As we previously showed, NK cells are depleted in patients treated with daratumumab, therefore, in this study, we focused on monocyte/macrophages as the macrophage number is not altered by daratumumab treatment.30 First, we assessed the effects of cyclophosphamide on the expression of the daratumumab target CD38 on MM cells. and increased CD32 and CD64 Fc receptor expression on their cell surface. Daratumumab-specific tumor clearance was increased by conditioning macrophages with CTX-TCS in a dose-dependent manner. This effect was impeded by pre-incubating macrophages with Cytochalasin D (CytoD), an inhibitor of actin polymerization, indicating macrophage-mediated ADCP as the mechanism of clearance. CD64 expression on macrophages directly correlated with MM cell clearance and was essential to the observed synergy between cyclophosphamide and daratumumab, as tumor clearance was attenuated in the presence of a FcRI/CD64 blocking agent. Cyclophosphamide independently enhances daratumumab-mediated killing of MM cells by altering the tumor microenvironment to promote macrophage recruitment, polarization to a pro-inflammatory phenotype, and directing ADCP. These findings support the addition of cyclophosphamide to existing or novel monoclonal antibody-containing MM regimens. KEYWORDS: Multiple myeloma, daratumumab, cyclophosphamide, macrophages, ADCP Introduction Multiple Myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow (BM). MM remains an incurable disease, however, with treatment regimens evolving, this dogma is being challenged.1,2 The difficulty in treating MM can in part be attributed to the supportive role of the BM microenvironment to malignant plasma cell differentiation, migration, clonal expansion, survival and resistance to therapies.3,4 It is thought that transformation to MM requires the development of a permissive tumor microenvironment (TME), which facilitates immune escape.5,6 Current MM therapies include proteasome inhibitors (e.g. bortezomib), immunomodulatory agents (e.g. lenalidomide), and monoclonal antibodies, particularly the IgG1 kappa (IgG1) CD38 monoclonal antibody daratumumab.7,8 The anti-MM activity of these therapies relies upon S-Ruxolitinib the presence of an intact immune system.9 Thus, an improved understanding of the mechanisms underlying the immune-escape observed in MM could provide new insights into disease pathogenesis and opportunities for therapeutic intervention. With progression to MM, there are an increasing number of tumor-associated macrophages (TAMs) detectable in the BM.10 These are predominantly of an anti-inflammatory phenotype and promote tumor survival and immune suppression, which enables disease progression.11 A high number S-Ruxolitinib of anti-inflammatory TAMs in the BM has been associated with inferior survival in MM.12 TAMs likely accumulate in the BM under the influence of chemokines such as CCL2 (MCP-1), CCL3 (MIP-1) and CCL5 (RANTES) secreted by the myeloma cells.13 CCL5, in conjunction with other chemokines including CCL2, promotes macrophage recruitment and survival and may act as a pro-survival factor.14 Circulating monocytes are attracted into the BM along this chemokine gradient where they are polarized toward an anti-inflammatory macrophage phenotype under the influence of factors such as prostaglandin E2 (PGE2) and interleukin (IL)-10 (reviewed in15). Indeed, many of these chemoattractants and polarizing factors are known to be produced by MM cells, and have been reported to be associated with adverse outcomes.16 The presence of a large number of TAMs is generally considered to be undesirable.17 Some therapeutic approaches, such as antibodies targeting colony stimulating factor (CSF)-1 receptor, have been designed to eliminate macrophages from the TME, although this approach has had limited success.18,19 An alternative approach is to activate or reprogram the cells to harness their anti-tumor potential. Repolarization of TAMs to an anti-tumor phenotype has been achieved by reprogramming TAMs using, for example, anti-CD47 antibodies,20 histone deacetylase inhibitors21 and Toll-like receptor agonists.22 In the tumor microenvironment, anti-tumor macrophages have the capacity to clear tumor cells by several mechanisms of cytotoxicity including: direct cytotoxicity by releasing cytotoxic agents e.g. reactive oxygen species, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).23 Macrophages are thought to be S-Ruxolitinib critical effectors of monoclonal antibody (mAb) therapy, with reduced therapeutic efficacy recorded when macrophage levels were depleted in the TME .24 Monoclonal antibodies bind to effector macrophages via Fc gamma (Fc) receptors, a family of glycoproteins which bind to the Fc portion of IgG antibodies. 25 These Rabbit Polyclonal to HSP105 receptors can be activating or inhibitory. The activating Fc receptors in humans are; FcRI/CD64, FcRIIa/CD32a, FcRIIc/CD32c and FcRIIIa/CD16a.26 Unlike NK cells which predominantly only express FcRIIIa/CD16a, macrophages express all types of Fc receptors.26 Their depletion has been associated with reduced efficacy of antibodies and improved outcome has been seen in conjunction with high affinity polymorphisms of FcRIIa/CD32a, which are not expressed on NK cells.27 This may be particularly important in the context of treatment with daratumumab. Originally, it was thought that ADCC mediated by NK cells would constitute one of the most important mechanisms of action of daratumumab.28 However, with the benefit of careful correlative studies from clinical trials, we now know that treatment with.