Multiplex Immuno-Liquid Chromatography–Mass Spectrometry–Parallel Reaction Monitoring (LC–MS–PRM) Quantitation of CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2 in Frozen Human Tissues

The immune status of tumors critically influences their responsiveness to PD1 blockades and other immune-based therapies. Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is a clinically validated predictive biomarker of response to checkpoint-inhibitor therapy in a limited number of clinical settings but is poorly predictive in most. With emerging evidence that multiple pathways and immune-checkpoint proteins may coordinately contribute to the adaptive immune resistance, the identification and quantitation of multiple immune markers in tumor tissue could help identify the controlling pathways in a given patient, guide the selection of optimal therapy, and monitor response to treatment. We developed and validated a sensitive and robust immuno-liquid chromatography–parallel reaction monitoring assay to simultaneously quantify the expression levels of six immune markers (CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2) using as little as 1–2 mg of fresh frozen tissue. The lower limit of quantitation rang...