Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors.

Abstract

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work. FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5-20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

• Authors: Jeongsup Shim, Ally Huang, Aaron S Miller
• Journal: Journal of immunological methods (J Immunol Methods) Vol. 448 Pg. 26-33 (09 2017) ISSN: 1872-7905 [Electronic] Netherlands
• PMID: 28506821 (Publication Type: Evaluation Study, Journal Article)
• Copyright: Copyright © 2017 Elsevier B.V. All rights reserved.

Chemical References

• Antibodies, Monoclonal
• Antibodies, Monoclonal, Humanized
• Antineoplastic Agents
• Receptors, IgG
• Receptors, TNF-Related Apoptosis-Inducing Ligand
• TNFRSF10B protein, human
• drozitumab

Topics

• Antibodies, Monoclonal (chemistry, metabolism, pharmacology)
• Antibodies, Monoclonal, Humanized
• Antineoplastic Agents (chemistry, metabolism, pharmacology)
• Apoptosis (drug effects)
• Coculture Techniques
• Dose-Response Relationship, Drug
• Drug Stability
• HEK293 Cells
• Hot Temperature
• Humans
• Immunoassay (methods)
• Jurkat Cells
• Microscopy
• Protein Binding
• Protein Denaturation
• Protein Interaction Domains and Motifs
• Protein Stability
• Receptors, IgG (chemistry, immunology, metabolism)
• Receptors, TNF-Related Apoptosis-Inducing Ligand (antagonists & inhibitors, immunology, metabolism)
• Reproducibility of Results
• Signal Transduction (drug effects)
• Surface Plasmon Resonance