Abstract A116: Characterization of micvotabart pelidotin target binding properties and extracellular payload release

Abstract Micvotabart pelidotin (micvo) is an antibody–drug conjugate (ADC) that targets the non-cellular extra-domain B of fibronectin (EDB+FN), an extracellular matrix (ECM) protein highly expressed in tumors compared to normal adult tissues. Micvo consists of a fully human IgG1 monoclonal antibody with site-specific conjugation to a cleavable mcValCitPABC linker and optimized auristatin payload Aur0101, with a homogeneous drug-to-antibody ratio (DAR) of four. Micvo is designed to bind the EDB splice variant of fibronectin (FN) in the tumor ECM where extracellular proteases cleave the linker and release the cytotoxic payload to diffuse into and kill tumor cells. Recent data showed an enzyme gene signature, including multiple proteases, was elevated in patient derived xenograft (PDX) models with very strong response to micvo compared to models which did not respond. Here, in vitro studies were performed to characterize the binding properties of micvo and also identify the proteases that can cleave micvo’s linker to release its payload extracellularly. The binding specificity and avidity of micvo were studied using FN proteins with or without the EDB domain and an EDB-Fc dimeric protein by Octet analysis. Several proteases known to be elevated in the tumor ECM were investigated for the ability to cleave the linker and release the payload from micvo by chromatography-based analysis and cell-based assays. The protease-mediated micvo linker cleavage and DAR changes were analyzed by hydrophobic interaction chromatography (HIC). The EDB+FN-negative tumor cell line HT-29 was used for the in vitro cytotoxicity assay to confirm protease-mediated extracellular payload release of micvo. In vitro studies showed that micvo specifically bound to the EDB-containing variants of FN and did not bind to non-EDB-containing FN. Additionally, micvo bound to dimeric EDB+FN with much stronger affinity than monomeric EDB+FN attributed to the significantly reduced dissociation rate. Recombinant proteases were tested in vitro and protease-mediated reduction of DAR by HIC analysis was observed. For certain proteases, the optimal conditions for extracellular linker cleavage of micvo were determined to be pH dependent. Finally, using an in vitro cytotoxicity assay with a cell line that is not directly killed by micvo, extracellularly released payload-mediated tumor cell death was observed, further elucidating the contribution of the extracellular proteases to micvo’s bystander effect and anti-tumor activity. These studies demonstrate that micvo specifically binds to the EDB splice variant of FN in an avidity driven manner and that proteases known to be present extracellularly in the tumor ECM can cleave the linker payload of micvo to enable tumor cell killing. These findings further elucidate the non-cellular targeting and mechanism of action of micvo and support its utility as an investigational therapy for patients with difficult-to-treat cancers in the ongoing clinical trials as a monotherapy (NCT05720117) and in combination with pembrolizumab (NCT06795412). Citation Format: Chuan Shen, Matthew Iovino, Amanda Facklam, Anthony B. Rodriguez, Nicolas Severe, Frank Wang, Marsha Crochiere. Characterization of micvotabart pelidotin target binding properties and extracellular payload release [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A116.