ORIGINAL RESEARCH article
Front. Immunol.
Sec. Vaccines and Molecular Therapeutics
This article is part of the Research TopicAdvances in Immunogenicity Risk Assessment, Monitoring and Mitigation of BiologicsView all 11 articles
Development and Implementation of a Gyrolab-Based Generic AntiDrug Antibody Assay for Antibody-Drug Conjugates in Cynomolgus Monkey Studies
Provisionally accepted
Runzhong Fu1,2*
Christine O'Day2*Razieh Esmaeili2Stavros Zinonos3Arlan Martin2Lisa McCulloch2Charles Y Tan3- 1Pfizer Andover, Andover, United States
- 2Pfizer, Bothell, WA, United States
- 3Pfizer Inc, New York, United States
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Non-clinical immunogenicity yields valuable insights into pharmacokinetics, efficacy, and safety. Given the complex nature of ADC therapeutics, early detection of anti-drug antibodies (ADAs) is critical for elucidating exposure and toxicity issues that may be translated into the clinic. A universal ADA assay employing generic reagents and a standardized cut point in nonclinical studies can enhance cost efficiency, expedite development, and address limitations in drug tolerance associated with traditional bridging assays. Gyrolab-based generic ADA assay was carried out by initially spiking ADC into cynomolgus samples at a concentration of 300 μg/mL. Drug-ADA complexes were isolated using an anti-human Fc antibody, followed by detection with an anti-cynomolgus detection antibody. A total of 22 distinct ADCs were assessed across 50 cynomolgus subjects, each evaluated in six replicates to determine the cut point. Sensitivity and positive control (PC) assessments were performed by titrating an anti-human light chain generic surrogate positive control. A Gyrolab-based ADA assay with a universal cut point of 2.64 was developed, with 16 out of 22 ADCs acceptable for producing a standardized cut point. The average sensitivity of the positive control was 99.5 ng/ml and drug tolerant up to 1 mg/mL. Factors such as various linkers, drug antibody ratio (DAR), or backbone variations (variable region, Fc mutants and engineered cysteines) had minimal effect; however, one antibody variable region gave elevated signals. A workflow for assay used in Good Laboratory Practice (GLP) study was established, and its application demonstrated in a case study. We successfully developed a generic cynomolgus ADA assay for ADC therapeutics with a universal cut point of 2.64. A workflow guiding the assay utility and assessment was established.
Keywords: Anti-drug antibodies, Immunogenicity, non-clinical, Antibody drug conjugates, Bioanalytical assays, Gyrolab
Received: 23 Sep 2025; Accepted: 30 Oct 2025.
Copyright: © 2025 Fu, O'Day, Esmaeili, Zinonos, Martin, McCulloch and Tan. 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:
Runzhong Fu, christina.fu@pfizer.com
Christine O'Day, odayc01@gmail.com
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.