title: High yield, site-specific coupling of N-terminally modified beta-lactamase to a proteolytically derived single-sulfhydryl murine Fab'.
abstract: The preparation of bispecific protein conjugates capable of performing diverse biological functions is an area of active investigation. Such conjugates are routinely prepared using techniques which employ random derivatization of lysine residues, but the overall utility of these methods is limited due to poor yields and heterogeneous conjugates. In this report we describe the development of site-specific linkage methodology for the chemical synthesis of a homogeneous enzyme-antibody Fab' conjugate with coupling efficiencies of at least 72%. The N-terminal threonine residue of beta-lactamase from the P99 strain of Enterobacter cloacae was oxidized to an aldehyde functional group under mild conditions with a 5-fold molar excess of sodium periodate. The murine Fab' with a single sulfhydryl at the hinge region was generated by further digestion of the peptic Fab' fragment with lysyl endopeptidase to remove a decapeptide containing two of the three cysteine residues. Coupling of the two modified proteins was accomplished through a bifunctional coupling reagent containing meleimide and aminooxy functional groups. Synthesis of the linker is described. Yields of 1:1 enzyme-Fab' were at least three times higher than for comparable random derivatization methods. Immunoreactivity and enzymatic activity were unaffected. Biodistribution studies showed a more favorable tumor to blood ratio with the site-specifically linked conjugate.
authors.affiliation: Hybritech Incorporated, San Diego, California 92196-9006.
authors.forename: S D
authors.initials: SD
authors.lastname: Mikolajczyk
authors.forename: D L
authors.initials: DL
authors.lastname: Meyer
authors.forename: J J
authors.initials: JJ
authors.lastname: Starling
authors.forename: K L
authors.initials: KL
authors.lastname: Law
authors.forename: K
authors.initials: K
authors.lastname: Rose
authors.forename: B
authors.initials: B
authors.lastname: Dufour
authors.forename: R E
authors.initials: RE
authors.lastname: Offord
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002272:Carcinoembryonic Antigen / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D008301:Maleimides / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010434:Pepsin A / Q000378:metabolism; D010504:Periodic Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D002272:Carcinoembryonic Antigen; D007140:Immunoglobulin Fab Fragments; D008301:Maleimides; D013438:Sulfhydryl Compounds; D010504:Periodic Acid; C043592:maleimide; D013912:Threonine; C009288:metaperiodate; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D010434:Pepsin A; D001618:beta-Lactamases