Quantitative Mass Spectrometry Analysis Of Immune Checkpoint Protein Expression And N-Glycosylation In Human Melanoma

Quantitative assessment of key proteins that control the tumor-immune interface is one of the most formidable analytical challenges in immunotherapeutics. We developed an targeted mass spectrometry (MS) platform to quantify programmed cell death-1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death 1 ligand 2 (PD-L2) at fmol/microgram protein levels in formalin fixed, paraffin-embedded sections from 22 human melanomas. PD-L1 abundance ranged 50-fold, from approximately 0.03 to 1.5 fmol/microgram protein and the PRM data were largely concordant with total PD-L1-positive cell content, as analyzed by immunohistochemistry (IHC) with the E1L3N antibody. PD-1 was measured at levels up to 20-fold lower than PD-L1, but the abundances were not significantly correlated (r2 = 0.062, p = 0.264). PD-1 abundance was weakly correlated (r2 = 0.3057, p = 0.009) with the fraction of lymphocytes/histiocytes in sections. PD-L2 was measured from 0.03 to 1.90 fmol/microgram protein and the ratio of PD-L2 to PD-L1 abundance ranged from 0.03 to 2.58. In 8 samples, PD-L2 was present at more than half the level of PD-L1, which suggests that PD-L2, a higher affinity PD-1 ligand, is sufficiently abundant to contribute to T-cell downregulation. We also identified five branched mannose and N-acetylglucosamine glycans at PD-L1 position N192 in all 22 samples. Extent of PD-L1 glycan modification varied by approximately 10-fold and the melanoma with the highest PD-L1 protein abundance and most abundant glycan modification yielded a very low PD-L1 IHC estimate, thus suggesting that N-glycosylation may affect IHC measurement and PD-L1 function. Additional PRM analyses quantified immune checkpoint/co-regulator proteins LAG3, IDO1, TIM-3, VISTA and CD40, which all displayed distinct expression independent of PD-1, PD-L1 and PD-L2. Targeted MS can provide a next-generation analysis platform to advance cancer immuno-therapeutic research and diagnostics. (Supported by NIH grant U24CA159988) Citation Format: Carlos Morales Betanzos, Hyoungjoo Lee, Paula I. Gonzalez Ericsson, Justin M. Balko, Douglas B. Johnson, Lisa J. Zimmerman, Daniel C. Liebler. Quantitative mass spectrometry analysis of immune checkpoint protein expression and N-glycosylation in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-056. doi:10.1158/1538-7445.AM2017-LB-056