Abstract Background: Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potent cytotoxicity of small-molecules payloads. While classical ADCs rely on antigen binding, internalization and intracellular linker cleavage in the lysosomes recent studies show that certain tumors exhibit extracellular cathepsin activity capable of cleaving ADC linkers in the tumor microenvironment (TME) and creating a peritumoral “drug halo” that enhances bystander killing even in antigen-low or antigen-negative TME. For this reason, identifying tumors with high extracellular cathepsin activity may be critical for optimizing ADC efficacy and patient stratification. Methods: We developed and investigated the potential of C3C, an ELISA-based biomarker that measures type III collagen fragments generated by cathepsins. Since the same cathepsins responsible for cleavage type III collagen and generating C3C also cleave commonly used peptide ADC linkers, we hypothesize that C3C could provide and non-invasive readout of cathepsin activity in the TME. We measured C3C in serum from patients with colorectal cancer (CRC) (n = 10), pancreatic ductal adenocarcinoma (PDAC) (n = 10) and non-small cell lung cancer (NSCLC) (n = 10) and compared to healthy controls (n = 26). To assess biological specificity, we also measured C3-HNE, a biomarker reflecting neutrophil elastase-driven inflammation. Results: We found significantly elevated levels of C3C compared to healthy individuals in patients with CRC ( p = 0.03), PDAC ( p = 0.006) and NSCLC (p 0.001). In contrast, C3-HNE showed no correlation with C3C in any indication, suggesting that C3C does not simply reflect general inflammation but instead captures cathepsin activity implicated in extracellular ADC linker cleavage. Conclusion: The results suggest that C3C is a promising biomarker for identifying cathepsin-rich TMEs that may support payload release and potent bystander activity of ADCs. Furthermore, C3C could be used to select patients more likely to benefit from cleavable-linker ADCs representing a potential tool for both drug development and clinical precision oncology. Citation Format: Marina Crespo-Bravo, Nicholas Willumsen, Morten Karsdal. C3C: A biomarker for extracellular cathepsin activity and ADCs abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3736.
Crespo-Bravo et al. (Fri,) studied this question.