Distinct in vitro binding properties of the anti-CD20 small modular immunopharmaceutical 2LM20-4 result in profound and sustained in vivo potency in cynomolgus monkeys

• Published in: Rheumatology (Oxford, England) Vol. 50; no. 6; pp. 1033 - 1044
• Main Authors: NICKERSON-NUTTER, Cheryl, TCHISTIAKOVA, Lioudmila, WAHL, Alan, HERBER, Deborah, VUGMEYSTER, Yulia, WENSEL, David, WOLFMAN, Neil M, GILL, Davinder, COLLINS, Mary, DUNUSSI-JOANNOPOULOS, Kyri, SETH, Nilufer P, KASAIAN, Marion, SIBLEY, Barbara, OLLAND, Stephane, ZOLLNER, Richard, BRADY, William A, MOHLER, Kendall M, BAUM, Peter
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01-06-2011
• Subjects: Animals; Antibodies, Monoclonal - pharmacology; Antibodies, Monoclonal, Murine-Derived - pharmacology; Antibody-Dependent Cell Cytotoxicity - drug effects; Antibody-Dependent Cell Cytotoxicity - immunology; Antigens, CD20 - drug effects; Antigens, CD20 - immunology; Autoimmune diseases; B-Lymphocytes - immunology; B-Lymphocytes - metabolism; Basic Science; Binding Sites; Biological and medical sciences; Bone marrow; CD20 antigen; Cells, Cultured; Confocal microscopy; Cynomolgus; Cytotoxicity; Disease Models, Animal; Diseases of the osteoarticular system; Flow cytometry; Humans; Immunologic Factors - pharmacology; In Vitro Techniques; Linear Models; Lipid rafts; Lymph nodes; Lymphocytes B; Macaca fascicularis; Medical sciences; Peripheral blood; Plasma membranes; Random Allocation; Rituximab; Sensitivity and Specificity; Single-Chain Antibodies - pharmacology; Toxicity; Immunopathology; Rheumatology; Monkey; Autoimmune disease; B-Lymphocyte; In vitro; In vitro properties; In vivo properties; In vivo; Vertebrata; B-cell depletion; Mammalia; Animal; Primates; Anti-CD20 small modular immunopharmaceutical; β Cell; Autoimmune diseases
• ISSN: 1462-0324; 1462-0332
• DOI: 10.1093/rheumatology/keq423

To 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.
Direct 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.
2LM20-4 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.
SMIP 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.