Best practices for optimization and validation of flow cytometry‐based receptor occupancy assays

Hilt, Ed; Sun, Yongliang; McCloskey, Thomas W.; Eck, Steve; McIntosh, Thomas; Grugan, Katharine D.; Lanham, David; Standifer, Nathan; Green, Cherie; Litwin, Virginia; Stewart, Jennifer J.

doi:10.1002/cyto.b.21970

Cited by 8 publications

(4 citation statements)

References 30 publications

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“…These assays are largely applied it evaluating therapies directed at immune conditions as well as targeted oncology therapy, making flow cytometry an ideal instrument for performing RO assays. Moreover, with flow cytometry, target engagement on specific cell subsets within heterogeneous mixtures can be achieved (4).…”

Section: Receptor Occupancy Assaysmentioning

confidence: 99%

“…The need for better assays and tools for evaluation and characterization of these drug candidates is still present and flow cytometry is one of the essential instruments in drug screening studies. Flow cytometry can be applied to various stages and aspects of drug discovery and development such as target identification, screening, mechanisms of action, biomarker discovery, toxicity testing and clinical trials (2)(3)(4)(5).…”

Section: Introductionmentioning

confidence: 99%

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Flow Cytometry: A Versatile and Powerful Tool for Drug Discovery and Development

Aru,

Yanikkaya Demirel

2024

Pharmedicine J.

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Flow cytometry, a pivotal tool in clinical and research labs since the discovery of cell markers in the mid-1970s, plays a crucial role across all phases of drug discovery. Modern flow cytometers can detect rare cell types relevant to disease pathogenesis, measure numerous parameters simultaneously, thus, offer versatility in drug screening. In drug discovery studies, flow cytometry contributes to the assessment of drug pharmacokinetics, pharmacodynamics and safety in animal models and clinical trials. It can also be used to monitor drug efficacy and identify biomarkers for diagnosis and prognosis. In essence, flow cytometry is a versatile, instrumental technique that supports drug discovery from target identification through to clinical development, limited only by the creativity of the researcher and the availability of fluorescent labels or specific size/scatter related findings. This review article focuses on the use of flow cytometry in drug discovery and drug development studies, summarizing not only conventional assays such as immunophenotyping, measurement of programmed cell death pathways and cell division to provide insights into drug effects and patient responses, but also novel approaches including mass cytometry, spectral cytometry, and droplet cytometry. Keywords: Flow cytometry, drug discovery, drug development, mass cytometry, spectral cytometry

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Section: Receptor Occupancy Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Flow Cytometry: A Versatile and Powerful Tool for Drug Discovery and Development

Aru,

Yanikkaya Demirel

2024

Pharmedicine J.

View full text Add to dashboard Cite

show abstract

“…Best practice guidelines have been published to guide flow cytometry data evaluation in a standardized manner ( 109 , 113 – 117 ). Guidelines for general method validation ( 114 , 118 – 120 ), as well as recommendations for specific applications are widely available ( 121 , 122 ). Furthermore, additional, unsupervised flow cytometry data evaluation methods have been developed in recent years ( 123 – 125 ) as the number of parameters that can be simultaneously interrogated by flow cytometry has increased ( 125 – 129 ).…”

Section: Measurements Of Cellular Immunitymentioning

confidence: 99%

Cellular Immunity Is Critical for Assessing COVID-19 Vaccine Effectiveness in Immunocompromised Individuals

Paramithiotis¹,

Sugden²,

Papp³

et al. 2022

Front. Immunol.

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COVID-19 vaccine clinical development was conducted with unprecedented speed. Immunity measurements were concentrated on the antibody response which left significant gaps in our understanding how robust and long-lasting immune protection develops. Better understanding the cellular immune response will fill those gaps, especially in the elderly and immunocompromised populations which not only have the highest risk for severe infection, but also frequently have inadequate antibody responses. Although cellular immunity measurements are more logistically complex to conduct for clinical trials compared to antibody measurements, the feasibility and benefit of doing them in clinical trials has been demonstrated and so should be more widely adopted. Adding significant cellular response metrics will provide a deeper understanding of the overall immune response to COVID-19 vaccination, which will significantly inform vaccination strategies for the most vulnerable populations. Better monitoring of overall immunity will also substantially benefit other vaccine development efforts, and indeed any therapies that involve the immune system as part of the therapeutic strategy.

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“…The sample stability will be tested in unstimulated and stimulated samples, to evaluate the expression of markers that are not present in naïve cell populations. Sample stability can be assessed holding at least three different donors freshly collected at the temperature required for the assay for a defined number of time points, established based on the sample type, on the cell population expressing the target and on the shipment condition-in case of international shipments time points for sample stability assessment can reach up to 4 days [39].…”

Section: Ro In Pre-clinical and Clinical Settingsmentioning

confidence: 99%

Flow cytometry and receptor occupancy in immune-oncology

Audia

Bannish

Bunting

et al. 2021

Expert Opinion on Biological Therapy

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Introduction: Immunotherapies are focused on strategies that alter immune responses, using antibodies that binds to receptors on different immune cell subsets and either activate or suppress their functions depending on the immune response being targeted. Hence, the necessity of developing assays that assess the functional and biological effect of a therapeutic on its target. When incorporated into high-parameter flow cytometry panels, receptor occupancy assay can simultaneously evaluate receptor expression and drug occupancy on defined cell subsets, which can provide information related to functional effects, and safety. Areas covered: This review focuses on the importance of developing, optimizing, and validating a robust Receptor Occupancy Assay (ROA) to improve dose selection, pharmacology monitoring and safety mainly in clinical settings. Expert opinion: The designing of an ROA can be challenging and can lead to exaggerated pharmacology if not accurately developed, optimized, and validated. However, improvements in our understanding of epitopes, binding, affinities, and pharmacological effects may lead to improved antibody drug targeting and receptor evaluation.

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Best practices for optimization and validation of flow cytometry‐based receptor occupancy assays

Cited by 8 publications

References 30 publications

Flow Cytometry: A Versatile and Powerful Tool for Drug Discovery and Development

Flow Cytometry: A Versatile and Powerful Tool for Drug Discovery and Development

Cellular Immunity Is Critical for Assessing COVID-19 Vaccine Effectiveness in Immunocompromised Individuals

Flow cytometry and receptor occupancy in immune-oncology

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