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255 ExPRESSTM: An accelerated process for the manufacture of KSQ-001, a CRISPR/Cas9-edited eTILTM product
  1. Sharon Lin,
  2. Leila Williams,
  3. Mitali Ghose,
  4. Hugh Gannon,
  5. Conor Calnan,
  6. Angelina Pizzo,
  7. Pei-Lun Kao,
  8. Mallory Brady,
  9. Katri Sofjan,
  10. Anne Dodson,
  11. Sol Shenker,
  12. Michael Schlabach,
  13. Fiona Sharp,
  14. Frank Stegmeier,
  15. Micah Benson and
  16. Karrie Wong
  1. KSQ Therapeutics, Cambridge, MA, USA


Background Adoptive cell therapy with ex vivo expanded tumor infiltrating lymphocytes (TIL) offers a potentially curative treatment for cancer. To improve the clinical durability of TIL, we have developed KSQ-001, a CRISPR/Cas9 engineered TIL (eTIL) product with inactivation of SOCS1 gene. SOCS1 is a negative regulator of cytokine signaling in T cells that we previously identified as a top target restraining T cell in viv anti-tumor function and long-term persistence in genome-wide in vivo CRISPR screens. The clinical manufacture of TIL has historically required a complex multi-step baseline process involving a pre-Rapid Expansion Protocol (pre-REP) followed by a Rapid Expansion Protocol (REP) with feeder cells. To simplify and shorten the manufacture of KSQ-001, we developed a next-generation ExPRESS manufacturing process involving fewer steps and eliminating the use of feeder cells. We demonstrated that ExPRESS can robustly manufacture KSQ-001 with high functional potency at clinical scale from tumor starting material in 21 days or less across multiple solid tumor types.

Methods Viably-cryopreserved-tumor-fragments (VCTF) from different solid tumor indications were used as starting materials for eTIL manufacture. T cells in the tumor fragments are activated to propagate for 7-11 days, after which TIL were electroporated with ribonucleoprotein (RNP) complexes containing the SOCS1-targeting guide RNA (gRNA). Following electroporation, TIL were further expanded for an additional 7-11 days prior to cryopreservation. The editing level, phenotypic characteristics, and functionality of KSQ-001 was assessed by NGS, flow cytometry, and in vitro functional assays.

Results KSQ-001 was successfully manufactured from 10/11 VCTF starting materials from melanoma and NSCLC samples to clinically relevant doses, demonstrating robust viability at the time of cryopreservation and following thaw. KSQ-001 manufactured using ExPRESS showed >90% editing of the SOCS1 gene as well as complete knockdown of SOCS1 protein in all donors tested. Importantly, KSQ-001 manufactured using ExRPESS exhibited heightened anti-tumor function when compared to un-engineered TIL, including increased production of IFNγ and anti-tumor potency in an in vitro TIL/tumor co-culture system. KSQ-001 also retained high diversity of the TCR repertoire and specificity for autologous tumor. Lastly, adoptively transferred eTIL showed enhanced persistence and anti-tumor efficacy in immunodeficient mouse tumor models.

Conclusions We describe a shortened and streamlined process for the manufacture of CRISPR/Cas9-edited eTIL. KSQ-001 can be manufactured at clinically relevant doses in 21 days or less and displays enhanced functional potency. These data support evaluating KSQ-001 manufactured by the ExPRESS process in the treatment of patients with metastatic treatment-refractory solid tumors.

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