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Article

Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain

1
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
2
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA
3
Department of Chemistry, University of Georgia, Athens, GA 30602, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Itai Benhar
Received: 3 August 2021 / Revised: 31 August 2021 / Accepted: 13 September 2021 / Published: 29 September 2021
Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells. View Full-Text
Keywords: human immunoglobulin 1 (hIgG1); human Fc gamma receptor IIIa (FcγIIIa); EndoS2; truncated N-glycan human immunoglobulin 1 (hIgG1); human Fc gamma receptor IIIa (FcγIIIa); EndoS2; truncated N-glycan
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MDPI and ACS Style

Shenoy, A.; Yalamanchili, S.; Davis, A.R.; Barb, A.W. Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain. Antibodies 2021, 10, 38. https://0-doi-org.brum.beds.ac.uk/10.3390/antib10040038

AMA Style

Shenoy A, Yalamanchili S, Davis AR, Barb AW. Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain. Antibodies. 2021; 10(4):38. https://0-doi-org.brum.beds.ac.uk/10.3390/antib10040038

Chicago/Turabian Style

Shenoy, Anjali, Srisaimaneesh Yalamanchili, Alexander R. Davis, and Adam W. Barb. 2021. "Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain" Antibodies 10, no. 4: 38. https://0-doi-org.brum.beds.ac.uk/10.3390/antib10040038

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