CAR-T cell drug targeting prostate cancer secreting IL-23 antibody
A technology for IL-23 and prostate cancer, applied in the field of immunocytology, can solve problems such as poor prognosis, side effects of bone health, and unmet medical needs for treatment
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Embodiment 1
[0068] Construction of a plasmid vector containing an expression cassette expressing a chimeric antigen receptor and a single-chain antibody specifically binding to IL-23, and the structure and positional relationship of each element on the expression cassette for reference figure 1 , plasmid vector backbone reference figure 2 .
[0069] Specific steps are as follows:
[0070] The first expression cassette (PSMA-CAR) was synthesized by GenScript to express the chimeric antigen receptor targeting prostate-specific membrane antigen. The first expression cassette (PSMA-CAR) includes: CD8α signal peptide, PSMA single-chain antibody heavy chain variable region , Linker1, PSMA scFv light chain, CD8 hinge region, CD8α transmembrane domain, intracellular costimulatory element of 4-1BB and intracellular domain of CD3ζ ( figure 1 In A), the above sequences are connected sequentially, and the Kozac sequence and the corresponding enzyme cutting site are introduced at the front end. Th...
Embodiment 2
[0112] (1) Construction of viruses expressing chimeric antigen receptors and anti-IL-23 scFv
[0113] The method is as follows: use Escherichia coli to amplify the above-mentioned IL-23ab-CAR plasmid and the lentiviral packaging helper plasmid psPAX2 (see Figure 8 ) and pMD2.G (see Figure 9 ), after the plasmid was extracted, agarose gel electrophoresis and sequencing were performed to identify the correctness of the plasmid. Select the 293T cells in good condition and the first generation as the lentivirus packaging cells, and use the transfection reagent PEI to transfect the 293T cells with the above three plasmids. The transfection is completed in a 10cm culture dish with a total system of 10mL, and the cell transfection mixture in each dish should be prepared into a 1mL system with serum-free DMEM, so that psPAX2 plasmid: pMD2.G plasmid: IL-23ab-CAR plasmid: PEI=5μg: 3 μg: 5 μg: 72 μl, mix the transfection mixture at room temperature, let it stand for 20 minutes, then ...
Embodiment 3
[0120] (1) Construction of T cells expressing chimeric antigen receptors and anti-IL-23 scFv
[0121] Methods as below:
[0122] Use lymphatic separation fluid to separate PBMC from human blood, and then use CD4 and CD8 magnetic bead sorting method to separate T cells. After being activated by CD3 / CD28 complex for 48 hours, the packaged PSMA-CAR and IL-23ab- CAR virus was infected by centrifugation at MOI=10 for 2h, and replaced with fresh medium (XVIVO+10%FBS+IL-2) after 24h. The two kinds of CART cells were named PSMA-CART cells and IL-23ab-CART cells respectively.
[0123] After 48 hours of infection, the CAR expression levels of the above two CARTs were detected according to the same method of titer detection.
[0124] see results Figure 4 It was shown that the CAR positive rates of IL-23ab-CART and PSMA-CART were 83% and 96%, respectively.
[0125] (2) Expression detection of IL-23-scFv:
[0126] For IL-23ab-CART, it is not only necessary to detect the expression of ...
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