Quantitative Mass Spectrometry Analysis Of The Pd-L1 And Tigit Signaling Axes In Primary Nonsmall Cell Lung Cancers And Lymph Node Metastases.

Abstract 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.