457 Engineered tumor-infiltrating lymphocytes (TIL) with co-regulated membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) generate significant antitumor efficacy in fibrotic tumor models

<h3>Background</h3> Non-engineered TIL cell therapy requiring co-administration of high-dose IL2 has shown promising activity, but concerning safety, in melanoma. To develop IL2-independent TIL, cytoTIL15^TM cells are engineered to express regulatable membrane-bound IL15 (mbIL15) using an FDA-approved small-molecule drug, acetazolamide (ACZ), which binds to the drug-responsive-domain (DRD) carbonic anhydrase 2 (CA2), driving expansion, persistence, and antitumor activity (OBX-115: NCT05470283, NCT06060613). To additionally address challenges inherent in immunologically cold tumors, another proinflammatory cytokine, LIGHT, was added to the cytoTIL15 backbone. LIGHT, a tumor necrosis factor family member (TNFSF14), interacts with lymphotoxin beta receptor (LTβR) and herpes virus entry mediator (HVEM) found on various tumor microenvironment (TME) cell types, including cancer-associated fibroblasts (CAF). Preclinically, intratumoral LIGHT expression via viral or cell therapy leads to tertiary lymphoid structure (TLS) formation¹ and vascular normalization,² both associated with better outcomes.³ Autologous TIL with co-regulated mbIL15 and LIGHT demonstrated increased tumor killing and efficacy in the B16-F10 syngeneic model.⁴ Herein, efficacy of mbIL15- and LIGHT-engineered TIL was tested in fibroblast-rich patient-derived human xenograft (PDX) and syngeneic tumor models. <h3>Methods</h3> A PDX model of human colorectal cancer was established in NSG mice. To develop a highly fibrotic syngeneic tumor model, MC38 cells engineered to express gp100 were inoculated subcutaneously into C57BL/6 mice and allowed to reach an average tumor size ~300 mm³ before adoptive cell transfer (ACT) of murine transgenic CD8+ Pmel TCR T cells (gp100-targeted), without preconditioning. Flow cytometry was performed to analyze the phenotype and immune profiling of both the exogenous cell product and host immune compartments. Spatial characterization of the TME was performed using immunofluorescence imaging. <h3>Results</h3> The combination of mbIL15 and LIGHT in autologous TIL yielded greater tumor growth inhibition compared to TIL expressing mbIL15 alone (p=0.0260, Day 54 post-ACT) in a fibroblast-rich CRC PDX model. Likewise, murine mbIL15- and LIGHT-transduced Pmel cells showed superior antitumor activity versus those expressing mbIL15 alone (p=0.0160, Day 26 post-ACT) against large, CAF-developed MC38-gp100 tumors. Moreover, compared to mbIL15 alone, Pmel cells expressing mbIL15 and LIGHT induced more B-cell aggregation and TLS formation in the tumor along with increased epitope spreading (p=0.0123, Day 21 post-ACT). <h3>Conclusions</h3> These preclinical results show that engineering TIL with regulatable mbIL15 and LIGHT has the potential to enhance their efficacy against challenging-to-treat tumors with a fibrotic and immunologically cold TME, opening the possibility to treat tumors currently unresponsive to non-engineered TIL cell therapy. <h3>References</h3> Zhang. <i>Molecular Therapy</i> 2023. Ramachandran. <i>Cancer Cell</i> 2023. Sautès-Fridman. <i>Nat Rev Cancer</i> 2019. Koscso, <i>et al. AACR</i> 2024 (Abstract LB065). <h3>Ethics Approval</h3> Mice were housed at Obsidian Therapeutics in accordance with approved IACUC protocols overseen annually by the Cambridge Commissioner of Laboratory Animals (Cambridge, MA) and an attending board-certified veterinarian.