Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate.
A murine antibody FvCys fragment with a single additional cysteine residue at the C terminus of the VH domain was expressed in Escherichia coli from a modified expression plasmid containing the structural genes for the VH and VL domains derived from the anti-lysozyme hybridoma D1.3. Chemical cross-linking between the introduced sulfhydryl groups of two FvCys fragments by means of bis-maleimidohexane was used to generate a bisFvCys conjugate. The stability of the bisFvCys conjugate and an FvCys analogue that had been reacted with N-ethyl-maleimide to block the free sulfhydryl group, FvCys(BL), were compared after 125I-labeling. The bisFvCys conjugate was completely stable to incubation in solution at 37 degrees C for 24 h whereas only 60% of the FvCys(BL) fragment remained soluble. After i.v. administration to normal Wistar rats, both Fv proteins were rapidly cleared from the circulation with biphasic kinetics that were best fitted to a two-compartment open pharmacokinetic model. The alpha-phase half-life of the bisFvCys conjugate, 0.32 h, was significantly longer than that of the FvCys(BL) fragment, 0.15 h (p less than 0.001) whereas there was no significant difference between the beta-phase half-lives, 1.4 to 1.6h. No chain cleavage or covalent attachment to serum protein was detected by SDS-PAGE analysis of serum samples. However, gel permeation HPLC revealed that both Fv proteins associated with serum proteins in vivo and in vitro.
Cumber
A J
AJ
Drug Targeting Laboratory, Institute of Cancer Research, Sutton, U.K.
Ward
E S
ES
Winter
G
G
Parnell
G D
GD
Wawrzynczak
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine; D004252:DNA Mutational Analysis; D005803:Genes, Immunoglobulin; D007128:Immunoglobulin Fragments / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D013329:Structure-Activity Relationship
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009113:Muramidase; D003545:Cysteine