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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Steeland S, Libert C, Vandenbroucke RE (2018) A new venue of TNF targeting. Int J Mol Sci 19:E1442. https://doi.org/10.3390/ijms19051442
Sedger LM, McDermott MF (2014) TNF and TNF-receptors: from mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev 25:453–472. https://doi.org/10.1016/j.cytogfr.2014.07.016
Metcalfe C, Dougall T, Bird C et al (2019) The first World Health Organization international standard for infliximab products: a step towards maintaining harmonized biological activity. mAbs 11:13–25. https://doi.org/10.1080/19420862.2018.1532766
Horiuchi T, Mitoma H, Harashima S et al (2010) Transmembrane TNF-alpha: structure, function and interaction with anti-TNF agents. Rheumatology (Oxford) 49:1215–1228. https://doi.org/10.1093/rheumatology/keq031
Mitoma H, Horiuchi T, Tsukamoto H, Ueda N (2018) Molecular mechanisms of action of anti-TNF-alpha agents—comparison among therapeutic TNF-alpha antagonists. Cytokine 101:56–63. https://doi.org/10.1016/j.cyto.2016.08.014
Velasco-Velazquez MA, Salinas-Jazmin N, Hisaki-Itaya E et al (2017) Extensive preclinical evaluation of an infliximab biosimilar candidate. Eur J Pharm Sci 102:35–45. https://doi.org/10.1016/j.ejps.2017.01.038
Zhang F, Yu W, Hargrove JL et al (2002) Inhibition of TNF-alpha induced ICAM-1, VCAM-1 and E-selectin expression by selenium. Atherosclerosis 161:381–386. https://doi.org/10.1016/S0021-9150(01)00672-4
Vidarsson G, Dekkers G, Rispens T (2014) IgG subclasses and allotypes: from structure to effector functions. Front Immunol 5:520. https://doi.org/10.3389/fimmu.2014.00520
Mitoma H, Horiuchi T, Tsukamoto H et al (2008) Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor alpha-expressing cells: comparison among infliximab, etanercept, and adalimumab. Arthritis Rheum 58:1248–1257. https://doi.org/10.1002/art.23447
Nesbitt A, Fossati G, Bergin M et al (2007) Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents. Inflamm Bowel Dis 13:1323–1332. https://doi.org/10.1002/ibd.20225
Arora T, Padaki R, Liu L et al (2009) Differences in binding and effector functions between classes of TNF antagonists. Cytokine 45:124–131. https://doi.org/10.1016/j.cyto.2008.11.008
Salinas-Jazmin N, Gonzalez-Gonzalez E, Vasquez-Bochm LX et al (2017) In vitro methods for comparing target binding and CDC induction between therapeutic antibodies: applications in biosimilarity analysis. J Vis Exp. https://doi.org/10.3791/55542
Shealy DJ, Cai A, Staquet K et al (2010) Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor alpha. mAbs 2:428–439. https://doi.org/10.4161/mabs.12304
Camacho-Sandoval R, Sosa-Grande EN, Gonzalez-Gonzalez E et al (2018) Development and validation of a bioassay to evaluate binding of adalimumab to cell membrane-anchored TNFalpha using flow cytometry detection. J Pharm Biomed Anal 155:235–240. https://doi.org/10.1016/j.jpba.2018.03.057
Perfetto SP, Chattopadhyay PK, Lamoreaux L et al (2006) Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 313:199–208. https://doi.org/10.1016/j.jim.2006.04.007
Perfetto SP, Chattopadhyay PK, Lamoreaux L et al (2010) Amine-reactive dyes for dead cell discrimination in fixed samples. Curr Protoc Cytom. https://doi.org/10.1002/0471142956.cy0934s53
Scallon B, Cai A, Solowski N et al (2002) Binding and functional comparisons of two types of tumor necrosis factor antagonists. J Pharmacol Exp Ther 301:418–426. https://doi.org/10.1124/jpet.301.2.418
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1450-1_17
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1449-5
Online ISBN: 978-1-0716-1450-1
eBook Packages: Springer Protocols