title: An anti-C3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20+ cells by rituximab.
abstract: We investigated deposition of the complement protein fragment C3b and its breakdown products (collectively designated as C3b(i)) on CD20-positive cells treated with rituximab (RTX) in the presence of normal human serum (NHS). Radioimmunoassay (RIA) demonstrates that about 500 000 C3b(i) molecules deposit per cell, and fluorescence microscopy reveals that C3b(i) colocalizes with bound RTX. Use of mAb 3E7, specific for C3b(i) bound to substrates, enhances C3b(i) deposition; > 1 million C3b(i) deposit when cells are incubated with NHS, RTX and mAb 3E7. Treatment of Raji cells in NHS plus RTX leads to robust cell killing (95%) after 24 to 48 hours, and mAb 3E7 significantly enhances RTX-mediated killing of Raji and DB cells. A cynomolgus monkey model based on intravenous infusion of RTX followed by mAb 3E7 demonstrated that RTX rapidly binds to B cells and promotes complement activation and C3b(i) deposition; fluorescence microscopy analyses revealed the same pattern of colocalization of C3b(i) on cell-bound RTX in vivo as observed in vitro. Preliminary in vitro studies with blood samples from patients with chronic lymphocytic leukemia lead to similar findings. These experiments suggest that complement plays a key role in the mechanism of action of RTX; moreover, the in vivo molecular form of RTX (and possibly other antitumor mAbs) in the circulation or in tissues may include C3b(i) molecules covalently bound to the therapeutic mAb, thus allowing it to interact with cells containing both Fc and complement receptors.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Center for Comparative Medicine, University of Virginia Health Sciences Center, Charlottesville 22908-0733, USA.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Kennedy
authors.forename: Michael D
authors.initials: MD
authors.lastname: Solga
authors.forename: Theodore A
authors.initials: TA
authors.lastname: Schuman
authors.forename: Amos W
authors.initials: AW
authors.lastname: Chi
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Lindorfer
authors.forename: William M
authors.initials: WM
authors.lastname: Sutherland
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Foley
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Taylor
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20 / Q000276:immunology*; D001402:B-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D003167:Complement Activation / Q000187:drug effects*; D003179:Complement C3b / Q000276:immunology* / Q000378:metabolism; D004357:Drug Synergism; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology; D008252:Macaca fascicularis; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D000069283:Rituximab; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000069283:Rituximab; D003179:Complement C3b