Abstract B046: Enhanced immune cell engineering utilizing a novel, effective intracellular delivery method for introduction of diverse cargo types

Abstract Engineered immune cell therapies have great potential to revolutionize cancer therapeutics and progress has been made in developing both engineering and manufacturing methodologies. However, limitations remain in some areas including long manufacturing times, potential for cell exhaustion, and limited access to the disease site as in the case for solid tumors. In addition, some immune cell types such as monocytes, B cells, and gamma delta T cells have remained difficult to engineer.Portal has developed a novel implementation of mechanoporation technology using a silicon membrane with pores which enables deformation of the cell and poration of the cell membrane, allowing for diffusion of any cargo, charged or uncharged, into the cell. Mechanoporation is a gentle intracellular delivery technique which preserves cell viability and function in cells (DiTommaso, et al PNAS 2018) and has the potential to shorten manufacturing time as well as allow for introduction of key therapy enhancing factors via transient modifications shortly before therapy administration. Portal has established methodology for cytoplasmic introduction of many diverse cargo including mRNA, circular RNA, siRNA, proteins, peptides, and CRISPR RNPs to primary immune cells, including both unstimulated and activated T cells, B cells, NK cells, monocytes and iPSCs. The Portal technology is easily scalable, enabling both research scale (1e6 - 5e7 cells) and process scale (over 1e9 cells) deliveries. Editing and RNA expression efficiencies are above 80% with viability maintained at 70% or above. We have demonstrated sustained expression of circular RNA, and have successfully delivered an mRNA CAR. Cargo can easily be multiplexed with equivalent delivery efficiencies across cargo classes. Portal’s methodology is gentle and effective enough to be utilized multiple times within a therapeutic workflow. For example, we have demonstrated high editing efficiencies in unstimulated T cells, followed by rapid expansion for 7 days and subsequent mRNA delivery at clinical scale - yielding over 50% knockout and 90% mRNA expression. Portal’s technology and approach has the potential to further advance immune cell therapy developments, particularly in multiplexed engineering approaches involving simultaneous delivery of multiple cargos. Coupled with Portal’s compatibility with diverse immune cell types and next generation cargos, such as circRNA, we believe this simple approach to intracellular delivery can dramatically reduce manufacturing time and cost, easily integrate into existing clinical equipment and enable unique enhancements that underpin a new generation of therapeutics. Citation Format: Zhihui Song, Eleni Rogers, Sophia Hirsch, Andrew Larocque, Darby Kreienberg, Rachel Conover, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei. Enhanced immune cell engineering utilizing a novel, effective intracellular delivery method for introduction of diverse cargo types [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B046.