Bondza, S, Foy, E, Brooks, J et al. (4 more authors) (2017) Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells. Frontiers in Immunology, 8. 455. ISSN 1664-3224
Abstract
Understanding molecular interactions on immune cells is crucial for drug development to treat cancer and autoimmune diseases. When characterizing molecular interactions, the use of a relevant living model system is important, as processes such as receptor oligomerization and clustering can influence binding patterns. We developed a protocol to enable time-resolved analysis of ligand binding to receptors on living suspension cells. Different suspension cell lines and weakly adhering cells were tethered to Petri dishes with the help of a biomolecular anchor molecule, and antibody binding was analyzed using LigandTracer. The protocol and assay described in this report were used to characterize interactions involving eight cell lines. Experiments were successfully conducted in three different laboratories, demonstrating the robustness of the protocol. For various antibodies, affinities and kinetic rate constants were obtained for binding to CD20 on both Daudi and Ramos B-cells, the T-cell co-receptor CD3 on Jurkat cells, and the Fcγ receptor CD32 on transfected HEK293 cells, respectively. Analyzing the binding of Rituximab to B-cells resulted in an affinity of 0.7–0.9 nM, which is similar to values reported previously for living B-cells. However, we observed a heterogeneous behavior for Rituximab interacting with B-cells, which to our knowledge has not been described previously. The understanding of complex interactions will be facilitated with the possibility to characterize binding processes in real-time on living immune cells. This provides the chance to broaden the understanding of how binding kinetics relate to biological function.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2017 Bondza, Foy, Brooks, Andersson, Robinson, Richalet and Buijs. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
Keywords: | affinity, kinetics, therapeutic antibody, B-cells, T-cells, CD20, Fcγ receptor |
Dates: |
|
Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Institute of Rheumatology & Musculoskeletal Medicine (LIRMM) (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 13 Jun 2017 11:33 |
Last Modified: | 13 Jun 2017 11:33 |
Status: | Published |
Publisher: | Frontiers Media |
Identification Number: | 10.3389/fimmu.2017.00455 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:117688 |