Potent Ex Vivo Anti-Tumor Activity in Relapsed Refractory Multiple Myeloma Using Novel DR5-Specific Antibodies with Enhanced Capacity to Form Hexamers upon Target Binding

Hyperclustering of Death Receptor 5 (DR5) after binding of its ligand TRAIL (TNF-related apoptosis-inducing ligand), induces apoptosis. As cancer cells are more sensitive for TRAIL-induced apoptosis than nonmalignant cells, targeting DR5 with agonistic monoclonal antibodies (mAbs) has been evaluated as a potentially effective therapy for several cancer types. Unfortunately, clinical results with conventional DR5-targeting mAbs have been disappointing due to lack of efficacy.

To improve antibody-mediated clustering of DR5 on cancer cells we applied the novel HexaBody® technology, which is based on the natural concept that, upon binding to antigens on a cell surface, immunoglobulins (IgGs) can organize into ordered hexamers through Fc-Fc interactions. By introducing a single point mutation at specific residues in the IgG1 Fc domain, hexamer formation could be significantly enhanced. Accordingly, Hx-DR5-01/05 (GEN1029) was developed. Hx-DR5-01/05 is a mixture of two non-competing DR5-specific antibodies, with enhanced capacity to form hexamers upon target binding due to an E430G mutation in their Fc domains. Thus, Hx-DR5-01/05 induces potent DR5 agonist activity through a combination of hexamer formation and dual-epitope targeting. In contrast to conventional DR5 specific antibodies, Hx-DR5-01/05-mediated tumor cell kill is independent of Fc gamma receptor-mediated crosslinking.

Here, we explored the potential utility of Hx-DR5-01/05 in multiple myeloma (MM) by analyzing cytotoxic activity against MM cell lines and a large panel of primary MM cells derived from patients with newly diagnosed or relapsed refractory disease. Hx-DR5-01/05 induced effective cytotoxicity in MM cell lines, whereas the same antibodies without the E430G mutation did not, underscoring the important contribution of hexamer formation for DR5 agonist activity of Hx-DR5-01/05.

Similarly, ex vivo assays using primary MM cells present in the bone marrow mononuclear cells of MM patients revealed superior lytic activity of Hx-DR5-01/05 against primary MM cells as compared to its wild-type counterpart. Overall, the lysis levels of primary MM cells displayed a weak but significant correlation with their DR5 expression levels. Importantly, MM cell lysis levels equal or above 20% were observed in a significantly higher fraction of relapsed refractory patients (74%) compared to those who are newly diagnosed (30%), which correlated with significantly higher DR5-expression levels of MM cells from relapsed/refractory compared to newly-diagnosed patients. In conclusion, our ex vivo results indicate superior therapeutic potential of Hx-DR5-01/05 as compared to conventional DR5 antibodies, with the highest activity in relapsed/refractory MM patients.