Creative Biolabs is excited to greet you at the upcoming international conferences. Meet our team at the 13th Annual World ADC San Diego and the 13th Annual World Multispecific Summit in September (booth number to be updated) for expert consultation on your drug discovery. Shoot an email to arrange an in-person meeting!
Immunoglobulin G carries a highly conserved asparagine 297 (N297) glycosylation site in the CH2 domain (Figure 1), allowing site-specific glycan modification with glycosyltransferases. The antibody may carry other N-glycans if the Fab domain of the antibody contains the Asn-X-Ser / Thr (X≠Pro) sequence. Therefore, careful selection of monoclonal antibodies lacking Fab glycosylation is very important for Fc-specific glycan modification.
Fig.1 The IgG-Fc N-glycan. Schematic depiction of IgG with two fully processed IgG-Fc N-glycans (left) and composition of the glycan (right).
The Fc region requires specific glycan modifications to mediate IgG effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is affected by many factors. The terminal sialic acid residues of glycoconjugates show significant functional and structural diversity. They affect the biological activity of glycoproteins, serum half-life and structural stability. It has recently been shown that increased Fc sialylation can lead to reduced binding to immobilized antigens and certain Fcγ receptors, as well as reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of the antibody. Fc sialylation may also affect antibody binding to cell surface bound antigen and sensitivity to proteases.
The efficacy of many therapeutic monoclonal antibodies (mAbs) depends on their Fc-dependent effector functions. To produce antibodies with enhanced effector functions, Creative Biolabs has developed an advanced GlycoDegree™ platform that allows limiting the degree of sialylation of recombinantly expressed therapeutic antibodies. Low or high sialylated sialic acid antibodies have been verified to have binding affinity similar to that of soluble antigens, and they have pharmacokinetic properties comparable to their sialylated counterparts.
Most of the N-glycans present in the Fc region of IgGs are complex biantennary structures, which are heterogeneous in terminal sialylation, galactose, bisecting GlcNAc, GlcNAc and core fucose. Human IgG only contains 2-6 linked sialic acid residues. Recombinant IgG expressed in CHO cells mainly contains a2,3-linked sialic acid residues, while recombinant IgGs derived from mouse myeloma cells contain both a2,3-linked and a2,6-linked sialic acid residues. In addition to the diversity in a2,3-linked and a2,6-linked sialic acid residues, IgG can also contain structural isomers. Diversification of terminal sialic acid significantly increases the heterogeneity of the Fc glycan oligosaccharide pool.
Fig.2 Biosynthesis of sialic acids and their activated derivatives in mammals.
Sialylation affects the biological activities and physical and chemical properties of glycolipids, glycoproteins and polysaccharides. Sialic acid residues are involved in cell signal transduction, cell adhesion, interaction between host cells and pathogens, structural stability, solubility, etc. The increase in terminal sialylation can improve the serum half-life of many glycoproteins. In nature, sialic acid residues promote beneficial and harmful activities. This is also the case with monoclonal antibodies, where the N-glycans present in the CH2 domain of Fc play a key role in defining antibody effector functions. Fc sialylation has attracted the attention of scientists due to it’s attribution to the anti-inflammatory response to intravenous Ig (IVIG) to treat autoimmune and inflammatory diseases. Sialylated human IgG has higher anti-inflammatory activity than desialylated IgG. In summary, sialylated glycans generally have positive and negative effects on IgG effector functions. Therefore, it is important to quantify the sialylation of mAbs entering the clinic, especially for the treatment of autoimmune diseases. To date, the function of different Fc-sialylated glycans in approved antibody-based therapeutics has not been studied clinically.
Creative Biolabs has developed many cutting-edge technologies in the field of glycan Engineering Service based on our GlycoDegree™ platform. We can produce sialylation modified antibodies in a variety of bioengineered mammalian or non-mammalian cell line systems by using in vitro enzyme or chemical enzyme methods. Our antibody glycan analysis system will ensure the quality of our service.
Send us your project description and requirements, and you will get tailored all-in-one services to meet your R&D or cGMP production needs.
Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details.
All products and services are for Research Use Only. Do Not use in humans.
ADCC Assay WT vs AfucoTM Mabs Visit
Antibody Fc Engineering: Towards Better Therapeutics Visit
Creative Biolabs has established a team of customer support scientists ready to discuss ADCC/CDC optimization strategies, antibody production, bioinformatics analysis and other molecular biology/biotechnology issues.