Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors
| Autor: | Ally Huang, Aaron Miller, Jeongsup Shim |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Protein Denaturation Hot Temperature medicine.drug_class Immunology Antineoplastic Agents Apoptosis Monoclonal antibody Antibodies Monoclonal Humanized Jurkat cells 03 medical and health sciences Jurkat Cells Drug Stability In vivo medicine Immunology and Allergy Humans Protein Interaction Domains and Motifs Receptor Immunoassay Microscopy biology Dose-Response Relationship Drug Protein Stability Receptors IgG Antibodies Monoclonal Reproducibility of Results Surface Plasmon Resonance Molecular biology In vitro Coculture Techniques Cell biology Receptors TNF-Related Apoptosis-Inducing Ligand 030104 developmental biology Cell killing HEK293 Cells biology.protein Antibody Signal transduction Protein Binding Signal Transduction |
| Zdroj: | Journal of immunological methods. 448 |
| ISSN: | 1872-7905 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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