title: Efficient generation of useful monoclonal antibodies reactive with globotriaosylceramide using knockout mice lacking Gb3/CD77 synthase.
abstract: Efficient generation of useful monoclonal antibodies (mAbs) with high performance in cancer therapeutics has been expected. Generation of mAbs reactive with globotriaosylceramide (Gb3/CD77) was compared between A/J mice and Gb3/CD77 synthase-deficient (A4GalT-knockout) mice by immunizing Gb3-liposome. Specificity and functions of established antibodies were examined by ELISA, TLC- immunostaining, cytotoxicity of cancer cells and immunoblotting. Compared with results with conventional mice, better generation of mAbs with higher functions has been achieved with A4GalT-knockout mice, i.e. acquisition of IgG class antibodies, activities in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity toward a Burkitt's lymphoma line Ramos. Binding of mAb k52 induced tyrosine phosphorylation of several proteins in Ramos cells. One of the strongest phosphorylation bands turned out to be c-Cbl. Pretreatment of B cell lines with mAbs resulted in the attenuation of BCR-mimicking signaling. All these results suggested that A4GalT-knockout mice are very useful to generate mAbs against globo-series glycolipids, and that suppressive signaling pathway driven by endogenous Gb3-ligand molecules might be present in B cells.
authors.affiliation: Department of Biochemistry II, Nagoya University Graduate School of Medicine, Tsurumai, Showa-ku, Nagoya, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kondo
authors.forename: Noriyo
authors.initials: N
authors.lastname: Tokuda
authors.forename: Keiko
authors.initials: K
authors.lastname: Furukawa
authors.forename: Reiko
authors.initials: R
authors.lastname: Ando
authors.forename: Makoto
authors.initials: M
authors.lastname: Uchikawa
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Fan
authors.initials: F
authors.lastname: Xiaoyan
authors.forename: Koichi
authors.initials: K
authors.lastname: Furukawa
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D055104:Infusions, Subcutaneous; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D014281:Trihexosylceramides / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D014281:Trihexosylceramides; C018549:globotriaosylceramide; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase