Remodeling Antibodies with G1 Glycoforms
Antibody glycan remodeling strategies generate monoclonal antibodies with homogeneous glycoforms to improve therapeutic efficacy. CD BioGlyco is proficient at remodeling antibodies with G1 glycoforms according to our client's special needs.
Immunoglobulin G (IgG) is a glycoprotein composed of two heavy chains and two light chains. The heavy chains and light chains together constitute the antigen-binding fragment (Fab) and the crystallizable fragment (Fc). The conserved asparagine (Asp) of IgG is glycosylated at position 297 in the CH2 constant domain of the Fc region. Glycans are important players in Fc effector function and are flexible and highly dynamic in IgG. Glycans in the Fc region can be modified by glycosidases to enhance the interaction of Fc receptors with glycans. The specific glycoforms of IgG are related to the function of antibody molecules. For example, fully sialylated IgG glycans are associated with increased anti-inflammatory properties. Deletion of core fucose residues in Fc glycans significantly improves antibody binding to Fcγ receptor IIIa (FcγRIIIa) and antibody-dependent cellular cytotoxicity (ADCC) activity.
Glycosidase is an enzyme that catalyzes the hydrolysis of glycosidic bonds in complex sugars. Glycosidase is divided into exoglycosidases and endoglycosidases. Endoglycosidases generally have glycoform-specific activity and have been widely used in the study of human glycoproteins and glycoforms. EndoH is an endoglycosidase that specifically hydrolyzes high-mannose glycans and partially hybrid glycans. EndoD from Streptococcus pneumoniae cleaves the chitobiose core of N-glycans. EndoS, EndoS2, EndoSe, and EndoSd endoglycosidases from Streptococcus are highly specific for IgG. Therefore, EndoS and EndoS2 have become valuable tools for antibody research.
Fig.1 IgG glycan remodeling by endoglycosidase. (Sjögren, et al., 2020)
Because the specific glycoform of the glycan in the Fc region of IgG is related to the function of the antibody molecule. To obtain therapeutic antibodies with consistent and homogenous glycoforms for enhanced therapeutic efficacy, it is necessary to modify the Fc region glycans of IgG through Glycoengineering strategies. CD BioGlyco has developed an efficient and specific antibody glycan remodeling service. We provide clients with human IgG (including human IgG1, IgG2, and IgG4) carrying homogenous G1 glycoforms through IgG-specific endoglycosidase. Our workflow is as follows:
To precisely control the glycan structures on antibodies, a crucial initial step involves the enzymatic modification of the Fc region. Fc glycans are trimmed to the innermost GlcNAc by IgG-specific endoglycosidases, such as EndoS or EndoS2. These enzymes specifically cleave the β-1,4 glycosidic bond between the two GlcNAc residues in the Fc glycan core, leaving a single GlcNAc attached to the asparagine residue at position 297 (Asn297). This enzymatic deglycosylation provides a uniform, truncated glycan substrate, which is essential for subsequent enzymatic glycoengineering steps, allowing for the defined attachment of specific glycan structures.
Following the initial trimming, glycosynthase catalyzes the reaction of pre-assembled G1 glycan oxazoline with the core GlcNAc to generate antibodies with homologous G1 glycoforms. Glycosynthases are engineered glycosidases that facilitate the formation of glycosidic bonds rather than their hydrolysis. In this specific application, a pre-activated G1 glycan (a galactose-containing biantennary glycan) in an oxazoline form is used as a donor substrate. The glycosynthase precisely transfers this G1 glycan to the exposed GlcNAc residue on the deglycosylated Fc region of the antibody. This enzymatic ligation results in a highly homogeneous population of antibodies, all bearing the desired G1 glycoform. This method offers a powerful approach to developing antibodies with specific and consistent glycosylation patterns, enabling researchers to investigate and optimize the impact of defined glycan structures on antibody efficacy, affinity, and interaction with immune effector cells.
Journal: Sci Rep
IF: 3.8
Published: 2016
Results: This article introduces multi-level glyco-engineering techniques for generating IgG with defined Fc-glycans. It notes that the conserved N-linked glycan at Asn297 in IgG's Fc domain affects its structure and interactions. Methods like using decoy substrates, overexpressing glycosyltransferases, and in vitro sialylation are developed to manipulate specific glycosylation traits (fucosylation, bisection, galactosylation, sialylation) in eukaryotic cells, effective in HEK cells. These tools create at least 20 glycoforms to explore biological effects, applicable to other N-linked glycoproteins, with plans to study combined trait impacts.
Fig.2 Glycosylation profile analysis. (Dekkers, et al., 2016)
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