Chimeric antigen receptor targeting HLA-A, encoding gene, CAR-Tregs cells, and preparation method and application of CAR-Tregs cells
A chimeric antigen receptor and HLA-A technology, applied in the field of molecular genes, can solve the problems of increased risk of tumors and infections, and low specificity.
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[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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