Control of Recombinant Monoclonal Antibody Effector Functions by Fc N‐Glycan Remodeling in Vitro
Abstract
N-Glycans at Asn(297) in the Fc domain of IgG molecules are required for Fc receptor-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study we have specifically remodeled the Fc N-glycans of intact recombinant IgG(1) therapeutic monoclonal antibody (Mab) products, Rituxan and Herceptin, with a soluble recombinant rat beta-1,4-N-acetylglucosaminyltransferase III (rGnTIII) produced by baculovirus-infected insect cells. N-Glycan remodeling in vitro permitted a controlled and selective transfer of a bisecting beta1,4-linked GlcNAc to the core beta-linked mannose of degalactosylated Mab N-glycans to yield Mabs varying in bisecting GlcNAc content from 31% to 85%. This was confirmed by analysis of N-glycans by both normal phase HPLC and MALDI-MS, the latter yielding the expected mass increase of 203.2 Da with no other oligosaccharide modifications evident. ADCC of remodeled Rituxan and Herceptin Mabs was determined using peripheral blood mononuclear cells as effectors and either CD20(+) (SKW6.4 and SU-DHL-4) or Her2(+) (SKBR-3) target cells, respectively. A conserved 10-fold increase in ADCC was observed for both remodeled therapeutic Mabs with high (>80%) bisecting GlcNAc content. In contrast, although the presence of a bisecting GlcNAc had minimal effect on CDC, degalactosylation of Rituxan reduced CDC by approximately half, relative to unmodified (variably galactosylated) control Mab. In…
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Enriching the abundance of mAb N-glycoforms with β1,4-linked galactose can enhance complement-dependent cytotoxicity (CDC).
“Enriching the abundance of mAb N-glycoforms with β1,4-linked galactose (Gal) can also be desirable as it has been shown to enhance complement-dependent cytotoxicity (CDC), another mechanism for the selective depletion of tumour cells by Fc activation of the complement pathway [16–21].”
From Glycoengineering Chinese hamster ovary cells: a short history (2021) · cites this paper as [17]
Addition of bisecting β1,4-linked GlcNAc by GnT-III or in vitro glycan remodelling is linked to increased ADCC in IgGs, albeit to a lesser extent than afucosylation.
“The addition of bisecting β1,4-linked N-acetylglucosamine (GlcNAc) by endogenous or recombinant N-acetylglucosaminyltransferase III (GnT-III in YB2/0 and CHO cells respectively) or via in vitro glycan remodelling has also been linked to increased ADCC in IgGs, albeit to a lesser extent than afucosylation [15–17].”
From Glycoengineering Chinese hamster ovary cells: a short history (2021) · cites this paper as [17]
UDP-Gal provides the mechanistic link between extracellular galactose and uridine supplementation and galactosylation, a key quality attribute of monoclonal antibodies.
“It provides the mechanistic link between extracellular galactose and uridine supplementation and galactosylation, a key quality attribute of monoclonal antibodies [13,50].”
From Soft sensing of intracellular states for CHO cell bioprocessing with Ensemble Kalman Filters (2026) · cites this paper as [13]
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Soft sensing of intracellular states for CHO cell bioprocessing with Ensemble Kalman Filters
Yu, L., del Rio Chanona, A., & Kontoravdi, C. (2026) · Journal of Process Control · doi:10.1016/j.jprocont.2026.103875
- Authors
- Hodoniczky, J., Zheng, Y. Z., & James, D. C.
- Affiliations
- The University of Queensland (AU)
- Corresponding author
- James, David C.
- ORCID
- James, David C.
- Venue
- Biotechnology Progress
- Publisher
- Wiley
- Published
- 2005-01
- Volume
- 21
- Issue
- 6
- Pages
- 1644-1652
- ISSN
- 1520-6033 (ISSN-L)
- 8756-7938
- OpenAlex
- W2052491267
- Cited by
- 393
- References
- 3
- Citations by year
- 2026: 11 · 2025: 14 · 2024: 15 · 2023: 15 · 2022: 18 · 2021: 34 · 2020: 25 · 2019: 20 · 2018: 32 · 2017: 23 · 2016: 28 · 2015: 20 · 2014: 22 · 2013: 31 · 2012: 24
- FWCI
- 2.76
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biotechnology Progress (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Centers for Disease Control and Prevention
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- bisecting GlcNAc, therapeutic monoclonal antibodies, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, trastuzumab, humanized monoclonal antibody
- MeSH terms
- Trastuzumab, Rituximab, Animals, Antibodies, Monoclonal, Antibody-Dependent Cell Cytotoxicity, Biotechnology, Cell Line, Chromatography, High Pressure Liquid, Cytotoxicity, Immunologic, Glycosylation, Humans, Immunoglobulin Fc Fragments, Polysaccharides, Receptors, Fc, Recombinant Proteins, N-Acetylglucosaminyltransferases, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Rats, Antibodies, Monoclonal, Murine-Derived, Antibodies, Monoclonal, Humanized, In Vitro Techniques
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