Anti-EpCAM chimeric antigen receptor encoding gene, preparation method, plasmid and immune cell with gene and application of encoding gene

A technology of chimeric antigen receptor and coding gene, applied in the field of genes

Inactive Publication Date: 2018-11-27
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Several CAR-Ts are currently approved for the treatment of liquid malignancies, but for solid tumors, it is difficult to find an effective method to solve the problems that occur in patients, especially digestive system tumors

Method used

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  • Anti-EpCAM chimeric antigen receptor encoding gene, preparation method, plasmid and immune cell with gene and application of encoding gene
  • Anti-EpCAM chimeric antigen receptor encoding gene, preparation method, plasmid and immune cell with gene and application of encoding gene
  • Anti-EpCAM chimeric antigen receptor encoding gene, preparation method, plasmid and immune cell with gene and application of encoding gene

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Experimental program
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Embodiment 1

[0064] The anti-EpCAM chimeric antigen receptor coding gene, wherein at least includes the EpCAM binding region and the CTLA4 binding region, and simultaneously includes the T2A nucleic acid artificial sequence between the two; also includes a transmembrane-stimulatory domain; the said The transmembrane-stimulatory domain is selected from CD8, CD27, CD28, CD226, 4-1BB (CD137), CD134 (OX40), all or partial fragments of ICOS molecules.

[0065] In this embodiment, the transmembrane-stimulatory domain is described by taking CD226 and 4-1BB as examples.

[0066] Such as figure 1As shown, the anti-EpCAM chimeric antigen receptor coding gene of the present embodiment includes the Leader nucleic acid artificial sequence (SEQ ID NO.2) connected in sequence, the EpCAM binding region nucleic acid artificial sequence (SEQ ID NO.3), and the CD8 Hinge region Nucleic acid artificial sequence (SEQ ID NO.5), CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO.6), CD226 intra...

Embodiment 2

[0068] Example of preparation of anti-EpCAM chimeric antigen receptor coding gene.

[0069] The preparation method of the anti-EpCAM chimeric antigen receptor coding gene of the present embodiment comprises the following steps:

[0070] According to the order of the fusion gene fragment Leader-scFv (EpCAM)-CD8-CD226-4-1BB-CD3ζ-T2A-Leader-scFv (CTLA4)-CD8-CD3ζ, we commissioned Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the entire expression box, inserted into the T vector pUC57 to obtain pUC57-scFv(EpCAM)-T2A-scFv(CTLA4).

[0071] (2) Carry out double digestion of pUC57-scFv(EpCAM)-T2A-scFv(CTLA4), use agar gel electrophoresis to cut out the agar part containing the scFv(EpCAM)-T2A-scFv(CTLA4) DNA fragment, pass through the sol solution Treat, pass through the DF column and discard the filtrate, rinse the DF column, empty, elute the DF column, collect the centrifuge, and obtain the purified scFv (EpCAM)-T2A-scFv (CTLA4) DNA fragment, which is the anti-EpCAM chime...

Embodiment 3

[0081] Example of a plasmid with an anti-EpCAM chimeric antigen receptor encoding gene.

[0082] The plasmid with the anti-EpCAM chimeric antigen receptor coding gene is prepared by a method comprising the following steps: respectively according to the fusion gene fragment Leader-scFv (EpCAM)-CD8-CD226-4-1BB-CD3ζ-T2A-Leader-scFv ( The sequence of CTLA4)-CD8-CD3ζ was inserted into the NotI-AsiSI site of the pLent-C-GFP carrier, and transformed into E.coli Top10. After identification by bacterial liquid PCR, the plasmid was extracted using a plasmid extraction kit. After the plasmid was sequenced correctly, the The recombinant plasmid with correct sequencing results is named pLent-scFv(EpCAM)-T2A-scFv(CTLA4), such as figure 2 shown.

[0083] In this embodiment, the more detailed steps are:

[0084] Insert the NotI-AsiSI site of the pLent-C-GFP vector in the order of the fusion gene fragment Leader-scFv (EpCAM)-CD8-CD226-4-1BB-CD3ζ-T2A-Leader-scFv (CTLA4)-CD8-CD3ζ respectively...

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Abstract

The invention discloses an anti-EpCAM chimeric antigen receptor encoding gene, a preparation method, plasmid and immune cells with the gene and application of the encoding gene. The encoding gene at least comprises an EpCAM bond zone and a CTLA4 bond zone, and comprises a T2A nucleic acid artificial sequence between the two bond zones as well; the anti-EpCAM chimeric antigen receptor encoding genealso comprises a transmembrane-stimulation structure domain; and the transmembrane-stimulation structure domain is selected from all or partial segments of CD8, CD27, CD28, CD226, 4-1BB, OX40 and ICOS molecules. By adopting the anti-EpCAM chimeric antigen receptor encoding gene disclosed by the invention, the killing effects of T cells upon tumor cells can be improved.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to an anti-EpCAM chimeric antigen receptor coding gene, a preparation method, a plasmid with the gene, immune cells and applications thereof. Background technique [0002] EpCAM (Epithelial cell adhesion molecule) is a glycoprotein expressed on the surface of some normal human epithelial cells and most malignant epithelial tumor cells, and has a strong antigenic epitope. EpCAM gene can regulate the functions of cell proliferation, differentiation and migration. On the other hand, it can promote Th2 differentiation and tumor immune escape function, and can block the major histocompatibility complex II-restricted antigen presentation of dendritic cells and promote tumorigenesis. . In cancer, EpCAM can switch between two functions, in the first case, EpCAM prevents strong cell adhesion and thus promotes cell migration and metastasis. In the second case, EpCAM mediates cell adhesion, th...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85C12N5/10A61P35/00
CPCA61P35/00C07K14/705C07K14/7051C07K2319/03C12N5/0636C12N15/85
Inventor 刘明录刘敏王立新万磊冯建海卢永灿王亮
Owner SHANDONG XINRUI BIOTECH CO LTD
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