ORIGINAL RESEARCH article
Front. Immunol.
Sec. Cancer Immunity and Immunotherapy
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1659966
This article is part of the Research TopicIntegrating Molecular Mechanisms, Immunotherapy, and Drug Sensitivity in Cancer Immunology and OncologyView all 33 articles
Physicochemical and biological characterization of a bispecific antibody in a CrossMab/KIH format that targets EGFR and VEGF-A
Provisionally accepted
- US Food and Drug Administration, Silver Spring, United States
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Bispecific antibodies (BsAbs) are a class of antibody therapeutics engineered in various molecular formats to bind two distinct antigens and potentially mediate multiple biological effects. These molecular formats are tailored to mediate specific mechanisms of action and possess unique physicochemical and biological properties that are necessary to assure product quality. In ovarian cancer (OC), both EGFR-and VEGF-A-mediated signaling pathways are often upregulated and cooperate to promote tumor growth and angiogenesis. Thus, inhibiting of EGFR-and VEGF-A pathways with a BsAb may provide synergistic anti-tumor activity. Using publicly available sequences and applying immunoglobulin domain crossover (CrossMab) and knobs-into-holes (KIH) technologies, we generated a BsAb to simultaneously bind EGFR and VEGF-A (designated as anti-EGFR/VEGF-A BsAb). This BsAb served as a model for physiochemical and biological characterization of quality attributes that would be critical (CQAs) for the BsAb's mechanisms of action. Our goal was to gain fundamental insights into BsAbs designed to target a receptor with one arm and a soluble ligand with the other, to support bioassay development and inform quality control strategies. Our data demonstrated that the CrossMab/KIH platform successfully produced a correctly assembled BsAb during cell culture. Characterization confirmed that the anti-EGFR/VEGF-A BsAb bound both EGFR and VEGF-A with comparable activity and affinity to the respective parental monoclonal antibodies. Functionally, the BsAb disrupted both EGF/EGFR and VEGF-A/VEGFR2 signaling pathways in OC and human umbilical vein endothelial cell (HUVEC) models. Furthermore, the BsAb effectively blocked angiogenic signaling driven by VEGF-A secreted from OC cells in a paracrine manner. Based on the combinatorial mechanism of action and our characterization findings, we concluded that two or more bioassays may be needed to accurately assess the activity of both arms of this type of BsAb.
Keywords: bispecific antibody, CrossMab, knobs-into-holes (KiH), epidermal growth factor receptor (EGFR), Vascular endothelial growth factor A (VEGF-A), ovarian cancer
Received: 04 Jul 2025; Accepted: 14 Aug 2025.
Copyright: © 2025 Ayinde, Dutta, Mohan and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Wen Jin Wu, US Food and Drug Administration, Silver Spring, United States
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