Mononuclear cells (MNC) from the bone marrow (BM) were isolated by Ficoll-Hypaque density-gradient centrifugation and contained 2%C35% MM cells as detected by flow cytometry

Mononuclear cells (MNC) from the bone marrow (BM) were isolated by Ficoll-Hypaque density-gradient centrifugation and contained 2%C35% MM cells as detected by flow cytometry. 1.8% of all cancer mortality worldwide.2 Despite encouraging improvements in the survival of MM patients over the last decade, the disease remains incurable, even with combination therapies with effective novel pharmacological agents.2C5 An attractive novel alternative to these treatments is the targeting of MM with therapeutic antibodies, as already standard-of-care in several other hematologic malignancies. Therefore, we generated the CD38-specific human monoclonal antibody, daratumumab (DARA), which induces MM cell death via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).6 Based on these preclinical data, DARA is currently being evaluated in patients with relapsed/refractory MM, with encouraging results.7 In previous studies, we demonstrated that DARA-mediated ADCC can be significantly improved by lenalidomide (LEN), mainly due to the potent capacity of LEN to activate NK cells.8,9 Based on these observations, we hypothesized that the efficacy of DARA-induced, NK cell-mediated ADCC may be further enhanced via modulation of NK-cell regulatory signals transmitted via the inhibitory and activating NK receptors [killer-cell immunoglobulin-like receptors (KIRs)].10,11 Since the signals transmitted by inhibitory KIRs may prevent NK cell-mediated ADCC, even in the presence of an activating receptor-ligand interaction,12 we set out to test the possibility of improving DARA efficacy by blocking inhibitory KIRs. IPH2102 (formerly 1-7F9 and IPH2101) is a hinge-stabilized, human IgG4 monoclonal antibody that blocks the interaction of the three main inhibitory KIR receptors (KIR2DL-1, -2, -3) with their ligands, the human leukocyte antigen-C (HLA-C) molecules. The predecessor of IPH2102, IPH2101 (a wild-type IgG4 version of the antibody), was shown to increase NK-cell cytotoxicity against MM cells, but not against normal healthy cells.13,14 Clinical trials conducted with IPH2101 in patients with relapsed/refractory MM and smoldering myeloma revealed that the clinical use of IPH2101 is safe and tolerable at doses that achieve full inhibitory KIR saturation, with disease stabilization as the best observed response to IPH2101.15,16 This suggested Rabbit Polyclonal to APOL2 that this antibody likely requires inclusion in a combination regimen such as with a potent ADCC-inducing antibody and/or with NK-cell activating agents like LEN. Hence, we explored in a series of assays the potential benefits of combining DARA with IPH2102 and LEN. We demonstrate that DARA-induced killing of primary MM cells increases synergistically when combined with these NK-enhancing agents. Methods Bone marrow mononuclear cells from MM patients All patients samples were collected and stored under protocols approved by the Institutional Review Board. All procedures involving bone marrow material were in accordance Leflunomide with the Declaration of Helsinki and approved by the local medical ethical committee. Mononuclear cells (MNC) from the bone marrow (BM) were isolated by Ficoll-Hypaque density-gradient centrifugation and contained 2%C35% MM cells as detected by flow cytometry. Freshly isolated Leflunomide BM-MNC from patients Leflunomide were immediately used in experiments (DARA + IgG4. ns: non-significant; *values were calculated by a paired t-test comparing DARA + IPH2102 DARA + IgG4. ns: non-significant; *flow cytometry-based cytotoxicity assays, in which we measure the survival of primary CD138+ MM cells in patients BM-MNC, without separating malignant cells from their microenvironment and autologous effector cells.8 In this setting, incubation of 10 BM-MNC in serial dilution (0C10 g/mL) of DARA and IPH2102 in a checkerboard fashion, confirmed the dose-dependent induction.