Phagocytosis Is A Mechanism of Action for Daratumumab

Abstract 4054

Daratumumab (DARA) is a human IgG1 CD38 antibody with broad-spectrum killing activity. DARA induces killing of CD38-expressing tumor cells, including fresh cells from multiple myeloma (MM) patient samples, via diverse mechanisms. These prominently include the Fc-dependent effector mechanisms complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers et al. J. Immunol. 2011). In this study we show that DARA is also able to induce tumor cell killing via antibody-dependent cellular phagocytosis (ADCP) as an additional Fc-dependent effector mechanism.

In a first set of experiments, we studied ADCP with human macrophages as effector cells. Calcein-AM-labeled Daudi tumor cells, a Burkitt's lymphoma cell line, were mixed with human macrophages in the absence or presence of DARA. Specific DARA-induced phagocytosis was analyzed in flow cytometry by measuring the percentage of calcein-AM+/CD11b+ double-positive (DP) macrophages. Both classical GM-CSF activated and alternative M-CSF activated macrophages mediated DARA-specific ADCP of the Burkitt's lymphoma cells. To further explore the in vivo contribution of ADCP in the mechanism of action (MoA) of DARA, we studied DARA mediated phagocytosis with murine macrophages. In vitro ADCP with M-CSF-stimulated bone marrow-derived murine macrophages showed a dose-dependent DARA-specific effect on the Burkitt's lymphoma cell lines Ramos and Daudi, resulting in up to 24% and 43% DP macrophages and a 25% and 50% tumor cell reduction, respectively. Furthermore, dose-dependent DARA-specific phagocytosis was observed with patient-derived MM cell lines L363 and UM9, which were transduced with CD38 to obtain levels CD38 expression as they are generally observed in primary MM patient samples. With life-cell imaging we found that ADCP of Daudi and Ramos cells occurred very rapid and efficiently. Interestingly, our recordings document that single macrophages could engage multiple target cells and that they were able to engulf up to six tumor cells sequentially in a 30 min period. This suggests that ADCP might be a very potent MoA of DARA in vivo, which we are currently studying in a mouse xenograft model.

In conclusion, in addition to CDC and ADCC, we now show that DARA can also induce killing of CD38 expressing tumor cells via phagocytosis. This very fast and potent MoA might contribute to the treatment efficacy of DARA in hematological tumors, especially at sites where high numbers of macrophages reside, such as the bone marrow.