The University of Southampton
University of Southampton Institutional Repository

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity
Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity.

ADCC, ciliate, monoclonal antibody, N-glycosylation pattern, recombinant expression, rituximab
1942-0862
1498-1511
Calow, Jenny
a7761dcc-ac48-4923-9fd5-3c8884e0ee66
Behrens, Anna Janina
ed584c40-79cb-4de9-bb9c-bd68c71d6a68
Mader, Sonja
d084e147-ef2b-4e21-b2e9-c616be8804f7
Bockau, Ulrike
11d2b2f5-3f84-4683-a988-ffe679d5d841
Struwe, Weston B.
16a348b1-3921-4a2d-b5fb-d341fccea65f
Harvey, David J.
8bb24417-3852-4b1f-827b-0d5d2c176744
Cormann, Kai U.
7b363d5e-eba9-4ae8-abdb-ad5a350fa4d1
Nowaczyk, Marc M.
fb039104-833e-4447-8260-98f233bba264
Loser, Karin
5e2ff509-2787-48f1-ba3c-646793a2c8b2
Schinor, Daniel
29e3f00d-5b3d-446e-86fe-3524523ffccf
Hartmann, Marcus W.W.
e8ecb956-ef1f-44ec-bb77-bc3f2be1788d
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Calow, Jenny
a7761dcc-ac48-4923-9fd5-3c8884e0ee66
Behrens, Anna Janina
ed584c40-79cb-4de9-bb9c-bd68c71d6a68
Mader, Sonja
d084e147-ef2b-4e21-b2e9-c616be8804f7
Bockau, Ulrike
11d2b2f5-3f84-4683-a988-ffe679d5d841
Struwe, Weston B.
16a348b1-3921-4a2d-b5fb-d341fccea65f
Harvey, David J.
8bb24417-3852-4b1f-827b-0d5d2c176744
Cormann, Kai U.
7b363d5e-eba9-4ae8-abdb-ad5a350fa4d1
Nowaczyk, Marc M.
fb039104-833e-4447-8260-98f233bba264
Loser, Karin
5e2ff509-2787-48f1-ba3c-646793a2c8b2
Schinor, Daniel
29e3f00d-5b3d-446e-86fe-3524523ffccf
Hartmann, Marcus W.W.
e8ecb956-ef1f-44ec-bb77-bc3f2be1788d
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9

Calow, Jenny, Behrens, Anna Janina, Mader, Sonja, Bockau, Ulrike, Struwe, Weston B., Harvey, David J., Cormann, Kai U., Nowaczyk, Marc M., Loser, Karin, Schinor, Daniel, Hartmann, Marcus W.W. and Crispin, Max (2016) Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity. mAbs, 8 (8), 1498-1511. (doi:10.1080/19420862.2016.1228504).

Record type: Article

Abstract

Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity.

Text
26_09_2017_Antibody p - Version of Record
Download (2MB)

More information

Accepted/In Press date: 19 August 2016
e-pub ahead of print date: 3 September 2016
Published date: 16 November 2016
Keywords: ADCC, ciliate, monoclonal antibody, N-glycosylation pattern, recombinant expression, rituximab

Identifiers

Local EPrints ID: 414331
URI: https://eprints.soton.ac.uk/id/eprint/414331
ISSN: 1942-0862
PURE UUID: 9845a2ee-c137-4220-b851-53788533944b
ORCID for Max Crispin: ORCID iD orcid.org/0000-0002-1072-2694

Catalogue record

Date deposited: 26 Sep 2017 16:30
Last modified: 14 Mar 2019 01:25

Export record

Altmetrics

Contributors

Author: Jenny Calow
Author: Anna Janina Behrens
Author: Sonja Mader
Author: Ulrike Bockau
Author: Weston B. Struwe
Author: David J. Harvey
Author: Kai U. Cormann
Author: Marc M. Nowaczyk
Author: Karin Loser
Author: Daniel Schinor
Author: Marcus W.W. Hartmann
Author: Max Crispin ORCID iD

University divisions

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×