A novel tumor-microenvironment-responsive pro-TCE therapy minimizes cytokine release for safer solid tumor treatment

Background: T-cell engagers (TCEs) represent a promising therapeutic modality, however their clinical translation to solid tumors is severely hampered by Cytokine Release Syndrome (CRS). This dose-limiting toxicity has contributed to the failure of numerous TCE trials. Previous conditional activation strategies, such as MMP-cleavable, pH-dependent, or polymer-masked pro-TCEs, were limited by tumor microenvironment heterogeneity, inconsistent activation, and immunogenicity. Here we report a first-in-class, beta-glucuronidase-activatable pro-TCE platform featuring an optimal TCE-Mask ratio (TMR) designed to overcome these limitations through complete tumor-microenvironment activation. Methods: Utilizing site-specific conjugation, we generated a library of TMRx-pro-TCEs by attaching 2~10 beta-glucuronidase-cleavable masking peptides to various TAA/CD3 bispecific and trispecific antibodies and screened for optimal masking efficiency. in vitro TCE activity assessments included TAA-engaged T-cell activation and cytotoxicity against different types of tumor cells. In vivo efficacy and safety were evaluated in hPBMC-reconstituted tumor-bearing mice by monitoring tumor growth inhibition, plasma cytokine levels (IL-6, IFN-gamma), body weight and survival. Results: Masking efficiency was precisely controlled by TMR, with optimal TMR values conferring the strongest suppression of nonspecific T-cell activation. Following beta-glucuronidase cleavage of the masking peptides, pro-TCEs completely restored activities to the level of unmasked TCEs and demonstrated potent, target-specific cytotoxicity. Talatamab is a market approval TCE for DLL3-expressing small cell lung cancer treatment. As a POC example, an optimized TMR4-pro-Talatamab was constructed and demonstrated markedly attenuation of CRS in hPBMC-reconstituted mice. The same pro-Talatamab also inhibited tumor growth to the level equivalent to that of unmasked Talatamab, demonstrating full retention of TCE capability. Conclusions: This novel pro-TCE platform shows potential as a solution to the central challenge of CRS in TCE therapy. By enabling precise tumor-microenvironment activation, it preserves full antitumor efficacy while fundamentally improving systemic safety which may offer a transformative strategy for the effective translation of TCEs to solid tumor indications.