ORIGINAL RESEARCH article
Front. Immunol.
Sec. Antigen Presenting Cell Biology
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1632302
This article is part of the Research TopicAdvances in Immunogenicity Risk Assessment, Monitoring and Mitigation of BiologicsView all 9 articles
A Real-Time Imaging Approach to Quantify Dendritic Cell Internalization for Immunogenicity Risk Assessment of Biotherapeutics
Provisionally accepted
- Genentech Inc., San Francisco, United States
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The presence of treatment-emergent anti-drug antibodies (ADAs) can pose challenges during biotherapeutic development by compromising drug safety and efficacy. Early assessment of immunogenicity risk is essential to mitigate these risks. Dendritic cells (DCs) are crucial for priming CD4 T cells and necessary for effective antibody production. Therefore, DC internalization has been investigated as a predictive tool for evaluating the immunogenicity risk of biotherapeutics. Previously reported flow cytometry-based DC internalization assays, including our own, have faced several limitations. These limitations included low throughput due to a restricted DC supply from healthy donors, restriction to Fc-containing antibody-based biotherapeutics, and offering only endpoint data. To address these limitations, we explored both direct and indirect labeling approaches using the IncuCyteⓇ real-time imaging platform. Our findings revealed that indirect labeling approach with the commonly used Fab anti-Fc reagents was unsuitable for DC internalization assays due to significant assay background noises and assay bias when evaluating biotherapeutics of different frameworks. In contrast, using direct labeling with Biotracker Orange demonstrated improved predictability, required fewer DCs, and was suitable for high-throughput screening. Additionally, this approach facilitates constant monitoring of the internalization process, offering a comprehensive understanding of cell morphology changes and internalization kinetics. Using a panel of 25 test therapeutic antibodies with known clinical ADA results, the IncuCyteⓇ-based direct labeling assay demonstrated significant improvements in predicting the immunogenicity risk of the tested molecules. The assay demonstrated a high correlation between internalization and clinical immunogenicity risk, outperforming our previous flow cytometry-based results. Overall, the IncuCyteⓇ-based direct labeling assay using Biotracker Orange represents a significant advancement compared to our previous flow cytometry assay. This new technique is a valuable addition to our immunogenicity risk assessment toolbox, and will ultimately lead to more informed decision-making in the early development of biotherapeutics.
Keywords: Anti-drug antibody, dendritic cell, immunogenicity prediction, internalization assay, real-time imaging
Received: 20 May 2025; Accepted: 25 Aug 2025.
Copyright: © 2025 Yin, Tran, Guerrero, Low, Xie and Peng. 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: Zhaojun Yin, Genentech Inc., San Francisco, United States
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.