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Background T cell engagers (TCEs) have been shown to treat various solid tumors. However, the clinical efficacy of TCEs varies and is associated with poor T cell function, e.g. T cell infiltration and tumor-associated T cell anergy owing to lack of concurrent TCR and co-stimulatory activation of T cells. Here, we present a novel tri-specific T cell engager (TJ-CP3B) that in addition to its tumor associated antigen, CDCP1, a transmembrane glycoprotein over-expressed in various malignancies while minimally expressed in normal epithelial tissues, is armed with CD3 (’signal 1’) and 4-1BB (’signal 2’) to enable adequate concurrent T cell activation within tumors. Furthermore, the 4-1BB arm of TJ-CP3B is designed with our proprietary platform for conditional activation to allow local activation of intra-tumor T cells and minimize systemic cytokine release and liver toxicities, providing durable tumor-specific T cell responses.Methods TJ-CP3B was engineered to incorporate a monovalent, low-affinity anti-CD3 scFv, bivalent anti-4-1BB VHHs, and bivalent anti-CDCP1. The in vitro functionality of TJ-CP3B was assessed using ELISA, FACS, signaling reporter assays, primary T-cell cytotoxicity and cytokine release assays. In vivo efficacy and PK profile of TJ-CP3B were evaluated in murine models.Results TJ-CP3B exhibited strong binding affinity for both intact and protease-cleaved forms of CDCP1 and displayed superior CDCP1 dependent cytotoxicity and cytokine release in vitro across a range of CDCP1 expression levels compared to the CDCP1 and CD3 bispecific TCE (BiTCE-C1), owing to synergistic concurrent T cell activation provided by the 4-1BB arm. Notably, TJ-CP3B did not mediate cytotoxicity and cytokine release in PBMC and CDCP1-negative cells in a co-culture experimental system. At low effector-to-target (E/T) ratios that represent limited intra-tumoral T cell infiltration typical of solid tumors, TJ-CP3B demonstrated enhanced and sustained tumor-specific cytotoxicity and cytokine release compared to BiTCE-C1. Furthermore, TJ-CP3B showed significant dose-dependent antitumor efficacy and a favorable mAb-like pharmacokinetic (PK) profile in mice models.Conclusions TJ-CP3B is designed to enable robust agonistic properties for CD3 and 4-1BB in a CDCP1-dependent manner, whose molecular properties are demonstrated extensively in our experimental system. The novel addition of 4-1BB conditional activation, as compared to conventional CD3-TCEs, provides a unique synergism that enables adequate intra-tumor T cell activation even in the condition where there is very limited T cell infiltration. TJ-CP3B is currently in development as a promising drug candidate for cancer immunotherapy targeting CDCP1-positive malignancies.