Antitumor activity of Avaren-Fc, a novel high-mannose-glycan-targeting immunotherapy, in ovarian cancer

Ovarian cancer (OVCA) is the most lethal and eighth most common gynecological cancer worldwide. Current standard-of-care treatments lack tumor specificity and fail to effectively manage chemoresistant disease recurrence, highlighting the need for new therapeutics. Avaren-Fc (AvFc) is a novel lectin-Fc fusion protein designed to target aberrant N -linked high-mannose-type glycans, an underutilized OVCA biomarker. After binding to cancer-associated high-mannose glycans, AvFc can activate Fcgamma receptors and induce cytotoxic effector functions. Our previous work showed that AvFc binds to and induces antibody-dependent cell-mediated cytotoxicity (ADCC) activity against multiple human OVCA cells lines (A2780, CAOV3, SKOV3, and SW626) in an in vitro luciferase-based ADCC reporter assay using a FcgammaRIIIa-expressing cell line. AvFc also selectively recognized stage I high-grade serous OVCA tumors over adjacent normal tissue by immunohistochemistry. In the present study, we assessed the anti-OVCA activity of AvFc using the murine ID8 cell line in in vitro experiments and a syngeneic orthotopic challenge model in C57BL/6 mice. Flow cytometry analysis demonstrated that AvFc binds to ID8 cells in a dose-dependent manner at nanomolar concentrations, with negligible binding to isolated murine reproductive cells. AvFc binding to ID8 cells significantly increased following treatment with kifunensine, a class I alpha-mannosidase inhibitor, and this enhanced binding was abolished following high-mannose glycan digestion with endoglycosidase H, confirming high-mannose dependence. AvFc also induced surrogate ADCC activity against ID8 cells by activation of FcgammaRIIIa. AvFc's antitumor efficacy was assessed in the ID8 OVCA model, where mice were intraperitoneally (i.p.) challenged with 2x10 6 ID8 cells and treated with 15 doses of AvFc (25 mg/kg, i.p., every other day), a non-sugar binding AvFc mutant, or vehicle beginning one-week post-challenge. AvFc treatment significantly prolonged median survival from 46 to 70 days compared with the vehicle control (p adj=0.0216, Log-rank [Mantel-Cox] test with Bonferroni's correction). Cytometry by time-of-flight (CyTOF) analysis performed one day after the final dose revealed that AvFc treatment uniquely led to the expansion of total spleen leukocytes, including Tbet+ Th1 cells, and increased CD8 + T cells expressing the degranulation marker CD107a in the peritoneal fluid, suggesting that AvFc's anticancer efficacy was partially mediated by adaptive immunity. Imaging mass cytometry analysis of ID8 tumors is ongoing. Taken together, these findings highlight AvFc's promising potential as an immunotherapeutic agent for OVCA.