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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
  1. Karrie Wong,
  2. Christopher Wrocklage,
  3. Sharon Lin,
  4. Isabelle Le Mercier,
  5. Caroline Bullock,
  6. Louise Cadzow,
  7. Anja Hohmann,
  8. Sol Shenker,
  9. Katri Sofjan,
  10. Leila Williams,
  11. Gregory Kryukov,
  12. Conor Calnan,
  13. Frank Stegmeier,
  14. Micah Benson and
  15. Michael Schlabach
  1. KSQ Therapeutics, Cambridge, MA, USA


Background The application of CRISPR/Cas9-gene-editing to enhance the anti-tumor activity of T cell Adoptive Cell Therapies (ACT) is a promising approach in the treatment of patients with solid tumors. We developed an in vivo CRISPR^2 screening approach and interrogated the top dual-edit combinations enhancing T cell anti-tumor function. We discovered that across all possible dual-edit combinations of T cell targets, inactivation of Regnase-1 and SOCS1 led to the greatest enhancement in anti-tumor T cell potency in vivo. We applied these findings to discover KSQ-004, a Regnase-1/SOCS1 dual-edited human CRISPR/Cas9-engineered TIL (eTIL) therapy currently under development for therapeutic use.

Methods We generated randomly paired CRISPR guide libraries (CRISPR2) targeting top hits from previous single-gene Immune CRISPRomics® screens. CRISPR^2 libraries with over 1200 gene pairs were screened in primary mouse OT1 and PMEL-TCR-Tg-T cells in the relevant syngeneic tumor models. Top dual-edit combinations were then evaluated in the immunotherapy-refractory B16F10 metastatic lung tumor model. The efficacy of the top combo was further evaluated in a mouse TIL model wherein TIL from B16-Ova tumors were expanded and engineered ex vivo and adoptively transferred into tumor bearing hosts for efficacy assessment.

Results Of the 1200+ combinations tested in the CRISPR^2 screens, the Regnase-1/SOCS1 combination ranked amongst the top dual-edits, with this combination enhancing T cell infiltration into tumors >3500-fold in comparison to controls. Studies conducted in the checkpoint therapy refractory B16F10 lung metastasis model revealed that Regnase-1/SOCS1 dual-edited PMEL-TCR-Tg-T cells conferred remarkable survival benefit relative to controls, significantly extending median survival of animals from 21 days to 53 days. Furthermore, Regnase-1+SOCS1-edited mouse TIL isolated and expanded from B16-Ova tumors exerted complete control of tumors upon re-infusion into hosts, suggesting rejuvenation of tumor-experienced TILs by this edit combination. To apply these insights for therapeutic use, we discovered KSQ-004, a human Regnase-1/SOCS1 dual-edited CRISPR/Cas9-engineered TIL (eTIL). Methods were developed to manufacture KSQ-004 from melanoma and NSCLC tumor samples, with eTIL demonstrating robust expansion and viability comparable to unedited control TIL with over 90% knockout of both targets. Importantly, KSQ-004 produced elevated IFNγ upon autologous tumor stimulation and exerted greater control of tumor spheroids in vitro.

Conclusions We used a novel CRISPR^2 screen approach to identify Regnase-1/SOCS1 as the top dual edit combination enhancing T cell function in the tumor microenvironment. We translated these findings to therapeutic use with the discovery of KSQ-004, a dual-edited eTIL therapy engineered that possesses enhanced anti-tumor potency and persistence against solid tumors.

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