Chimeric antigen receptor and its gene and recombinant expression vector, CD19-CD20 dual targeting T cell and application thereof

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

Inactive Publication Date: 2019-02-01
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 CD20 dual-target Tropical T cells and their preparation methods and applications. Chimeric antigen receptor-modified T cells have stronger anti-tumor activity when treating B-cell malignant hematological tumors, and significantly reduce the risk of recurrence after treatment

Method used

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  • Chimeric antigen receptor and its gene and recombinant expression vector, CD19-CD20 dual targeting T cell and application thereof
  • Chimeric antigen receptor and its gene and recombinant expression vector, CD19-CD20 dual targeting T cell and application thereof
  • Chimeric antigen receptor and its gene and recombinant expression vector, CD19-CD20 dual targeting T cell and application thereof

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preparation example Construction

[0039] The preparation method of the lentiviral expression vector is not particularly limited, and can be various methods that can be imagined by those skilled in the art. Preferably, the preparation method of the lentiviral vector includes the following steps:

[0040] (1) Amplify the hinge and transmembrane domains of CD8, the intracellular signal domain of CD137, and the intracellular signal domain of CD3ζ from T cell cDNA, and clone them into the vector pRRLsin (starter of lentiviral expression vector) Sub CMV promoter was replaced with EF1a promoter), pRRLsin-CD8-CD137-CD3ζ was constructed;

[0041] (2) Synthesize the nucleotide sequence encoding CD8a signal peptide and CD20ScFv-L-CD19ScFv, and clone them into pRRLsin-CD8-CD137-CD3ζ, and obtain the correct sequence of lentiviral expression vector pRRLsin-CD20ScFv-L after sequencing verification -CD19ScFv-CD8-CD137-CD3ζ.

[0042] In step (1), the method for amplifying the hinge and transmembrane domains of CD8, the intracellular...

Embodiment 1

[0084] This example is used to illustrate the preparation of T cells

[0085] (1) Take human venous blood in a vacuum tube containing heparin. Using lymphocyte separation fluid, mononuclear cells (PBMCs) were obtained by density gradient centrifugation.

[0086] (2) After washing PBMCs three times, use X-VIVO 15 containing 0.5% by volume of human autologous serum TM Adjust the final cell concentration of serum-free T cell culture medium to 2×10 6 Cells / mL; Inoculate the cells in 75cm pre-coated with retronectin with a final concentration of 10μg / mL and 50ng / ml CD3 monoclonal antibody 2 Cell culture flask. Then add recombinant human interleukin 2 with a final concentration of 300 U / mL to the medium, and the CO at 37°C and saturated humidity of 5% 2 Cultivate in an incubator.

[0087] (3) On the fourth day of culture, transfer the cells to an uncoated culture flask, and add T cell culture medium X-VIVO 15 according to the number of cells grown every 2 days TM , The control cell co...

Embodiment 2

[0089] This example is used to illustrate the construction of lentiviral expression vector

[0090] (1) Preparation of T cell cDNA

[0091] Centrifuge the T cells cultured in Example 1, extract the total RNA of the cells with the total RNA extraction kit RNAiso Reagent, and store them at -80°C for later use. RevertAid, a reverse transcription kit for extracted total RNA TM First Strand cDNA Synthesis Kit reverse transcribed T cell cDNA, stored at -20℃ for later use.

[0092] (2) Preparation of lentiviral expression vector (pRRLsin-CD20ScFv-L-CD19ScFv-CD8a-CD137-CD3ζ)

[0093] Design and synthesize the following primer sequences (wherein, the underlined mark is the protective base, and the box is the restriction site):

[0094]

[0095] Using the T cell cDNA in step (1) as a template, PCR amplification was performed with primers P1 and P2 to obtain the hinge region and transmembrane region of CD8 with a length of 227 bp. The nucleotide sequence is shown in SEQ ID NO. 4, and the two e...

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Abstract

The invention belongs to the field of tumor biological products, in particular to a chimeric antigen receptor and its gene and a recombinant expression vector, CD19-CD20 dual targeting T cells and anapplication thereof. The chimeric antigen receptor is CD20ScFv-L-CD19ScFv-CD8-CD137-CD3 zeta, which includes CD20ScFv, ligation peptide L, CD19ScFv, a hinge region and a transmembrane region of CD8, an intracellular signal domain of CD137, and an intracellular signal domain of CD3 zeta which are connected in series in order. In the treatment of B cell line hematological malignancy, the chimeric antigen receptor-modified T cells of the present invention can not only specifically recognize tumor cells expressed by a single target of CD19 antigen or CD20 antigen, but also to recognize tumor cellsco-expressed by two targets of CD19 antigen and CD20 antigen, compared with single target CAR T, dual-target CAR T has stronger anti-tumor activity, which avoids immune escape of tumor cells with lowabundance antigen expression, and the risk of recurrence 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, CD19-CD20 dual-targeting T cell and application thereof. Background technique [0002] The incidence of hematological malignancies represented by acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin’s lymphoma, etc., has a rising trend in my country, and young and middle-aged adults are the main disease group, which seriously threatens our population Health and life. Although chemotherapy, radiotherapy, hematopoietic stem cell transplantation, and molecular targeted drugs and therapeutic antibodies are the main treatment methods that have greatly improved the remission rate of the disease and prolonged the survival period of patients, 20-50% of patients Relapse after remission, and about 30% of patients have primary resistance to the existing methods of inducing remission. In r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61K35/17A61P35/00A61P35/02C07K14/7051C07K14/70517C07K14/70578C07K16/2803C07K16/2887C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2501/2302C12N2501/515C12N2510/00C12N2533/56C12N2740/15043C12N2800/107
Inventor 伍志强韩为东王瑶佟川张文英王晓慧
Owner GENERAL HOSPITAL OF PLA
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