Differences in the phenotypes and transcriptomic signatures of chimeric antigen receptor T lymphocytes manufactured via electroporation or lentiviral transfection.

Chimeric antigen receptor (CAR)-T cell therapy is an innovative treatment for CD19-expressing lymphomas. CAR-T cells are primarily manufactured via lentivirus transfection or transposon electroporation. While anti-tumor efficacy comparisons between the two methods have been conducted, there is a current dearth of studies investigating the phenotypes and transcriptome alterations induced in T cells by the two distinct manufacturing methods. Here, we established CAR-T signatures using fluorescent imaging, flow cytometry, and RNA-sequencing. A small fraction of CAR-T cells that were produced using the PiggyBac transposon (PB CAR-T cells) exhibited much higher expression of CAR than those produced using a lentivirus (Lenti CAR-T cells). PB and Lenti CAR-T cells contained more cytotoxic T cell subsets than control T cells, and Lenti CAR-T cells presented a more pronounced memory phenotype. RNA-sequencing further revealed vast disparities between the two CAR-T cell groups, with PB CAR-T cells exhibiting greater upregulation of cytokines, chemokines, and their receptors. Intriguingly, PB CAR-T cells singularly expressed IL-9 and fewer cytokine release syndrome-associated cytokines when activated by target cells. In addition, PB CAR-T cells exerted faster in vitro cytotoxicity against CD19-expressing K562 cells but similar in vivo anti-tumor efficacy with Lenti CAR-T. Taken together, these data provide insights into the phenotypic alterations induced by lentiviral transfection or transposon electroporation and will attract more attention to the clinical influence of different manufacturing procedures.

Development of a novel reporter gene assay for platelet-derived growth factor-BB bioactivity.

Platelet-derived growth factors (PDGFs) are involved in various physiological and pathological processes, making them important targets for drug development. However, current methods for measuring PDGF bioactivity do not meet the rapidly growing requirements of pharmaceutical research. Here, we describe a novel reporter gene assay (RGA) for PDGF-BB activity measurement. RGA was developed with engineered cells expressing a modified luciferase protein under the control of an SRE element. With PDGF-BB stimulation, cells produced stable dose-dependent signals with a correlation coefficient of R2 > 0.97 within several hours. The relative accuracy of the assay, represented by the relative bias for five independent samples with different bioactivity levels, was less than 4.67%. Variations in RGA caused by intra- and inter-assay factors were smaller than 10% and 15%, respectively. RGA not only yielded consistent results for estimating PDGF-BB activity, but also presented significantly lower variations than did the traditional colorimetric assay. Moreover, RGA could be completed within 2 days, showing a much higher efficiency than the WST method, which requires 4-5 days. Furthermore, RGA is suitable for other PDGF species besides PDGF-BB. Our results demonstrate that RGA could be a powerful tool for screening and identifying PDGF-related drug candidates in pharmaceutical applications.