Abstract C003: A quantitative mass spectrometry workflow for highly multiplexed measurement of immunomodulatory proteins to support immunotherapy clinical trials

Abstract

Abstract Background: Since the approval of the first immune checkpoint-directed immunotherapies, it has been well-known that the efficacy of these therapies has been limited to a subset of cancer patients. As the number of new potential immunotherapy targets has grown, including novel combination therapy strategies, improved immune-related biomarker measurement has played a key role in understanding novel mechanisms of action and response to drugs. To support these efforts, we have developed an analytically validated assay for the multiplexed quantitation of up to 113 immunomodulatory proteins in various matrices (e.g., human plasma, PBMC, cells, or tissue) based on anti-peptide immunoaffinity capture followed by LC-MRM mass spectrometry analysis. Methods: Remnant samples from commercial biobanks were acquired in a variety of solid tumor indications. Briefly, matrices were denatured, reduced, alkylated, and digested using trypsin. Immunoaffinity capture of the peptide targets consisted of overnight incubation of the digested sample with anti-peptide antibodies coupled to protein G magnetic beads, followed by automated washing and elution of the peptides from the antibodies using the KingFisher platform. The peptides were analyzed in a multiplexed method by LC-MRM mass spectrometry. Endogenous levels for each target were measured relative to a stable isotopically labeled peptide standard. Results: In panel experiments, we found that the targets can be quantified precisely and accurately over 3 orders of magnitude with an intra-/inter-assay precision and accuracy of less than 20%. In total, 107 peptide targets were detected endogenously in cell lines. We expand on these data with analysis of solid tumor FFPE specimens where the results will be compared to immunohistochemical staining for selected targets. Conclusions: This study shows how multiplex immunoaffinity methods can complement classical methods of tissue-based protein expression for simultaneous and quantitative interpretation of protein levels. Citation Format: Ons Ousji, Anne Jang, Luca Genovesi, Nicholas Dupuis, Timon Geib, Gwenael Pottiez, Michael Schirm. A quantitative mass spectrometry workflow for highly multiplexed measurement of immunomodulatory proteins to support immunotherapy clinical trials [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C003.