Abstract A112: Micvotabart pelidotin induces immunogenic cell death markers and activates tumor immune cells in pre-clinical studies

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 protein highly expressed in tumors compared to normal adult tissues. Micvo’s antitumor activity is hypothesized to be driven by 3 mechanisms- 1) direct tumor cell killing, 2) bystander activity and 3) immunogenic cell death (ICD). Recent studies have demonstrated that a mouse analog of micvo enhanced T cell infiltration in EMT6 tumors and induced strong tumor regression when combined with anti–PD1, supporting clinical development of micvo as a monotherapy (NCT05720117) and in combination with pembrolizumab (NCT06795412). ICD elicits an immune response through the release of damage-associated molecular patterns (DAMPs) and tumor antigens, contributing to anti-tumor activity and sensitizing cancers to PD1/PDL1 targeting. Here, both in vitro and in vivo assays were used to better characterize micvo’s mechanism of inducing ICD. EDB+FN+ Caki2 cells were treated with micvo for 3 days and High Mobility Group Box 1 (HMGB1) protein and extracellular ATP (eATP) were measured in the supernatants via luminescence-based plate assays. Micvo pre-treated Caki2 cells were co-cultured with myeloid cells and the induction of their activation markers was measured by flow cytometry. Immunocompetent mice bearing subcutaneous EMT6 tumors were treated with a mouse analog of micvo (Q4Dx3) or saline buffer, and tumors were collected post-treatment and enzymatically digested. Dissociated tumors were stained with a T cell flow cytometry panel to assess their activation status. DAMPs were first analyzed to assess induction of ICD on cancer cell lines. HMGB1 and eATP levels increased after in vitro micvo treatment compared to control. Additionally, it was observed that micvo treated Caki2 cells induced expression of activation and co-stimulatory markers on myeloid cells including HLA-A2, HLA-DR and CD80 while CD163 was downregulated in a co-culture system, indicating a more inflammatory and antigen presenting phenotype. Flow cytometry analysis of EMT6 tumors from mice treated with a mouse analog of micvo showed an increase of tumor infiltrating T cells expressing several activation markers including Slamf6, CD69, CD38 as well as PD1, demonstrating a stimulation of immune response post-treatment. Together, these findings provide additional evidence for the mechanism of action of micvo related to ICD, contributing to the overall anti-tumor activity of the ADC. In addition to payload-mediated cancer cell killing, the occurrence of ICD induced by micvo stimulates an immune response likely driven by DAMPs and tumor antigens which enhance anti-tumor activity and activate immune cells. Ongoing studies aim to further characterize the activation profile of both myeloid and T cell populations in vitro and in vivo. These data further provide a rationale for the on-going clinical evaluation of micvo as a monotherapy (NCT05720117) and in combination with pembrolizumab in patients with difficult-to-treat cancers (NCT06795412). Citation Format: Matthew Iovino, Anthony B. Rodriguez, Amanda Facklam, Chuan Shen, Nicolas Severe, Marsha Crochiere. Micvotabart pelidotin induces immunogenic cell death markers and activates tumor immune cells in pre-clinical studies [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 A112.