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High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds
Published on: August 18, 2023
Development of a simple new flow cytometric antibody-dependent cellular cytotoxicity (ADCC) assay with excellent
Miho Tanaka1, Akiko Ishige1, Masami Yaguchi1
1Drug Discovery Antibody Platform Unit, RIKEN Center for Integrative Medical Science (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Abstract:
Antibody-based therapeutic strategies have become recognized as useful clinical options in several types of cancer, often with the expectation that such therapies will trigger target cell elimination via antibody-dependent cellar cytotoxicity (ADCC) by natural killer cells. The successful development of therapeutic monoclonal antibodies (mAbs) requires an assay system that permits a critical evaluation of their physicochemical and biological characteristics. At present a number of ADCC assay systems have been reported, however, there is still room for improvement in terms of usability, operability and sensitivity. Here we report a novel flow cytometric ADCC assay that uses a human natural killer cell line stably transfected with mouse FcγRIII, and Fc receptor common-γ chain (FcRγ) and a reporter gene as effector cells. This assay relies on discriminating effector and target cells by their differential immunofluorescence, which allows for clear-cut gating and accurate calculation of the number of surviving cells in a target population. This assay is easy and quick to perform and provides reliable data even for low frequency target cells in assay samples and with low concentrations of mAbs. Furthermore, our approach allows us to identify synergistic ADCC activity of mAbs with different epitope specificities on the same target antigen.
Insights
A new flow cytometric assay enhances antibody-dependent cellular cytotoxicity (ADCC) evaluation for cancer therapeutics. This improved method offers greater sensitivity and accuracy for assessing monoclonal antibodies (mAbs) and their synergistic effects.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Antibody-based therapies are crucial in cancer treatment, often relying on antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer cells.
- Developing effective therapeutic monoclonal antibodies (mAbs) necessitates robust assay systems for evaluating their characteristics.
- Existing ADCC assay systems have limitations in usability, operability, and sensitivity.
Purpose of the Study:
- To develop a novel, highly sensitive, and user-friendly flow cytometric assay for evaluating antibody-dependent cellular cytotoxicity (ADCC).
- To enable accurate assessment of monoclonal antibodies (mAbs) and their potential synergistic activities.
- To improve the evaluation of therapeutic antibody efficacy in cancer treatment.
Main Methods:
- Development of a novel flow cytometric ADCC assay using engineered human natural killer cells as effector cells.
- Effector cells were stably transfected with mouse FcγRIII, Fc receptor common-γ chain (FcRγ), and a reporter gene.
- Discrimination of effector and target cells using differential immunofluorescence for precise cell counting and analysis.
Main Results:
- The novel assay demonstrated ease of use, speed, and reliable data acquisition, even with low target cell frequencies and low mAb concentrations.
- The assay allows for clear-cut gating and accurate quantification of surviving target cells.
- The system successfully identified synergistic ADCC activity between mAbs with different epitope specificities against the same target antigen.
Conclusions:
- This flow cytometric ADCC assay represents a significant advancement in evaluating therapeutic mAbs for cancer treatment.
- The assay's sensitivity, accuracy, and ability to detect synergistic effects offer improved tools for drug development.
- This method facilitates a more comprehensive understanding of mAb-mediated anti-cancer immune responses.
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