Journal Article

A masking clamp for conditional activation of therapeutic antibodies

Habermann, Jan, Ullrich, Evelyn, Zimmermann, Jan Felix, Bloch, Adrian, Kolmar, Harald

2025

Overview

Therapeutic monoclonal antibodies (mAbs) constitute cornerstone therapeutics in oncology, yet their clinical utility is often limited by on-target, off-tumor toxicity due to shared antigen expression in both tumor and healthy tissues. To counteract this issue, various approaches, including pH-dependent, as well as affinity-based and steric hindrance-based masked antibodies, have been developed. Several steric hindrance-based masking strategies have been proposed utilizing non-human proteins, potentially leading to an immunogenic response. To address this challenge, we engineered a modular protein-based masking platform leveraging the high-affinity interaction between human calmodulin (CaM) and a calmodulin-binding peptide (CBP). This strategy enables conditional activation of antibodies via tumor microenvironment (TME)-associated proteases (e.g., MMP-9), minimizing systemic off-tumor binding. The CaM-CBP peptide clamp, composed exclusively of human-derived protein domains, was fused to the amino termini of heavy and light chains of trastuzumab and cetuximab. On-cell binding assays demonstrated up to a 410-fold reduction in EC 50 for masked constructs across multiple antigen-antibody systems. Functional validation using a reporter-cell-based antibody-dependent cellular cytotoxicity (ADCC) assay confirmed that masking abrogated effector cell activation, leading to up to 78-fold reduction of EC 50 and no ADCC activation at concentrations corresponding to the onset of maximal ADCC activation by unmodified antibodies. Demasking via MMP-9-mediated linker hydrolysis restored antigen binding and ADCC potency. Structural optimization revealed that linker length and clamp positioning critically influenced masking efficiency. This human-derived, modular masking platform mitigates immunogenicity risks while enabling tumor-selective antibody activation. Its adaptability across antibody scaffolds underscores broad applicability for improving the therapeutic index of antibodies.

• Publisher: Frontiers Media SA,Frontiers Media S.A
• Subject: Antibodies, Monoclonal - immunology / antibody engineering / antibody masking / Antigens / Antineoplastic Agents, Immunological - pharmacology / Breast cancer / Calmodulin / Calmodulin - chemistry / Calmodulin - genetics / Calmodulin - immunology / Calmodulin - metabolism / Cancer therapies / Cell activation / Cell Line, Tumor / Cetuximab - genetics / Cetuximab - immunology / Cetuximab - pharmacology / Colorectal cancer / conditional antibody activation / Cytotoxicity / Gelatinase B / Humans / Immunogenicity / Kinases / Light / Light chains / Matrix Metalloproteinase 9 - metabolism / MMP-9 / Modularity / Monoclonal antibodies / off-target toxicity / Oncology / Peptides / pH effects / Plasmids / Proteins / Toxicity / Trastuzumab / Trastuzumab - genetics / Trastuzumab - immunology / Trastuzumab - pharmacology / Tumor microenvironment / Tumor Microenvironment - immunology / Tumors