In a series of experiments using purified multiple myeloma cells, multiple myeloma cell lines, full bone marrow mononuclear cell suspensions derived from multiple myeloma patients and peripheral blood mononuclear cells isolated from lenalidomide-treated patients, we show that the combination of lenalidomide and daratumumab significantly increases the lysis of multiple myeloma cells as compared to either of the single agents. Obviously, the increased lysis of multiple myeloma cells by the combination of lenalidomide and daratumumab is to a certain extent due to the separate actions of these agents. pre-treatment of the effector cells derived from peripheral blood mononuclear cells from healthy individuals. More importantly, we demonstrated a clear synergy between lenalidomide and daratumumab-induced antibody-dependent cell-mediated cytotoxicity directly in the bone marrow mononuclear cells of multiple myeloma patients, indicating that lenalidomide can also potentiate the daratumumab-dependent lysis of myeloma cells by activating the autologous effector cells within the natural Rabbit Polyclonal to SIAH1 environment of malignant cells. Finally, daratumumab-dependent cell-mediated cytotoxicity was significantly up-regulated in peripheral Benzenepentacarboxylic Acid blood mononuclear cells derived from 3 multiple myeloma patients during lenalidomide treatment. Conclusions Our results indicate that Benzenepentacarboxylic Acid powerful and complementary effects may be achieved by combining lenalidomide and daratumumab in the Benzenepentacarboxylic Acid clinical management of multiple myeloma. multiple myeloma effects after allogeneic stem cell transplantation.11 In several initial studies, lenalidomide has been frequently used alone or in combination with other chemotherapeutical agents.12C15 Nonetheless, such strategies are possibly not exploiting the full immunomodulatory capacities of lenalidomide. In particular, its Natural Killer cell stimulatory properties suggest that lenalidomide could be highly effective in combination with therapeutic antibodies capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).12C14 Supporting this idea, a number of earlier studies showed that both thalidomide and lenalidomide can enhance rituximab-mediated antibody-dependent cell-mediated cytotoxicity.15C17 Furthermore, multiple myeloma cell lysis was significantly improved when lenalidomide was combined with a humanized CD40 antibody.13,14 Indeed, promising results are being reported from a number of recently started clinical trials combining lenalidomide with rituximab or CD40 antibodies for the treatment of chronic lymphoid leukemia, lymphoma and multiple myeloma.16C18 A highly interesting target for antibody therapy in multiple myeloma is the CD38 molecule, a 46 kDa type II trans-membrane glycoprotein with a short N-terminal cytoplasmic tail (20 amino acids) and a long extracellular domain (256 amino acids).19,20 CD38 is expressed at low or moderate levels on various hematopoietic cells and in some solid tissues; but the extremely bright and uniform expression of CD38 on all multiple myeloma cells suggests that this molecule is an optimal therapeutic target for antibody therapy.21,22 Recently we have developed a new human CD38 antibody, daratumumab (DARA), and we have shown that it induces killing of tumor cells via anti-Fc-mediated effector functions, e.g. complement-dependent cytotoxicity, Natural Killer cell-mediated antibody-dependent cell-mediated cytotoxicity and apoptosis upon secondary cross-linking. Therefore, we now investigated the possibility that combining lenalidomide with daratumumab would significantly enhance the killing of multiple myeloma tumor cells. In a series of experiments using a CD38+ multiple myeloma cell line, purified multiple myeloma cells and full bone marrow mononuclear cells (BM-MNC) of multiple myeloma patients containing 2C50% malignant plasma cells, we demonstrate that lenalidomide significantly improves daratumumab-dependent lysis of multiple myeloma cells, mainly by activating the effector cells of antibody-dependent cell-mediated cytotoxicity. Furthermore, peripheral blood mononuclear cells (PBMC) isolated from patients during or just after lenalidomide treatment show an increased capacity of mediating daratumumab-dependent antibody-dependent cell-mediated cytotoxicity against multiple myeloma cells, emphasizing the potential clinical benefits that can be obtained by combination of daratumumab with lenalidomide in the clinical setting. Design and Methods Primary multiple myeloma cells and multiple myeloma cell lines After obtaining written informed consent, primary CD138+ multiple myeloma cells were isolated from bone marrow of multiple myeloma individuals using anti-CD138 (Becton Dickinson) coated rabbit-anti-mouse microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturers protocol. Isolated main multiple myeloma cells were immediately used in experiments after determining CD138 and CD38 manifestation. The CD38+ multiple myeloma cell collection UM9 was generated and managed as previously explained.23,24 Peripheral blood mononuclear cells from healthy donors and multiple myeloma individuals All procedures including material from healthy donors and multiple myeloma individuals were authorized by the institutional medical ethical committee. After obtaining written educated consent peripheral blood was from healthy volunteers and from multiple myeloma individuals. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density-gradient centrifugation.11 Freshly isolated peripheral blood mononuclear cells from healthy individuals were used either immediately or after culturing with lenalidomide (Cellgene, 3mol/L) for three days as effector cells in antibody-dependent cell-mediated cytotoxicity assays. Peripheral blood mononuclear cells from multiple myeloma individuals were frozen prior to use as effector cells in antibody-dependent cell-mediated cytotoxicity assays. Bone marrow mononuclear cells from multiple myeloma individuals All procedures including bone marrow material were authorized by the institutional medical honest committee. Mononuclear cells from bone marrow were isolated by Ficoll-Hypaque density-gradient centrifugation11 and contained 2C50% malignant plasma cells. Freshly isolated bone marrow mononuclear cells from individuals were treated immediately with lenalidomide.