Higher-order death receptor 5 (DR5) clustering can induce tumor cell death; however, therapeutic compounds targeting DR5 have achieved limited clinical efficacy. We describe HexaBody-DR5/DR5, an... Show moreHigher-order death receptor 5 (DR5) clustering can induce tumor cell death; however, therapeutic compounds targeting DR5 have achieved limited clinical efficacy. We describe HexaBody-DR5/DR5, an equimolar mixture of two DR5-specific IgG1 antibodies with an Fc-domain mutation that augments antibody hexamerization after cell surface target binding. The two antibodies do not compete for binding to DR5 as demonstrated using binding competition studies, and binding to distinct epitopes in the DR5 extracellular domain was confirmed by crystallography. The unique combination of dual epitope targeting and increased IgG hexamerization resulted in potent DR5 agonist activity by inducing efficient DR5 outside-in signaling and caspase-mediated cell death. Preclinical studies in vitro and in vivo demonstrated that maximal DR5 agonist activity could be achieved independent of Fc gamma receptor-mediated antibody crosslinking. Most optimal agonism was observed in the presence of complement complex C1, although without inducing complement-dependent cytotoxicity. It is hypothesized that C1 may stabilize IgG hexamers that are formed after binding of HexaBody-DR5/DR5 to DR5 on the plasma membrane, thereby strengthening DR5 clustering and subsequent outside-in signaling. We observed potent antitumor activity in vitro and in vivo in large panels of patient-derived xenograft models representing various solid cancers. The results of our preclinical studies provided the basis for an ongoing clinical trial exploring the activity of HexaBody-DR5/DR5 (GEN1029) in patients with malignant solid tumors. Show less
Tetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function... Show moreTetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function for killing hematological cancer cells, CD37-specific antibodies are generally poor inducers of CDC. To enhance CDC, the E430G mutation was introduced into humanized CD37 monoclonal IgG1 antibodies to drive more efficient IgG hexamer formation through intermolecular Fc-Fc interactions after cell surface antigen binding. DuoHexaBody-CD37, a bispecific CD37 antibody with the E430G hexamerization-enhancing mutation targeting two non-overlapping epitopes on CD37 (biparatopic), demonstrated potent and superior CDC activity compared to other CD37 antibody variants evaluated, in particular ex vivo in patient-derived chronic lymphocytic leukemia cells. The superior CDC potency was attributed to enhanced IgG hexamerization mediated by the E430G mutation in combination with dual epitope targeting. The mechanism of action of DuoHexaBody-CD37 was shown to be multifaceted, as it was additionally capable of inducing efficient antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. Finally, potent anti-tumor activity in vivo was observed in cell line- and patient-derived xenograft models from different B-cell malignancy subtypes. These encouraging preclinical results suggest that DuoHexaBody-CD37 (GEN3009) may serve as a potential therapeutic antibody for the treatment of human B-cell malignancies. Show less
CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and... Show moreCD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo. Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations. Show less
