ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues by drug linker reagents, which results in a highly heterogeneous mixture. With the development of the fields of bioorthogonal chemistry and protein engineering, there is a growing interest in site-specific conjugation. CD BioGlyco provides ADC development services through glycosylation site-specific conjugation strategy for our clients, such as glycoengineering-based ADC development.
ADCs have both the targeting ability of antibody macromolecules and the anti-tumor activity of small molecule toxins and use the characteristics of antibody binding to target antigens to carry small molecule toxins into tumor cells to play an anti-tumor effect. The structure of ADCs is generally composed of three parts: 1) Antibodies, which are generally specific monoclonal antibodies designed for target antigens on the surface of tumor cells; 2) Highly active cytotoxins; 3) Linkers, which make toxins and antibodies stable connection by covalent bond. Due to the existence of many functional groups on the antibody molecule, the coupling reaction may be carried out at multiple coupling sites. This leads to the fact that ADCs are highly heterogeneous mixtures of conjugates with different drug-antibody ratios (DAR) and random conjugation sites, which affect the safety and efficacy of the drugs. In recent years, researchers have used unnatural amino acid mutation or used steric hindrance to block other conjugation sites, so that the linker-toxin is site-specific conjugated with specific amino acids of antibodies, which greatly reduces the heterogeneity of ADCs products.
Fig.1 The general structure of an ADC and the role of each component. (Bargh, et al., 2019)
There is a conserved glycosylation site N297 in IgG-type antibodies, which is located in the CH2 domain and is far from the functional domain of the antibody that binds the antigen. Therefore, the use of this site for coupling will not affect the binding function of the antibody. The glycans of the side chains are targeted for site-specific conjugation. This coupling is mainly by introducing a new reactive group on the sugar group and then using related reagents for site-specific coupling.
In addition to the engineering of antibody glycans using in vitro chemical and enzymatic modification strategies, ADCs are also developed by introducing saccharide analogs containing bioorthogonal reactive groups through metabolic engineering. Introduction of 6-thiofucose into the expression medium, which replaces fucose in antibody glycans with 60-70% efficiency, yields antibodies with thiol-functionalized glycans. Antibodies to the thiol-functionalized glycans are conjugated to maleimide-linked drugs to produce ADCs. CD BioGlyco provides ADC development services using this strategy for our clients worldwide.
Case: Metabolic incorporation of 6-thiofucose followed by conjugation with maleimide to produce ADC.
Fig.2 Metabolic incorporation of 6-thiofucose-based site-specific conjugation. (Agarwal & Bertozzi, 2015)
CD BioGlyco is committed to providing ADC development services utilizing glycosylation site-specific conjugation technology to meet the research needs of our clients. If you are interested in our glycoengineering-based ADC development services, please feel free to contact us for more information.