Introduction to Antibody Conjugation

A Platform Technology for Site-specific Conjugation

Antibody conjugation is a powerful tool with applications ranging from biomedical research to the development of diagnostic methods and antibody-drug conjugates (ADCs). A reliable conjugation technology is crucial to obtain a homogenous and reproducible conjugate while preserving the stability and immunoreactivity of the final product. The GlyCLICK technology is a platform for glycan-specific labeling of native IgG when the primary sequence is unknown or engineering is not feasible.

 
 

Antibody conjugation - Glyclick Workflow

A New Generation of Antibody Conjugates

Conjugation is conventionally performed using random strategies by labeling at the ~20 readily available amines on lysine residues or at the 4-8 solvent accessible inter-chain cysteines. Although versatile, such methods often require suboptimal pH conditions and offer limited control of the labeling excess and site-occupancy, thus they are prone to influence stability, immunoreactivity and reproducibility of the final product. Site-specific strategies have become an attractive option to overcome these limitations, but often require engineering to control and stabilize the process. Conjugation at the Fc glycan sites using GlyCLICK is not only site-specific (Fig. 1) but also decreases off-target effects by disrupting the binding to Fc receptors (FcYRs) on immune cells due to the Fc glycan modification.

Antibody Conjugation fig1

 

Figure 1. Schematic representation of IgG with different site occupancies and degree of labeling according to the conjugation technology, a) lysine based conjugation (DOL = varied), b) cysteine based conjugation (DOL=0-8) and c) GlyCLICK conjugation (DOL=2.0).

Preserved Immunoreactivity After Conjugation

 

Antibodies used for applications in imaging, bioassays or the development of antibody-drug conjugates (ADCs) can suffer from impaired immunoreactivity due to poor conjugation quality, resulting in unreliable and heterogenous antibody constructs. To evaluate the antigen-binding capacity of GlyCLICK conjugated antibodies, trastuzumab was conjugated with the AlexaFluor®488 label and compared to its native counterpart or a randomly labeled product. The analysis of the binding capacity after labeling was carried out using SPR analysis, the results showing that association and dissociation to the HER2 antigen was unaffected by the GlyCLICK conjugation, indicating a fully preserved immunoreactivity (Fig. 2).

Preserved Immunoreactivity After Antibody Conjugation

Figure 2. SPR analysis. Anti-human IgG (Fc) captured trastuzumab: native (control), DyLight®488 (random) conjugated to AlexaFluor®488 or GlyCLICK conjugated to different alkyne-carrying labels (DOL=2.0). HER2 was injected in a range to ensure sufficient curvature. All data were fitted against a 1:1 mathematical model.

Customized Labels for Versatile Applications

The combination of enzymatic remodeling and click chemistry (SPAAC) enables GlyCLICK to site-specifically conjugate antibodies using any alkyne-carrying label of choice. To demonstrate the homogenous material obtained using GlyCLICK, trastuzumab was conjugated with different alkyne-carrying labels and analyzed at the F(ab´)2 and Fc/2 subunit level by RP-HPLC. The resulting separation shows shifts in retention detected only for the labeled Fc/2 subunits when compared to the native counterpart, showing that GlyCLICK specifically conjugates at the Fc-glycan sites independently of the label type being used (Fig. 3).

GlyCLICK World ADC 2019 Poster

Figure 3. RP-HPLC analysis of panitumumab unmodified and conjugated with Desferroxamine (DFO), AlexaFlour®488, Biotin or Monomethyl auristatin E (MMAE) digested with FabRICATOR. RP-HPLC was performed on an Agilent 1290 LC using Water Acquity UPLC BEH C4, 1.7 um, 2.1x100 mm column.

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