Coding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with gene, immune cell and application thereof

A chimeric antigen receptor, B7-H3 technology, applied in the field of genes, can solve the problems of lack of safe and effective specific tumor antigen targets, obstacles, and poor results

Inactive Publication Date: 2020-01-14
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, CAR-T cells targeting CD19 and CD22 have made significant progress in the treatment of hematological tumors, and the curative effect is remarkable, but the progress of

Method used

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  • Coding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with gene, immune cell and application thereof
  • Coding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with gene, immune cell and application thereof
  • Coding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with gene, immune cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The anti-B7-H3 chimeric antigen receptor coding gene at least contains artificial nucleotide sequences expressing IL15 and CCL21. It also includes antigen binding region, transmembrane structure region, co-stimulatory signal transduction region and T cell signal transduction region functional domain.

[0056] In this embodiment, the anti-B7-H3 chimeric antigen receptor includes sequentially linked

[0057] (1) artificial sequence of leader nucleic acid (SEQ ID NO.2)

[0058] (2) Anti-B7-H3 antigen antibody single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO.3)

[0059] (3) CD8 Hinge region nucleic acid artificial sequence (SEQ ID NO.4)

[0060] (4) CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO.5)

[0061] (5) CD226 intracellular nucleic acid artificial sequence (SEQ ID NO.6)

[0062] (6) CD3ζ intracellular region nucleic acid artificial sequence (SEQ ID NO.7)

[0063] (7) T2A-IL15-CCL21 nucleic acid artificial sequence...

Embodiment 2

[0066] Example of preparation of anti-B7-H3 chimeric antigen receptor coding gene.

[0067] The preparation method of the anti-B7-H3 chimeric antigen receptor coding gene of this embodiment comprises the following steps:

[0068] (1) According to the artificial nucleic acid sequence of the fusion gene fragment guider Leader, the artificial nucleic acid sequence of Anti-B7-H3, the artificial nucleic acid sequence of CD8, the artificial nucleic acid sequence of linker, the artificial nucleic acid sequence of CD8TM, and the artificial nucleic acid sequence of CD226 , CD3ζ nucleic acid artificial sequence, and T2A-IL15-CCL21 nucleic acid artificial sequence entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the entire expression cassette, insert it into pLent-EF1α-FH-CMV-GFP-P2A-Puro, and obtain pLent-EF1α-CAR(B7-H3)-IL15-CCL21;

[0069] (2) Carry out double enzyme digestion of pLent-EF1α-CAR(B7-H3)-IL15-CCL21, use agar electrophoresis to cut out the agar part of th...

Embodiment 3

[0079] Example of a plasmid with an anti-B7-H3 chimeric antigen receptor encoding gene.

[0080] A plasmid with an anti-B7-H3 chimeric antigen receptor coding gene is prepared by a method comprising the following steps: respectively according to the fusion gene fragment Leader-scFv(B7-H3)-CD8-CD226-4-1BB-CD3ζ-T2A- The sequence of Leader-scFv(CTLA4)-CD8-CD3ζ was inserted into the NotI-AsiSI site of pLent-EF1α-FH-CMV-GFP-P2A-Puro vector (Invitrogen), transformed into E.coli(DH5α), and identified by bacterial liquid PCR Finally, use the plasmid extraction kit of Qiagen company to extract the plasmid, after the plasmid is sequenced correctly, the recombinant plasmid with the correct sequencing result is named pLent-EF1α-CAR(B7-H3)-IL15-CCL21, as figure 1 shown.

[0081] In this embodiment, the more detailed steps are:

[0082] Insert the pLent-EF1α-FH-CMV-GFP-P2A-Puro vector (Invitrogen) NotI-AsiSI in the order of the fusion gene fragment Leader-scFv(Anti-B7-H3)-CD8-CD226-CD3ζ-T...

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Abstract

The invention discloses a coding gene of an anti-B7-H3 chimeric antigen receptor, a preparation method, a plasmid with the gene, an immune cell and an application thereof, and the coding gene of the anti-B7-H3 chimeric antigen receptor is characterized by at least comprising an artificial nucleotide sequence for expressing IL15 and CCL21. The coding gene of the anti-B7-H3 chimeric antigen receptorprovided by the invention enhances the killing effect of CIK cells on solid tumor cells.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to an anti-B7-H3 chimeric antigen receptor coding gene, a preparation method, a plasmid with the gene, immune cells and applications thereof. Background technique [0002] In the treatment of malignant tumors, traditional therapies include surgery, radiotherapy, chemotherapy, traditional Chinese medicine and immunotherapy. Immunotherapeutic approaches to anticancer treatments are currently in clinical trials around the world, such as leukemia or lymphoma. Two have been approved by the FDA for marketing. At present, CAR-T cells targeting CD19 and CD22 have made significant progress in the treatment of hematological tumors, and the curative effect is remarkable, but the progress of CAR-T cell therapy against solid tumors is slow and the effect is not good, and there is a lack of safe and effective specific tumor antigen targets is one of the major hindrances. [0003] Therefore, the ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/70C12N15/867C12N5/10
CPCC07K14/521C07K14/5443C07K14/7051C07K16/2827C07K2319/02C07K2319/03
Inventor 刘明录许淼冯建海金海锋张传鹏王亮
Owner SHANDONG XINRUI BIOTECH CO LTD
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