IgG antibodies contain a conserved N-glycosylation site (N-297) on the Fc fragment. Glycans attached to this site vary in their composition, leading to uneven antibody performance. Having worked in the field of antibody engineering for many years, Creative Biolabs is able to provide our worldwide customers with high-quality Fc glycoengineering services, helping guide the development of future Fc-based therapeutics (e.g., monoclonal antibodies and Fc-fusion proteins). Our professional scientists will do their best to meet every customer's requirements.
Different Fc glycoforms impact their effector function, pharmacokinetics (PK), stability, aggregation, safety, and immunogenicity. Fc glycoforms affect monoclonal antibody (mAb) effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by modulating the Fc-FcγRs and Fc-C1q interactions. Plenty of cell glycoengineering and chemoenzymatic glycoengineering strategies are emerging to produce IgGs with homogenous glycoforms.
Fig.1 Structure of an antibody and the N-glycan attached to Asn297. (Kiyoshi, 2017)
Since different glycoforms have distinct impacts on antibody effector function, it is important to control antibody glycoforms. Strategies used for the production of homogeneously glycosylated antibodies with tailored effector function include host cell based glycoengineering involving manipulation of biosynthetic pathways and in vitro chemoenzymatic glycosylation remodeling.
Recent years, host cell glycoengineering has been highly pursued to produce recombinant IgGs with desired glycoforms. Cell glycoengineering strategies are classified into the following four types, (i) selection of cell type, environmental factors, and cell culture conditions, (ii) using enzyme inhibitors to intervene host biosynthesis pathway, (iii) genetic modifications of the host biosynthesis pathway, and (iv) “humanization” of N-glycosylation pathway of non-mammalian cells.
Fig.2 Use of the endoplasmic reticulum α-mannosidase inhibitors, kifunensine, to produce the high mannose glycoform with low fucose. (Li, 2017)
Despite much progress in cell glycoengineering, it remains challenging to produce IgGs with highly homogenous glycoforms in host cells. Thus, the in vitro chemoenzymatic glycosylation remodeling provides an attractive alternative for the production of therapeutic mAbs with predefined and homogeneous glycoforms. This method normally contains three steps. Firstly, deglycosylation of IgG by an endo-β-N-acetylglucosaminidase, simultaneously leaving the innermost GlcNAc at N297. Secondly, preparation of oxazoline derivatives of customized N-linked glycan structures as sugars donors by chemical methods. Lastly, transglycosylation of the glycan oxazoline donor to the innermost GlcNAc acceptor.
Fc N-Glycan Specific ADC
The methods for conjugating the small molecule drugs onto IgG represent one of the key technologies in antibody-drug conjugate (ADC) development. The traditional conjugation approaches involve random addition of drugs onto Lys or reduced Cys residues, which leads to highly heterogeneous mixtures with different drug-antibody ratios and inconsistent yield. This heterogeneity negatively impacts the in vivo stability, efficacy, and PK of ADCs. Conjugation through IgG-Fc N-glycans represents one of the most widely used site-specific conjugation methods. Glycosite-specific conjugation proceeds with the introduction of a chemically active moiety onto the Fc N-glycans followed by reacting with payloads carrying another chemically active group.
By using the above two approaches, Creative Biolabs is able to offer the following antibody glycoengineering services:
IgG N-glycans affect their CDC and ADCC activity by altering Fc conformations and modulating the non-covalent interactions between oligosaccharides and CH2 domains. Creative Biolabs has been focusing on Fc glycoengineering for many years. With pleasure, we can tailor high-quality glycoengineering services to exactly suit your research/project demands. If you are interested in our service, please do not hesitate to contact us for more details.
References
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