Development of PureQuant Real-Time PCR-Based Assay for Assessment of Immune Cell Identity and Purity in CAR-T Workflows

Abstract

Abstract Significant progress has been made in harnessing the power of immune system, in particular, T cells, to treat certain kinds of cancers. One of the key challenges in developing immune cells as therapeutic agents is the accurate estimation of their identity and purity. Current methods used for the characterization of immune cell types rely on flow cytometry. Flow cytometry can accurately estimate CD8+ T lymphocytes and other surface markers, but the method is challenging to implement in a GMP manufacturing environment posing logistical challenges such as requirement for live cells, variability leading to difficult in standardizing and high throughput. In addition, cytometric methods are not accurate for specific intracellular targets that positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Therefore, there is an emerging need for alternative assay methods. Methylation state is known to be unique for specific cell types and can thus be used as an identifier in heterogeneous population of cells. Exploiting differences in cell type-specific methylation signatures, we developed assay kits that quantify the percentage of Treg and Th17 by detecting methylation status of FoxP3 and IL17A via qPCR of bisulfite converted genomic DNA. In contrast to flow analysis, sample requirement is minimal and the assay works well with fresh/frozen cells or genomic DNA. This assay has been implemented to accurately identify and estimate different T cell population in Chimeric Antigen Receptor (CAR)-modified T cells. The combination of accuracy, low sample requirement and flexibility provides an ideal measurement system for confirmation of identify and purity of T cell types critical for therapeutic applications.