title: Construction and expression of a mouse-human chimeric antibody against human tumor necrosis factor-alpha.
abstract: A mouse anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (MoAb), designated as 3B10, has previously been produced and characterized by our laboratory. We report here the construction and the expression of mouse-human chimeric antibody derived from the MoAb. cDNAs encoding variable regions of heavy and light chains were prepared from 3B10 cells by polymerase chain reaction, and introduced to mammalian expression vectors containing cDNA for human gamma1 and kappa constant regions, respectively. Cotransfection of the vectors into CHO cells resulted in production of antibody reacting with human TNF-alpha. In SDS-PAGE analysis, the chimeric antibody, c3B10, migrated at 170 kDa under a nonreducing condition, whereas two bands with 58 and 28 kDa appeared following treatment with 2-mercaptoethanol. Both c3B10 and mouse 3B10 neutralized the cytotoxic activity of human TNF-alpha to the same level, indicating that c3B10 holds the binding activity of its original MoAb. These findings suggest that the introduced genes for chimeric heavy and light chains are transcribed and translated to produce the chimeric heavy and light chain peptides, and that the peptides are assembled to form native IgG molecule. The chimeric anti-TNF-alpha antibody described in this study is expected to be less immunogenic and thus more suitable for possible clinical use.
authors.affiliation: Suntory Institute for Biomedical Research, Osaka, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nagahira
authors.forename: Y
authors.initials: Y
authors.lastname: Fukuda
authors.forename: T
authors.initials: T
authors.lastname: Nasu
authors.forename: H
authors.initials: H
authors.lastname: Kawashima
authors.forename: C
authors.initials: C
authors.lastname: Noguchi
authors.forename: T
authors.initials: T
authors.lastname: Kurihara
authors.forename: S
authors.initials: S
authors.lastname: Oikawa
authors.forename: T
authors.initials: T
authors.lastname: Nakanishi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha