Abstract 4867: Enhancing TCR-T with a Fas-based switch receptor boosts T cell engraftment, persistence, and anti-tumor activity in models of hard-to-treat PRAME solid tumor indications

Abstract Background: Adoptive cell therapies have shown promising efficacy in melanoma and synovial sarcoma, but success in other tumor types remains limited. A significant challenge is the hostile tumor microenvironment (TME) that restricts T cell infiltration, function and persistence. Here, we developed a switch receptor (SwR) tailored to PRAME positive solid tumors. Methods: Analysis of TIGIT, Fas and PD1 ligand expression was performed using the TempusTM bulk RNA database and publicly available single-cell RNA sequencing (scRNAseq) data. T cells were retrovirally transduced to express different SwRs in combination with an HLA-A*0201 restricted PRAME-specific TCR and a CD8 co-receptor, and tested for proliferation, viability, cytotoxicity and cytokine secretion in models reflecting the TME of PRAME positive tumors. Results: Analysis of the ligands of the common inhibitory receptors PD1, Fas, and TIGIT in (1) 4, 622 stage III/IV patient samples across 10 PRAME-expressing indications, (2) scRNAseq of 7, 000 - 176, 000 cells covering 98 patients over 5 tumors, and (3) healthy tissue scRNAseq data, demonstrated high expression of TIGIT ligands in tumors and healthy tissue, raising potential safety concerns. PD-L1 and FasL had moderate expression across all target indications, with FasL having the most promising expression profile. Expression in tumors is generally stable across tumor sites and lines of treatment and increased upon CPI treatment. In healthy tissue, FasL is expressed mainly in the lymph nodes (LNs), enabling a Fas-SwR to support TCR-T engraftment and persistence during trafficking. Eight SwR candidates were designed combining the extracellular domain of Fas receptor with different intracellular activating motifs. The SwR combining CD40 and OX40 motifs (Fas-TNFR) was selected based on high cell surface expression and superior protection against FasL-induced apoptosis. When testing in models reflecting the TME of PRAME positive indications, PRAME TCR-T co-expressing Fas-TNFR showed improved T cell fitness as demonstrated by earlier memory phenotype, higher cytokine production and long-term cytotoxic capacity. Notably, Fas-TNFR significantly outperformed a Fas-41BB SwR currently in the clinic. To mimic trafficking through the draining LNs before tumor homing, PRAME TCR-T cells expressing Fas-TNFR were pre-stimulated with FasL. This led to improved T cell fitness and a trend for increased cytotoxic activity upon encountering cancer cells. Conclusions: Extensive characterization of the TME of PRAME-expressing solid tumors guided the development of a best-in-class switch receptor. Fas-TNFR increased fitness of PRAME TCR-T cells before encountering cancer cells, and boosted long-term persistence and anti-tumor activity, indicating the potential of deeper clinical responses at lower doses. Citation Format: Laure Twyffels, Alvaro Haroun-Izquierdo, Friederike Knipping, Mikhail Steklov, Panagiota A. Sotiropoulou, Marleen M. Van Loenen. Enhancing TCR-T with a Fas-based switch receptor boosts T cell engraftment, persistence, and anti-tumor activity in models of hard-to-treat PRAME solid tumor indications [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 4867.