Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate

Cumber, Alan J., Ward, E. Sally, Winter, Greg, Parnell, Geoffrey D. and Wawrzynczak, Edward J. (1992) Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate. Journal of Immunology, 149 (1), 120-126.

Abstract

A murine antibody FvCys fragment with a single additional cysteine residue at the C terminus of the V_H domain was expressed in Escherichia colt from a modified expression plasmid containing the structural genes for the V_H and V_L domains derived from the anti-lysozyme hybridoma D 1.3. 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-ethylmaleimide 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°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 α-phase half-life of the bisFvCys conjugate, 0.32 h, was significantly longer than that of the FvCys(BL) fragment, 0.15 h (p < 0.001) whereas there was no significant difference between the β-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.

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Contributors

Author: E. Sally Ward

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