204 KSQ-004: Unbiased pair-wise discovery of SOCS1 and Regnase-1 as the top CRISPR/Cas9 dual-edit combination enhancing in vivo TIL potency against solid tumors

Background The application of CRISPR/Cas9-gene-editing to enhance the anti-tumor activity T cell Adoptive Cell Therapies (ACT) is a promising approach in treatment patients with solid tumors. We developed an vivo CRISPR^2 screening and interrogated top dual-edit combinations enhancing function. discovered that across all possible targets, inactivation Regnase-1 SOCS1 led greatest enhancement potency vivo. applied these findings discover KSQ-004, Regnase-1/SOCS1 dual-edited human CRISPR/Cas9-engineered TIL (eTIL) therapy currently under development for therapeutic use. Methods generated randomly paired CRISPR guide libraries (CRISPR2) targeting hits from previous single-gene Immune CRISPRomics® screens. over 1200 gene pairs were screened primary mouse OT1 PMEL-TCR-Tg-T cells relevant syngeneic tumor models. Top then evaluated immunotherapy-refractory B16F10 metastatic lung model. efficacy combo was further model wherein B16-Ova tumors expanded engineered ex adoptively transferred into bearing hosts assessment. Results Of 1200+ tested screens, combination ranked amongst dual-edits, this infiltration >3500-fold comparison controls. Studies conducted checkpoint refractory metastasis revealed conferred remarkable survival benefit relative controls, significantly extending median animals 21 days 53 days. Furthermore, Regnase-1+SOCS1-edited isolated exerted complete control upon re-infusion hosts, suggesting rejuvenation tumor-experienced TILs by edit combination. To apply insights use, we (eTIL). manufacture KSQ-004 melanoma NSCLC samples, eTIL demonstrating robust expansion viability comparable unedited 90% knockout both targets. Importantly, produced elevated IFNγ autologous stimulation greater spheroids vitro. Conclusions used novel screen identify as dual function microenvironment. translated use discovery possesses enhanced persistence against