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Binding affinity determination of therapeutic antibodies to membrane protein targets: Kinetic Exclusion Assay using cellular membranes for anti-CD20 antibody

Vaish, Amit and Lin, Joanne S. and McBride, Helen J. and Grandsard, Peter J. and Chen, Qing (2020) Binding affinity determination of therapeutic antibodies to membrane protein targets: Kinetic Exclusion Assay using cellular membranes for anti-CD20 antibody. Analytical Biochemistry, 609 . Art. No. 113974. ISSN 0003-2697. https://resolver.caltech.edu/CaltechAUTHORS:20201012-163632580

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Abstract

Antibody-based therapeutics targeting membrane proteins have evolved as a major modality for the treatment of cancer, inflammation and autoimmune diseases. There are numerous challenges, ranging from desired epitope expression to reliable binding/functional assays which are associated with developing antibodies for this target class. Specifically, having a robust methodology for characterizing antibody interaction with a membrane protein target is essential for providing guidance on dosing, potency and thus expected efficacy. Fluorescence-activated cell sorting (FACS) has been commonly used to characterize antibodies binding to membrane protein targets. FACS provides information about the antibody-receptor complex (antibody bound to cells) and the apparent equilibrium dissociation constant (K'_D) is elucidated by fitting the antibody-receptor binding isotherm as a function of total antibody concentration to a nonlinear regression model. Conversely, Kinetic Exclusion Assay (KinExA) has been used to measure solution-based equilibrium dissociation constant (K_D) of antibodies. Here, K_D is determined by measuring the free antibody concentration at equilibrium in a series of solutions in which the antibody is at constant concentration and the receptor (either in the membrane or the cell) is titrated. We measured the binding affinity of the anti-CD20 antibody, Rituximab, using both FACS and KinExA. There was ~25-fold difference in the binding affinity measured by these two techniques. We have explored this discrepancy through additional experiments around the mathematical framework involved in the analysis of these two different binding assays. Finally, our study concluded that KinExA enables accurate measurement of the K_D for strong protein-protein interactions (sub-nanomolar values) compared to FACS.


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https://doi.org/10.1016/j.ab.2020.113974DOIArticle
Additional Information:© 2020 Elsevier. Received 22 January 2020, Revised 15 September 2020, Accepted 18 September 2020, Available online 30 September 2020. We thank Larry Daugherty for assistance with flow cytometry and Tom Glass from Sapidyne for providing insight on KinExA technology and specific binding analysis of cell membrane targets. CRediT authorship contribution statement. Amit Vaish: wrote the manuscript.: analyzed the data. Joanne S. Lin: performed the experiments, analyzed the data. Helen J. McBride: Writing - original draft, critically reviewed and helped writing the manuscript. Peter J. Grandsard: Writing - original draft, critically reviewed and helped writing the manuscript. Qing Chen: wrote the manuscript, performed the experiments, analyzed the data. Disclosure of potential conflicts of interest. Amit Vaish, Peter Grandsard, and Qing Chen are employees of and own stock of Amgen, Inc. Joanne Ho and Helen McBride are former employees of Amgen, Inc. No other potential conflicts of interest were disclosed.
Record Number:CaltechAUTHORS:20201012-163632580
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201012-163632580
Official Citation:Amit Vaish, Joanne S. Lin, Helen J. McBride, Peter J. Grandsard, Qing Chen, Binding affinity determination of therapeutic antibodies to membrane protein targets: Kinetic Exclusion Assay using cellular membranes for anti-CD20 antibody, Analytical Biochemistry, Volume 609, 2020, 113974, ISSN 0003-2697, https://doi.org/10.1016/j.ab.2020.113974.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106009
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:13 Oct 2020 16:45
Last Modified:13 Oct 2020 16:45

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