Carbohydrate Based Conjugation

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.

• Approach #1: aldehydes on galactose residues can be generated by treating the carbohydrate moiety with neuraminidase that removes the terminal sialic acid residues followed by galactose oxidase treatment. Subsequently, hydrazide- or primary amine functionalized molecules react with the aldehyde groups to afford relatively homogenous ADCs. Comparing with the classical chemical oxidation approach, this type of glycan enzymatic modification has been proven to be more specific.
• Approach #2: galactosyltransferases are used to generate homogeneous antibody glycans with full galactosylation at the glycan G2 positions. The glycans are subsequently treated with sialyltransferase to incorporate sialic acid residues onto the galactose units. The sialic acid residues are easily oxidized under mild conditions to yield aldehyde-functionalized antibodies. This strategy combine the enzymatic method with chemical oxidation, and is thus referred to as glycan chemo-enzymatic modification.

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).

• Approach #3: Creative Biolabs has adopted another enzymatic strategy for antibody modification and conjugation via the insertion of “unnatural” monosaccharide moieties. The terminal galactose residues are selectively cleaved by a galactosidase and the resulted glycan is treated with a mutant galactosyltransferase to incorporate N-acetylgalactosamineazide (GalNAz) onto the exposed GlcNAc residues. Cyclooctyne-functionalized drugs or drug-linker complexes are conjugated at the GalNAz sites via Cu-free click chemistry to produce ADCs with controlled DAR.

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

• Efficient approaches for the development of site-specific ADCs
• Suitable for the conjugation to glycosylated full length IgG
• Does not require antibody protein sequence modification or engineering
• Versatile approaches, including chemical, enzymatic, chemo-enzymatic, and metabolic engineering methods
• Conjugation is achieved without reducing antigen binding affinity

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:

1. Agarwal, P.; et al. Site-specific antibody−drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 2015, 26: 176−192.
2. Dennler, P.; et al. Antibody conjugates: from heterogeneous populations to defined reagents. Antibodies. 2015, 4: 197-224.