SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via several mechanisms in vitro. These include induction of cytotoxic ligands of TNFR family and suppression of IL-6-induced proliferative and antiapoptotic effects, as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Since > 80% of primary patient MM cells express CD40, targeting CD40 using SGN-40 presents a potential novel treament strategy, and a phase I clinical study of SGN-40 in patients with refractory or recurrent MM is ongoing. We recently reported that Thalidomide and immunomodulatory drugs (IMiDs) target both MM cells and the bone marrow (BM) microenvironment, and activate NK cells via induction of IL-2 production. In the present study, we therefore evaluated the effects of IMiD3 on the direct antiproliferative and apoptotic effects of SGN-40, as well as on ADCC against both MM cell lines and patient MM cells (CD40+CD138++). SGN-40 and IMiD3 induced synergistic growth inhibition, assayed by [3H] thymidine uptake, in dexamethasone (Dex)-sensitive MM.1S and Dex-resistant MM.1R lines, 2 other CD40-positive MM cell lines, as well as 2 patient MM cells. The temporal sequence of SGN-40 and IMiD3 treatment did not alter growth inhibition. The combination of SGN-40 and IMiD3 significantly increased MM apoptosis, evidenced by enhanced cleavage of caspase 3/8/PARP and increased subG0 cells compared with either single agent. The addition of IMiD3 to target cells and effector cells moderately increased specific lysis in any MM cell line, whereas pretreatment of target cells with IMiD3 significantly augmented sensitivity of all MM lines to ADCC and pretreatment of effector cells also improved specific MM cell lysis. In addition, preincubation of both effector and tumor cells with IMiD3 greatly enhanced specific lysis of MM cell lines and 2 patient MM cells in ADCC assay, associated with a significant increase 38+3% in natural killer cells (CD56+CD16+ and CD14-CD3-) following IMiD3 treatment. IMiD3 not only improved natural cytotoxicity of NK cells, but also significantly induced the CD56dimCD16+CD3- NK subset, which is a more potent mediator of ADCC against MM than the CD56bright NK subset. Moreover, IMiD3 treatment upregulates CD40L expression on CD56+CD3- NK effectors: IMiD3 (2 μM) induces CD40L upregulation equivalent to IL-2 (1000 unit/ml). Finally, combined SGN-40 and IMiD3 augments NK cell proliferation, which is associated with enhanced AKT/NF-kB and ERK activation. Taken together, our studies show that the addition of IMiD3 to SGN-40 results in synergistic cytotoxicity mediated via direct antiproliferative and apoptotic effects therefore increased sensitivity of MM cells to ADCC by inducing the cytotoxic NK subset. These studies establish the framework for the development of SGN-40 and IMiD3 in a new treament paradigm to both target MM cells directly and to induce immune effectors against MM.