The carbohydrate moiety on an antibody is located at the hinge region of the Fc domain, with a sufficient distant from the variable region that regulates antigen interaction. Therefore, conjugation involving carbohydrate moiety is unlikely to impair the antigen binding affinity and could potentially serve as an excellent alternative for site-specific antibody drug conjugate (ADC) generation. With years of experience in antibody engineering and ADC development, Creative Biolabs now offers clients three elaborate strategies to target carbohydrate moiety for the conjugation of payload drugs: (1) glycan chemical oxidation, (2) glycan enzymatic and chemo-enzymatic modification, and (3) carbohydrate moiety metabolic engineering. Using our well-established carbohydrate-based conjugation methods, Creative Biolabs is dedicated to develop customized ADCs to fit your requirements, timeline, and R&D budget.
Glycan Chemical Oxidation
Human IgG molecules are glycosylated at the conserved N297 residue in the heavy chain CH2 domain. Antibody glycans usually contain vicinal diol moieties that can be oxidatively cleaved to yield aldehydes. Therefore, the conserved glycans can serve as valuable sites for payload conjugation. In order to introduce bio-orthogonal functionalities onto the antibody carbohydrate moiety, classic chemical methods utilizing sodium periodate (NaIO4) as an oxidation reagent is commonly used to oxidize certain glycosyl residues in the native glycans to aldehydes. The generated aldehydes are subsequently conjugated with hydrazide- or primary amine functionalized molecules. Scientists at Creative Biolabs can control the rate of oxidation by adjusting the concentration of the oxidation reagent, reaction temperature, pH… to achieve semi-selective oxidation.
Sodium periodate mediated oxidation of fucose followed by hydrazone condensation, conjugating a drug-linker molecule onto the N-glycans of an IgG (Bioconjugate Chem, 2015).
Glycan Enzymatic and Chemo-enzymatic Modifications
This set of approaches are mediated by different carbohydrate modifying enzymes.
Conjugation based on chemo-enzymatic modification of glycans: enzymatic transfer of galactose and sialic acid followed by periodate oxidation and oxime condensation to yield antibody-drug conjugates (Bioconjugate Chem, 2015).
Enzymatic removal of terminal galactose followed by the transfer of GalNAz via mutant GalT and drug conjugation using Cu-free click reactions (Bioconjugate Chem, 2015).
Metabolic Engineering of the Carbohydrate Moiety
This strategy provides an alternative for carbohydrate based conjugation by glycol-engineering of the antibody during its expression. By introducing thiofucose into the expression media, the host cells will metabolically incorporate the unnatural saccharide moieties into the antibody glycan during post-translational modification. This process generates antibodies with thiol-functionalized glycans that can serve as “orthogonal handles” for the attachment of drugs or drug-linker sets containing for instance, maleimide functional groups.
Metabolic incorporation of 6-thiofucose followed by maleimide conjugation (Bioconjugate Chem, 2015).
Advantages of carbohydrate based conjugation
Creative Biolabs is committed in utilizing various new technologies to provide comprehensive antibody modification and conjugation services to help our clients with various ADC development projects, please contact us for more information and a detailed quote.
References:
For Research Use Only. NOT FOR CLINICAL USE.
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