Abstract Background IMV-M is a MUC16 × DR5 bispecific antibody has demonstrated MUC16-selective anti-tumor activity. However, it remained unclear whether multisite binding to MUC16 is required for cytotoxicity, and whether circulating CA125 (soluble MUC16) or anti-drug antibodies could affect efficacy or cause toxicity. Methods A comparative analysis of three bispecific antibodies, IMV-M (sofituzumab×DR5), 11D10 × DR5, and fluor×DR5, sharing an identical IgG1–anti-DR5 scFv architecture, was performed. Sofituzumab binds to multiple epitopes on a single MUC16 molecule, whereas 11D10 binds a single MUC16 epitope, and fluor does not bind any human antigen. Antibody binding to shed and cell-surface MUC16 was evaluated by ELISA and flow cytometry. Cytotoxicity was assessed in a MUC16+/DR5+ tumor cell line and MUC16−/DR5+ hepatic cell lines. Additional studies examined the effects of soluble CA125 and Fc-directed polyclonal antibodies on IMV-M activity. Results IMV-M bound MUC16 to a markedly higher extent than the 11D10 × DR5 comparator, consistent with its multivalent engagement, while binding of fluor×DR5 to MUC16 was negligent. Only IMV-M induced potent cytotoxicity in MUC16+ tumor cells, whereas 11D10 × DR5 and fluor×DR5 control antibodies were inactive, consistent with the notion that multivalent clustering on MUC16 is required for apoptosis. IMV-M showed no significant cytotoxicity toward hepatic cell lines, even in the presence of Fc-directed polyclonal antibodies or clinically relevant concentrations of soluble CA125. Conclusions These findings suggest that IMV-M cytotoxic activity requires clustering on MUC16, that CA125 at clinically relevant concentrations does not mediate IMV-M neutralization, and that aggregate formation with secondary antibodies or soluble MUC16 does not induce off-target toxicity.
Goldmacher et al. (Fri,) studied this question.