Digital Receptor Occupancy Assay in Quantifying On- and Off-Target Binding Affinities of Therapeutic Antibodies
Year: 2020
Type: article
Abstract: While monoclonal antibodies are the fastest-growing class of therapeutic agents, we lack a method that can directly quantify the on- and off-target binding affinities of newly developed therapeutic antibodies in crude cell lysates. As a result, some therapeutic antibody candidates could have a moderate on-target binding affinity but a high off-target binding affinity, which not only gives a reduced efficacy but triggers unwanted side effects. Here, we report a single-molecule counting method that precisely quantifies antibody-bound receptors, free receptors, and unbound antibodies in crude cell lysates, termed digital receptor occupancy assay (DRO). Compared to the traditional flow cytometry-based binding assay, DRO assay enables direct and digital quantification of the three molecular species in solution without the additional antibodies for competitive binding. When characterizing the therapeutic antibody, cetuximab, using DRO assay, we found the on-target binding ratio to be 65% and the binding constant ( K d ) to be 2.4 nM, while the off-target binding causes the binding constant to decrease by 0.3 nM. Other than cultured cells, the DRO assay can be performed on tumor mouse xenograft models. Thus, DRO is a simple and highly quantitative method for cell-based antibody binding analysis which can be broadly applied to screen and validate new therapeutic antibodies.
Source: ACS Sensors
Language: English
Subfield: Radiology, Nuclear Medicine and Imaging
Field: Medicine
Domain: Health Sciences
Open Access status: green
Funders: Welch Foundation, Breast Cancer Research Foundation, Cancer Prevention and Research Institute of Texas, National Breast Cancer Foundation, Ministry of Health and Welfare, Ministry of Science and Technology, Taiwan, China Medical University, University of Texas MD Anderson Cancer Center, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, Division of Chemistry
Awards: Turning a sequence barcode into a spectral barcode for single-cell analysis., G1716122260, A Randomized Clinical Trial Platform with Translational Studies to Overcome Resistance in Triple Negative Breast Cancer, G2426134809, Lung Cancer Organoid-Based High-Content Screening Platform for Evaluation and Prediction of Tumor Response to Treatment Using Machine Vi Sion and Deep Learning( I ), G3020008655, G3351836676, Training Grant in Cancer Biology, Recruitment of Established Investigators, Negative regulation of C-type lectin receptor signaling in response to fungal infection, Rational Combination Therapy with PARP Inhibitors in Triple Negative Breast Cancer, G7870490674, Mechanical Drugs: Harnessing Cancer Aggressiveness to Overcome Its Resistance, Cancer Center Support Grant, G912456358, G955703449
OpenAlex ID: W3007877619