-15 factor into cell of transduceing equally can promote the Effective multiplication of CAR-T cell in vitro, eliminating needs repeatedly to be added IL-7 the and IL-15 factor in experimentation, simplify experimental procedure, and can reduce the cost of CAR-T preparation during the preparation process.
Description
Technical field
The present invention relates to cellular immunotherapy technical field, in particular to a kind of system for improveing Chimeric antigen receptor T cell
Standby and its application.
Background technique
Chimeric antigen receptor (Chimeric antigen receptor) T cell technology is not by the limit of immune system MHC
Property processed, can be to tumour cell special target, so that the one kind killed to tumour is quickly grown by genetic modification technology
Cell therapy technology.Have approved the Tisagenlecleucel's and Kite of Novartis Co., Ltd from FDA in 2017
Since two kinds of CAR-T cell products listings of Axicabtegene Ciloleucel, it is respectively used to treatment children and young man B
Cell acute lymphoblastic leukaemia and adult recurrent or intractable large B cell lymphoid tumor, CAR-T immunotherapy are being directed to blood
The treatment aspect that liquid tumor is related to B-lineage Acute Lymphocyte Leukemia, non Hodgkin lymphom and Huppert's disease etc. obtains
Remarkable effect.And U.S.'s medical insurance and medical assistant service centre (Centers for Medicare and Medicaid
Services, CMS) on 2 15th, 2019, formal publication proposal determined memorandum: approval CAR-T cell therapy is formally included in
Medical insurance.This enables CAR-T therapy faster to apply to clinic, brings glad tidings for patient.
Number of the infected and death toll according to " 2018 global cancer statistical data " display Cancer in China are the world the
One.Lung cancer, breast cancer and colorectal cancer are the highest three kinds of cancers of global incidence, and it is then respectively lung that the death rate is highest
Cancer, colorectal cancer and gastric cancer.Although CAR-T therapy obtains significant progress in blood tumor, and its treatment for being directed to solid tumor
Especially with regard to colorectal cancer treatment still have it is to be solved.
In order to obtain preferable clinical effectiveness, it is ensured that the quality that CAR-T feeds back preparation is particularly important, including CAR-T preparation
Cell concentration, virus transduction efficiency etc..This need in vitro in incubation slow virus can high efficiency transduction, and cell energy
Enough effective proliferation.The currently used factor for promoting the amplification of CAR-T cell has IL-2 or IL-7/IL-15.In naive T
After cell is activated, effector T cell can secrete IL-2 and increase the expression of IL-2 receptor, to promote the proliferation of T cell.Mesh
Preceding IL-2 has been the cell factor of generally acknowledged promotion T cell proliferation.But itself is to internal existing resting naive
The proliferation of T cell and memory T cell is limited.On the one hand IL-7 can be pressed down by Jak-STAT access and pI3K-AKt access
Apoptosis processed improves cell survival；The expression of another aspect induction duration albumen promotes cell cycle progress, to promote
The proliferation of naive T cell and memory T cell.IL-15 in conjunction with its receptor after mainly pass through JAK1/STAT3, JAK3/STAT5
And tri- signal paths of Ras/MAPK play a role, by the enhancing of proliferation signals and the decrease of apoptotic signal, to increase
Add the immune response of CD8+T cell.
In CAR-T cell preparation preparation process, the factor of above-mentioned promotion cell Proliferation is often additionally added to T cell
Cultivating system in.And the IL-7 factor 1mg price of commercially available recombinant humanized is at 4680 dollars, the IL-15 of recombinant humanized because
Sub- 1mg price is at 4000 dollars or so, in T cell incubation, needs repeatedly to be added the corresponding factor, the use of cell factor
Measure it is larger, greatly improve CAR-T cell preparation cost, and it also requires ensuring the matter of different batches cell factor product
Amount.CN109055430A discloses a kind of preparation for co-expressing IL-18 and CCL19 albumen and targeting MUC1 gene C AR-T cell
Method.IL-18 and CCL19 gene can be added in the design process of CAR, thus make CAR-T cell coexpression IL-18 and
The CCL19 factor, both can promote T cell proliferation, can also raise more T cells and DC cell to tumor tissues together kill swell
Tumor.Although slow virus carrier can stablize expression, the capacity of carrier is small, and maximum can only accommodate the target gene of about 8Kb.
And at present in the design process of CAR, it further include inducing CAR-T dead other than the antibody of specific recognition tumour antigen
Suicide gene etc..If multiple genes are additionally added again, it may be more than the capacity of slow virus carrier, influence viral packaging.
Therefore, the present invention provides a kind of preparations and its application for improveing Chimeric antigen receptor T cell.In gene containing CAR
During viral transduction, while IL-7, IL-15 slow virus of building are transferred to T cell, the T for obtaining three gene co-expressings is thin
Born of the same parents.While reducing cost, endogenic IL-7 and IL-15 can equally effectively facilitate cell Proliferation and carry out killing tumor cell.
To make CAR-T cellular immunotherapy preferably be promoted and applied.
Summary of the invention
The purpose of the present invention is to provide a kind of Chimeric antigen receptor T cells of improvement, it is characterised in that while will building
Three including CAR kind slow virus corotation import T cell, on the one hand can both reduce life in CAR-T formulation process
Cost is produced, does not on the other hand influence the proliferative conditions of CAR-T in vitro again.
To achieve the goals above, the present invention adopts the following technical scheme that:
(1) healthy human peripheral blood is acquired, peripheral blood mononuclear cells is obtained through density gradient separation, is purified after sorting
T cell；
(2) after t cell activation, three kinds of slow virus is taken out and carry out centrifugation cotransduction, inhaled abandon viral supernatants immediately, replacement culture
Liquid continues to cultivate；
(3) T cell after step (2) transduction is subjected to flow cytometer showed, the Chimeric antigen receptor T improved after culture
Cell.
Preferably, sorting described in step (1) uses magnetic bead feminine gender separating method.
Preferably, activation described in step (2) is activated using the magnetic bead of anti-CD3/anti-CD28 coupling, is activated
The magnetic bead of Shi Caiyong and the additional proportion of T cell are 1:1~3:1.
Preferably, three kinds of slow virus respectively include IL-7, IL-15 and target tri- kinds of slow virus of CAR of solid tumor, wherein
The MOI of IL-7 slow virus is 5-25, and the MOI of IL-15 slow virus is 5-25, and the MOI of CAR slow virus is 5-25.
Preferably, cotransduction is centrifuged in step (2), the centrifugal rotational speed used is 2000-3000rpm, centrifugation time 1-
3h。
Preferably, 48h carries out flow cytometer showed transduction efficiency after transduction in step (3).
Preferably, used complete medium is RPMI-1640 base to improvement Chimeric antigen receptor T cell during the preparation process
Basal culture medium, including 10% fetal calf serum, 1% Pen .- Strep is dual anti-and the IL-2 of 500IU/mL.
Since IL-7 and IL-15 remarkably promotes effect to what T cell was proliferated, by IL-7 and IL-15 gene packet in the present invention
Dressing up slow virus, corotation imports T cell together with the slow virus for carrying CAR gene later.
Compared with prior art, the beneficial effects of the present invention are be transformed existing CAR-T cell, simultaneously will
Three kinds of slow virus corotation import T cell, on the one hand can both play CAR and act on the special target of tumour, and carry out to tumour
Killing；On the other hand, IL-7 the and IL-15 factor into cell of transduceing equally can promote the Effective multiplication of CAR-T cell in vitro,
Eliminating needs repeatedly to be added IL-7 the and IL-15 factor in experimentation, simplify experimental procedure, and can reduce CAR-T preparation and make
Cost during standby.
Detailed description of the invention
Fig. 1 is the flow diagram for improveing the preparation of Chimeric antigen receptor T cell；
Fig. 2 is the schematic diagram before and after Healthy People T cell activation, and wherein A is activation pre-T cell, and B is T cell after activation；
Fig. 3 is the transduction positive rate figure after three kinds of slow virus cotransductions；
Fig. 4 is cells expanded figure after cotransduction；
Fig. 5 is the schematic diagram of CAR；
Specific embodiment
In order to which the present invention is further explained, technological means used, this is explained further below by way of specific embodiment
The technical solution of invention, but the present invention is not limited in scope of embodiments.
The present invention provides a kind of three kinds of slow virus by including CAR to transduce simultaneously into the improvement inosculating antibody of T cell
The preparation method of original receptor T cell, the preparation of acquisition, IL-7 and IL-15 and tri- kinds of slow virus of CAR including purified T cell are total to
The acquisition for Chimeric antigen receptor T cell of transduceing, the Characteristics Detection for improveing CAR-T cell.
Embodiment 1
The acquisition of purified T cell
The peripheral blood of Healthy People is taken with EDTA anticoagulant tube, and isometric DPBS is then added and is diluted.Later with dilution
The ratio that peripheral blood and lymphocyte separation medium volume ratio afterwards is 3:2, is added lymphocyte separation medium, and 400g is centrifuged 40min；
Cell can be divided into 4 layers from top to bottom, draw the tunica albuginea layer of the second layer, and it is thin to can be obtained the single core of peripheral blood after DPBS is washed
Then born of the same parents can obtain the T cell of purifying according to the Pan T Cell Isolation Kit specification operation of U.S. day Ni.
It is 1,000,000/mL by cell concentration is adjusted using complete medium after the T cell counting of purifying, according to magnetic bead and carefully
As a result born of the same parents see Fig. 2 then by T cell activation than the magnetic bead that anti-CD3/CD28 coupling is added in the ratio for 1:1~3:1.
Embodiment 2
The preparation of tri- kinds of slow virus of IL-7, IL-15 and CAR
First by tri- kinds of plasmids of IL-7, IL-15, CAR [three kinds of plasmid purchases are in Takara] and slow virus packaging system
Helper plasmid pLp1, pLp2, pLp/VSVG expanded through Escherichia coli after, utilize endotoxin-free plasmid extracts kit extract
Plasmid is for virus packaging.Ten thousand 293FT cell of 1000-2000 is spread into T75 Tissue Culture Flask on the day before virus packaging, when
Viral packaging is carried out when cell length to 80% density.Cell is preheated to FreeStyle without double antibody before packingTM293 expression
Culture medium changes liquid.The packaging of tri- kinds of slow virus of IL-7, IL-15 and CAR is carried out under the action of lipo2000 promotees transfection reagent.Turn
Culture solution is replaced after dye after 6h.Viral supernatants are collected after culture 48h, are dispensed after 0.45 μm of membrane filtration in -80 DEG C of refrigerators
It saves.The measurement of virus titer is carried out using the control slow virus EGFP virus of packaging, concrete outcome is shown in Table 1.
The virus titer of 1 three kinds of slow virus of table
| Slow virus | Virus titer (TU/mL) |
| IL-7 | 9.7*10^6 |
| IL-15 | 1.1*10^7 |
| CAR | 1.3*10^7 |
Embodiment 3
Improve the preparation of Chimeric antigen receptor T cell
Before cotransduction, the three kinds of slow virus frozen are taken out from -80 DEG C of refrigerators, are dissolved rapidly in 37 DEG C of water-baths.From training
The T cell progress cell count that case takes out activation is supported, 320g is centrifuged 5min, abandons supernatant.Three kinds of slow virus are 15 calculating with MOI
Required virus liquid volume.By virus liquid and after promoting transfection reagent incubation at room temperature 5min, cell precipitation is resuspended, with 2000-
The revolving speed room temperature of 3000rpm is centrifuged 1-3h.It after centrifugation, inhales and abandons supernatant, continue at training after cell precipitation is resuspended with culture solution
Support case culture.
48h after the transduction utilizes IL-7-FITC streaming antibody, IL-15-AF647 streaming antibody, and the PE for CAR
Dyestuff detects the transduction efficiency of virus, as a result sees Fig. 3.It can be obtained the Chimeric antigen receptor T cell of improvement, wherein turn of CAR
Leading the transduction efficiency that efficiency is 20.60%, IL-7 and IL-15 is respectively 13.55% and 12.78%.Three kinds of slow virus turn jointly
Lead T cell concept feasible.Furthermore the cell proliferative conditions cultivated 20 days are investigated, as a result see Fig. 4.Know the expansion of cotransduction group cell
It is higher than the amplification times of individually transduction CAR slow virus to double number, there are significant differences for two groups of cell Proliferations.
In conclusion the invention discloses the preparations and its application of a kind of Chimeric antigen receptor T cell of improvement.In CAR
During lentiviruses transduction, while two kinds of lentiviruses transductions for carrying IL-7 gene and IL-15 gene are entered into cell, wherein slowly
MOI has significant effect transduction efficiency during viral cotransduction.Groped by many experiments, what we were suitable for
MOI, to make it possible three kinds of slow virus while to transduce into cell.And the Chimeric antigen receptor T cell improved does not influence
The Effective multiplication of CAR-T cell in vitro, with good application prospect.
The Applicant declares that technological means involved in the present invention and scheme are elaborated above, but it is only
It is as example, protection scope of the present invention is not limited to above-mentioned specific example.The personnel of technical field are to the present invention
Any equivalents done or improvement are included in protection scope of the present invention and the open scope.
Claims (7)
1. a kind of preparation method for improveing Chimeric antigen receptor T cell, it is characterised in that: in T cell during viral transduction,
It will transduce simultaneously into T cell containing tri- kinds of slow virus of IL-7, IL-15 and CAR.
2. according to the method described in claim 1, it is characterized by: the method is realized by following steps:
(1) healthy human peripheral blood is acquired, peripheral blood mononuclear cells is obtained through density gradient separation, the T of purifying is obtained after sorting
Cell；
(2) after t cell activation, tri- kinds of slow virus of IL-7, IL-15 and CAR is taken out and carry out centrifugation cotransduction, inhales abandon virus immediately
Supernatant, replacement culture solution continue to cultivate；
(3) T cell after step (2) transduction is subjected to flow cytometer showed, the Chimeric antigen receptor T improved after culture is thin
Born of the same parents.
3. according to the method described in claim 2, it is characterized by: method for separating described in step (1) is using magnetic bead feminine gender point
Select method.
4. according to the method described in claim 2, it is characterized by: t cell activation technology described in step (2) is to use
The magnetic bead of anti-CD3/anti-CD28 coupling is activated, when activation the magnetic bead used and the additional proportion of T cell for 1:1 ~
3:1。
5. according to the method described in claim 2, it is characterized in that step (2) in be centrifuged cotransduction, the centrifugal rotational speed used for
2000-3000 rpm, centrifugation time are 1-3 h.
6. -2 method obtained according to claim 1, it is characterised in that 48h carries out flow cytometer showed after transduction in step (3)
Transduction efficiency.
7. method described in -6 any one according to claim 1, it is characterised in that: the MOI of the IL-7 slow virus is 5-
The MOI of 25, IL-15 slow virus is 5-25, and the MOI of CAR slow virus is 5-25.
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| CN110964121A (en) * | 2019-12-20 | 2020-04-07 | 山东省齐鲁细胞治疗工程技术有限公司 | CD19-CAR-T cell secreting IL-7 and IL-15 factors and application thereof |
| CN112251466A (en) * | 2020-10-29 | 2021-01-22 | 江苏拓弘生物科技有限公司 | CAR-T preparation method with low virus dosage |
| CN112375743A (en) * | 2020-11-20 | 2021-02-19 | 山东省医学科学院附属医院 | Secretory HER2 antigen-targeted chimeric antigen receptor T cell and application thereof |
| WO2021052496A1 (en) * | 2019-09-20 | 2021-03-25 | 科济生物医药（上海）有限公司 | Immune effector cell in which expression is regulated by cytokines |
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| CN112251466A (en) * | 2020-10-29 | 2021-01-22 | 江苏拓弘生物科技有限公司 | CAR-T preparation method with low virus dosage |
| CN112375743A (en) * | 2020-11-20 | 2021-02-19 | 山东省医学科学院附属医院 | Secretory HER2 antigen-targeted chimeric antigen receptor T cell and application thereof |
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Application publication date: 20190507 |