Abstract 4-1BB (CD137) is a member of the TNFR superfamily involved in stimulation of several immune cell types, including T cells and NK cells. CD137 is well validated pre-clinically, as agonism with anti-CD137 antibodies is effective in vivo [1], however, clinical utility to date has been limited by dose dependent hepatotoxicity. Bicycles® are a new therapeutic modality - fully synthetic, constrained bicyclic peptides with high affinity and excellent target selectivity. We hypothesized that Bicycle CD137 agonists that lack Fc domains and exhibit rapid renal elimination may induce CD137 mediated anti-tumor activity while avoiding liver toxicity. Initially, we identified and optimized CD137 specific Bicycle agonists which, when multimerized together, induced CD137 mediated anti-tumor activity [2]. We have built on this initial approach and developed second generation molecules to enable potent stimulation of immune cells exclusively at the tumor site. These tumor-targeted immune cell agonists (TICAsTM) comprise CD137 binding Bicycles coupled to tumor antigen binding Bicycles. Erythropoietin-producing hepatocellular A2 receptor (EphA2) is a tumor target overexpressed in several human cancers and its overexpression correlates with poor clinical prognosis. Here, we present new preclinical data demonstrating the potent immunomodulatory activity of dual targeting EphA2/CD137 TICAs. EphA2/CD137 TICAs engage EphA2 and CD137 with high affinity resulting in picomolar potency in co-culture assays consisting of cancer cell lines endogenously expressing EphA2, and CD137 Jurkat NF-kB/luciferase reporter cells. Moreover, EphA2/CD137 TICAs potentiate cytokine secretion (e.g. IFNγ) in immune cell co-culture experiments and promote caspase activity in T cell mediated cell killing assays. In vivo, EphA2/CD137 TICA (BCY9173), when dosed at 15 mg/kg (BID) to PBMC-humanized mice bearing HT29 xenografts, increased CD8+ T cells in tumors but not in plasma, suggesting local tumor target specific stimulation of T cells without systemic CD137 agonism. Intermittent dosing with various regimens of BCY12491, an EphA2/CD137 TICA, demonstrated robust anti-tumor activity including complete responses (CR) in 10/12 animals in a syngeneic humanized CD137 MC38 mouse model. Importantly, CR mice are resistant to re-challenge with MC38 tumor cells demonstrating a memory response, a phenomenon which has been previously reported for CD137 agonists [1]. Our EphA2/CD137 TICAs have shown the potential to precisely and potently stimulate immune cells in tumors without systemic immune activation. This provides a strong rationale to further develop first-in-class Bicycle TICAs to potentially treat EphA2 expressing cancers.