Development of a Highly Sensitive Hybrid LC/MS Assay for the Quantitative Measurement of CTLA-4 in Human T Cells

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a check point protein expressed on the surface of T cells and plays a central role in regulating the immune response. In recent years, CTLA-4 has become a popular target for cancer immunotherapy in which blocking CTLA-4 can restore T-cell function and enhance the immune response against cancer. Currently, there are many CTLA-4 inhibitors in a variety of modalities, including cell therapies, which are being developed in both preclinical and clinical stages to further harness the potential of the target for the treatment of certain types of cancer. In drug discovery research, measuring the level of CTLA-4 in T cells is important for drug discovery and development because it provides key information for quantitative assessment of the pharmacodynamics, efficacy, and safety of the CTLA-4-based therapies. However, to our best knowledge, there is still no report of a sensitive, specific, accurate, and reliable assay for CTLA-4 measurement. In this work, an LC/MS-based method was developed to measure CTLA-4 in human T cells. The assay demonstrated high specificity with an LLOQ of 5 copies of CTLA-4 per cell when using 2.5 million T cells for analysis. As shown in the work, the assay was successfully used to measure CTLA-4 levels in subtype T-cell samples from individual healthy subjects. The assay could be applied in supporting the studies of CTLA-4-based cancer therapies.