Generation of Highly Potent ADCs using GlyCLICK®

Application

Site-specific ADC generation with defined DAR, preserving antigen binding and delivering potent, controlled cytotoxicity in target cells.

Site-specific conjugation at the Fc glycan sites using GlyCLICK preserves the antigen-binding capacity and stability after conjugation. The defined DAR that is obtained enhances controlled cytotoxicity while minimizing instability and off-target effects, otherwise often observed for randomly conjugated ADCs.

In collaboration with Glykos Finland, we have conjugated the toxin PNU to panitumumab using the GlyCLICK ADC Kit. The PNU-conjugated ADC molecules were digested using FabRICATOR before LC-MS analysis to characterize the scFc domain. The deconvoluted mass spectra confirm complete and efficient deglycosylation with subsequent PNU conjugation using GlyCLICK demonstrating reliable generation of a panitumumab-PNU ADC molecules with a DAR of 2 (Fig.1).

 Reliable ADC generation with DAR of 2 using GlyCLICK

Instability in serum and biotransformation events due to poor ADC quality can cause premature drug loss in circulation and affect the conjugates distribution in tissue, potentially limiting dose-response toxicity. To demonstrate the functionality and cytotoxic effect of GlyCLICK conjugated ADCs, SK-BR-3 HER2+ cells were incubated with trastuzumab-PNU ADC molecule. The killing curve shows the in vitro cytotoxic effect on cells with an IC50-value in the pM range, indicating the generation of a functional and potent ADC (Fig 2).

Generation of functional and highly potent ADC molecule

Figure 1. LC/MS analysis of panitumumab conjugated with the PNU linker-payload using  GlyCLICK. The antibody was analyzed after FabRICATOR digestion determine the native Fc/2 glycan profile (top) and site-specific labeling after conjugation using GlyCLICK ADC kit PNU (bottom).

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