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Background T cell engagers (TCEs) have demonstrated clinical success in hematological malignancies, however, many compelling TCE targets have remained out of reach due to toxicity limitations. Aberrant protease expression in solid tumors presents an opportunity for tumor-selective activation of biotherapeutic molecules. We are developing masked TCE programs, including bispecific molecules designed using our ATACR (Advanced Tumor-Activated Cell Engager) format and tri-specific molecules designed using our SEECR (Selective Effector Enhanced Cell Engager) format. Both formats incorporate protease-cleavable masking domains and conditional half-life modulation to restrict activity to the tumor microenvironment (TME), and the SEECR format builds upon the ATACR format by adding co-stimulatory signaling designed to further enhance potency and T cell activation.Methods ATACR and SEECR molecules were engineered with masking domains linked via protease cleavage sites to prevent peripheral T cell engagement. Protease-dependent activity was evaluated across multiple tumor-associated antigens using biochemical and cell-based assays. Molecule activation and functional activity was confirmed in primary human tumor samples ex vivo. Anti-tumor activity, pharmacodynamics and tolerability were characterized in humanized mouse xenograft models.Results Both ATACR and SEECR formats demonstrated effective masking of T cell engagement across multiple tumor antigens, with >100-fold reduction in CD3 binding and T cell activation in the masked state. Ex vivo studies using primary human tumor samples showed efficient proteolytic activation and functional activity. Additionally, SEECR molecules were able to promote a polyfunctional effector phenotype in endogenous tumor infiltrating lymphocytes in primary tumor samples cultured ex vivo. In adoptive transfer xenograft models, masked TCE molecules, in both the ATACR and SEECR formats, showed significantly improved tolerability compared with non-masked TCEs, with reduced systemic cytokine release and body weight loss. In addition, both the ATACR and SEECR formats maintained potent anti-tumor activity capable of driving complete tumor regression in multiple models.Conclusions Our ATACR and SEECR formats represent next-generation masked TCEs designed to overcome the challenges associated with current, systemically active TCEs. By leveraging TME-specific protease activity for conditional activation, these molecules maintained potent anti-tumor activity while reducing systemic toxicity in murine models. The incorporation of co-stimulatory signaling in the SEECR format further enhanced T cell function within tumors in these studies. Together, these preclinical data support the potential of our masked TCEs to expand the therapeutic window for solid tumor antigens.Ethics Approval All animal studies were conducted under IACUC-approved protocols in compliance with AAALAC regulations