title: Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab.
abstract: We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.
authors.affiliation: Laboratory of Cellular Therapy G. Lanzani, Division of Hematology, Ospedali Riuniti, 24128 Bergamo, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Bologna
authors.forename: Elisa
authors.initials: E
authors.lastname: Gotti
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Manganini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Rambaldi
authors.forename: Tamara
authors.initials: T
authors.lastname: Intermesoli
authors.forename: Martino
authors.initials: M
authors.lastname: Introna
authors.forename: Josée
authors.initials: J
authors.lastname: Golay
mesh_terms: D000074323:Alemtuzumab; D000911:Antibodies, Monoclonal / Q000097:blood / Q000494:pharmacology* / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived / Q000097:blood / Q000494:pharmacology* / Q000627:therapeutic use; D000912:Antibodies, Neoplasm / Q000097:blood / Q000494:pharmacology* / Q000627:therapeutic use; D018951:Antigens, CD20 / Q000276:immunology*; D003165:Complement System Proteins / Q000502:physiology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood / Q000276:immunology* / Q000628:therapy*; D015202:Protein Engineering / Q000379:methods*; D000069283:Rituximab; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D000912:Antibodies, Neoplasm; D018951:Antigens, CD20; D000074323:Alemtuzumab; D000069283:Rituximab; D003165:Complement System Proteins; C543332:obinutuzumab