CN108841792A - Target the T cell and the preparation method and application thereof of CD19 and EBNA1 gene modification - Google Patents

Target the T cell and the preparation method and application thereof of CD19 and EBNA1 gene modification Download PDF

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CN108841792A
CN108841792A CN201810615864.2A CN201810615864A CN108841792A CN 108841792 A CN108841792 A CN 108841792A CN 201810615864 A CN201810615864 A CN 201810615864A CN 108841792 A CN108841792 A CN 108841792A
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cell
ebna1
ser
targeting
leu
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钱文斌
陆哲明
张超亭
雷文
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Beijing Inst Of Tumor Prevention & Cure
Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The present invention relates to molecular biology field, tool discloses gene modification T cell that is a kind of while targeting CD19 and EBNA1.The T cell has the TCR of the CAR and targeting EBNA1 of targeting CD19 simultaneously, its tumour cell that CD19 can be not only expressed with targets identification, and in CD19 low expression or when not expressing, the tumour cell of EBNA1 can be expressed with targets identification, so that the tumour for EBV positive B-cells source provides a kind of new treatment means.Moreover, improving the ratio of gene modification T cell identification EBNA1 present invention optimizes the incubation of the gene modification T cell.

Description

Target the T cell and the preparation method and application thereof of CD19 and EBNA1 gene modification
Technical field
The present invention relates to molecular biology fields, repair specifically, being related to gene that is a kind of while targeting CD19 and EBNA1 Adorn T cell and the preparation method and application thereof.
Background technique
In recent years, the CAR-T for targeting CD19 is recurred in treatment, drug resistance, is obtained in terms of refractory B cell leukemia and lymthoma Remarkable achievement, causes the great interest of countries in the world clinical tumor doctor.But only target the CAR-T treatment of CD19 B cell source tumour will lead to tumour cell CD19 low expression or not express, and generate so as to cause immunosurveillance escape Recurrence and drug resistance.Therefore the TCR for targeting the CAR and targeting EBNA1 of CD19 simultaneously can target the EBV sun in B cell source simultaneously Property tumour two target spots of CD19 and EBNA1, therefore for B cell source EBV positive tumor especially CD19 CAR-T treat The patient of failure, double target gene modification T cell expections can obtain preferable therapeutic effect.CD19 is the cell of B cell system, packet Include normal B cell, pre B cell, B cell lymphoma and leukaemia cell, specific expressed memebrane protein, other cells and group It knits and does not express CD19；And EBNA1 is the albumen that all EBV positive tumors are expressed.Therefore, for the B cell source EBV positive The patient of tumour especially CD19 CAR-T treatment failure, double target gene modification T cells can obtain preferable therapeutic effect.
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide it is a kind of target simultaneously CD19 and Gene modification T cell of EBNA1 and the preparation method and application thereof.
In order to achieve the object of the present invention, technical scheme is as follows：
In a first aspect, a kind of gene modification T cell for targeting CD19 and EBNA1 simultaneously of culture of the present invention.
The CAR for targeting CD19 is implemented in slow virus carrier and then is transferred to patient T cells by the present invention, makes the gene modification T cell can express the tumour cell of CD19 with targets identification.But only target treatment of the CAR in B cell source tumour of CD19 In easily lead to the low expression of tumour cell CD19 or even do not express, therefore easily lead to progression of disease or even recurrence.But due to part B Cell origin tumour can infect EBV, and the tumour cell for infecting EBV can express EBNA1, therefore the CAR for targeting CD19 is infected After peripheral blood in patients T cell, two cycle in vitro stimulations are carried out to it using the EBNA1 Antigenic Peptide of commercialization, to target CD19 CAR-T also can specific recognition EBNA1, the final gene modification T cell for targeting CD19 and EBNA1 simultaneously is expected have it is stronger Therapeutic effect.
Second aspect, the present invention provide the preparation method of above-mentioned double targeting T-cells：EBNA1 Antigenic Peptide is loaded into DC cell, Obtain the DC cell of Loading peptides；The CAR-T of the DC cell of Loading peptides and targeting CD19 is co-cultured, that is, is obtained simultaneously Target the gene modification T cell of CD19 and EBNA1.
First, the constructing plan of the CAR-T of targeting CD19 is：The CAR for targeting CD19 is implemented in slow virus carrier, in turn It is transferred to T cell.Specific step is as follows：
(1) nucleotide sequence of the CAR of synthesis expression targeting CD19, separately includes when synthesizing the gene at its both ends XbaI and SalI restriction enzyme site, and be loaded on plasmid；
(2) XbaI and SalI double digestion step (1) described plasmid, gel extraction target gene fragment are utilized；
(3) XbaI and SalI double digestion slow virus initial carrier, the carrier segments of gel extraction are utilized；
(4) carrier segments of the target gene fragment and step (3) recycling that are recycled step (2) using DNA ligase are connected Connect to get.
Since the efficiency of slow-virus infection periphery blood T cell is not generally high, in order to promote the CAR for targeting CD19 in T cell In can efficiently be expressed, the further preferred slow virus carrier of the present invention starts subcategory, best in the hope of playing T cell Efficiency of infection.Promoter EF1 α so that the slow virus carrier generate slow virus can with efficient infection periphery blood T cell, and And the CAR for targeting CD19 can be with high efficient expression in the T cell surface.
More specifically：The slow virus carrier carries the CAR gene of targeting CD19, first passes through transfection reagent transfection 293ft cell generates slow virus, then slow-virus infection periphery blood T cell, and then by the gene integration into T cell genome, To realize that the gene is expressed in T cell.
Further, the slow virus carrier preferably is selected from the slow virus carrier that promoter is EF1 α.In this way, then without to slow The promoter of viral vectors is replaced, the EF1 α promoter directly carried using slow virus carrier.Included EF1 α starting The slow virus carrier of son can be bought by commercial sources and be obtained.For example, in a specific embodiment of the invention, it is used from Slow virus carrier with EF1 α promoter is pCDH-EF1-Luc2-T2A-tdTomato, limited purchased from the vast clever biotechnology in Wuhan Company.
The amino acid sequence of the CAR of the targeting CD19 is as shown in SEQ ID NO.1, the nucleotide sequence of encoding gene As shown in SEQ ID NO.2.
Second, the CAR-T of the DC cell of Loading peptides and targeting CD19 are co-cultured, two are carried out by EBNA1 Antigenic Peptide Cycle in vitro stimulation, to the not only massive amplification gene modification T cell, but also makes the ratio of the T cell of specific recognition EBNA1 Significant increase.
More specifically：Dendritic cells (DC) load EBNA1-mix antigen (Human, MiltenyiBiotec), Then the CAR-T cell of itself and targeting CD19 are incubated for altogether, every 3 days culture mediums of the supplement comprising cell factor, and at one After week, the dendritic cells for loading above-mentioned Antigenic Peptide carry out total incubation with the T cell of stimulation one week again, then supplement every 3 days Culture medium comprising cell factor, and cultivate and complete after a week.Post-stimulatory CAR-T cell not only specific recognition twice CD19 has simultaneously more at high proportion can be with specific recognition EBNA1.
Third, the application that the present invention provides aforementioned slow virus carriers in antineoplaston and it is anti-swollen in preparation Application in tumor medicine.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified This field routine operation.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can be combined with each other, obtain specific embodiment party Formula.
The beneficial effects of the present invention are：A kind of gene of targets identification CD19 and EBNA1 simultaneously of culture of the present invention is repaired T cell is adornd, which can identify two target spots of CD19 and EBNA1 of the EBV positive tumor in B cell source simultaneously, therefore right In the patient of B cell source EBV positive tumor especially CD19 CAR-T treatment failure, double target gene modification T cells can be taken Obtain preferable therapeutic effect.Further, the preferred efficient promoter of the present invention, so that the CAR of targeting CD19 is in human T cells Middle high efficient expression.
Detailed description of the invention
Fig. 1 is the 4 kinds of promoter schematic diagrames for the high efficient expression CD19 CAR that the present invention attempts.
Fig. 2 is that the corresponding slow virus carrier of 4 kinds of promoters infects expression targeting CD19CAR-T cell after periphery blood T cell Percentage, wherein ordinate indicate expression target gene T cell percentage.
The final preferred slow virus carrier structural schematic diagram of Fig. 3.
Fig. 4 is the T cell quantity of specific recognition EBNA1 before and after two period stimulating CD19 CAR-T cell of EBNA1 Antigenic Peptide. ELISPOT test result, each point represent the T cell of a specific recognition EBNA1.
Fig. 5 is specific killing expression EBNA1 target cell before and after two period stimulating CD19 CAR-T cell of EBNA1 Antigenic Peptide Ratio.CD107 fragmentation test expresses the T cell ratio of the target cell of the T cell proportional representation killing expression EBNA1 of CD107.
Fig. 6 is while targeting the gene modification T cell (CD19&EBNA1 CAR-T) of CD19 and EBNA1, only targeting CD19 Gene modification T cell (CD19 CAR-T) and without gene modification T cell for the recognition effect figure of EBNA1 Antigenic Peptide；
Fig. 7 is while targeting the gene modification T cell (CD19&EBNA1CAR-T) of CD19 and EBNA1, only targeting CD19 Gene modification T cell (CD19 CAR-T) and without gene modification T cell for the recognition effect of Raji cell (expression CD19) Figure；
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
1 the present embodiment of embodiment is used for the construction method for illustrating to target CD19 CAR-T cell.
Initial carrier pCDH-EF1-Luc2-T2A-tdTomato：Purchased from Wuhan Miao Ling Biotechnology Co., Ltd.XbaI With SalI restriction endonuclease：Purchased from New England Biolabs (Beijing) LTD..
The construction method of targeting CD19 CAR-T cell includes the following steps：
(1) synthesis targets the nucleotide sequence of CD19CAR and separately includes XbaI and SalI restriction enzyme site at its both ends, and It is loaded on pUC57 carrier；
(2) XbaI and SalI double digestion includes the pUC57 carrier of target gene, gel extraction target gene fragment；
(3) XbaI and SalI double digestion initial carrier pCDH-EF1-Luc2-T2A-tdTomato is utilized, gel extraction is about The carrier segments of 6.5kb；
(4) with DNA ligase connection recycling target gene fragment and carrier segments to get.
(5) MSCV, PGK, CMV3 kind objective gene sequence are synthesized, by constructing successfully in ClaI and XbaI double digestion (4) Carrier, the carrier built in slow virus carrier that 3 kinds build and (4) is subjected to slow virus packaging respectively, and then feel Periphery blood T cell is contaminated, the antibody for targeting CD19CAR carries out streaming dyeing, is analyzed by flow cytometer.As a result such as table 1 With shown in Fig. 2.
It should be noted that working as use since pCDH-EF1-Luc2-T2A-tdTomato carrier carries promoter EF1 α When other promoters, need to introduce the restriction enzyme site sequence comprising ClaI and XbaI at the both ends of other promoter sequences first, so The nucleotide sequence synthesized afterwards by ClaI and XbaI double digestion, while using the two enzyme double digestions pCDH-EF1-Luc2- T2A-tdTomato carrier obtains the carrier segments of about 9kb, then by the promoter sequence after double digestion and the carrier after digestion Segment is attached by DNA ligase, so that building includes the slow virus carrier of different promoters.
CD19 CAR positive T cell ratio after the corresponding slow virus carrier infection periphery blood T cell of 14 kinds of promoters of table
By comparing experimental result, it is found that No. 4 carriers can be realized high efficient expression of the targeting CD19 CAR in T cell, And have been surprisingly found that its expression ratio is significantly higher than other promoters.
Therefore, the preferred EF1 α of the present invention is the slow virus promoter (Fig. 3) for targeting CD19 CAR, which carries out slow virus Then packaging infects T cell, i.e., the successfully CAR-T of building targeting CD19.
2 the present embodiment of embodiment is for illustrating the thin cultured and amplified in vitro of the CAR-T of specific recognition EBNA1
X-VIVO15 culture medium：Purchased from imperial husky (China) Investment Co., Ltd.GM-CSF and IL-4 cell factor：It is purchased from PeproTech company (U.S.).
The preparation of Dendritic Cells and the load of Antigenic Peptide
(1) it is resuspended fresh peripheral blood mononuclear cells with serum free medium X-VIVO15, adjustment cell concentration 2.5 × 106/ml；
(2) culture dish is put in 37 DEG C, 5%CO2In incubator, 90min is cultivated, keeps cell adherent.
(3) not adherent cell is collected in new 15ml sterile centrifugation tube, and the culture of T cell is used for after centrifugation；
(4) it is cleaned attached cell 2 times with X-VIVO15 serum free medium, then is carefully added into appropriate X-VIVO15 serum-free Culture medium continues to cultivate 60min, avoids blowing afloat adherent cell；
(5) not adherent cell is discarded, the DC culture medium containing 800U/ml GM-CSF and 400U/mlIL4 is replaced, is placed in 37 DEG C, 5%CO2It is cultivated in environment.
(6) the 3rd days, fresh DC cell culture medium is suitably supplemented, notices that operation is soft, avoids blowing afloat culture dish bottom surface Cell；
(7) the 6th days, the DC cell of inductive formation is collected, EBNA1 Antigenic Peptide is added, adjusts the final concentration of of Antigenic Peptide 10ug/ml, in 37 DEG C, 5%CO215-18h is co-cultured in incubator, stimulates the maturation of DC cell；It is being trained altogether with Autologous T cells Before supporting, with the DC cell of 40Gy radioactive ray vertical irradiation Loading peptides, for use.
The preparation of EBNA1 specific T-cells
(1) radioactive ray process is cleaned 2 times with 1 × PBS after the DC cell of Loading peptides, is resuspended in serum-free In X-VIVO culture medium；
(2) it stimulates for the first time：By the cultured CAR-T cell of embodiment 1 and DC cell, according to E:T=10:1 ratio, In 37 DEG C, 5%CO2It co-cultures, is denoted as the 1st day in incubator；
(3) the 4th days, it is complete suitably to supplement corresponding T cell for observation Antigenic Peptide first time post-stimulatory T cell growing state Full culture medium；
(4) the 7th days, T cell after Antigenic Peptide stimulation for the first time is collected, 1 × PBS is washed one time；
(5) it stimulates for second：Post-stimulatory T cell for the first time is taken, according to E:T=10:1 ratio, it is negative with maturation again The DC cell for carrying corresponding peptides co-cultures；
(6) the 10th days, second of post-stimulatory T cell growing state of observation suitably supplemented corresponding T cell and cultivates completely Base；
It (7) the 14th days, collects second of Antigenic Peptide 1 × 10 after stimulating6A T cell, and carry out cell phenotype and function Detection.
Fig. 6 is while targeting the gene modification T cell (CD19&EBNA1 CAR-T) of CD19 and EBNA1, only targeting CD19 Gene modification T cell (CD19 CAR-T) and without gene modification T cell and load EBNA1 Antigenic Peptide DC be incubated for altogether after, ELISA detects IFN-γ secretion situation, the results show that only CD19&EBNA1 CAR-T could specific recognition EBNA1 Antigenic Peptide.
Fig. 7 is while targeting the gene modification T cell (CD19&EBNA1 CAR-T) of CD19 and EBNA1, only targeting CD19 Gene modification T cell (CD19 CAR-T) and after being incubated for altogether without gene modification T cell and Raji cell (expression CD19), ELISA detects IFN-γ secretion situation, and CD19 CAR-T and CD19&EBNA1 CAR-T can specific recognition Raji as the result is shown Cell, it was demonstrated that CD19&EBNA1 CAR-T can express the cell of CD19 with specific recognition.
When understanding, after the dosage of above-described embodiment agents useful for same or raw material is carried out equal proportion expansion or is reduced Technical solution, it is substantially identical with above-described embodiment.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
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Beijing Inst of Tumor Prevention and Treatment
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Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
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Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly
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Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
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Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
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Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
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ctgcacaacc actatacaca gaagtccctg agcctgagcc tgggaaaaat gttctgggtc 1500
ctcgtcgtgg tgggaggcgt gctcgcttgc tactccctgc tcgtgaccgt cgccttcatc 1560
atcttctggg tgaagagagg caggaaaaag ctgctctata tcttcaagca gcccttcatg 1620
aggcccgtgc agacaacaca agaggaggac ggctgcagct gcagatttcc tgaggaggag 1680
gagggaggct gcgagctcag agtcaagttt agcaggagcg ctgacgcccc tgcctatcag 1740
cagggccaga accagctcta caatgagctg aacctgggaa ggagggaaga gtacgacgtg 1800
ctcgacaaaa gaaggggaag ggaccccgag atgggcggaa aacccagaag gaagaatcct 1860
caggagggcc tgtataatga gctgcagaag gacaagatgg ccgaggccta cagcgagatt 1920
ggcatgaaag gagagaggag aaggggcaaa ggccatgacg gactgtacca aggcctgtcc 1980
accgccacca aggataccta tgatgccctg cacatgcagg ctctgcctcc caggtga 2037

Claims (5)

1. a kind of gene modification T cell for targeting CD19 and EBNA1 simultaneously.
2. the preparation method of T cell as described in claim 1, which is characterized in that this method is：EBNA1 Antigenic Peptide is loaded DC cell obtains the DC cell of Loading peptides；The CAR-T of the DC cell of Loading peptides and targeting CD19 is co-cultured, i.e., Obtain while targeting the gene modification T cell of CD19 and EBNA1.
3. according to the method described in claim 2, it is characterized in that, the CAR-T of the targeting CD19 is by that will target CD19 CAR be implemented in and slow virus carrier and then be transferred to T cell and obtain.
4. according to the method described in claim 3, it is characterized in that, slow virus carrier preferably is selected from the slow virus that promoter is EF1 α Carrier.
5. T cell application in preparation of anti-tumor drugs as described in claim 1.
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