P07.01 Targeting cancer antigens with de novo designed protein binders as bispecific T cell engagers

Background Pancreatic ductal adenocarcinoma (PDAC) remains refractory to immunotherapy despite major advances in the treatment of several solid tumors.This resistance is driven by a hostile tumour microenvironment and reinforced by tumour-intrinsic immune resistance genes (IRGs) that protect cancer cells from T cell-mediated killing, enabling immune evasion and survival.Identifying and therapeutically targeting such intrinsic resistance mechanisms is critical to improving immunotherapy efficacy in PDAC.Methods High-throughput small interfering RNA (siRNA)based genetic screens were performed in PDAC cells to identify IRGstumour-intrinsic regulators of immune resistance.Candidate genes were ranked based on enhanced susceptibility to cytotoxic T cell-mediated killing following gene knockdown, quantified using luciferase-based tumour cell viability assays.A kinase anchoring protein 1 (AKAP1) emerged as a top candidate.AKAP1 function was evaluated by genetic depletion in PDAC cells subjected to immune-mediated stress, including co-culture with cytotoxic T cells or exposure to tumour necrosis factor alpha (TNF alpha).Downstream signalling events were analysed by immunoblotting, focusing on protein kinase A-dependent phosphorylation of Bcl-2-associated death promoter, mitochondrial translocation, and caspase activation.Synthetic peptides designed to disrupt AKAP1 anchoring to the outer mitochondrial membrane were generated, and their activity was validated by assessing mitochondrial localisation and Caspase 9 activation.Findings obtained by genetic depletion were revalidated using peptide-mediated AKAP1 inhibition.Functional relevance was assessed in patient-derived PDAC organoids exposed to immune-mediated stress.Mitochondrial metabolism under immune challenge was analysed using Seahorse extracellular flux assays, measuring oxygen consumption rate and extracellular acidification rate.Given the broad physiological role of AKAP1, a tumour-restricted targeting strategy was pursued using chimeric antigen receptor T cells capable of delivering AKAP1 inhibitory peptides upon tumour cell recognition.Peptide localisation and transfer were visualised by fluorescence and confocal microscopy.Results AKAP1 was identified as a mitochondrial immune resistance regulator in PDAC.AKAP1-regulated protein kinase A suppressed apoptosis by phosphorylating the pro-apoptotic protein Bcl-2-associated death promoter, thereby limiting mitochondrial translocation and downstream caspase activation.siRNA-mediated depletion of AKAP1 sensitised PDAC cells to cytotoxic T cells and T cell-derived effector molecules, includ-