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Chimeric antigen receptor targeting HLA-A, coding gene, CAR-Tregs cell and preparation method and application thereof

A chimeric antigen receptor, HLA-A technology, applied in the field of molecular genes, can solve problems such as low specificity, increased risk of tumors and infections, achieve good binding effect, inhibit specific immune responses in the body, and improve survival. The effect of quality and time

Active Publication Date: 2021-05-28
CHENGDU SHIKANGMEI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of immunosuppressants at this stage can effectively reduce the risk of rejection and improve the survival rate of grafts, because it reduces the overall immune function of the body, the specificity is not high, which leads to an increase in the risk of tumors, infections, etc.
At the same time, because immunosuppressants have certain drug toxicity, long-term use is likely to cause some chronic diseases, such as chronic kidney injury, hyperuricemia, etc.

Method used

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  • Chimeric antigen receptor targeting HLA-A, coding gene, CAR-Tregs cell and preparation method and application thereof
  • Chimeric antigen receptor targeting HLA-A, coding gene, CAR-Tregs cell and preparation method and application thereof
  • Chimeric antigen receptor targeting HLA-A, coding gene, CAR-Tregs cell and preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0057] The present invention also provides a CAR-Tregs cell, and the CAR-Tregs cell is the Treg cell modified by the above-mentioned chimeric antigen receptor. The present invention also provides a method for preparing the above-mentioned CAR-Tregs cells, comprising: transfecting the chimeric antigen receptor into Treg cells for expression through its encoded nucleic acid sequence.

[0058] Preferably, the preparation method specifically includes:

[0059] (1) Construction of a chimeric antigen receptor lentiviral expression plasmid targeting HLA-A;

[0060] (2) Preparation of chimeric antigen receptor lentivirus targeting HLA-A;

[0061] (3) Transfect Treg cells with the chimeric antigen receptor lentivirus targeting HLA-A prepared in step (2).

[0062] The present invention provides the application of the above-mentioned CAR-Tregs cells in the preparation of drugs for treating or preventing transplant rejection and graft-versus-host disease, or in the preparation of drugs ...

Embodiment 1

[0065] 1. Construction of lentiviral expression plasmid

[0066] 1. Preparation of coding gene sequence of chimeric antigen receptor

[0067] Preparation of CD8 signal peptide, single-chain antibody targeting HLA-A11, CD8α hinge region, CD8 transmembrane region, 4-1BB hinge region, 4-1BB transmembrane region, 4-1BB intracellular signaling domain and CD3ζ signaling domain coding genes;

[0068] The single-chain antibody targeting HLA-A11 includes a tandem HLA-A11 single-chain antibody heavy chain VH, a connecting peptide, and an HLA-A11 single-chain antibody light chain VL; the HLA-A11 single-chain antibody heavy chain VH is located in the target HLA - the N-terminal of the single-chain antibody of A11, the light chain VL of the HLA-A11 single-chain antibody is located at the C-terminal of the single-chain antibody targeting the HLA-A11; the gene encoding the heavy chain VH of the HLA-A11 single-chain antibody is as shown in SEQ ID NO The nucleotide sequence shown in .1, the ...

Embodiment 2

[0118] The only difference between this example and Example 1 is that the HLA-A33-targeting single-chain antibody is used to replace the HLA-A11-targeting single-chain antibody, and finally HLA-A33-CAR-Tregs are obtained.

[0119] Wherein, the single-chain antibody targeting HLA-A33 includes a tandem HLA-A33 single-chain antibody heavy chain VH, a connecting peptide, and an HLA-A33 single-chain antibody light chain VL; the HLA-A33 single-chain antibody heavy chain VH is located at the target To the N-terminus of the single-chain antibody of HLA-A33, the light chain VL of the HLA-A33 single-chain antibody is located at the C-terminus of the single-chain antibody targeting the HLA-A33; the gene encoding the heavy chain VH of the HLA-A33 single-chain antibody is as shown in SEQ The nucleotide sequence shown in ID NO.3, the nucleotide sequence shown in SEQ ID NO.4 of the HLA-A33 single-chain antibody light chain VL coding gene; the connecting peptide coding gene is shown in SEQ ID ...

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Abstract

The invention discloses a chimeric antigen receptor targeting HLA-A, a coding gene, a recombinant expression vector, a CAR-Tregs cell and a preparation method and application thereof. The chimeric antigen receptor comprises a signal peptide, an antigen binding structural domain, a hinge region, a transmembrane region, a costimulatory factor and an intracellular signal transduction domain. The antigen binding domain is a binding structural domain of an anti-HLA-A11 antibody or an anti-HLA-A33 antibody, and the CAR-Tregs cell is a Treg cell modified by a chimeric antigen receptor targeting HLA-A11 or HLA-A33, can inhibit specific immune response, and can be used for inhibiting rejection reaction after a solid organ transplantation operation.

Description

technical field [0001] The invention relates to the field of molecular gene technology, in particular to a chimeric antigen receptor targeting HLA-A, a coding gene, a recombinant expression vector, CAR-Tregs cells, a preparation method, and an application. Background technique [0002] There are approximately 300,000 solid organ transplant recipients in China, who face a huge risk of rejection after surgery. Although the use of immunosuppressants at this stage can effectively reduce the risk of rejection and improve the survival rate of grafts, because it reduces the overall immune function of the body and has low specificity, it leads to an increase in the risk of tumors and infections. At the same time, because immunosuppressants have certain drug toxicity, long-term use is likely to cause some chronic diseases, such as chronic kidney injury, hyperuricemia and so on. Contents of the invention [0003] In view of this, the present application provides a chimeric antigen ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C12N15/62C12N15/867A61K35/17A61P37/06
CPCC07K16/2833C07K14/7051C12N15/86A61K35/17A61P37/06C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2740/15043C12N5/0637
Inventor 魏亮
Owner CHENGDU SHIKANGMEI BIOTECHNOLOGY CO LTD
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