title: Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.
abstract: Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies.
authors.affiliation: German Primate Centre (DPZ) GmbH, Department of Infection Biology, Goettingen, Germany.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Mueller
authors.forename: Lars
authors.initials: L
authors.lastname: Heinig
authors.forename: Sanja
authors.initials: S
authors.lastname: Ramljak
authors.forename: Astrid
authors.initials: A
authors.lastname: Krueger
authors.forename: Reiner
authors.initials: R
authors.lastname: Schulte
authors.forename: Arne
authors.initials: A
authors.lastname: Wrede
authors.forename: Andreas W
authors.initials: AW
authors.lastname: Stuke
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000276:immunology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D011328:Prions / Q000235:genetics / Q000276:immunology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D013752:Tetracycline; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011328:Prions; D013752:Tetracycline