Abstract 2142: Development and validation of multicolor flow cytometric tetramer assays for use in multi-year clinical cancer immunotherapeutics studies
Authors: Chengsen Xue, Christina D. Swenson, Simone Di Maulo, Weiguo Zhong, Thomas W. Mc Closkey
Journal: Cancer Research (2025)

Abstract Major histocompatibility complex (MHC) tetramers recognize antigen-specific cytotoxic T lymphocytes (CTL). As such, fluorescently labeled tetramers may be used to precisely monitor targeted T cell responses in infectious diseases and autoimmune diseases, like type 1 diabetes. As new biomedical technologies such as T-cell receptor (TCR) therapy gain traction, antigen specific CTL tetramer assays become even more important in current oncology clinical research projects, cancer vaccine development and cancer immunotherapeutics. Tetramer assays are unique and clearly differentiated from more routine applications of flow cytometry. Their specificity and sensitivity allow for the detection of specific cellular CTL, despite their extremely low frequency. We monitor broad anti-tumor immune responses. To do so, along with other phenotypic and functional analyses of T cells using monoclonal antibodies against surface and intracellular biomarkers, we have developed and validated tetramer assays. In practice, flow cytometry-based tetramer assays measure the percentage (%), and median fluorescence intensity (MFI) of target cells. During the development of these assays, to be able to measure rare events and test for binding specificity, we spiked positive CTLs into healthy blood samples and determined the assay’s lower limit of quantification (LLOQ). After a feasibility study, we measured the LLOQ using different dose levels of CTL. Other assay validation parameters assessed included post-collection sample stability and intra- and inter-assay precision, and a comparison of results in analyst-to-analyst and instrument-to-instrument assessments. The general acceptance criteria for these validation parameters are to achieve a 10%-30%CV between replicate results, or a similar percent difference between comparative measurements. The LLOQ of a tetramer assay can be determined during assay development when precision is at a CV of &amp;lt; 35% near the low end. In order to achieve the desired precision in rare event measurements, we established that it is critical to acquire a sufficient number of CD8+ cytotoxic T cells. For example, one single test sample can easily reach more than 2.5 million cell counts to precisely quantify the CTL of interest. Our results indicate that the developed tetramer assays are robust and reproducible, meeting the clearly pre-defined acceptance criteria during validation processes. More importantly, we found that the measurement of CTL as a percentage of CD8+ T lymphocytes in peripheral blood samples are very consistent in complex clinical trial settings over many years. In conclusion, the tetramer assay is suitable for use to accrue highly specific data in the measurement of CTL in multi-year oncology clinical studies. Citation Format: Chengsen Xue, Christina D. Swenson, Simone Di Maulo, Weiguo Zhong, Thomas W. Mc Closkey. Development and validation of multicolor flow cytometric tetramer assays for use in multi-year clinical cancer immunotherapeutics studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2142.