Reliable quantitative assays for protein targets of immune checkpoint (IC) therapeutics are lacking and this complicates clinical development of IC drug combinations. Biomarker-blind clinical development of drug combinations may help explain high profile failures of late-stage trials for combination immunotherapies. Targeted mass spectrometry (MS) of IC drug targets in tumors offers a solution to this problem through precise quantitation of multiple IC targets, immune co-regulators and immune cell markers in the same tissue sample. Combination immunotherapies targeting PD-1/PD-L1 and TIGIT are in clinical development in solid tumors, but without the use of TIGIT biomarkers. We used MS to precisely quantify PD-1, PD-L1, TIGIT and their co-regulators in 100 primary non-small cell lung cancers (NSCLC) and matched tumor draining lymph node metastases (TDLN). PD-1, PD-L1 and PD-L2 were more abundant in TDLN than in tumors for approximately two-thirds of the cases. PD-L1 and PD-L2 were co-expressed in all tumors and TDLN and were quantified across a 46-fold and 15-fold abundance range, respectively. PD-L1 was more abundant than PD-L2 in approximately 80% of tumors and TDLN, potentially explaining differential responsiveness to PD-1 compared to PD-L1-targeted therapies. In contrast to PD-1/PD-L1, TIGIT was quantifiable in only 4% of tumors and 60% of TDLN, whereas the activating CD226 receptor was quantified in 47% of tumors and 93% of TDLN. All TDLN that expressed TIGIT also co-expressed CD226 at approximately 2- to 3-fold greater abundance than TIGIT. TIGIT ligands CD112 and CD155 were co-expressed in nearly all tumors and TDLN, with combined CD112/CD155 in greater abundance than TIGIT in TDLN. Quantitation of CD4, CD8, FOXP3 and CD56 in the same analyses enabled comparison of the IC targets and their co-regulators with abundance of cytotoxic T-cells, Tregs and NK cells. PD-1, PD-L1, and PD-L2 were measured in approximately a 15-fold, 8-fold, and 5-fold abundance range, respectively, in TIGIT-expressing TDLN. The data indicate that clinical biomarker assays for TIGIT should ideally include both TDLN and primary tumors. Verified co-expression of TIGIT and PD-1 or PD-L1 in TDLN provide a rationale for combination therapy, whereas lack of target expression should have negative predictive value. Targeted MS assay panels offer a powerful platform for quantifying IC drug targets and their functional partners, defining their co-expression in tumor specimens. This deployment of a new generation of biomarkers will enable a systematic prioritization and development of combination immunotherapies to transform therapeutic development from serendipity to predictability.

Citation Format: Daniel C. Liebler, Matt Westfall, Salisha Hill, Ryan D. Morrison, Kevin J. Horgan, Alexander Haragan. Quantitative mass spectrometry analysis of the PD-L1 and TIGIT signaling axes in primary non-small cell lung cancers and lymph node metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB042.