PD1-knockout chimeric antigen receptor T cell targeting EGFR, preparation method and application thereof

A chimeric antigen receptor and targeting technology, applied in the field of medical biology

Inactive Publication Date: 2019-06-04
SHENZHEN BINDEBIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on chimeric antigen receptor T cells targeting EGFR that can prevent tumor cells from escaping immune surveillance

Method used

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  • PD1-knockout chimeric antigen receptor T cell targeting EGFR, preparation method and application thereof
  • PD1-knockout chimeric antigen receptor T cell targeting EGFR, preparation method and application thereof
  • PD1-knockout chimeric antigen receptor T cell targeting EGFR, preparation method and application thereof

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Embodiment 1

[0093] A method for preparing chimeric antigen receptor T cells targeting EGFR that knocks out PD1, comprising the following steps:

[0094] (1) Prepare the gene sequence of chimeric antigen receptor CAR-EGFR targeting EGFR

[0095] The genes encoding the signal peptide, the single-chain antibody targeting EGFR, the hinge region of CD8α, the transmembrane region of CD8, the signal region of 4-1BB and the signal region of CD3ζ were prepared respectively, and the genes encoding the signal peptide were shown in SEQ ID NO: 8 The coding gene of the single-chain antibody targeting EGFR is shown in SEQ ID NO: 5, the coding gene of the CD8 α hinge region is shown in SEQ ID NO: 10, and the coding gene of the CD8 transmembrane region As shown in SEQ ID NO: 12, the coding gene of the 4-1BB signal region is shown in SEQ ID NO: 14, and the coding gene of the CD3ζ signal region is shown in SEQ ID NO: 16, wherein the targeting The single-chain antibody to EGFR is a humanized single-chain an...

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PUM

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Abstract

The invention provides a preparation method PD1-knockout chimeric antigen receptor T cell targeting EGFR. The method comprises: linking an encoding gene of the chimeric antigen receptor CAR-EGFR and targeting EGFR to a vector, and constructing the recombinant lentivirus, performing transfection on CD3 positive T lymphocytes, and performing knockout of the PD1 gene of the transfected T cells to obtain the PD1-knockout chimeric antigen receptor T cell targeting EGFR. The knockout of the PD1 gene is realized by the chimeric antigen receptor T cell, which is in favor of amplification of T cells inthe patient, and avoids the escape of immunosurveillance of the tumor cells, and the T cell has the performance of killing tumor cells with high efficiency and specificity, which does not cause damage to normal cells. At the same time, the chimeric antigen receptor T cell targeting EGFR prepared by a humanized single-chain antibody can better maintain the viability and lethality of chimeric antigen receptor T cells.

Description

technical field [0001] The present invention relates to the field of medical biology, in particular to a chimeric antigen receptor T cell targeting EGFR knocked out of PD1 and its preparation method and application. Background technique [0002] Human epidermal growth factor receptor (EGFR) is the main receptor for human epidermal growth factor. Upon activation, EGFR undergoes homodimerization or heterodimerization with other members of the human epithelial growth factor receptor family, thereby inducing autophosphorylation of EGFR intracellular structures and activating downstream pathways such as mitogen activation The protein kinase (MAPK) pathway, etc., and mediate cell expansion. Studies have shown that EGFR is a cell surface antigen that is expressed or abnormally expressed in various solid tumor cells such as lung cancer, but weakly expressed in ordinary cells. EGFR is related to the proliferation of tumor cells, tumor invasion, metastasis and inhibition of apoptosi...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/867C12N15/90C12N5/10A61P35/00
Inventor 曾滢杨忠华
Owner SHENZHEN BINDEBIOTECH CO LTD
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