Abstract C010: A novel T-cell engager prodrug platform achieves dual masking by a single VHH domain for balancing efficacy and safety in solid tumor therapy

Abstract Background: Despite their therapeutic potential, T-cell engagers (TCEs) encounter significant hurdles in solid tumor therapy, primarily manifesting as on-target off-tumor toxicities, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS). To address these limitations, we have developed a novel TCE prodrug platform that utilizes a proprietary cleavable substrate linker and a VHH masking domain (VHHm) specific for SP34-derived anti-CD3 antibodies. Dual masking is achieved through the binding of VHHm to anti-CD3 scFv, which simultaneously induces conformational 'locking' of tumor-associated antigen (TAA)-targeting Fab domains, ensuring minimal off-tumor activity. An albumin-binding domain (ABD) is fused to the N-terminus of VHHm to prolong the half-life of prodrug, with a single-site cleavage releasing ABD-VHHm in circulation. This platform eliminates the need to screen specific masking peptides for different TAA-targeting antibodies during dual masking, distinguishing it from previously reported TCE prodrugs. Leveraging these advantages, three prodrugs (DR510 targeting hEGFR, DR507 targeting hTROP-2, and DR508 targeting hEpCAM) have been characterized. Methods: Binding interactions of DR510, DR507 and DR508 to hCD3 and to hEGFR/hTROP-2/hEpCAM were evaluated via flow cytometry. Enzymatic cleavage-induced T-cell activation was evaluated via T-cell-dependent cytotoxicity (TDCC) reporter bioassays using different tumor cell lines. Then, the in vivo efficacy of DR510 was evaluated in human PBMC-engrafted mice harboring HT29, PC9, and HNSCC PDX tumors. ELISA and Western blotting (WB) were conducted to identify the protease cleavage efficiency of DR510 in serum and tumors. A single-dose pharmacokinetic (PK) study and a pilot toxicity study (once-weekly dosing with two doses of 0.65 mg/kg followed by two doses of 1.3 mg/kg) were performed in cynomolgus monkeys, respectively. Results: Flow cytometry analysis confirmed negligible binding of the prodrugs to hCD3 and target antigens (hEGFR, hTrop-2, and hEpCAM). Consistent with this binding profiles, TDCC reporter assays demonstrated minimal basal bioactivity but revealed a significant functional restoration following enzymatic cleavage. In all mouse models, DR510 exhibited dose-dependent tumor growth inhibition that outperformed a clinical-stage comparator across the 0.066 to 0.2 mg/kg dose range without any impact on body weight. In cynomolgus monkey studies, DR510 exhibited favorable PK profiles and good tolerability at two doses of 0.65 mg/kg and 1.3 mg/kg, respectively, accompanied by minimal IL-6 release and undetectable levels of other cytokines including IL-2, IL-4, IL-10, TNF-α, and IFN-γ, suggesting a reduced risk of CRS. Conclusions: These preclinical data highlight our platform's ability to balance the therapeutic efficacy of TCEs with safety. Supported by these data, DR510 is now advancing toward clinical development for the treatment of EGFR-positive solid tumors. Citation Format: Xiaofang Wen, Gaofeng Yao, Wenwen Duan, Zhenxing Zhou, Yonglu Chen, Xin Zhang, Jingjin Fang, Changkui Li, Mingjie Shao, Chengcheng Yang, Dan Cheng, Yanshan Huang. A novel T-cell engager prodrug platform achieves dual masking by a single VHH domain for balancing efficacy and safety in solid tumor therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C010.