一种新颖多功能的前药T细胞衔接器平台及展现强效且肿瘤限制性活性的新候选分子

T-cell engagers (TCEs) have emerged as a powerful modality of immunotherapies. However, their clinical application, particularly in solid tumors, is limited by on-target, off-tumor toxicities and potentially life-threatening systemic immune activation, such as severe cytokine release syndrome (CRS). To address these challenges, conditionally activated prodrug TCEs (ProTCEs) represent a promising next-generation strategy. Recent efforts have focused on masking either the CD3 binding domain or both CD3 and TAA (tumor associated antigen) binding domain of TCEs. The masking peptide is fused to ProTCE molecules usually through a cleavable linker that can be cleaved by tumor-specific proteases. Despite recent progress, there is still a challenge for balancing efficient masking and tumor-specific activation to achieve a wider therapeutic window. Here, we present a modular ProTCE design that includes a masking peptide screening platform, allowing rapid identification of masking peptides that effectively suppress the antibody binding ability. Moreover , we engineered a proprietary peptide linker that can be recognized and cleaved by multiple tumor specific proteases. This novel linker exhibits exceptional stability in serum to prevent systemic activation, while demonstrating enhanced cleavage efficiency by tumor-associated proteases, ensuring tumor-specific drug activation and exposure. In addition, our platform is flexible to generate single masking (mask CD3 binding domain) or dual masking (mask both TAA and CD3 binding domains) ProTCEs. With this platform, we have generated a pipeline of ProTCE molecules targeting various TAAs. Most notably, we have developed a ProTCE, EM33, targeting a TAA that is highly expressed on multiple solid tumors but also expressed on normal tissues at a low level. This molecule was designed to have both TAA and CD3 binding domains masked. EM33 exhibits minimal activity in its prodrug form; however, upon exposure to proteases, its activity is fully restored, demonstrating a 1000-fold therapeutic window. In a high density PBMC assay, our ProTCE does not induce T-cell activation and cytokine release, in contrast to its unmasked variant. In vivo, this molecule demonstrated significant tumor growth inhibition in multiple PBMC engrafted tumor cell xenograft models. EM33 also exhibited comparable anti-tumor activity to the unmasked TCE while inducing significantly less cytokine release, indicative of its potential enhanced benefit/risk ratio in human.Our ProTCE platform enables the rapid development of conditionally activated TCEs with an improved therapeutic window. The preclinical data for our first candidate highlights the potential of our platform to generate new TCE therapies for cancer patients targeting TAAs, that currently have posed significant safety and/or efficacy challenges to the classical T-cell engaging approach.