Multivalent
antibodies, either monospecific or bispecific, may improve the efficacy of current therapeutic interventions involving a single
monoclonal antibody (mAb). We have applied the Dock-and-Lock (DNL) method, a new platform technology for the site-specific and covalent assembly of modular components into stably tethered complexes of defined composition, to prepare a hexavalent, anti-CD20 antibody, designated Hex-hA20, which comprises six Fabs with one Fc. We show that Hex-hA20 retains the binding activity of all six Fabs, associates with CD20 in
lipid rafts, affects antibody-dependent cell-mediated cytotoxicity, but not
complement-dependent cytotoxicity, and inhibits proliferation of Daudi, Raji, and Ramos cells in vitro at subnanomolar concentrations without the need for a cross-linking antibody. In addition, Hex-hA20 induces strong homotypical adhesion and is inefficient in stimulating
calcium mobilization. Thus, Hex-hA20 exhibits
biological properties attributable to both type I and type II anti-CD20 mAbs, as exemplified by
rituximab and tositumomab, respectively. Although Hex-hA20 has a short serum half-life, it shows antitumor efficacy in
tumor-bearing mice comparable with
veltuzumab at equivalent doses. The versatile DNL method was also applied to generate two other multivalent anti-CD20
antibodies without the Fc region, Tri-hA20 and Tetra-hA20, comprising three and four Fabs of
veltuzumab, respectively. Similar to Hex-hA20, these were purified to near homogeneity and shown to have potent antiproliferative activity in vitro, thus indicating the need for clustering three or more CD20 molecules on the cell surface to induce growth inhibition.