XTX601的发现与优化：一种被遮蔽的靶向claudin 18.2的T细胞衔接器

T cell engagers (TCEs) are bispecific molecules engineered to bind to tumor-associated antigens on cancer cells and simultaneously engage T cells via CD3 interaction. Multiple TCEs have been approved for treating hematological malignancies, but approvals against solid tumor antigens have been limited to-date due to on-target, off-tumor toxicities. Aberrant protease dysregulation in solid tumors presents an opportunity for tumor-selective activation of biotherapeutic molecules including TCEs. To optimize the therapeutic index of TCEs by maximizing tumor exposure and minimizing peripheral activity, we have developed a masked TCE format we call ATACR (Advanced Tumor-Activated Cell Engager). In the ATACR format, peripheral engagement of CD3 is limited by a masking domain that is designed to prevent the TCE's anti-CD3 domain from binding CD3. By incorporating a protease cleavable linker between the half-life extension domain and the anti-CD3 domain, cleavage of the ATACR molecule by tumor associated proteases results in the release of a short half-life, fully active TCE locally within the tumor microenvironment. Here we report preclinical data for XTX601, a masked TCE in the ATACR format designed to target the clinically validated tumor associated antigen Claudin 18.2 (CLDN18.2), which is aberrantly expressed in gastrointestinal cancers, such as gastric, esophageal, and pancreatic cancer. A comprehensive CLDN18.2 TCE discovery and optimization effort yielded XTX601, which was found to be 1) highly selective for CLDN18.2; and 2) demonstrated efficient masking with a significant reduction in the levels of CD3 binding compared to the proteolytically activated molecule. Consistent with the CD3 binding data, intact XTX601 was effectively masked relative to proteolytically activated XTX601 when tested in T cell-dependent cellular cytotoxicity (TDCC) assays across multiple cancer cell lines that endogenously express CLDN18.2. XTX601 demonstrated robust anti-tumor activity in humanized, murine xenograft models adoptively transferred with human T cells. In both mice and non-human primates, XTX601 exhibited favorable pharmacokinetics and tolerability, highlighting the potential for an improved therapeutic index compared to systemically active TCEs targeting CLDN18.2. Collectively, these preclinical data demonstrate the effectiveness of our ATACR masked T cell engager format in reducing systemic activation of T cells while retaining potent anti-tumor activity. XTX601 is the first masked CLDN18.2-targeting TCE and has the potential to address a significant unmet need for patients with CLDN18.2 expressing malignancies including gastric, esophageal, and pancreatic cancers.