A new method of constructing CD20/CD22 bispecfic antibody fusion proteins with improved direct lymphoma cytotoxicity compared to rituximab

2536 Background: Anti-CD20 and anti-CD22 monoclonal antibodies (MAbs) have been shown to have antitumor activity in non-Hodgkin’s lymphoma (NHL) patients. Since preclinical and clinical trials also suggested that combining CD20 and CD22 MAbs, which have different mechanisms of action, could improve antitumor activity without a commensurate increase in toxicity, we hypothesized that a therapeutic advantage may be achieved with bispecific MAbs that bind simultaneously to both CD20 and CD22. Methods: A new platform technology, termed the Dock and Lock method (DNL), was successfully applied to produce a trivalent bispecific antibody, named TF3, which comprises two recombinant Fab fragments of hA20 (humanized anti-CD20 MAb; IMMU-106) stably tethered to one recombinant Fab fragment of epratuzumab (humanized anti-CD22 MAb; IMMU-103) via the specific interaction between a dimerization-and-docking domain and an anchoring domain appended to hA20 and epratuzumab, respectively. The cytotoxicity of TF3 was evaluated by cell-based assays using NHL cell lines. Results: TF3 is stable in both human and mouse sera andexhibitsthe samebinding affinity as hA20 IgG or epratuzumab Fab by competitive ELISA. With a 3-day MTT assay, TF3 at 10 nM inhibited 50% and 60% growth of Daudi and Ramos cells, respectively. Further, the observed anti-proliferative activity increased synergistically to >90% in the presence of anti-IgM (0.1 μg/mL). Results from a cell counting assay also demonstrated the ability of TF3 at 1 μM to completely inhibit the growth of Daudi and the potency of TF3 at 1 nM was comparable to that of rituximab at 1 μM under the same experimental conditions, reflecting a 1000-fold enhancement. Conclusions: These findings, to be extended by ongoing in vivo studies, suggest that the new DNL platform technology for making bispecific antibody fusion proteins provided a CD20/CD22 binding protein that is significantly more potent than rituximab in an in vitro NHL direct cytotoxicity assay. [Table: see text]