452 Development of a gene editing platform for the manufacture of CRISPR/Cas9 engineered TIL (eTIL)

<h3>Background</h3> To improve the clinical activity of Tumor Infiltrating Lymphocyte (TIL) therapy, we developed engineered TIL (eTIL®) using CRISPR/Cas9 gene editing: KSQ-001EX is a single-edit eTIL product with SOCS1 inactivated; KSQ-004EX is a dual-edit eTIL product with inactivation of both SOCS1 and Regnase-1. Inactivation of SOCS1 and Regnase-1 is anticipated to enhance anti-tumor efficacy of TIL. These eTIL products will be evaluated in the clinic for the treatment of solid tumors. To streamline and shorten the manufacture of eTIL, we established a next-generation ExPRESS manufacturing process. The ExPRESS process is initiated with either surgically resected tumors or core biopsies as starting material, with a pre-editing phase, followed by electroporation of CRISPR/Cas9 ribonucleoproteins for gene editing, and a post-editing expansion phase. This process eliminated the Rapid Expansion Phase (REP) as well as the requirement for feeder cells, resulting in a manufacturing process approximately 22 days in length. Due to the highly heterogeneous nature of tumor starting material, the number of TIL generated following the pre-editing expansion phase is highly variable, presenting a unique challenge to engineering of TIL with CRISPR/Cas9. Here, we describe the successful development of an eTIL editing platform. <h3>Methods</h3> To account for inherent patient to patient variability several process development studies were performed to optimize the eTIL engineering step of the ExPRESS process. Viably-cryopreserved-tumor-fragments (VCTF) from different solid tumor indications were used as starting materials, and key parameters were assessed during the expansion and electroporation stages. Different means of improving sterility assurance and a wide range of editing parameters were experimentally tested. Following electroporation, engineered TIL were further expanded for an additional 10 days prior to cryopreservation. The growth profile and editing efficiency and functionality of KSQ-001EX and KSQ-004EX were characterized. <h3>Results</h3> GMP-compatible gene editing process parameters were optimized and identified at full scale. Gene editing of eTIL using electroporation of Cas9/sgRNAs using a MaxCyte ExPERT GTx consistently achieved robust (&gt;90%) editing in both single and dual-edit settings within KSQ-001EX and KSQ-004EX eTIL, respectively. Robust eTIL editing efficiency was achieved while maintaining consistently high cell viability, in vitro functionality, and other process attributes. <h3>Conclusions</h3> With the goal of simplifying the manufacture of eTIL and improving sterility assurance, we report the development of a robust, scalable gene editing platform that accounts for tumor variability in the manufacture of eTIL using a next generation ExPRESS manufacturing process.