Human CAR-T Cells engineered using the Solupore Ex Vivo Cell Engineering Platform are highly cytotoxic and specific against CD19+ cells in vitro

Abstract

Background & Aim Solupore® is a non-viral ex vivo cell engineering platform that enables development and manufacture of cell therapies, using reversible permeabilization to achieve rapid intracellular delivery of cargos. The technology achieves intracellular delivery and subsequent reversal of cell permeabilization by precisely controlling the contact of the target cells with a solution containing the cargo. We have termed the method ‘soluporation’. Non-viral methods, such as soluporation, that enable intracellular delivery of various cargo types for clinical applications are attractive candidates for next-generation delivery modalities because of potential benefits for manufacture, safety and regulation compared with viral vectors. While electroporation is currently the most widely-used non-viral method used for cell engineering, it can be damaging to cells. In contrast, we have previously demonstrated that soluporation causes minimal perturbation of human T cells. Methods, Results & Conclusion Here we have further investigated the impact of soluporation and nucleofection on T cells by carrying out gene expression profiling in transfected cells. In nucleofected cells, 263 out of 574 immune related genes were misregulated at 6 h post-transfection. These genes clustered to several immune-related pathways including T cell activation, metabolism and exhaustion. In contrast, 8 genes were misregulated in soluporated cells. This indicates that soluporation maintains cellular function similar to untreated control cells, unlike nucleofection. We also assessed whether Solupore® could be used to engineer T cells to express a chimeric antigen receptor (CAR) and if these cells could effectively kill cancer cells in vitro. CD19 CAR mRNA was delivered to primary human T cells with on average 60% CD19 CAR expression detected at 24 h post-transfection. These CD19-CAR T cells were able to kill target cancer cells in a density-dependent manner. These studies demonstrate that the Solupore® technology is a gentle modality for efficient engineering of immune cells. The technology is highly reproducible, automated, and scalable and has the potential to enable a broad range of T cell engineering applications.