ADC Development
Site-specific Generation of Custom ADCs
Antibody-drug conjugates (ADCs) comprise a new generation of antibody-based biologics that carry drug payloads directly into target cells, allowing for a broadened therapeutic window. The drawbacks of conventional antibody conjugation strategies are rapidly being surpassed by site-specific methods, where conjugation at the Fc-glycan sites using GlyCLICK® has proven to be an attractive option for labeling of native antibodies without genetic engineering.
GlyCLICK ADC kits enable the generation of site-specific ADCs using native IgG and unique linker-payloads.
Two-step Cleavable Linkers
The combination of antibody and payload consisting of both the linker and toxin is a fundamental part of ADC development to obtain optimal performance in vivo. By combining potent toxins with linkers susceptible to enzymatic digestion, release of the payload is achieved once the antibody has entered the target cell, resulting in controlled drug release and reduced off target toxicity. The cleavable glycopeptide linker provided with GlyCLICK ADC is lysosomally activated in a two-step reaction for controlled drug release in targeted cells (Fig. 1). Once the antibody conjugate enters the cell by endocytosis, the linker-glycoside is hydrolyzed by the lysosomal β-glucuronidase. Removal of the glycoside exposes the peptide linker to proteolytic cleavage by the lysosomal protease cathepsin B, releasing the toxin within the cell.
Figure 1. Schematic representation of the cleavable linkers carrying toxic payloads and the cleavable sites subjected to digestion by lysosomal enzymes. Left) DBCO-Val-Ser(GlcA)-PAB-MMAE and Right) DBCO-Val-Ser(GlcA)-EDA-PNU.
Payloads PNU and MMAE
The GlyCLICK conjugation technology results in site-specific incorporation of 2.0 drugs per antibody, for this reason the GlyCLICK ADC kits offer conjugation with highly potent payloads functionalized with DBCO to enable click-chemistry to azide activated antibodies. GlyCLICK ADC kits can be used to combine the native IgG of choice with a two-step cleavable linker carrying either MMAE or PNU for the desired cytotoxic effect on targeted cells (Fig 2).
- MMAE is a potent cytotoxin that inhibits microtubule assembly and tubulin dependent GTP hydrolysis, causing cell cycle arrest and apoptosis.
- PNU is an anthracycline based DNA-alkylating toxin causing apoptosis with significantly higher potency that tubulin-targeting payloads.
Figure 2. Schematic representation of the lysosomal uptake, linker-payload digestion and cytotoxic mechanisms of GlyCLICK ADC.
Site-specific ADC Generation
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. The conjugation performance of GlyCLICK ADC kits was evaluated on two different monoclonal antibodies and analyzed using LC-MS (Fig. 3,4), the results showing the mass shifts from native antibody (top) to complete conjugation with two linker-payloads per antibody (bottom).
Figure 3. LC/MS analysis of trastuzumab conjugated with the MMAE (monomethyl auristatin E) 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 MMAE (bottom).
Figure 4. 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).
Potent and Functional ADCs
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 ADCs carrying either MMAE or PNU. The killing curves show the in vitro cytotoxic effect on cells with IC50-values in the pM range, indicating functional and potent ADC (Fig 4, 5).
Figure 4. In vitro cytotoxicity analysis of trastuzumab conjugated with the MMAE linker-payload using GlyCLICK ADC kit MMAE. Her2+ SK-BR-3 cells were incubated with the ADC and cell viability measured with PrestoBlue after 5-6 days. Curves represent the average result from three replicates.
Figure 5. In vitro cytotoxicity analysis of trastuzumab conjugated with the PNU linker-payload using GlyCLICK ADC kit PNU. Her2+ SK-BR-3 cells were incubated with the ADC and cell viability measured with PrestoBlue after 5-6 days. Curves represent the average result from three replicates.
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Linker-payloads included in GlyCLICK®
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SiteClick™ included in GlyCLICK®
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