title: Gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties.
abstract: We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine residues. In most cases, enzymatic activity in vitro was unaltered by this modification. The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65 Fab and F(ab')2. Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells than those made with linker-modified rGel, whereas others were less potent. In the rat, the in vivo clearance rates of whole antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody and rGel was the predominant clearance mechanism. Fab conjugates to rGel analogs which differed in their in vitro disulfide bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate from the serum, and is less dependent on deconjugation. Our results demonstrate that rGel analogs with a single cysteine at various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position of the cysteine greatly influences the potency and stability of the resulting immunoconjugates.
authors.affiliation: XOMA Corporation, Santa Monica, California 90404.
authors.forename: M
authors.initials: M
authors.lastname: Better
authors.forename: S L
authors.initials: SL
authors.lastname: Bernhard
authors.forename: D M
authors.initials: DM
authors.lastname: Fishwild
authors.forename: P A
authors.initials: PA
authors.lastname: Nolan
authors.forename: R J
authors.initials: RJ
authors.lastname: Bauer
authors.forename: A H
authors.initials: AH
authors.lastname: Kung
authors.forename: S F
authors.initials: SF
authors.lastname: Carroll
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D003545:Cysteine; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000378:metabolism; D000922:Immunotoxins / Q000378:metabolism*; D008297:Male; D008657:Metabolic Clearance Rate; D010940:Plant Proteins / Q000096:biosynthesis / Q000276:immunology*; D015202:Protein Engineering; D011500:Protein Synthesis Inhibitors / Q000493:pharmacokinetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D054789:Ribosome Inactivating Proteins, Type 1; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D000922:Immunotoxins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum; D003545:Cysteine