Clinical-Scale Non-Viral Based Engineering and Feeder-Free Production of Car-Nk Cells for Cancer Immunotherapy

Background & AimThe use of engineered primary human natural killer (NK) cells holds great promise in immuno-oncology due to their demonstrated safety and potent anti-tumor properties, especially in the case of solid tumors. Unlike primary T cells, NK cells do not rely on a matching human leukocyte antigen to function, reducing the risk of graft-versus-host diseases in allogeneic therapies. Gene delivery into NK cells is a crucial tool for studying NK cell biology and developing NK cell-based immunotherapies. However, efficient, safe, non-viral gene delivery methods for primary NK cells remain limited. In this study, we optimized a scalable, non-viral, closed-system gene delivery method for production of CAR-NK cells.Methods, Results & ConclusionFirst, we investigated the effects of different electroporation parameters, buffers and DNA concentrations on gene delivery efficiency and cell viability. We isolated PBMCs using CTSTM RoteaTM, a closed counterflow centrifugation system. Isolated NK cells from PBMCs, were then expanded in feeder-free culture using CTS™ NK-Xpander™ Medium for 6 days before proceeding to gene engineering using CRISPR/Cas9 RNP and relevant donor DNA delivery via electroporation. We conducted optimization of electroporation conditions at a small scale (100 µl) using the Neon™ NxT Electroporation System, and successfully translated the optimized conditions to a clinically relevant scale using the CTS™ XenonTM Electroporation System. Our optimized non-viral NK cell engineering workflows yielded efficient knockout (>80%) of different genes tested and successful site-specific CAR knock in at AAVS1 locus using a single electroporation step to co-deliver CRISPR-Cas9 RNP and single-stranded DNA (ssDNA) template for homology-directed repair. The genome edited NK cells were expanded after electroporation and achieving clinically relevant levels of cell expansion. We performed functional assays and observed that gene delivery using Electroporation System did't impair the cytotoxicity of CAR-NK cells against target cells.In conclusion, this study presents closed automated cell processing and scalable, non-viral gene delivery methods for primary NK cell engineering that enables efficient transgene expression without compromising NK cell viability or function. The ability to efficiently modify primary NK cells will facilitate further studies on NK cell biology and enable the development of NK cell-based immunotherapies.