title: Galactosylation of human IgG monoclonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity.
abstract: Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B cell lines grown in serum-free medium in low density (LD) static cultures or high density (HD) hollow fiber bioreactors. Glycosylation analysis of the purified IgG was determined by the binding of anti-GlcNAc monoclonal antibody (GN7) and by analysis of oligosaccharides released by hydrazinolysis. The LD MAbs had only trace levels of agalactosyl oligosaccharides (G0), the major species (> 70%) being digalactosyl structures (G2). The HD MAbs, by contrast, contained about 10% G0 and relatively high levels (over 50%) of monogalactosyl (G1) oligosaccharides. beta 1-4 galactosyltransferase activity in the LD cell lines was similar to that found previously for other EBV-transformed B cell lines. The predominant oligosaccharides of an IgG3 anti-D, BRAD-3, contained bisecting N-acetylglucosamine. In functional assays with Fc receptor (Fc gamma R) positive effector cells, the highly galactosylated LD form of BRAD-3 was more active than the HD form in monocyte (Fc gamma RI) and K cell (Fc gamma RIII) mediated lysis of erythrocytes in ADCC assays, although these preparations showed no difference in Fc gamma RI-mediated rosette formation with U937 cells. One MAb, JAC10, was over 10-fold less active than two other IgG1 MAbs, 2B6 and BRAD-5, at mediating lysis of erythrocytes by Fc gamma RIII+ K cells; differences in sialylation may have contributed to this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: International Blood Group Reference Laboratory, Bristol, UK.
authors.forename: B M
authors.initials: BM
authors.lastname: Kumpel
authors.forename: T W
authors.initials: TW
authors.lastname: Rademacher
authors.forename: G A
authors.initials: GA
authors.lastname: Rook
authors.forename: P J
authors.initials: PJ
authors.lastname: Williams
authors.forename: I B
authors.initials: IB
authors.lastname: Wilson
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000920:Antibody-Dependent Cell Cytotoxicity; D001402:B-Lymphocytes / Q000276:immunology*; D002240:Carbohydrate Sequence; D002460:Cell Line; D005690:Galactose / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000502:physiology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology; D009844:Oligosaccharides / Q000032:analysis; D011961:Receptors, Fc / Q000378:metabolism*; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D012397:Rosette Formation
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011961:Receptors, Fc; D012204:Rh-Hr Blood-Group System; D005690:Galactose