HM1.24/BST-2 (CD317) is a type II transmembrane protein that was originally identified as a cell surface antigen overexpressed on multiple myeloma (MM) cells. Our previous studies have demonstrated that HM1.24 has a potential as an attractive target molecule for antibody-based or cellular-mediated immunotherapy of MM. Subsequent studies have revealed that rat homologue of HM1.24 at the cell surface can be internalized and localized to the intracellular trans-Golgi network, suggesting that HM1.24 may play an important role in trafficking and signaling. To evaluate the potential effect of HM1.24-targeting therapy in MM, we generated internalizing fully human monoclonal antibodies specific for human HM1.24 by using KM mice. Among these monoclonal antibodies, b-76-8 had the highest affinity to HM1.24 and induced ADCC activity and complement-dependent cytotoxicity in the presence of immune effectors. Importantly, b-76-8 internalized rapidly after cell surface binding and approximately 80% of b-76-8 was delivered to the intracellular location within 30 min as determined by immunofluorescence staining of b-76-8-treated RPMI 8226 cells. We next developed the immunoconjugate of b-76-8 with the analog of the cytotoxic drug maytansine, DM1 [N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine] and further evaluated its potential impact on MM cells. The ratio of DM1 molecules linked per antibody molecule was 1.8–3.0. Several MM cell lines as well as primary MM cells were cultured with either b-76-8, DM1, b-76-8-DM1, or control IgG-DM1, and cell viability was determined by a WST-8 assay. Treatment with b-76-8-DM1 (2–10 nM, based on the concentration of DM1) induced 50–70% of cell death in RPMI 8226, KMS12-BM, and IM-9 cells. In contrast, the relevant amount of b-76-8, DM1 (10 nM), or control IgG-DM1 (10 nM) did not inhibit growth of these MM cells. Of interest, T-cell lines such as MOLT-4 and CEM also expressed HM1.24; however, these cells showed minimal cytotoxicity (<10%) to b-76-8-DM1 (10 nM) despite having a similar sensitivity to DM1. On the other hand, primary MM cells from 5 patients were relatively resistant to DM1, but maximal cytotoxicity (10–40%) was achieved at the concentration of 250 nM by b-76-8-DM1 but not by control IgG-DM1. This dose of b-76-8-DM1 did not mediate significant cytotoxicity against normal bone marrow mononuclear cells. We finally examined the anti-tumor activity of b-76-8-DM1 in an xenograft model of human MM. SCID mice were inoculated intravenously with IM-9 cells, and groups of 8 mice were treated with control IgG, b-76-8, b-76-8 mixed with free DM1, or b-76-8-DM1 on day 5–8. Treatment with b-76-8 (160 μg/dose) alone or b-76-8 together with DM1 (1.9 nmol/dose) significantly prolonged survival of these mice (p<0.01). In contrast, administration of b-76-8-DM1 (160 μg/dose) resulted in more significant tumor regression including complete remission in 3 of 8 mice (p<0.003). These results suggest that HM1.24 might be involved in trafficking between the cell surface and intracellular sites of MM cells, and that the immunoconjugate targeting HM1.24 provides a novel therapeutic approach in patients with MM.