Abstract 822: PureQuant Methylation Assay for the accurate quantification of immune cell populations

Abstract

Abstract A key challenge during the development and manufacturing of cell based immunotherapies is the reliable estimation of cell purity. Current flow cytometric methods, while effective for the detection of surface antigens such as CD8 that positively identifies Cytotoxic T lymphocytes, are not ideal for intracellular markers used to positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Additionally, characterization methods that utilize live cells pose a challenge in large scale GMP manufacturing environments due to complicated logistics, limited throughput and difficulty in standardization. Therefore, there is an emerging need for alternative assay methods that address some of these challenges. Here, we introduce our newly developed PureQuant™ Assays that specifically measure the unique DNA methylation status of specific immune cell types. Assays were developed specifically for Cytotoxic T Cells, Regulatory T cells and T helper 17 cells by detecting methylation status of CD8, FoxP3 and IL17A, respectively, via qPCR of bisulfite converted genomic DNA. These assays are robust with minimal sample requirement, utilizing fresh/frozen cells or isolated gDNA. Results are represented as a percentage of the total cell population similar to flow cytometry based methods. Comparison of the two methods for surface markers such as CD8 show comparable results. Our results demonstrate that this method which combines accuracy, low sample requirement and flexibility, is an ideal measurement system for confirmation of T cell identity and purity that is critical for cell therapy research and development. Citation Format: Suman Pradhan, Carl Dargitz, Jerry Guzman, Uma Lakshmipathy. PureQuant Methylation Assay for the accurate quantification of immune cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 822.