Effect of signal peptide optimized by site-directed mutagenesis on the function of chimeric antigen receptor T cells
Signal peptides(SP)are involved in regulating the secretion level and transmembrane translocation of chimeric antigen receptors(CAR),which is crucial for CAR-T cells.This study aimed to optimize the SP sequence by site-directed mutagenesis and investigate its impact on the killing function of CD19-CAR-T.Firstly,CAR vectors targeting CD19 containing wild-type SP(SP-wtY)or two mutant SP(SP-muK or SP-muR)were constructed using gene synthesis and molecular cloning techniques.The successfully constructed vector was packaged with lentivirus,and T cells were infected.The transfection efficiency of T cells was detected by flow cytometry,while the killing effect on target cells was assessed using the calcein release method.The secretion levels of cytokines interferon-γ(IFN-γ)and interferon-α(TNF-α)were measured using enzyme linked immunosorbent assay(ELISA).The results showed that successful construction of recombinant lentivirus plasmids with wild type and signal peptide mutation.After the transferring the lentivirus into T cells,the transfection efficiency of CD19-CAR carrying three signal peptides(SP-wtY,SP-muK,or SP-muR)were 33.9%,35.5%,and 36.8%,respectively.Further killing assay showed that the tumor-killing effect of SP-muR cells was significantly higher than that of SP-muK and SP-wtY cells.When the ratio of effector to target was 10:1,the secretion levels of cytokines IFN-γ and TNF-α of CAR-T cells of the SP-muR group were significantly higher than those in SP-muK and SP-wtY groups.In summary,this study revealed that increasing the N-terminal positive charge of the signal peptide can improve the expression efficiency of CAR and promote the killing of CD19+target cells.These findings provide a scientific basis the optimization and clinical application of CAR structure.
point mutationsignal peptidechimeric antigen receptorCD19-CAR-T cellskilling tumor in vitrocytokines
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维普
