Abstract 6109: Dual targeting of cancer and stromal cells, by combining a FAP-CAR and a PRAME TCR, enhances T cell infiltration and efficient eradication of a wide range of hard-to-treat solid tumors

Abstract Background: The immunosuppressive tumor microenvironment (TME) is a significant barrier to improving the clinical efficacy of T cell receptor (TCR) T cell therapies in solid tumors. The TME largely consists of stromal cells such as cancer associated fibroblasts (CAF) that support tumor growth and hamper immune responses by forming a physical barrier preventing T cell infiltration and exerting immunosuppressive activity. A common cell surface antigen of CAFs with limited expression in healthy tissue is fibroblast activating protein (FAP). Dual targeting T cells expressing an anti-FAP CAR together with a PRAME-specific TCR and CD8 co-receptor (CoR) were generated using non-viral gene editing. This construct enables depletion of CAFs and disruption of the tumor’s supportive stroma and immune barriers, thereby enhancing TCR-T cell infiltration and tumor eradication. Methods: Ten scFvs against FAP were engineered into 2nd gen 4-1BB CARs and retrovirally transduced into PMBCs. FAP-CAR T cells were selected based on CAR expression using flow cytometry and cytotoxicity against FAP positive target cells. T cells were non virally gene edited with selected FAP-CARs in combination with a HLA-A*02 restricted PRAME-specific TCR and a CD8 CoR. Functionality was tested under repeated antigen stimulation in 2D and 3D in vitro cancer models expressing physiological levels of FAP and PRAME. Results: Ten anti-FAP CARs bearing different scFvs were screened based on T cell surface expression and functional activity against FAP overexpressing BT-549 cancer cells. An in vitro multi-cellular model was developed consisting of GFP-labelled cancer cells expressing physiological levels of PRAME and immortalized RFP positive fibroblasts expressing physiological levels of FAP. T cells co-expressing a PRAME TCR, CD8 CoR and 4 selected FAP-CARs were generated and tested for cytotoxicity upon repeated stimulation. Two candidates enabled multiple rounds of killing. Then, a multicomponent 3D spheroid model of non-small cell lung cancer (NSCLC) was developed combining cancer cells, monocytes, CAFs and extracellular matrix, mimicking the dual action of TME, raising a physical barrier against T cell infiltration and mediating immune suppression. Constructs containing an anti-FAP-CAR demonstrated a superior infiltration, cytotoxicity and cytokine secretion profile compared to TCR-T cells without CAR armoring. Conclusion: Dual targeting T cells incorporating both a FAP-CAR and a PRAME TCR demonstrated successful infiltration and effective eradication of both cancer cells and stromal cells within our proprietary, multicomponent 3D spheroid model mimicking the harsh NSCLC TME. This approach holds promise to tackle a major challenge in solid tumor therapy, increasing T cell infiltration and eliciting potent, durable anti-tumor responses. Citation Format: Paul Najm, Kathrin Skibbe, Mikhail Steklov, Panagiota A. Sotiropoulou, Marleen M. Van Loenen. Dual targeting of cancer and stromal cells, by combining a FAP-CAR and a PRAME TCR, enhances T cell infiltration and efficient eradication of a wide range of hard-to-treat solid tumors [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 6109.