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Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies

Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies
Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan-protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man 5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan-protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein-carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

0002-7863
9723-9732
Yu, Xiaojie
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Baruah, Kavitha
a02f59f3-5e0f-4735-9fdd-639bdbb8f563
Harvey, David J.
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Vasiljevic, Snezana
17e075b4-520d-4b9b-a4a7-08ac394ae5e1
Alonzi, Dominic S.
6c724aed-60fc-4f01-8ace-98fc3e64d863
Song, Byeong Doo
8570f719-c8b1-4772-9e4e-d541f5fcb0a7
Higgins, Matthew K.
17871712-96d5-4862-a9db-ec8ee7e25262
Bowden, Thomas A.
4b17a588-ac01-4112-807a-8b99a6c20d0f
Scanlan, Christopher N.
04dd1b57-b6fc-414c-8595-08310dbb3d32
Crispin, Matthew
cd980957-0943-4b89-b2b2-710f01f33bc9
Yu, Xiaojie
44d52374-eacc-4e23-b7da-c881e6d3a5dd
Baruah, Kavitha
a02f59f3-5e0f-4735-9fdd-639bdbb8f563
Harvey, David J.
8bb24417-3852-4b1f-827b-0d5d2c176744
Vasiljevic, Snezana
17e075b4-520d-4b9b-a4a7-08ac394ae5e1
Alonzi, Dominic S.
6c724aed-60fc-4f01-8ace-98fc3e64d863
Song, Byeong Doo
8570f719-c8b1-4772-9e4e-d541f5fcb0a7
Higgins, Matthew K.
17871712-96d5-4862-a9db-ec8ee7e25262
Bowden, Thomas A.
4b17a588-ac01-4112-807a-8b99a6c20d0f
Scanlan, Christopher N.
04dd1b57-b6fc-414c-8595-08310dbb3d32
Crispin, Matthew
cd980957-0943-4b89-b2b2-710f01f33bc9

Yu, Xiaojie, Baruah, Kavitha, Harvey, David J., Vasiljevic, Snezana, Alonzi, Dominic S., Song, Byeong Doo, Higgins, Matthew K., Bowden, Thomas A., Scanlan, Christopher N. and Crispin, Matthew (2013) Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies. Journal of the American Chemical Society, 135 (26), 9723-9732. (doi:10.1021/ja4014375).

Record type: Article

Abstract

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan-protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man 5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan-protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein-carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

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2013-Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.
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e-pub ahead of print date: 7 June 2013
Published date: 3 July 2013

Identifiers

Local EPrints ID: 414597
URI: http://eprints.soton.ac.uk/id/eprint/414597
ISSN: 0002-7863
PURE UUID: 555f1cd1-478a-43be-93ef-b72a8a4dc30d
ORCID for Matthew Crispin: ORCID iD orcid.org/0000-0002-1072-2694

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Date deposited: 04 Oct 2017 16:30
Last modified: 06 Jun 2024 01:59

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Contributors

Author: Xiaojie Yu
Author: Kavitha Baruah
Author: David J. Harvey
Author: Snezana Vasiljevic
Author: Dominic S. Alonzi
Author: Byeong Doo Song
Author: Matthew K. Higgins
Author: Thomas A. Bowden
Author: Christopher N. Scanlan
Author: Matthew Crispin ORCID iD

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