Abstract 2196: Characterizing the impact of SOCS1 and Regnase-1 inactivation on tumor reactive T cells present in CRISPR/Cas9-engineered TIL (eTIL®) therapies derived from melanoma and NSCLC tumors through functional and single cell analyses
Authors: David Monteiro, Meghan Travers, Tyler G. Searles, Gustavo J. Martinez, Sharon Lin, Ashish Yeri, Alex Smashnov, Mitali Ghose, Seamus McKenney, Paul Dunbar, Charles H. Yoon, Dipen Sangurdekar, Karrie Wong, Joseph D. Phillips, Mary Jo Turk, Micah J. Benson
Journal: Cancer Research (2025)

Tumor Infiltrating Lymphocyte (TIL) therapy involves the isolation and ex vivo expansion of TIL for the treatment of solid tumors. The T cell receptor (TCR) repertoires present within TIL drug products (DPs) are highly patient unique and include TCRs specific against tumor antigens at variable frequencies. The clinical activity of TIL is thought to be mediated in part by the anti-tumor functionality profile of TIL and the frequency of tumor reactive T cells (TRTs) within the TIL DP. Seeking to improve the clinical activity of TIL therapy, we developed KSQ-001EX and KSQ-004EX, two CRISPR/Cas9-engineered TIL (eTIL®) therapies wherein the genes encoding SOCS1 or SOCS1 in combination with Regnase-1 are inactivated, respectively. SOCS1 is a negative regulator of cytokine signaling while Regnase-1 is an RNase that degrades transcripts encoding proteins involved in regulating T cell anti-tumor function. We have shown in preclinical models that inactivation of SOCS1 and SOCS1/Regnase-1 enhance the anti-tumor functionality of TIL; however, the incidence of TRTs in eTIL DP and how inactivation of SOCS1 and Regnase-1 impact their functionality remains unclear.To identify and characterize TRT clones in eTIL, we generated KSQ-001EX and KSQ-004EX using TIL derived from patients with melanoma or non-small cell lung cancer (NSCLC) and analyzed them with distinct but complimentary methods. We functionally characterized melanoma-derived DPs via coculture with paired autologous melanoma tumor cell lines, with samples exhibiting cognate reactivity against tumor through induction of CD69 and CD137. Consistent with its biology, we observed strong SOCS1-driven enhancement of IFNγ and CD25 in this setting. We extended these insights to NSCLC using single cell RNA/TCR sequencing performed on DPs and tumor starting material. In tumor starting material, we identified CD8+ T cell clusters aligning to IL7R+ circulating/stem, GZMB+ effector, and CXCL13+ resident memory (Trm) states. Predicted TRT (predTRT) clones were identified using a custom gene set constructed from published TRT signatures and cells from these clones were overrepresented in Trm. The majority of predTRT clones were also detected in DPs, where they preferentially aligned to effector memory and proliferating states. Further demonstrating the impact of SOCS1 and Regnase-1 edits on predTRT clones, we observed enrichment of an activated interferon-stimulated gene signature in eTIL DPs, with predTRT clones in dual-edited KSQ-004EX uniquely enriched for heightened expression of a Regnase-1 transcriptional signature.To conclude, we observe that eTIL possess reactivity against tumor through retention of TRT clones, with SOCS1 and Regnase-1 inactivation enhancing the anti-tumor functionality of TRT clones within eTIL DP. Citation Format: David Monteiro, Meghan Travers, Tyler G. Searles, Gustavo J. Martinez, Sharon Lin, Ashish Yeri, Alex Smashnov, Mitali Ghose, Seamus McKenney, Paul Dunbar, Charles H. Yoon, Dipen Sangurdekar, Karrie Wong, Joseph D. Phillips, Mary Jo Turk, Micah J. Benson. Characterizing the impact of SOCS1 and Regnase-1 inactivation on tumor reactive T cells present in CRISPR/Cas9-engineered TIL (eTIL®) therapies derived from melanoma and NSCLC tumors through functional and single cell analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2196.