Abstract: Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells have emerged as a promising platform for next-generation immunotherapy, offering a homogeneous, scalable and versatile approach for consistent large-scale manufacturing of off the shelf allogeneic therapies. This approach involves multiple steps including iPSC culturing and banking followed by gene editing and differentiation to tumor specific iNK cells to enhance cytotoxicity, persistence, and tumor-targeting specificity, while minimizing risks of graft-versus-host disease. Building closed modular automated workflows will help minimize risks associated with manual processes associated with these methods. Through this work we built closed automated cell harvest, cell wash and buffer exchange, gene delivery and editing protocols that can enable iNK-based cell therapy manufacturing. In our current workflow, we cultured and expanded iPSCs up to a billion cells in a 10-layer cell factory system for master cell bank preparation. iPSC manual process and harvest in 10-layer cell factory system is very labor intensive and prone to contamination. Utilizing CTS Rotea counterflow centrifugation system minimized human intervention at multiple stages iPSC large scale process. Using this protocol, we processed the harvesting of entire iPSC culture in 10-layer cell factory system in a single batch to create a master bank for cell therapy development. The iPSCs prepared using this method retain pluripotency characteristics with good viability and expansion rate. We then successfully used these iPSC banks and carried out CRISPR-based non-viral gene editing using Neon NxT or CTS Xenon closed automated electroporation system for gene delivery system. Results show successful generation of engineered CAR-iPSC with reproducible KI efficiency of up to 15%. We established the optimal target gene and promoter combination to stabilize transgene expression during the differentiation of iPSCs to iNK cells. With methods developed through this work, we successfully generated potent CAR iNK cells. Together the workflows described here utilizing the clean room compliant closed automated cell processing and gene delivery platforms and the GMP compatibility iPSC and NK media systems enable clinical scale iNK cell therapy manufacturing.
