TY - CHAP
T1 - Bioassays for the Evaluation of Target Neutralization and Complement-Dependent Cytotoxicity (CDC) of Therapeutic Antibodies
AU - Salinas-Jazmín, Nohemí
AU - Medina-Rivero, Emilio
AU - Velasco-Velázquez, Marco Antonio
N1 - Publisher Copyright:
© 2022, Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
KW - CDC
KW - Flow cytometry
KW - Infliximab
KW - Target Neutralization
KW - Therapeutic antibody
KW - anti-TNFα
UR - http://www.scopus.com/inward/record.url?scp=85114618359&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-1450-1_17
DO - 10.1007/978-1-0716-1450-1_17
M3 - Capítulo
C2 - 34478145
AN - SCOPUS:85114618359
T3 - Methods in Molecular Biology
SP - 281
EP - 294
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -