CN110157680A - Improve the cell culture processes of Chimeric antigen receptor T cell curative effect and duration of action - Google Patents

Improve the cell culture processes of Chimeric antigen receptor T cell curative effect and duration of action Download PDF

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CN110157680A
CN110157680A CN201910380701.5A CN201910380701A CN110157680A CN 110157680 A CN110157680 A CN 110157680A CN 201910380701 A CN201910380701 A CN 201910380701A CN 110157680 A CN110157680 A CN 110157680A
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cell
car
culture
dasatinib
curative effect
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黄河
张�浩
徐玉林
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Zhejiang University ZJU
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Zhejiang University ZJU
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Priority to PCT/CN2019/094961 priority patent/WO2020224042A1/en
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Abstract

The present invention provides a kind of cell culture processes for improving Chimeric antigen receptor T cell curative effect and duration of action, the transmitting of CAR-T cell activation signal is reduced by addition tyrosine kinase inhibitor Dasatinib (dasatinib), inhibit the terminal differentiation of CAR-T cell, the ratio of the T cells and Central memory T cell in CAR-T cell product is improved, while the exhaustion of CAR-T cell being inhibited to be inclined to.The method of the present invention solves the problems, such as the terminal differentiation of CAR-T cell in Process of in vitro and exhausts tendency.The method of the present invention incubation is simple and easy, low in cost, and clinical application is safe and reliable；It cultivates obtained CAR-T cell T cells and Central memory T cell ratio is high, and avoid the exhaustion tendency of CAR-T cell, it is reproducible；It cultivates obtained CAR-T cell to show preferably treatment curative effect and maintain persistence in vivo, has a wide range of applications promotional value.

Description

Improve the cell culture processes of Chimeric antigen receptor T cell curative effect and duration of action
Technical field
The invention belongs to immunologys and cell therapy research field, are related to a kind of raising Chimeric antigen receptor T cell curative effect With the cell culture processes of duration of action, be using tyrosine kinase inhibition drug improve CAR-T cell in T cells and Central memory T cell reduces the cultural method of CAR-T cell depletion.
Background technique
Chimeric antigen receptor T (chimeric antigen receptor modified T, CAR-T) cellular immunotherapy In recent years become and a kind of most treatment means of development prospect are immunized in targeted therapy, action principle is to utilize genetic engineering skill Art will identify the antigen-binding portion of the specific single-chain monoclonal antibody of certain tumour antigen and the tyrosine of T cell receptor Activation motifs and costimulatory molecules are coupled in vitro makes T by the methods of viral vectors transfecting T cells for a chimeric protein Cell specific recognition tumour cell and can transmit signals to intracellular, cause the Proliferative Activated to passing through release of T cell Perforin/granzyme target killing tumor cell, and not by major histocompatibility complex (Major Histocompatibility Complex, MHC) limitation.CAR-T treatment in recent years has been applied to leukaemia, lymthoma, more The tumours such as hair property myeloma, glioma, melanoma, lung cancer, prostate cancer, cancer of pancreas and oophoroma.Wherein in blood system The achievement that research in system malignant tumour obtains is attracted attention the most.At present carried out for as CD19, CD22, CD20, CD33, The CAR-T cell clinical treatment of the target spots such as CD30, CD38, BCMA, CD138, CD123 is studied.Most notable one curative effect is main The application being embodied in the malignant neoplastic disease of B cell source, such as B-lineage Acute Lymphocyte Leukemia (B-ALL), chronic lymphatic is thin Born of the same parents' leukaemia (CLL), Huppert's disease (MM), B cell lymphoma (B-NHL) etc..The targeting CD19's reported in the world CAR-T (CART19) cell therapy is refractory/recurrence B-ALL, complete remission rate (CR) reaches 90%.CAR-T cell therapy is in blood Breakthrough in liquid oncotherapy brings invalid or multiple after new hope, especially traditional treatment to the research field Hair, in the case where clinical treatment policymaker is helpless, CAR-T cellular immunotherapy becomes best therapeutic choice.
But in the CAR-T cell therapy patient for receiving targeting CD19, the main barrier at CAR-T cell therapy is recurred Hinder, the patient of about 30-50% is recurred after receiving CAR-T cell therapy, and recurrence mostly occurs in and receives CAR-T cell therapy In 1 year；And most of solid tumors give targeting CAR-T cell therapy curative effect it is not good enough.It is now recognized that the CAR-T cell of targeting CD19 is controlled Protopathy recurrence is main reasons is that the loss of CAR-T cell in vivo after treatment, namely cannot maintain for a long time in vivo；Evidence is aobvious Show, the long-term disease-free survival of patient and CAR-T cell holding time in vivo are highly relevant after CAR-T cell therapy.And CAR-T cell depletion be not only targeting CD19 CAR-T cell therapy hematological system tumor disease after tumor recurrence it is important Factor, while being also the not good enough major reason for the treatment of solid tumor curative effect.Therefore exploitation improves CAR-T cell curative effect, extends CAR-T The method that cell maintains in vivo, to improve CAR-T cell therapy tumor disease curative effect, prevent palindromia and improve suffer from Person's long-term disease-free survival rate is most important.
CAR-T cell curative effect in vivo and persistence and its differentiation rank for obtaining cell product in incubation in vitro Section is significant related to depletion state.In Process of in vitro, it is produced from because of the mutual aggregation of CAR-T cell surface CAR molecule The activation signals for sending out lasting not only can make CAR-T cell stage differentiated to terminal, and can occur to exhaust phenomenon, especially with The CAR-T cell of CD28 costimulation structural domain.The differential period of CAR-T cell is divided into: T cells, and stemness center memory T is thin Born of the same parents, Central memory T cell, Effector memory T cell, effector T cell.T cells and center memory T in CAR-T cell product Cell shows long-term surviving ability in vivo, and the CAR-T broken up to terminal stage such as effect memory stage and effect is thin Born of the same parents act in vivo cannot be lasting；Simultaneously exhaust the high-caliber Inhibitory receptor of CAR-T cell continuous expression such as PD1, TIM3, LAG3, proliferative capacity is low, low cytokine release reduced capability, and Yi Fasheng apoptosis seriously limits CAR-T in vivo enduringly Play effector function.But there is no the terminal differentiations and exhaustion that solve CAR-T cell in Process of in vitro to incline both at home and abroad at present To method.
Summary of the invention
The object of the present invention is to provide a kind of cell culture for improving Chimeric antigen receptor T cell curative effect and duration of action Method is realized by step in detail below:
1. prepared by peripheral blood mononuclear cells
Periphery blood specimen is taken, anticoagulant heparin separates preparation peripheral blood mononuclear cells using human lymphocyte separating liquid.
The enrichment of 2.CD3 (+) T cell and t cell activation
It is given after mixing well with peripheral blood mononuclear cells and be incorporated into CD3 (+) T cell using anti-CD3/CD28 magnetic bead Magnetic frame is enriched with CD3 (+) T cell, and activates CD3 (+) T using the anti-CD3/CD28 antibody for being incorporated in magnetic bead surfaces simultaneously Cell.
3. carrying the CAR slow-virus transfection T cell of targeting CD19
By the slow virus of the CAR of the carrying targeting CD19 prepared according to MOI=10 transfection anti-CD3/CD28 magnetic bead activation CD3 (+) T cell.
4. adding tyrosine kinase inhibitor Dasatinib expands culture CAR-T cell
Take 3-5 days GFP+CAR-T cells of culture, the expression of flow cytometer detection CAR molecule, after confirming that CAR-T cell is successfully prepared, By continuous culture 9 days respectively of CAR-T cell points 2 groups: respectively using addition with do not add tyrosine kinase inhibitor Dasatinib Culture solution culture CAR-T cell.Divide experimental group and 2 groups of control group, Dasatinib 30nmol/L experimental group
(RPMI1640+10%FBS+IL-2200U/ml+ Dasatinib 30nM), control group: isometric DMSO control group
(the isometric DMSO of RPMI1640+10%FBS+IL-2200U/ml+).
The detection of 5.CAR-T cell subsets
It cultivates and samples within the 9th day this mark fluorescent antibody CD45RO, CD62L, flow cytomery CAR-T cell subsets；Through step Suddenly the CAR-T cell of (4) culture, cell differentiation can be continuously maintained in T cells and central memory T cell stage, effectively Ground inhibits CAR-T cell downstream and terminal is stage differentiated.
The detection of 6.CAR-T cell depletion relevant surfaces molecule
It cultivates and samples within the 9th day this mark fluorescent antibody PD1, TIM3, LAG3, flow cytomery CAR-T cell depletion is related Surface molecular；The CAR-T cell cultivated through step (4), low expression T cell exhaust associated inhibitory receptor PD1, TIM3, LAG3, It restrained effectively the exhaustion tendency of CAR-T cell.
7. assessing the CAR-T cell curative effect and persistence handled through Dasatinib
Prepare ALL-NSG mouse model, according to following grouping: 1. control group (the CAR-T cell of tail vein injection DMSO processing), 2. experimental group (the CAR-T cell of tail vein injection Dasatinib 30nM culture) is tested；Weekly using small animal living body at Picture instrument gives mouse to be imaged, and compares two groups of tumor load differences；Flow cytometry CAR-T cell proportion weekly；Record each group Mouse diing time draws survivorship curve.The CAR-T cell cultivated through step (4), treatment acute lymphoblastic leukemia are small Mouse can get better curative effect and life cycle.
The method of the present invention solves the problems, such as the terminal differentiation of CAR-T cell in Process of in vitro and exhausts tendency, leads to It crosses addition tyrosine kinase inhibitor Dasatinib (dasatinib) and reduces the transmitting of CAR-T cell activation signal, it is suppressed that CAR- The terminal differentiation of T cell improves the ratio of the T cells and Central memory T cell in CAR-T cell product, presses down simultaneously The exhaustion of CAR-T cell processed is inclined to, and obtains more remarkable treatment effect, longer CAR-T cell product of holding time in vivo.The present invention The method of foundation solves the problems, such as the terminal differentiation of CAR-T cell in Process of in vitro and exhausts tendency, by adding network Histidine kinase inhibitor Dasatinib reduces the transmitting of CAR-T cell activation signal, it is suppressed that the terminal differentiation of CAR-T cell mentions The ratio of T cells and Central memory T cell in high CAR-T cell product, while the exhaustion of CAR-T cell being inhibited to incline To obtaining more remarkable treatment effect, longer CAR-T cell product of holding time in vivo.Inventive method has the following characteristics that (1) Incubation is simple and easy, low in cost, and clinical application is safe and reliable；(2) the obtained CAR-T cell T cells of culture and Central memory T cell ratio is high, and avoids the exhaustion tendency of CAR-T cell, reproducible；(3) CAR-T that culture obtains is thin Born of the same parents show preferably treatment curative effect in leukemia mouse model and maintain persistence in vivo, have a wide range of applications promotion price Value.The present invention is directed to establish a kind of method system, CAR-T cell is prevented to terminal differentiation, to inhibit CAR-T thin during the cultivation process Born of the same parents exhaust, obtain high quality, and the CAR-T cell product of high T cells and Central memory T cell content not only can be improved The treatment curative effect of CAR-T cell, the recurrence after can more reducing CAR-T treatment, improves patient's long-term disease-free survival rate.
Detailed description of the invention
Fig. 1 .CAR-T cell in incubation, since CAR molecule is mutually assembled, causes CAR-T cell persistently to be lived in vitro Change, so that part CAR-T cell limits its persistence in vivo to effective stage differentiation；Addition reaches sand in incubation CAR-T cell can be substantially reduced for Buddhist nun downstream to break up, and keep cell in initial and center memory stage.
Fig. 2 leads to CAR-T cell continuous activation since CAR molecule is mutually assembled, so that part CAR-T cell consumes It exhausts, limits the clinical efficacy of CAR-T cell；Dasatinib is added in incubation can be reversed the exhaustion tendency of CAR-T cell, Cell low expression level PD1, TIM3, LAG3 are kept, the CAR-T cell product of high quality is obtained.
Fig. 3 treats the white blood of acute lymphoblastic using the CAR-T cell tail vein injection that Dasatinib 30nmol/L is cultivated After sick mouse, compared with the control group, mouse tumor load is significantly lower than control group.
The acute lymphoblastic leukemia mouse of the CAR-T cell therapy of Fig. 4 Dasatinib 30nmol/L culture, middle position Existence 55 days, and control group the median survival time 43 days, prompt the CAR-T cell of addition Dasatinib culture that can significantly extend acute leaching The life cycle of bar chronic myeloid leukemia mouse.
Fig. 5 CAR-T cell infusion mouse after a week, two groups of CAR-T cells of flow cytomery are in mouse peripheral blood Ratio in karyocyte, the CAR-T cell proportion of Dasatinib 30nmol/L culture is significantly higher than control group as the result is shown, mentions Show that the maintenance of the CAR-T cell of Dasatinib 30nmol/L processing in vivo is more lasting.
Specific embodiment
The present invention in conjunction with the accompanying drawings and embodiments, is further described.
Embodiment 1
1. the separation of mononuclearcell
(1) peripheral blood 10-20ml is acquired；
(2) peripheral blood is diluted using isometric PBS；
(3) 15ml centrifuge tube is taken, 5ml lymphocyte separation medium is added, using liquid-transfering gun by the blood sample 10ml after dilution along tube wall It is added slowly to the upper layer of separation agent, avoids the mixing of separation agent and blood sample；
(4) 400G is set by centrifuge, revolving speed rising is set as 4 grades, and revolving speed decrease speed is set as 0 grade, and room temperature is centrifuged 30 minutes；
(5) after being centrifuged, gently the mononuclearcell layer in serum and separation agent interface is drawn and is transferred to one In new centrifuge tube, PBS is washed cell 2 times.
The enrichment of 2.CD3 (+) T cell and t cell activation
(1) mononuclearcell obtained is counted, 1640 complete medium of RPMI is resuspended, adjustment cell concentration to 107/ml；
(2) it is washed anti-CD3/CD28 magnetic bead 2 times with 0.1%BSA/PBS solution；Washing methods: 5-10ml0.1%BSA/ is taken PBS solution is placed in 15ml centrifuge tube, and required anti-CD3/CD28 magnetic bead after calculating is added, mixes well and is placed on magnetic 1 minute is stood on power frame, magnetic bead is affixed on two sides centrifugation tube wall, and absorption discards 0.1%BSA/PBS solution.Repeated washing 1 time；
(3) according to magnetic bead: CD3 (+) T cell=3:1, by after washing anti-CD3/CD28 magnetic bead and mononuclearcell it is abundant It mixes, moves to culture bottle (selecting culture bottle specification depending on amount of liquid), set shaking table and jiggle 20 minutes, make magnetic bead and CD3 (+) T Cell sufficiently combines；
(4) cell suspension of magnetic bead and PBMC are transferred to centrifuge tube, are placed on magnetic frame and stand 1 minute, in conjunction with magnetic bead CD3 (+) T cell is affixed on two sides centrifugation tube wall, draws the cell suspension for discarding and being not associated with magnetic bead in centrifuge tube；
(5) CD3 (+) T cell for combining magnetic bead, adjustment are resuspended with 1640 complete medium of RPMI containing IL-2 (200IU/ml) Cell concentration is to 1 × 106/ ml, 37 DEG C, 5%CO2It is cultivated 24 hours in saturated humidity incubator.
3. carrying CAR slow-virus transfection T cell
(1) it is centrifuged and is counted CD3 (+) T cell in conjunction with magnetic bead, contains 1640 complete medium of RPMI of IL-2 (200IU/ml) It is resuspended, adjustment cell concentration to 4 × 106/ ml is inoculated in 12 orifice plates by the hole 500ul/；
(2) experiment is using the slow virus for carrying GFP/mCherry and CAR target gene, according to MOI=10, required for calculating Virus quantity.Calculation formula is as follows: required virus quantity=(MOI × cell quantity)/virus titer；
(3) after -80 DEG C of refrigerators taking-up virus, melt in 37 DEG C of water-baths rapidly.Above-mentioned calculating institute is added in 12 orifice plates The virus quantity obtained adds the polybrene of final concentration of 5 μ g/mL, mixes well and be placed on 37 DEG C, 5%CO2Incubator in, RPMI 1640 complete medium of the supplement containing IL-2 (200IU/ml) continues culture 24 hours to 2ml after 6-8 hours；
(4) 800rpm is centrifuged 8 minutes, is removed containing virulent culture medium supernatant, and cell precipitation is resuspended with fresh culture, will be thin Dysuria with lower abdominal colic moves in six orifice plates or culture bottle, continues culture 3-5 days；
(5) using CAR-T cell in 5ml liquid-transfering gun piping and druming culture bottle, and cell is moved into 50ml centrifuge tube, is placed in magnetic frame Upper standing 1min, magnetic bead are adsorbed in tube wall, cell suspension are transferred in new centrifuge tube, are centrifuged and add fresh containing IL-2 1640 complete medium of RPMI of (200IU/ml) continues to expand culture.
4. adding tyrosine kinase inhibitor Dasatinib expands culture CAR-T cell
(1) the CAR-T cell for taking culture 3-5 days is resuspended thin with the RPMI l640 complete medium of the 200IU/mL containing human IL-2 Born of the same parents are simultaneously counted using full-automatic cell calculating instrument, and press 5 × 105/ hole is seeded in 12 orifice plates；
(2) experimental group is handled: experimental group is Dasatinib 30nmol/L group, isometric DMSO control group；37 DEG C, 5%CO2 It is cultivated in saturated humidity incubator, liquid is changed in centrifugation in every 3 days, adds Dasatinib and DMSO again.
Cell differentiation detection in 5.CAR-T cell cultivation process
(1) it cultivates the 9th day and samples this flow cytometer detection；
1. sample collection and processing: mixing each group CAR-T cell in six orifice plates, pressed after respectively drawing appropriate cell suspension centrifuge washing According to 0.5-1 × 106A cell/pipe is added in streaming pipe, and born of the same parents are resuspended using 100ul PBS buffer solution；
2. labelled antibody: CD45RO, CD62L antibody of the corresponding fluorescent marker of addition 2.5ul into respective streams style quality control, 4 DEG C It is protected from light and is incubated for 30min；
3. washing: 2mLPBS buffer is added in every pipe, is centrifuged 5min after mixing under room temperature, abandons supernatant.It is repeated 2 times；
4. detection and analysis: the cell after marking is resuspended using 500ul PBS buffer solution, machine testing on flow cytometer uses The analysis of Flowj7.6 software is analysis object gating with GFP positive CAR-T, analyzes the ratio of each subgroup.
(2) each subgroup of cell is defined as: killer T cell CD8+, helper T lymphocyte CD4+, T cells CD45RO- CD62L+, Central memory T cell CD45RO+CD62L+, effect memory T thin CD45RO+CD62L-, effector T cell CD45RO+ CD62L- is indicated with ratio.
(3) it is compared with conventional culture methods (control group), this cultural method can substantially reduce CAR-T cell and downstream divide Change, keeps cell in initial and center memory stage, the result is shown in Figure 1.
The detection of cell depletion relevant surfaces molecule in 6.CAR-T cell cultivation process
(1) it cultivates the 9th day and samples this flow cytometer detection；
(2) sample is collected by preceding method, marks the fluorescent labeled antibody and isotype control Ab of PD1, TIM3, LAG3, washing After be resuspended, flow cytomery；
(3) data are analyzed: being used 7.6 software analysis data of Flowjo, be analysis object gating with GFP positive CAR-T, as a result It is indicated with positive cell ratio；
(4) exhaust that mark of correlation is indicated with PD1, TIM3, LAG3 positive ratio.It is compared with conventional culture methods (control group), this The exhaustion tendency of CAR-T cell can be reversed in cultural method, keeps cell low expression level PD1, TIM3, LAG3, as a result sees Fig. 2.
The CAR-T cell curative effect and persistence that assessment is handled through Dasatinib in 7.ALL-NSG Mice Body
(1) CAR-T cell prepares: preparation carries the CAR-T cell of mCherry, takes the CAR-T cell of culture 3-5 days, and streaming is thin The ratio of born of the same parents' instrument detection CAR-T cell.Dividing 2 groups to be cultivated 1. control group (isometric DMSO is added), 2. (addition reaches experimental group Sand replaces Buddhist nun 30nM), every 3 days replacement culture mediums simultaneously add drug again, continuous culture 9 days.
(2) ALL-NSG mouse model prepares: 4-5 week old NSG mouse is raised in SPF grades of Animal Research Centers.Take logarithm raw Long-term luciferase (+) Nalm6 cell strain prepares cell concentration to 1 × 106/ 200ul, by 1 × 106/ tail vein injection, Every mouse injects total volume 200ul.Small animal living body imager detects tumor load after 5 days, is randomly divided into 2 by fluorescence intensity Group adjusts 2 groups of average fluorescent strengths without significant difference, the CAR-T cell of next day tail vein injection different disposal.
(3) experimental group: experiment is divided into 2 groups, every group of 5 mouse, respectively 1. control group (tail vein injection DMSO processing CAR-T cell), the 2. experimental group CAR-T cell of processing (tail vein injection Dasatinib 30nM).
(4) CAR-T cell tail vein injection: according to 1 × 106CAR-T cell/mouse simultaneously calculates according to CAR-T cell proportion Total cell amount required for every mouse, PBS is resuspended after collecting the CAR-T cell centrifugation of culture, and configuration concentration is 1 × 106CAR-T Cell/200ul；It is small that the control group prepared and experimental group CAR-T cell suspension are seeded to NSG in a manner of tail vein injection In mouse body, volume injected is 200ul/ mouse.
(5) curative effect and existence observation:
1. being imaged using small animal living body imager to mouse weekly, compare two groups of tumor load differences；
2. mCherry (+) CAR-T cell proportion in Flow cytometry Mice Body weekly；
3. recording each group mouse diing time, survivorship curve is drawn；
(6) compared with the control group using the CAR-T cell of this cultural method acquisition, mouse tumor load is significantly lower than control As a result group is shown in Fig. 3.The CAR-T cell of this law culture can significantly extend the life cycle of acute lymphoblastic leukemia mouse, as a result See Fig. 4.
CAR-T cell infusion mouse after a week, two groups of CAR-T cells of flow cytomery are in mouse peripheral blood karyocyte In ratio, Dasatinib 30nmol/L culture CAR-T cell proportion be significantly higher than control group, as a result see Fig. 5.

Claims (4)

1. a kind of cell culture processes for improving Chimeric antigen receptor T cell curative effect and duration of action, which is characterized in that pass through Following steps are realized:
(1) prepared by peripheral blood mononuclear cells: taking periphery blood specimen, anticoagulant heparin is separated using human lymphocyte separating liquid and made Standby peripheral blood mononuclear cells；
(2) enrichment of CD3 (+) T cell and t cell activation:
It is given after mixing well with peripheral blood mononuclear cells and be incorporated into CD3 (+) T cell using anti-CD3/CD28 magnetic bead Magnetic frame is enriched with CD3 (+) T cell, and activates CD3 (+) T using the anti-CD3/CD28 antibody for being incorporated in magnetic bead surfaces simultaneously Cell；
(3) the CAR slow-virus transfection T cell of targeting CD19 is carried: by the slow virus of the CAR of the carrying targeting CD19 prepared According to CD3 (+) T cell of MOI=10 transfection anti-CD3/CD28 magnetic bead activation；
(4) addition tyrosine kinase inhibitor Dasatinib expands culture CAR-T cell
The CAR-T cell of culture 3-5 days is taken, the expression of flow cytometer detection CAR molecule will after confirmation CAR-T cell is successfully prepared 2 groups of CAR-T cell point is continuous respectively to be cultivated 9 days；
(5) CAR-T cell subsets detects: culture samples this mark fluorescent antibody CD45RO, CD62L, flow cytometer inspection on the 9th day Survey CAR-T cell subsets；
(6) detection of CAR-T cell depletion relevant surfaces molecule
It cultivates and samples within the 9th day this mark fluorescent antibody PD1, TIM3, LAG3, flow cytomery CAR-T cell depletion is related Surface molecular；
(7) the CAR-T cell curative effect and persistence handled through Dasatinib is assessed
Divide control group and experimental group, the CAR-T cell that control group DMSO is handled, experimental group is cultivated with Dasatinib 30nM CAR-T cell compares two groups of tumor load differences by imager, by Flow cytometry CAR-T cell proportion, draws Survivorship curve.
2. a kind of cell culture for improving Chimeric antigen receptor T cell curative effect and duration of action according to claim 1 Method, which is characterized in that step (4) it is described culture be respectively using addition with do not add tyrosine kinase inhibitor Dasatinib Culture solution culture CAR-T cell.
3. a kind of cell culture for improving Chimeric antigen receptor T cell curative effect and duration of action according to claim 1 Method, which is characterized in that described point 2 groups of step (4) are experimental group and experimental group, and experimental group is Dasatinib 30nmol/L group, Control group is isometric DMSO group.
4. a kind of cell culture for improving Chimeric antigen receptor T cell curative effect and duration of action according to claim 2 Method, which is characterized in that the culture solution of the addition Dasatinib is that RPMI1640+10%FBS+IL-2 200U/ml+ reaches sand For Buddhist nun 30nM, the culture solution for not adding Dasatinib is that RPMI1640+10%FBS+IL-2 200U/ml+ is isometric DMSO。
CN201910380701.5A 2019-05-08 2019-05-08 Improve the cell culture processes of Chimeric antigen receptor T cell curative effect and duration of action Pending CN110157680A (en)

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PCT/CN2019/094961 WO2020224042A1 (en) 2019-05-08 2019-07-07 Cell culture method for improving curative effect and durability of chimeric antigen receptor t cell

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110760481A (en) * 2019-10-30 2020-02-07 武汉科技大学 Improved RPMI1640 culture medium and application thereof
CN112852730A (en) * 2021-02-01 2021-05-28 河南省遗传资源细胞库有限公司 CART-20 cell amplification culture method based on CAR technology
CN113943710A (en) * 2021-09-17 2022-01-18 浙江大学医学院附属第一医院 Culture medium for CAR-T cell culture and application thereof
CN115702899A (en) * 2021-08-03 2023-02-17 上海优卡迪生物医药科技有限公司 Application of luccotinib to preparation of CAR-T medicine

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4309669A1 (en) * 2021-03-16 2024-01-24 Shanghai Iaso Biotechnology Co., Ltd. Universal chimeric antigen receptor t-cell and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107075482A (en) * 2014-04-07 2017-08-18 诺华股份有限公司 Use anti-CD19 Chimeric antigen receptors treating cancer
WO2017172981A2 (en) * 2016-03-29 2017-10-05 University Of Southern California Chimeric antigen receptors targeting cancer
WO2018183842A1 (en) * 2017-03-31 2018-10-04 The Board Of Trustees Of The Leland Standford Junior University Methods of treating t cell exhaustion by inhibiting or modulating t cell receptor signaling

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3344262A4 (en) * 2015-09-01 2019-03-20 Innate Tumor Immunity, Inc. Immune cells having increased immunity or resistance to an immunosuppressive cytokine and use of the same
WO2018106595A1 (en) * 2016-12-05 2018-06-14 Fate Therapeutics, Inc. Compositions and methods for immune cell modulation in adoptive immunotherapies
CN107098981B (en) * 2017-06-29 2020-05-01 青岛麦迪赛斯医疗技术有限公司 Chimeric antigen receptor modified T lymphocyte targeting CD19

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107075482A (en) * 2014-04-07 2017-08-18 诺华股份有限公司 Use anti-CD19 Chimeric antigen receptors treating cancer
WO2017172981A2 (en) * 2016-03-29 2017-10-05 University Of Southern California Chimeric antigen receptors targeting cancer
WO2018183842A1 (en) * 2017-03-31 2018-10-04 The Board Of Trustees Of The Leland Standford Junior University Methods of treating t cell exhaustion by inhibiting or modulating t cell receptor signaling

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
刘保池等: "《特殊感染外科新理念与新技术》", 31 December 2017, 上海科技教育出版社 *
赵富玺 许礼发: "《医学免疫学》", 30 April 2013 *
饶洪洋等: "《病原生物与免疫学基础》", 28 February 2015 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110760481A (en) * 2019-10-30 2020-02-07 武汉科技大学 Improved RPMI1640 culture medium and application thereof
CN110760481B (en) * 2019-10-30 2021-04-02 武汉科技大学 Improved RPMI1640 culture medium and application thereof
CN112852730A (en) * 2021-02-01 2021-05-28 河南省遗传资源细胞库有限公司 CART-20 cell amplification culture method based on CAR technology
CN115702899A (en) * 2021-08-03 2023-02-17 上海优卡迪生物医药科技有限公司 Application of luccotinib to preparation of CAR-T medicine
CN113943710A (en) * 2021-09-17 2022-01-18 浙江大学医学院附属第一医院 Culture medium for CAR-T cell culture and application thereof

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