title: Bacterially expressed Fabs of monoclonal antibodies neutralizing tumour necrosis factor alpha in vitro retain full binding and biological activity.
abstract: Antibody fragments specific for the human tumour necrosis factor alpha (TNF alpha) have been cloned from lambda combinatorial expression libraries using total RNA obtained from three different hybridoma cell lines of therapeutic interest. The previously described bacteriophage lambda vectors, lambda HC2 and lambda LC1, were modified to create unique antibody cloning sites in the combinatorial construct and a novel tag peptide was inserted at the C-terminal end of the expressed Fd chain. Sequence analysis of the cloned Fabs indicated that two of them were derived from a single B cell. Expression in E. coli showed that the amount of recovered Fab in the bacterial culture medium was related to the sequences of the variable coding regions. Hybrid Fabs created by chain exchange of similar antibodies were as active as the originally paired Fabs in binding assays. The relative affinities and the capacities of the bacterially expressed Fabs to neutralize TNF alpha cytotoxicity in vitro were identical to those of the parental antibodies. The results demonstrate that, using an in vitro approach, it is possible to generate from existing hybridoma cell lines high affinity Fabs which retain antigen specificity. The cloning sites incorporated into the C-terminal parts of these Fabs will now permit their further modification to include additional functional characteristics not possible with the original hybridoma antibodies.
authors.affiliation: Ecole Supérieure de Biotechnologie, Strasbourg, France.
authors.forename: G
authors.initials: G
authors.lastname: Orfanoudakis
authors.forename: B
authors.initials: B
authors.lastname: Karim
authors.forename: D
authors.initials: D
authors.lastname: Bourel
authors.forename: E
authors.initials: E
authors.lastname: Weiss
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D006825:Hybridomas / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha