Preparation method and application of controllable CD20 chimeric antigen receptor modified T cell

A chimeric antigen receptor and cell technology, which is applied in the field of preparation of controllable CD20 chimeric antigen receptor modified T cells, can solve the problems of reducing expression and restriction, and achieve the effect of fast proliferation and broad tumor killing spectrum

Inactive Publication Date: 2017-11-24
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, tumor cells may also use "immune evasion" strategies to deal with the killing effect of T cells, such as reducing the expression of MHC class I molecules on the surface of tumor cells, so that they cannot form complexes with surface antigens, and thus cannot be detected by T cell receptors. identify
The complex regulation of T cell activation process and the immune evasion mechanism of tumor cells greatly limit the full play of the potential of T cells to kill tumor cells

Method used

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  • Preparation method and application of controllable CD20 chimeric antigen receptor modified T cell
  • Preparation method and application of controllable CD20 chimeric antigen receptor modified T cell
  • Preparation method and application of controllable CD20 chimeric antigen receptor modified T cell

Examples

Experimental program
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Effect test

Embodiment 1

[0025] The construction of embodiment 1pLent-C-GFP-CD20 expression vector

[0026] Leader-CD20-CD8-4-1BB-CD3ζ-T2A-HSV-TK module opinion figure 1 (See appendix SEQ ID NO.1 for the complete nucleic acid sequence).

[0027] Each module sequence of Leader-CD20-CD8-4-1BB-CD3ζ-T2A-HSV-TK

[0028] (1) Leader (SEQ ID NO.2)

[0029] (2) Humanized anti-human B-cell lymphoma CD20 single-chain antibody (SEQ ID NO.3)

[0030] (3) CD8Hinge region (SEQ ID NO.4)

[0031] (4) CD8 transmembrane region (SEQ ID NO.5)

[0032] (5) 4-1BB intracellular region (SEQ ID NO.6)

[0033] (6) CD3ζ intracellular region (SEQ ID NO.7)

[0034] (7) Self-cleaving polypeptide T2A (SEQ ID NO.8)

[0035] (8) HSV-TK sequence (SEQ ID NO.9)

[0036] According to the above sequence from (1)-(8), entrust Beijing Biomed Gene Technology Co., Ltd. to synthesize its entire expression cassette, and insert it into the pLent-C-GFP vector (purchased from Vigene Company) AsiSI-NotI site (see figure 2 ), transformed int...

Embodiment 2

[0037] Example 2 Preparation of pLent-C-GFP-CD20 modified T cells

[0038] 1. Preparation of heterogeneous T cells

[0039] Take 75ml of autologous peripheral blood from the patient, and separate peripheral blood mononuclear cells with TBD sample density separation medium (purchased from Tianjin Haoyang Huake Biology). After inducing culture for 24 hours with a medium (purchased from CORNING Company, 88-551-CM) containing 1000 IU / ml of recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical), 1500 IU / ml of recombinant interleukin 2 ( purchased from Shenyang Sansheng Pharmaceutical), 50ng / ml of OKT-3 and 5% of the patient's autologous plasma to induce further culture. Doubling liquid was added every three days, cultured until the 14th day, and the positive expression rate of CD3+ and CD56+ in T cells was detected by flow cytometry (CD3-FITC, CD16 / CD56-PE antibodies were purchased from BECKMAN, A07735). CD3+ positive rate>80%, CD3+CD56+ double positive rate...

Embodiment 3

[0044] Example 3: Research on the killing activity of CAR-T cells with a suicide gene system

[0045] The human B lymphoma cell line RAMOS was used as target cells, and the effector cells were CAR-T cells and T cells infected with empty lentivirus.

[0046] 1. Control of CAR-T cell activity by suicide gene system

[0047] The CAR-T cells were divided into 6×10 4 cells / ml inoculated in 96-well plate, 100ul per well, placed in 5% CO 2 , Cultivate in a 37°C incubator for 24 hours; add 10 ng of ganciclovir (Wuhan Haite Biopharmaceutical Co., Ltd.), once every 24 hours, a total of 3 times. CAR-T cells without ganciclovir were used as negative control. Detect the amount of remaining cells in the 96-well plate by the MTT method, the cell survival rate=(experimental group OD-blank group OD) / (control group OD-blank group OD), the control rate of the suicide gene system to the modified T cell activity=( 1-cell viability)×100%. The control rate of the suicide gene system on the acti...

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Abstract

The invention discloses a preparation method and an application of a controllable CD20 chimeric antigen receptor modified T cell. A Leader-CD20-CD8-4-1BB-CD3 zeta-T2A-HSV-TK gene segment is synthesized and inserted into a pLent-C-GFP vector, lentivirus carrying recombinant pLent-C-GFP-CD20 is packaged, a monocyte induced heterogeneous T lymphocyte is infected with the recombinant pLent-C-GFP-CD20 lentivirus, and the controllable CD20 chimeric antigen receptor modified T cell is obtained. The prepared controllable CD20 chimeric antigen receptor modified T cell has the characteristics of high proliferation speed, tumor recognition mechanism, wide tumor killing spectrum and the like.

Description

technical field [0001] The invention relates to the technical field of biology and new medicine, more specifically, a preparation method and application of controllable CD20 chimeric antigen receptor modified T cells. Background technique [0002] Cancer, that is, malignant tumors, is a disease with a high mortality rate of human beings second only to cardiovascular diseases. Leukemia is a malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, differentiation disorder, and apoptosis inhibition, and infiltrate other non-hematopoietic tissues and organs, while inhibiting normal hematopoietic function. Different degrees of clinical anemia , bleeding, infection, fever, and liver, spleen, lymph node enlargement, and bone pain. According to reports, the incidence of leukemia in various regions of my country occupies the sixth place...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867A61K35/17A61P35/00
CPCA61K35/17C12N15/86C12N2740/15043
Inventor 刘明录冯建海王立新姜夕锋张传鹏强邦明金海锋万磊韩庆梅
Owner SHANDONG XINRUI BIOTECH CO LTD
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