Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions in vitro

Tisotumab vedotin is an antibody-drug conjugate composed of a human IgG targeting tissue factor (TF), a valine citruline linker, and the microtubule disrupting agent monomethyl auristatin E (MMAE). TF is aberrantly expressed in solid tumors and is thought to contribute to tumor progression by enhancing tumor growth, neo-angiogenesis and metastatic potential through local activation of coagulation and protease-activated receptor-2 (PAR-2) signaling. Tisotumab vedotin was previously shown to induce cytotoxicity through MMAE- and Fc-dependent mechanisms. In addition, tisotumab vedotin inhibited PAR-2 signaling in TF-positive tumor cells. Here we aimed to elucidate additional effector mechanisms by assessing the capacity of tisotumab vedotin to induce immunogenic cell death (ICD), bystander cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP) in vitro. ICD was assessed by incubating A431, MDA-MB-231, and HPAFII cells with tisotumab vedotin, and measuring ER stress by western blot, ATP secretion by reporter assay, and HMGB1 release by ELISA. Bystander cytotoxicity was assessed by incubating mixed cultures of TF+ (MDA-MB-231) and TF- (A549) tumor cells with tisotumab vedotin and quantifying viable cells by flow cytometry. To assess ADCP, PKH26-labeled BxPC-3 or A431 cells were incubated with human monocyte derived macrophages in presence of tisotumab vedotin. ADCP activity was analyzed by measuring the fraction of macrophages that double stained with PKH26 and anti-CD11c antibody using flow cytometry. Tisotumab vedotin induced key hallmarks of ICD including induction of ER stress, ATP secretion, and release of HMGB1, which have been shown to facilitate antitumor immunity. In co-cultures of TF+ and TF- tumor cells, tisotumab vedotin induced cytotoxicity in both cell types, even at a target-positive to target-negative cellular ratio of 1:15. This was mediated through bystander cytotoxicity, as demonstrated by the lack of cytotoxicity in monocultures of TF- cells. Finally, ADCP was observed with macrophages derived from multiple donors, with double-positive macrophages comprising up to 50% of total macrophages. In summary, ICD, bystander cytotoxicity, and ADCP were identified as novel effector mechanisms of tisotumab vedotin in vitro. Combining the present study with previous results, tisotumab vedotin induces TF-dependent anti-tumor activity through 1) MMAE-mediated effector mechanisms, including MMAE-mediated direct and bystander cytotoxicity, and induction of ICD; 2) Fc-mediated effector mechanisms, including ADCC and ADCP; and 3) Fab-mediated inhibition of PAR-2 dependent signaling. Tisotumab vedotin is currently being investigated in a variety of solid tumors (NCT03245736, NCT03485209, NCT03438396, NCT03657043). Citation Format: Stephen C. Alley, Jeffrey R. Harris, Anthony Cao, Elke Gresnigt-van den Heuvel, Jyoti Velayudhan, David Satijn, Sandra Verploegen, Teresa Dominguez, Esther C. Breij. Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 221.