Abstract Objective: On-target toxicity remains a major limitation of anti-EGFR antibodies, as exemplified by a 9.8% discontinuation rate in cetuximab-treated patients due to skin toxicity. Protease-activated pro-antibodies can improve tumor selectivity; however, their performance is limited by heterogenous clinical protease activity and antibody-specific CDR structural constraints. We aimed to develop an optimized EGFR Lock-antibody (PSM101) with improved blocking efficiency, high tumor-specific cleavability, and minimal normal-tissue binding. Methods: PSM101 was engineered by fusing the Antibody Lock™ masking domain, tumor-associated protease substrates, and optimized linkers to an anti-EGFR antibody. Clinical tumor tissues were profiled to identify highly active protease substrates across multiple cancer types. An automated structure-based simulation platform (MSCS 2.0) was established to predict cover rate and protease cleavability for variant designs. Lead PSM101 molecules were evaluated for (1) binding to human normal cells (flow cytometry), (2) protease activation in clinical tumor tissues (IHZ assay), and (3) antitumor efficacy in EGFR-positive CDX and PDX mouse models. Results: Clinical protease profiling from head and neck, lung, colon, and gastric tumors identified substrates with high and consistent tumor-associated cleavage activity. MSCS 2.0 successfully predicted variants with optimal masking coverage and cleavability, enabling rational selection of the final PSM101 design. PSM101 demonstrated a 100-200-fold reduction in binding to human normal cells compared with cetuximab, and showed robust activation in head and neck tumor tissues. In vivo, PSM101 achieved significant antitumor activity in both CDX and PDX models. Conclusion: We developed a protease-activated EGFR Lock-antibody, PSM101, with markedly improved tumor selectivity and strong therapeutic efficacy. MSCS 2.0 provides a predictive framework for early-stage optimization of pro-antibodies, which may accelerate next-generation antibody drug development and reduce clinical toxicity. Ongoing studies include safety evaluation of PSM101 in cynomolgus monkeys. Citation Format: Yi-An Cheng, Yun-Chi Lu, Yu-Chi Lee, Chen-Yung Hung. Integrative design of PSM101 Lock-antibody for EGFR-positive tumors through clinical protease profiling and structure-based computer simulation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4407.
Cheng et al. (Fri,) studied this question.