842 Unlocking precision: a next-generation multi-specific CD3 Switch-DARPin with enhanced function to tackle the current limitations of T cell engagers in ovarian cancer

<h3>Background</h3> Ovarian cancer (OC) is the leading cause of cancer mortality in women and the 5-year overall survival remains under 50% due to late diagnosis and high relapse rate. Current treatments such as chemotherapy and targeted therapies fail to achieve durable responses due to multiple parameters, including the absence of subtype-specific treatment, and tumor heterogeneity. Immunotherapy has also shown limited success, partially due to the absence of clean tumor-associated antigens (TAA), the presence of dysfunctional T cells, and the immunosuppressive tumor microenvironment. To tackle these challenges, our clinically validated Designed Ankyrin Repeat Protein (DARPin) platform was used to design a conditionally activated CD3 Switch-DARPin T cell engager (TCE) with a co-stimulatory effector function. This DARPin safely sustains anti-tumor efficacy and opens the space to new TAAs for OC. <h3>Methods</h3> Phenotypic analyses of OC samples were performed by in-house curated RNAseq data and validated using flow cytometry analysis of primary OC tumor tissues. The specific activity of our novel CD3 Switch-DARPin was assessed using Jurkat reporter cells and primary human T cells in co-culture assays with various target cell lines differing in expression levels of TAAs. Additionally, a repetitive T-cell activation assay was used to demonstrate the benefit of co-stimulation function. <h3>Results</h3> Our new DARPin therapeutic design allows a logic-gated CD3-mediated T cell activation, upon the co-engagement of two TAAs (the ‘Switch’). Additionally, a co-stimulatory function leads to the enhancement of antitumor activity. Such tumor-specific T-cell activation opens a safety window for a potent TCE with otherwise high risk of systemic toxicities. Phenotypic analysis confirmed that the selected TAAs are highly co-expressed in tumor samples (co-expression in &gt;70% of tumor cells) and that the co-stimulatory receptors are reduced on tumor-infiltrated lymphocytes (expression in &lt;30% T cells). We observed that the CD3 Switch-DARPin abrogated TAA-independent T-cell activation and increased the safety window against target cells expressing only one of the TAAs. Additionally, the engagement and activation of a co-stimulatory function sustained T cell response and prevented T cell dysfunction. Here we present a potent and safe anti-tumor T cell response, via conditional binding of CD3 and engagement of a co-stimulatory signal. <h3>Conclusions</h3> Our CD3 Switch-DARPin provides an innovative solution to overcome current TCE challenges, such as TAA heterogeneity and poor efficacy/toxicity profile, through multi-specific TAA-dependent targeting and sustained T cell response. Our approach may also expand the targetable TAA field by enabling T cell engagement/activation and tumor selectivity based on antigen co-expression (‘AND’ gate).