Abstract
Therapeutic monoclonal antibodies (mAbs) are complex bioengineered proteins that require to be routinely characterized with robust and reliable bioassays. Infliximab was the first anti-TNFα mAb approved for use in humans and its use has revolutionized the treatment TNF-mediated inflammatory disorders. The mechanism of action (MOA) of infliximab involves its binding to soluble (s) and membrane (m) TNFα. Here, we describe two simple in vitro bioassays for the assessment of key activities of infliximab. First, a bioassay for TNFα neutralization, which evaluates the Fab binding to sTNFα and the consequent reduction in the activation of TNFα receptors and TNFα-induced expression of adhesion molecules on endothelial cells. A second bioassay evaluates the triggering of Complement-Dependent Cytotoxicity (CDC) in cells expressing mTNFα, which requires the interaction of infliximab-Fc with proteins of the complement system. In both cases, the biological responses are measured by flow cytometry, which is accessible for most laboratories. The methods reported here can be easily adapted to other therapeutic mAbs with similar MOA.
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Acknowledgments
This project was partially supported by CONACYT A1-S-18285 and PAPIIT UNAM IN219719. The results reported here were generated at “Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+D+i) para Farmoquímicos y Biotecnológicos” (LANSEIDI-FarBiotec-CONACYT), which is supported by CONACYT.
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Salinas-Jazmín, N., Medina-Rivero, E., Velasco-Velázquez, M.A. (2022). Bioassays for the Evaluation of Target Neutralization and Complement-Dependent Cytotoxicity (CDC) of Therapeutic Antibodies. In: Houen, G. (eds) Therapeutic Antibodies. Methods in Molecular Biology, vol 2313. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1450-1_17
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DOI: https://doi.org/10.1007/978-1-0716-1450-1_17
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