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

Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate
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 colt from a modified expression plasmid containing the structural genes for the VH and VL 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.

0022-1767
120-126
Cumber, Alan J.
9e5cd21c-93ba-4b2d-9ac0-1c5f3e23e26c
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Winter, Greg
72106f71-2931-453a-861d-27625707b8fe
Parnell, Geoffrey D.
e5f9209c-22ea-449b-b560-348229f0ea61
Wawrzynczak, Edward J.
013daf71-9483-43f6-a6a3-445ef3a9c7e4
Cumber, Alan J.
9e5cd21c-93ba-4b2d-9ac0-1c5f3e23e26c
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Winter, Greg
72106f71-2931-453a-861d-27625707b8fe
Parnell, Geoffrey D.
e5f9209c-22ea-449b-b560-348229f0ea61
Wawrzynczak, Edward J.
013daf71-9483-43f6-a6a3-445ef3a9c7e4

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. The Journal of Immunology, 149 (1), 120-126.

Record type: Review

Abstract

A murine antibody FvCys fragment with a single additional cysteine residue at the C terminus of the VH domain was expressed in Escherichia colt from a modified expression plasmid containing the structural genes for the VH and VL 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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Published date: 1 July 1992

Identifiers

Local EPrints ID: 425261
URI: https://eprints.soton.ac.uk/id/eprint/425261
ISSN: 0022-1767
PURE UUID: 4b1311b4-78cd-4a04-9ec3-6d0eb88dbb4a
ORCID for E. Sally Ward: ORCID iD orcid.org/0000-0003-3232-7238

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Date deposited: 12 Oct 2018 16:30
Last modified: 14 Mar 2019 01:21

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Contributors

Author: Alan J. Cumber
Author: E. Sally Ward ORCID iD
Author: Greg Winter
Author: Geoffrey D. Parnell
Author: Edward J. Wawrzynczak

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