SHORT REPORT Recombinant humanized anti-CD40 monoclonal antibody triggers autologous antibody-dependent cell-mediated cytotoxicity against multiple myeloma cells Toshiaki Hayashi, 1 Steven P. Treon, 1 Teru Hideshima, 1 Yu-Tzu Tai, 1 Masaharu Akiyama, 1 Paul Richardson, 1 Dharminder Chauhan, 1 Iqbal S. Grewal 2 and Kenneth C. Anderson 1 1 Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, MA, and 2 Department of Immunology, Genentech Inc., South San Francisco, CA, USA Received 19 December 2002; accepted for publication 10 January 2003 Summary. Multiple myeloma (MM) is currently incurable, and novel therapies are needed. In this study, we examined a novel recombinant humanized monoclonal antibody against CD40 (rhuCD40 mAb) and demonstrate for the first time that rhuCD40 mAb induces antibody-dependent cell- mediated cytotoxicity (ADCC) against CD40-positive MM cells. Importantly, we show that rhuCD40 mAb induces autologous ADCC against primary patient MM cells, without triggering ADCC against normal B cells. This study, therefore, both demonstrates that rhuCD40 mAb triggers autologous ADCC against patient MM cells and provides the framework for the clinical evaluation of rhuCD40 mAb immunotherapy to improve patient outcome in MM. Keywords: ADCC, CD40, multiple myeloma. Multiple myeloma (MM) is currently incurable with con- ventional therapies; however, both biologically based and immune-based therapies have great promise for the improvement of patient outcome (Hayashi et al, 2003). In patients with non-Hodgkin’s lymphoma, the addition of chimaeric mouse human anti-CD20 monoclonal antibody (chCD20 mAb) immunotherapy to conventional chemo- therapy significantly prolongs survival (Coiffier et al, 2002). In MM, our recent clinical trial demonstrated that chCD20 mAb induced responses in a small subset of patients whose MM cells expressed CD20, however, most MM cells are CD20 negative (Treon et al, 2002). CD40 is a potential target for immunotherapy, as it is expressed broadly on MM cells (Urashima et al, 1995). Importantly, mouse anti-CD40 mAb blocks CD40 ligand (CD40L)-induced malignant B-cell growth in vitro (Francisco et al, 2000), inhibits the prolif- eration of B-cell lymphoma or leukaemia cells in vivo (Funakoshi et al, 1994; Dilloo et al, 1997), and prolongs the survival of severe combined immunodeficient (SCID) mice xenografted with human MM cells (Francisco et al, 2000). The anti-CD40 mAb also acts on host immunity to stimulate dendritic cells, resulting in the activation of CD8 + T cells in mice (van Mierlo et al, 2002). In this study, we demonstrate that recombinant humanized anti-CD40 (rhuCD40) mAb induces antibody- dependent cell-mediated cytotoxicity (ADCC) activity, analogous to the ADCC induced by chCD20 mAb therapy, in a mouse xenograft model (Clynes et al, 2000). Fur- thermore, we show that rhuCD40 mAb triggers autolo- gous ADCC against patient MM cells, providing the rationale for its clinical application to improve patient outcome in MM. MATERIALS AND METHODS Antibodies. Recombinant humanized anti-CD40 mAb [Immunoglobulin G1 (IgG1)] and control human mono- clonal IgG1 were provided by Genentech (South San Francisco, CA, USA). The murine anti-human CD40 mAb SGN-14 (Seattle Genetics, Seattle, WA, USA) was human- ized to construct rhuCD40 mAb. Heavy and light chain complementarity-determining regions (CDRs) from SGN-14 murine mAb were swapped into plasmids encoding a human light and human heavy immunoglobulin chain. Additional individual substitutions were made in the light Correspondence: Kenneth C. Anderson, MD, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA. E-mail: kenneth_anderson@dfci.harvard.edu British Journal of Haematology, 2003, 121, 592–596 592 Ó 2003 Blackwell Publishing Ltd