Chimeric antigen receptor, coding gene, expression vector and application thereof

A technology of chimeric antigen receptors and coding genes, which is applied in the field of chimeric antigen receptors, expression vectors, and coding genes to achieve the effects of enhancing specificity, enhancing killing activity, and enhancing activity

Active Publication Date: 2014-12-03
SHANGHAI YAKE BIOTECHNOLOGY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a chimeric antigen receptor, coding gene, expression vector and application thereof, aiming at solving the problems existing in the existing chimeric antigen receptor

Method used

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  • Chimeric antigen receptor, coding gene, expression vector and application thereof
  • Chimeric antigen receptor, coding gene, expression vector and application thereof
  • Chimeric antigen receptor, coding gene, expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Taking carcinoembryonic antigen (CEA) as an example, the CEA-specific ScFv (CEA)-CD8α-CD3ζ-2A-CD80-2A-4-1BBL gene fragment was obtained through in vitro synthesis and molecular cloning methods. Wherein, the nucleotide sequence of the ScFv (CEA) gene includes the signal leader region, the V H Fragment, Linker(GGGGS)3 and V LFragment, the nucleotide sequence of which is shown in SEQ NO.1-4. The nucleotide sequence of the CD8α transmembrane region gene is shown in SEQ NO.5. The nucleotide sequence of the CD3ζ signal region gene is shown in SEQ NO.6. The nucleotide sequence of the 2A-CD80-2A-4-1BBL gene sequentially includes a T2A fragment, a CD80 fragment, a F2A fragment, and a 4-1BBL fragment, and its nucleotide sequence is shown in SEQ NO.7-10.

Embodiment 2

[0036] The scFv (CEA)-CD8α-CD3ζ-2A-CD80-2A-4-1BBL gene fragment was introduced into the lentiviral vector pRRL to obtain a lentiviral vector carrying the Super-CAR gene: the ScFv-CD8α-CD3ζ- The 2A-CD80-2A-4-1BBL gene fragment and the lentiviral vector pRRL were double digested with the same restriction endonuclease to recover the DNA fragments; the recovered DNA fragments were ligated by ligase.

[0037] Similarly, use the same method to construct lentiviral vectors of the first, second and third generation CARs with the CEA single-chain antibody gene, such as figure 1 As shown, among them, 1 st G CAR lentiviral vector is the first generation chimeric antigen receptor, 2 nd G CAR (CD28) is a second-generation chimeric antigen receptor carrying CD28 co-stimulatory molecules, 2 nd G CAR (4-1BB) is a second-generation chimeric antigen receptor carrying 4-1BB co-stimulatory molecules, 3 rd G CAR is the third-generation chimeric antigen receptor, and Super-CAR is a novel chi...

Embodiment 3

[0042] The lentiviral vectors constructed above carrying different CAR genes were transiently transfected into 293T cells. After 48 h, the cells were lysed, and the expression of the CD3ζ intracellular signal region was detected by Western blot. The results were as follows: image 3 shown. Among them, 1: 293T cells transfected with empty vector; 2: transfected with 1 st G CAR vector cells; 3: transfection 2 nd Cells with G CAR (CD28) vector; 4: transfection 2 nd Cells with G CAR (4-1BB) vector; 5: transfection 3 rd G CAR vector cells; 6: cells transfected with Super-CAR vectors. 293T cells themselves do not express CD3ζ. After transfection, the intracellular signal region of CD3ζ that meets the expected size can be detected, and the size is correct, indicating that the recombinant lentiviral vector after transfection is correctly expressed in human cells.

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Abstract

The invention discloses a chimeric antigen receptor, a coding gene, an expression vector and application thereof. The chimeric antigen receptor comprises a single chain variable fragment, a CD8 alpha transmembrane domain, a CD3 zeta intracellular signal domain, a first co-stimulator ligand and a second co-stimulator ligand which are in successive tandem connection, wherein the CD8 alpha transmembrane domain is used for connecting the single chain variable fragment to a cell membrane, 2A short peptide is arranged between and in tandem connection with the CD3 zeta intracellular signal domain and the first co-stimulator ligand, and another 2A short peptide is arranged between and in tandem connection with the first co-stimulator ligand and the second co-stimulator ligand. According to the chimeric antigen receptor provided by the invention, the 2A short peptide is used to connect the chimeric antigen receptor with the two co-stimulator ligands, so the two co-stimulator ligands are expressed on the surface of an immune cell at the same time; once the chimeric antigen receptor specifically binds with antigen, the two co-stimulator ligands rapidly migrate to synapses formed by the combination of cells, so activity of immune cells is effectively enhanced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chimeric antigen receptor, coding gene, expression vector and application thereof. Background technique [0002] Chimeric antigen receptor (chimeric antigen receptor, CAR) is composed of single-chain variable region fragment (Single Chain Variable Fragment, ScFv) of monoclonal antibody connected with the signal region of T cells. Chimeric antigen receptors can be divided into three generations according to the number of intracellular signal domains. The intracellular part of the first-generation chimeric antigen receptor only contains a CD3ζ intracellular signaling region, so the modified immune cells cannot be effectively activated and the action time is relatively short. The second-generation chimeric antigen receptors add a co-stimulatory molecule receptor signaling region before the CD3ζ intracellular signaling region, such as CD28, 4-1BB, OX40, etc., although the clinical per...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10A61K35/14A61P35/00A61P31/20A61P1/16A61P31/00
Inventor 张鸿声
Owner SHANGHAI YAKE BIOTECHNOLOGY LTD
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