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A Human Skin Explant Test as a Novel In Vitro Assay for the Detection of Skin Sensitization to Aggregated Monoclonal Antibodies

Lookup NU author(s): Dr Ricardo Martins Gouveia, Arathi Kizhedath, Dr Shaheda Ahmed, Professor Jarka Glassey, Abbas Ishaq, Professor Anne Dickinson



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


© 2024 by the authors.Introduction: Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. Material and Methods: Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Results: Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II–III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. Conclusions: The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation.

Publication metadata

Author(s): Martins-Ribeiro A, Kizhedath A, Ahmed SS, Glassey J, Ishaq A, Freer M, Dickinson AM

Publication type: Article

Publication status: Published

Journal: Toxics

Year: 2024

Volume: 12

Issue: 5

Online publication date: 30/04/2024

Acceptance date: 22/04/2024

Date deposited: 03/06/2024

ISSN (electronic): 1996-2024

Publisher: Multidisciplinary Digital Publishing Institute (MDPI)


DOI: 10.3390/toxics12050332


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Funder referenceFunder name
European Union’s Horizon 2020
Marie Skłodowska-Curie