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Catabolism of the murine IgGl molecule: Evidence that both CH2‐CH3 domain interfaces are required for persistence of IgGl in the circulation of mice

Catabolism of the murine IgGl molecule: Evidence that both CH2‐CH3 domain interfaces are required for persistence of IgGl in the circulation of mice
Catabolism of the murine IgGl molecule: Evidence that both CH2‐CH3 domain interfaces are required for persistence of IgGl in the circulation of mice

Site‐directed mutagenesis of a recombinant Fc‐hinge fragment has previously been used to identify a region of the murine IgG 1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this ‘catabolic site’ has been further analysed using recombinant techniques. Fc‐hinge, CH2‐hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice. The CH2‐hinge has been analysed as both a monomer and dimer, and the dimer has a longer β phase half‐life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc‐hinge fragment comprising a heterodimer of one Fc‐hinge with the wild type (WT) IgGl sequence and a mutant Fc‐hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a β phase half‐life of 37.9 h and this is significantly shorter than that of the WT Fc‐hinge fragment (82.9 h). In contrast to the CH2‐hinge dimer, the CH3 domain is cleared rapidly (β phase half‐life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgGl molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.

0300-9475
457-465
Kim, J. ‐K
f2951688-2d00-4a39-9ca6-ccb2a96fc4c8
Tsen, M. ‐F
ee928224-ae90-4cf6-8a0a-263e7f8ec057
Ghetie, V.
b7b50946-2bd9-4896-b841-6bbfba8237c2
Ward, E. S.
b31c0877-8abe-485f-b800-244a9d3cd6cc
Kim, J. ‐K
f2951688-2d00-4a39-9ca6-ccb2a96fc4c8
Tsen, M. ‐F
ee928224-ae90-4cf6-8a0a-263e7f8ec057
Ghetie, V.
b7b50946-2bd9-4896-b841-6bbfba8237c2
Ward, E. S.
b31c0877-8abe-485f-b800-244a9d3cd6cc

Kim, J. ‐K, Tsen, M. ‐F, Ghetie, V. and Ward, E. S. (1994) Catabolism of the murine IgGl molecule: Evidence that both CH2‐CH3 domain interfaces are required for persistence of IgGl in the circulation of mice. Scandinavian Journal of Immunology, 40 (4), 457-465. (doi:10.1111/j.1365-3083.1994.tb03488.x).

Record type: Article

Abstract

Site‐directed mutagenesis of a recombinant Fc‐hinge fragment has previously been used to identify a region of the murine IgG 1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this ‘catabolic site’ has been further analysed using recombinant techniques. Fc‐hinge, CH2‐hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice. The CH2‐hinge has been analysed as both a monomer and dimer, and the dimer has a longer β phase half‐life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc‐hinge fragment comprising a heterodimer of one Fc‐hinge with the wild type (WT) IgGl sequence and a mutant Fc‐hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a β phase half‐life of 37.9 h and this is significantly shorter than that of the WT Fc‐hinge fragment (82.9 h). In contrast to the CH2‐hinge dimer, the CH3 domain is cleared rapidly (β phase half‐life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgGl molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.

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Published date: October 1994

Identifiers

Local EPrints ID: 425264
URI: https://eprints.soton.ac.uk/id/eprint/425264
ISSN: 0300-9475
PURE UUID: 62c3e9b2-17ac-4077-81c9-2b3d5002f02b
ORCID for E. S. 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: J. ‐K Kim
Author: M. ‐F Tsen
Author: V. Ghetie
Author: E. S. Ward ORCID iD

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