Remodeling Antibody with G0 Glycoforms
Glycans are complex carbohydrate structures attached to proteins, and their composition varies widely, leading to a heterogeneous mixture of glycoforms. Among these, the G0 glycoform, characterized by the absence of terminal galactose residues, is a key structure. The ability to precisely control and enrich an antibody with a homogenous G0 glycoform is a transformative approach in biopharmaceutical development. At CD BioGlyco, our advanced remodeling service is designed to deliver antibodies with a defined G0 glycoform profile, enabling enhanced functional properties for next-generation therapeutic antibodies.
The development of recombinant protein therapeutics is a major revolution in modern medicine. Over the past two decades, the number of therapy-based monoclonal antibodies (mAbs) has grown rapidly, bringing new therapeutic hope to cancer patients. Therapeutic mAbs are typically immunoglobulin G (IgG) containing four polypeptide chains. Their conserved asparagine (Asp) is glycosylated at position 297 in the CH2 constant domain of the crystallizable fragment (Fc) region. Fc glycosylation has a great influence on the structure, immunogenicity, pharmacokinetics, and pharmacodynamics of mAb. It has been found that the immunological properties of antibodies can be improved by modulating the fine structure of glycans, especially those linked to the Fc domain. For example, reducing Fc core fucosylation can significantly enhance the antibody-dependent cellular cytotoxicity (ADCC) of therapeutics, thereby improving anticancer activity.
Since different glycans have a significant impact on the function of antibodies. Therefore, researchers have modified the glycan structure in the Fc domain by glycoengineering strategies to improve the therapeutic effect. There are currently two commonly used glycoengineering strategies, namely host cell glycoengineering and in vitro chemoenzymatic glycosylation remodeling. Host cell glycoengineering is the modification of glycan structure by manipulating important mediators in the glycan biosynthetic pathway of different host expression systems. This method has been used to produce optimal glycoforms for some therapeutic antibodies. In vitro chemoenzymatic glycosylation remodeling provides a novel approach to remodeling Fc glycans from a heterogeneous N-glycosylation pattern to a homogeneous pattern. First, deglycosylating IgG antibodies is performed by specific enzymes and leaving the innermost layer of N-Acetylglucosamine (GlcNAc). Glycoengineered mAbs with homologous N-glycans are then prepared by transglycosylation.
Fig.1 In vitro chemoenzymatic glycosylation remodeling using endo-β-N-acetylglucosaminidase and glycosynthase. (Boune, et al., 2020)
Since natural and recombinant antibodies are often produced as a heterogeneous mixture of glycoforms, it is extremely difficult to isolate or enrich for pure glycans. Therefore, it is highly necessary to develop glycoengineering strategies to generate glycan-defined and site-selectively modified antibodies, improving their function and therapeutic efficacy. At CD BioGlyco, we have developed an advanced antibody glycoengineering platform. We provide clients with efficient and site-specific glycan remodeling services for human IgG, including human IgG1, IgG2, and IgG4. We generate human IgG carrying the G0 glycoform by an in vitro chemoenzymatic glycosylation remodeling technique. Both fucosylated G0 and afucosylated G0 glycoform are available. Our workflow is as follows:
This step relies on specialized enzymatic glycan trimming services, including the sourcing and production of highly purified endoglycosidases, such as Endo S or Endo F3, and optimizing reaction conditions to ensure complete and specific removal of glycans, leaving only the core GlcNAc.
This step involves specialized glycosynthetase technology, encompassing the engineering and production of these modifying enzymes, as well as the complex chemical synthesis of preassembled glycosyloxazolines (such as G0). This service focuses on optimizing the chemoenzymatic reaction between the core GlcNAc-modified antibody and the glycosyloxazoline, catalyzed by the glycosynthase, to generate an antibody with the homologous G0 glycoform. Advanced mass spectrometry (MS) and other techniques are used to confirm the glycan linkage.
Journal: Glycobiology
IF: 3.3
Published: 2020
Results: This review focuses on the enzymatic remodeling of IgG glycosylation, highlighting key enzymatic tools and their uses. It emphasizes that IgG glycosylation, especially Fc-glycans, modulates functions like ADCC and complement activation, with features such as sialylation affecting Fcγ receptor interactions. Streptococcus-derived endoglycosidases (e.g., EndoS) selectively target Fc-glycans, enabling applications like generating uniform glycoforms, site-specific antibody conjugation, improved assay specificity, simplified antibody analysis, and studying mAb mechanisms by blocking ADCC. It also touches on IgG3/O-glycosylation and new O-glycan tools, noting these enzymes advance antibody research.
Leveraging proprietary enzymatic technologies, advanced analytical platforms, and extensive glycobiology expertise, CD BioGlyco delivers highly pure, functionally optimized antibodies with homogeneous G0 glycoforms. We invite you to contact us to discuss your specific project requirements.
References
