Abstract 4287: Serological quantification of fibroblast activation protein (FAP) cleaved type III collagen: A biomarker for FAP activity

Abstract Introduction: Fibroblast activation protein (FAP) has unique proteolytic activity with very low expression in healthy tissues, while being upregulated in diseased tissues including many types of cancer for example non-small cell lung cancer (NSCLC). The disease specific expression and unique proteolytic activity have made FAP an interesting protein to be utilized for drug targeting purposes. Therefore, it is important to identify the patients with FAP activity. The aim of the study was to develop a tool to non-invasively quantify FAP activity and investigate its potential as a biomarker for patients with NSCLC. Methods: We developed an ELISA (named C3F), with monoclonal antibodies raised against a FAP generated fragment of type III collagen that was identified by MS, to quantify FAP-mediated cleavage of type III collagen in serum to reflect proteolytic activity of FAP. We confirmed that the biomarker reflects proteolytic activity of FAP by incubating type III collagen with or without FAP and measured C3F. In addition, we confirmed elevated FAP expression in NSCLC (adenocarcinoma and squamous cell carcinoma) compared to healthy tissue and correlation between FAP and COL3A1 expression using data from The Cancer Genome Atlas (TCGA). The comparisons between FAP expression in healthy and cancer tissue was done using one-way ANOVA and correlation between FAP and COL3A1 was done using Spearman correlation. Lastly, C3F was measured in serum samples from patients with NSCLC (n = 109) and healthy controls (n = 42) and the two groups was compared using Mann Whitney test and area under receiver operating characteristic curves (AUROCs). Results: A C3F signal only appeared after type III collagen incubation with FAP, supporting that C3F reflects FAP proteolytic activity. C3F was significantly elevated in serum from patients with NSCLC compared to healthy controls (p < 0.0001, AUROC = 0.78). In support, FAP expression (TCGA) was significantly elevated in NSCLC tissue compared to normal lung tissue (p < 0.0001) and FAP expression correlated with expression of COL3A1 (persons r = 0.83). Conclusion: The FAP-cleavage mediated type III collagen fragment C3F reflects FAP activity and can be quantified non-invasively in serum. In this study we show C3F as promising diagnostic biomarker in NSCLC, however as the biomarker reflects FAP activity the optimal use is more is potentially as a predicter of treatment response from FAP-based drug delivery systems. Citation Format: Rasmus Sund Pedersen, Morten Karsdal, Nicholas Willumsen. Serological quantification of fibroblast activation protein (FAP) cleaved type III collagen: A biomarker for FAP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4287.