Glycoengineering through biosynthetic pathway has enabled the production of certain pure or enriched monoclonal antibodies (mAbs) glycoforms for functional studies and therapeutic development. Nevertheless, the quality and diversity of glycoforms that can be modified through genetic manipulations are quite limited, owing to the complexity and restriction of biosynthetic pathways. In contrast to in vivo genetic approaches, in vitro chemoenzymatic glycan remodeling of mAbs using glycosidases and glycosyltransferases has been employed to extend the glycan chains in an intact antibody. With Ph.D. level scientists and extensive experience in the research of glycoengineering, Creative Biolabs is proficient in employing chemoenzymatic method for glycoengineering of mAbs. We are dedicated to serving every unique need of our clients by providing custom mAb glycoengineering service to meet our clients’ R&D timeline and budget.
As an important class of therapeutic glycoproteins, mAbs are widely used in the treatment of cancer, inflammation, and infectious diseases. Therapeutic mAbs possess several clinical actions including disruption of tumor cell signaling, activation of complement-dependent cytotoxicity (ADC), Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis, and induction of adaptive immunity. The immunoglobulin G (IgG) type mAbs consist of two heavy and two light chains, which are covalently associated to form three domains, two identical Fab domains, and the Fc domain. IgG mediates its immune functions through complement and cellular IgG-Fc gamma receptors (FcγR). The Fc domain of IgG is recognized by FcγR on myeloid and natural killer cells, providing target recognition and activation of immune effector cells. A highly conserved glycan at position Asn297 in the Fc-domain infers structural changes to the Fc-region required for binding to FcγR. This glycan consists of variable levels of fucose, sialic acid, galactoseand, bisecting N-acetylglucosamine. Subtle differences in this glycan can affect the effector functions of antibodies and may also change the interaction with FcγR. However, mAbs are usually generated as mixtures of Fc glycoforms, and the control of glycosylation in the homogeneous status in different host expression systems is still a challenge.
The significant impact of fine structures of Fc N-glycans on the biological functions of antibodies has stimulated more and more interest in exploring methods to control the Fc glycosylation of antibodies. Great efforts have been taken in mammalian cells, yeast cells, and plant cells aiming to control the glycosylation of mAbs in these expression systems. Those in vivo methods have resulted in the production of low-fucose or non-fucosylated monoclonal antibodies with improved therapeutic efficacy. However, complete control of the Fc glycosylation to a given specific homogeneous glycoform is still a challenge. In vitro chemoenzymatic method is another promising strategy for glycan remodeling via chemoenzymatic synthesis, which could lead to highly homogeneous antibody glycoforms. Scientists have used chemoenzymatic method to generate a panel of enriched Fc glycoforms, including G0F, G2F, and bisecting GlcNAc G2F of an intact antibody.
Fig.1 Glycosylation remodeling of Rituximab to prepare homogeneous natural and selectively modiﬁed glycoforms. (Huang, 2012)
With proven experience in glycoscience, Creative Biolabs employs a chemoenzymatic approach for Fc glycan remodeling of mAbs using endoglycosidase, an endo-β-N-acetylglucosaminidase that is highly specific for the Fc N-glycans of IgG. This chemoenzymatic strategy is dependent on the use of activated glycan oxazoline as the donor substrate which mimics the transition state of the hydrolysis reaction. Our chemoenzymatic glycoengineering approach provides a general platform to enable the optimization of therapeutic eﬃcacy and modulate the eﬀector functions of mAbs.
This chemoenzymatic glycoengineering method can be used for the Fc glycosylation remodeling of various mAbs to provide homogeneous Fc glycoforms for different applications. Creative Biolabs is fully competent and dedicated to serving as your one-stop-shop for custom mAb glycoengineering and characterization. For more detailed information, please feel free to contact us or directly send us an inquiry.