Abstract A026: Validation of an assay to identify regulatory T cells by flow cytometry in global clinical trials
Authors: Zhou Li , Buono Krista D. , Swenson Christina D. , Brien Michelle , Closkey Thomas W. Mc 
Published: May 2016

Abstract
The measurement of regulatory T cells (Treg) by flow cytometry provides important information in the assessment of new anti-tumor drug candidates, particularly in regards to an immuno-oncology mechanism of action. An assay was validated using a fit-for-purpose approach to characterize an optimized multi-parameter flow cytometry protocol for the identification and quantification of Treg from whole blood for use in global clinical trials, using standardized central laboratory sample testing. The method established at ILS-New York detects Treg by using fluorochrome-conjugated monoclonal antibodies which bind the cellular surface marker proteins CD45, CD3, CD4, CD25, and intracellular FOXP3. This method was determined to yield low non-specific labeling and increased Treg resolution with limited cell loss. Validation of the Treg assay included titration of monoclonal antibodies, assessment of stability of collected samples, inter- and intra- assay precision determinations, and analyst-to-analyst and instrument-to-instrument comparisons. In addition, global harmonization of the procedure was conducted across three ILS laboratories in New York, Dublin and Singapore. Samples were acquired on a BD FACSCanto II flow cytometer and results averaged across five donors. Samples, assessed at 0, 24, 48 and 72 hours post-collection, were stable up to 72 hours post-collection. The percent coefficient of variation calculated for intra- and inter- assay precision assessments, in the measurement of the percentage of CD25+FOXP3+ cells, was found to be &lt;10%. Comparisons of variability between analysts and instruments indicated, for the percentage of CD25+FOXP3+ cells, a percent difference of &lt;20%. Global laboratory assessments of the Treg phenotype were made between New York and Dublin and also between New York and Singapore, using aliquots from the same donor samples; the percent differences between laboratories were 13.43% and 24.19%, respectively.
In conclusion, in clinical trials analyzing cancer immunotherapy candidates that modulate lymphocyte immune responses, it is informative to have an established method for Treg analysis that is robust and reproducible. This is especially important as Treg are a low frequency subpopulation, ranging from 1% to 7% of CD4+ T lymphocytes in normal peripheral blood, and assessment of differences between low frequency populations can lead to increased variability. Despite the low frequency of these cells, our data indicate that a validated Treg assay allows for clinical trials to be carried out with multiple subjects that can be assessed at multiple time points. Importantly, this method has been validated at different sites around the world, with the ability to obtain comparable data from North America, Europe, and Asia. Applying complex scientific techniques such as multicolor flow cytometry, in a globally harmonized manner, will provide important data for drug development of immuno-oncology therapeutic candidates and ultimately serve to accelerate new therapeutic options to patients.
Citation Format: Li Zhou, Krista D. Buono, Christina D. Swenson, Michelle Brien, Thomas W. Mc Closkey. Validation of an assay to identify regulatory T cells by flow cytometry in global clinical trials. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A026.