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Chimeric antigen receptor, gene of chimeric antigen receptor, recombinant expression vector of chimeric antigen receptor, CD22-CD19 double-targeting T cell and application of cell

A technology of chimeric antigen receptors and expression vectors, which is applied in the field of tumor biological products, can solve the problems of high recurrence risk, low efficiency, and reduce recurrence risk, and achieve strong anti-tumor activity, avoid immune escape, and reduce recurrence risk.

Active Publication Date: 2019-04-26
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the defects of low efficiency and high risk of recurrence of CAR T in the process of tumor treatment in the prior art, and provide a chimeric antigen receptor and its gene and recombinant expression vector, engineered CD19 and CD22 dual-target Tropical T cells and their preparation methods and applications, chimeric antigen receptor modified T cells have stronger anti-tumor activity in the treatment of B-cell malignant hematological tumors, and significantly reduce the risk of recurrence after treatment

Method used

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  • Chimeric antigen receptor, gene of chimeric antigen receptor, recombinant expression vector of chimeric antigen receptor, CD22-CD19 double-targeting T cell and application of cell
  • Chimeric antigen receptor, gene of chimeric antigen receptor, recombinant expression vector of chimeric antigen receptor, CD22-CD19 double-targeting T cell and application of cell
  • Chimeric antigen receptor, gene of chimeric antigen receptor, recombinant expression vector of chimeric antigen receptor, CD22-CD19 double-targeting T cell and application of cell

Examples

Experimental program
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preparation example Construction

[0037] There is no particular limitation on the preparation method of the lentiviral expression vector, which can be various methods conceivable by those skilled in the art. Preferably, the preparation method of the lentiviral vector comprises the following steps:

[0038] (1) Amplify the hinge region and transmembrane domain of CD8, the intracellular signaling domain of CD137, and the intracellular signaling domain of CD3ζ from T cell cDNA, and clone them into the vector pRRLsin (the promoter of the lentiviral expression vector Substitute CMV promoter was replaced by EF1a promoter), and pRRLsin-CD8-CD137-CD3ζ was constructed;

[0039] (2) Synthesize the nucleotide sequence encoding CD8a signal peptide and encoding CD22ScFv-L-CD19ScFv, and clone it into pRRLsin-CD8-CD137-CD3ζ, and obtain the correct sequence lentiviral expression vector pRRLsin-CD22ScFv-L after sequencing verification - CD19ScFv-CD8-CD137-CD3ζ.

[0040] In step (1), there is no particular limitation on the me...

Embodiment 1

[0083] This example is used to illustrate the preparation of T cells

[0084] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.

[0085] (2) After PBMCs were washed three times, X-VIVO 15 containing 0.5% by volume of human autologous serum was used to TM Serum-free T cell culture medium adjusted the final cell concentration to 2×10 6 cells / mL; the cells were inoculated in a 75cm 2 in cell culture flasks. Then add recombinant human interleukin 2 with a final concentration of 300 U / mL to the culture medium, at 37°C, with a saturated humidity of 5% CO 2 cultured in an incubator.

[0086] (3) On the 4th day of culture, transfer the cells to an uncoated culture flask, and add T cell culture medium X-VIVO 15 every 2 days according to the number of cells growing TM , the control cell concentration was 1×10 8 cells / mL, and added recombinant human interle...

Embodiment 2

[0088] This example is used to illustrate the construction of lentiviral expression vector

[0089] (1) Preparation of T cell cDNA

[0090] The T cells cultured in Example 1 were centrifuged, and the total RNA of the cells was extracted with RNAiso Reagent, a total RNA extraction kit, and stored at -80°C for future use. Extracted total RNA with RevertAid Reverse Transcription Kit TM First StrandcDNASynthesis Kit reverse-transcribed T cell cDNA and stored at -22°C for future use.

[0091] (2) Preparation of lentiviral expression vector (pRRLsin-CD22ScFv-L-CD19ScFv-CD8a-CD137-CD3ζ)

[0092] The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

[0093]

[0094] Using the T cell cDNA in step (1) as a template, PCR amplification was carried out with primers P1 and P2 to obtain the hinge region and transmembrane region of CD8 with a length of 227bp. The nucleotide seque...

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Abstract

The invention belongs to the field of tumor biological products, and particularly relates to an chimeric antigen receptor, a gene of the chimeric antigen receptor, the recombinant expression vector ofthe chimeric antigen receptor, a CD22-CD19 double-targeting T cell and application of the T cell. The chimeric antigen receptor is CD22ScFv-L-CD19ScFv-CD8-CD137-CD3zeta and comprises CD22ScFv, connecting peptide L, CD19ScFv, a hinge region and a transmembrane region of CD8, an intracellular signal structural domain of CD137 and an intracellular signal structural domain of the CD3zeta which are connected in series. According to the chimeric antigen receptor, the gene of the chimeric antigen receptor, the recombinant expression vector of the chimeric antigen receptor, the CD22-CD19 double-targeting T cell and the application of the T cell, when B-cell line hematological malignant tumors are treated, the T cell modified by the chimeric antigen receptor not only can specifically recognize a tumor cell which is only expressed by a single target spot of a CD19 antigen or a CD22 antigen, but also can recognize tumor cells co-expressed by the two target spots of the CD19 antigen and the CD22antigen, compared with the single target spot CAR T, the double target spots CAR T have stronger anti-tumor activity, so that the immune escape of the tumor cells expressed by the low-abundance antigen is avoided, and therefore the recurrence risk is reduced.

Description

technical field [0001] The invention belongs to the field of tumor biological products, and specifically relates to a chimeric antigen receptor and its gene and recombinant expression vector, CD22-CD19 dual-targeting T cells and applications thereof. Background technique [0002] The incidence of hematological malignancies represented by acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin's lymphoma in my country is on the rise, and young and middle-aged people are the main affected groups, seriously threatening the population of our country. health and life. Although chemotherapy, radiotherapy, hematopoietic stem cell transplantation, and molecular targeted drugs and therapeutic antibodies have greatly improved the remission rate of the disease and prolonged the survival of patients, 20-50% of patients Relapse after remission, and about 30% of patients have primary resistance to the existing remission induction methods. For this situation, it is necessary...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00A61P35/02
CPCA61P35/00A61P35/02A61K35/17C12N5/0636C12N15/86C07K14/7051C07K14/70517C07K14/70578C07K16/2803C07K2317/31C07K2319/02C07K2319/33C07K2317/622C07K2319/00C07K2319/74C12N2510/00C12N2740/15043C12N2800/107
Inventor 伍志强韩为东王瑶代汉仁贾鹤静王晓慧
Owner GENERAL HOSPITAL OF PLA
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