Advancing iPSC derived CAR NK cell therapy for enhanced solid tumor treatment

Induced pluripotent stem cells (iPSCs) have become an essential research platform to study various human diseases and hold significant potential for clinical developments. iPSC-derived natural killer (iNK) cells are a groundbreaking platform for next-generation immunotherapy, providing ready-made allogeneic solutions. The manufacturing process involves culturing, banking, genome editing and differentiation into tumor-specific iNK cells to enhance cytotoxicity and tumor-targeting precision while minimizing graft-versus-host risks. In our current workflow, we utilized the Neon™ NxT Electroporation System with the 8-Channel Pipette for rapid optimization of electroporation parameters for editing iPSCs. We evaluated different electroporation buffers and achieved up to 30% CAR knock-in efficiency in iPSCs. Through systematic screening of voltage, pulse duration, and pulse number, we optimized the payload delivery while maintaining high post genome editing cell viability in presence of CultureCEPT™ supplement along with genomic integrity, and pluripotency. Using the CultureCEPT™ supplement for post-genome editing iPSC recovery resulted in improved cell viability and a two-fold increase in total CAR-iPSC number compared to widely used ROCK inhibitors alone. To streamline iNK cell therapy production for master cell bank preparation, we utilized CTS TM Rotea TM counterflow centrifugation system to minimize human intervention and to automate key steps such media removal, cell washing, detachment, collection and transfer. Upon establishment of the optimal target gene and promoter combination to ensure stable expression of transgene during iPSC to iNK differentiation steps, we successfully generated potent CAR-NK cells that were maintained and expanded in CTS TM NK-Xpander TM media. The resulting CAR-iNK cells demonstrated robust and significantly improved cytolytic capabilities to target ovarian tumor SKOV3 cell line via ex vivo killing assay setup. IPSC-based allogeneic cell therapy for cancer treatment is a developing field and has drawn unique interest for the clinical manufacturing of CAR-NK and CAR-T cells. Presented workflow incorporates Neon NxT Electroporation and CTS TM Rotea TM counterflow centrifugation systems to achieve rapid and efficient optimization of nonviral-based genome editing and closed automated cell processing to enable iNK-based cell therapy manufacturing. This capability significantly advances iPSC-based research and cell therapy applications, addressing scalability challenges and improving patient outcomes.