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Development and Validation of a Cell-Based Binding Neutralizing Antibody Assay for an Antibody–Drug Conjugate

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Abstract

The utilization of antibody–drug conjugates (ADCs) has gained considerable attention in the field of targeted cancer therapy due to their ability to synergistically combine the specificity of monoclonal antibodies (mAbs) and the potency of small molecular drugs. However, the immunogenic nature of the antibody component within ADCs warrants the need for robust immunogenicity testing, including a neutralizing antibody (NAb) assay. Since the mechanism of action (MOA) of the ADC is to first bind to the target cells and then release the payload intracellularly to kill the cells, the most relevant NAb assay format would be a cell-based killing assay. However, in this paper, we present a case where a cell-based killing assay could not be developed after multiple cell lines and NAb-positive controls (PC) had been tested. Surprisingly, contrary to our expectations, all NAb PCs tested exhibited an increased killing effect on the target cells, instead of the expected protective response. This unexpected phenomenon most likely is due to the non-specific internalization of drug/NAb complexes via FcγRs, as an excessive amount of human IgG1 and mouse IgG2a, but not mouse IgG1, greatly inhibited drug or drug/NAb complexes induced cell death. To overcome this obstacle, we implemented a novel cell-based binding assay utilizing the Meso Scale Discovery (MSD) platform. We also propose that an in vitro cell killing NAb assay is limited to at best monitoring the target binding and internalization induced cell death, but not by-stander killing induced by prematurely released or dead-cell released payload, hence cannot really mimic the in vivo MOA of ADC.

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There is no large data base utilized in this paper.

Change history

  • 12 April 2024

    A typo has been corrected to update the word "comparative" to "competitive" in the phrase "a cell-based-binding NAb assay or a competitive ligand binding NAb assay."

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Acknowledgements

We thank Mrs. Ketal Shah and Mrs. Marina Ichetovkin for their support of this project by facilitating communication among the collaborators and managing the overall time track of the project.

Funding

Funding and all the materials that are not commercially available for this research were provided by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

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Author notes

  1. Nazneen Bano, Olguitza Guzman-Valdes, Jessica Amberman, and Elisha Bandlamudi contributed equally to this work.

Authors and Affiliations

  1. Merck & Co., Inc., Rahway, New Jersey, 07065, USA

    Weifeng Xu, Nazneen Bano, Elisha Bandlamudi, Pooja Khanna & Roy Helmy

  2. PPD Clinical Research Business, Thermo Fisher Scientific, Richmond, Virginia, 23832, USA

    Olguitza Guzman-Valdes, Jessica Amberman & Rebecca Carmean

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  1. Weifeng Xu
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Contributions

Conception, design, or planning of the study: WX. Acquisition of the data: OG and JA. Analysis of the data: WX, NB, EB, OG, JA, and RC. Interpretation of the results: WX, NB, EB, OG, and RC. Drafting of the manuscript: WX, NB, and EB. Critically reviewing or revising the manuscript for important intellectual content: WF, NB, EB, PK, RH, OG, JA, and RC.

Corresponding author

Correspondence to Weifeng Xu.

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Conflict of Interest

WX, NB, EB, PK, and RH are current employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and may own stock and/or stock options in Merck & Co., Inc., Rahway, NJ, USA.

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Xu, W., Bano, N., Guzman-Valdes, O. et al. Development and Validation of a Cell-Based Binding Neutralizing Antibody Assay for an Antibody–Drug Conjugate. AAPS J 26, 37 (2024). https://doi.org/10.1208/s12248-024-00909-7

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