Characterization of Short Production Cycle and Nongenotoxic mRNA-Based CAR-T Cells Targeting CD19-Positive and...

The chimeric antigen receptor (CAR) T cell therapy targeting cluster of differentiation 19 (CD19)-positive malignancies has shown considerable efficacy in clinical settings. However, the potential genotoxicity of viral-based CAR-T therapy, their considerable manufacturing cycle and high costs, prompts questions about its safety and leads to limitations in clinical application. Aiming to explore a safe, efficient, and low-cost mRNA-based CAR-T therapy, we characterize a nonviral, mRNA-based approach utilizing a current good manufacturing practice (cGMP)-compatible electroporation (EP) platform to generate anti-CD19, anti-CD22, and tandem anti-CD19/CD22 CAR-T products with a turn-around time of less than 48 h. The protocol yields a more than 90% cellular viability with a CAR expression of exceeding 60% within the 24 h of transfection. The mRNA-based CAR-T products have a significantly enhanced T cell activation profile with CD69 upregulation, production of tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and Granzyme B, and higher Ki67 expression level. Robust in vitro tumor neutralization by the mRNA-based CAR-T was observed compared to control. Interestingly, we have observed that the short-term cell cryopreservation after EP resulted in a 50% reduction of cellular expansion. However, the cryopreservation and thaw did not have an observed negative impact on the in vitro tumor neutralization. Lastly, we have evaluated the metabolic activity of the mRNA-based CAR-T products, which has a clearly inducible and significant increase in basal respiration (by 60%) and a 2-fold spare respiratory capacity (SRC) upon exposure with the antigen positive target cells. We believe this approach holds promise as a viable alternative that addresses the limitations of current CAR-T therapies, offering a potential solution for future nonviral CAR-T clinical application.