• Cheryl Nickerson-Nutter, Lioudmila Tchistiakova, Nilufer P Seth, Marion Kasaian, Barbara Sibley, Stephane Olland, Richard Zollner, William A Brady, Kendall M Mohler, Peter Baum, Alan Wahl, Deborah Herber, Yulia Vugmeyster, David Wensel, Neil M Wolfman, Davinder Gill, Mary Collins, and Kyri Dunussi-Joannopoulos.
• Inflammation and Immunology, Pfizer Biotherapeutics Research and Development, Cambridge, MA 02140, USA.
• Rheumatology (Oxford). 2011 Jun 1; 50 (6): 1033-44.

ObjectivesTo characterize the in vitro binding and effector function properties of CD20-directed small modular immunopharmaceutical (SMIP) 2LM20-4, and to compare its in vivo B-cell depletion activity with the mutated 2LM20-4 P331S [no in vitro complement-dependent cytotoxicity (CDC)] and rituximab in cynomolgus monkeys.MethodsDirect binding is examined in flow cytometry, confocal microscopy, scatchard and lipid raft assays. Effector function assays include CDC and Fc-mediated cellular toxicity. In the 6-month-long in vivo B-cell depletion study, single i.v. dosages of 1 or 10 mg/kg of anti-CD20 proteins were administered to monkeys and B-cell counts were monitored in peripheral blood, bone marrow and lymph nodes.Results2LM20-4 has lower saturation binding to human primary B cells and recruits fewer CD20 molecules into lipid rafts compared with rituximab; however, it induces higher in vitro CDC. In competitive binding, 2LM20-4 only partially displaces rituximab, suggesting that it binds to a fraction of CD20 molecules within certain locations of the plasma membrane as compared with rituximab. In monkeys, 2LM20-4 had more sustained B-cell depletion activity than rituximab in peripheral blood and had significantly more profound and sustained activity than 2LM20-4 P331S and rituximab in the lymph nodes.ConclusionsSMIP 2LM20-4, which binds to a fraction of CD20 molecules as compared with rituximab, has more potent in vitro CDC, and more potent and sustained B-cell depletion activity in cynomolgus monkeys. Our work has considerable clinical relevance since it provides novel insights related to the emerging B-cell depletion therapies in autoimmune diseases.

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