ADC Development

An Elegant Platform for ADC Development

Antibody-Drug Conjugates (ADCs) comprise a new generation of antibody-based biologics that broaden the therapeutic window for better biopharmaceuticals. The antibody conjugation strategy is central for the development in order to preserve the immunoreactivity and stability of the final ADC, while a defined drug-antibody ratio (DAR) is key for an optimal dose-response cytotoxicity and in vivo efficacy. The drawbacks of conventional conjugation are rapidly being surpassed by site-specific methods, where labeling at the Fc-glycan sites using GlyCLICK® has proven to be an attractive option to label native antibodies without engineering.

 

GlyCLICK Workflow - ADC development

Homogenous and Site-specific ADCs

The heterogenous nature of randomly conjugated antibodies can restricts both the reproducibility and controlled cytotoxic dosage of the ADC, while increasing the hydrophobicity of the final product. Site-specific conjugation at the Fc glycan sites using the GlyCLICK technology circumvent these limitations and produce homogenous, more hydrophilic ADCs with minimal batch variations.

 


ADC development - GlyCLICK

 

Figure 1a. HIC separation of top) native trastuzumab (T), bottom) T-GlyCLICK-DM1 (DBCO-PEG4-Ahx-DM1). Separation on TSKgel@Butyl-NPR column (Tosoh Biosciences) in 25 nM NaP pH 7, 1.5 M ammonium sulfate. Salt gradient elution (20% isopropanol).

Here, trastuzumab was conjugated to DM1 using GlyCLICK and compared to approved ADCs by separation using HIC. The single elution peak observed demonstrates the homogenous and less hydrophobic material obtain with GlyCLICK, as compared to the unresolvable lysine conjugated ADC or the heterogenous material observed for cystine conjugation (Fig. 1).

 

 

 

Figure 1b. HIC separation of top) NHS-conjugated T-DM1 and bottom) brentuximab-vedotin. Separation on TSKgel@Butyl-NPR column (Tosoh Biosciences) in 25 nM NaP pH 7, 1.5 M ammonium sulfate. Salt gradient elution (20% isopropanol). Randomly conjugated trastuzumab was unresolved due to the sample heterogeneity.

A Defined Drug-Antibody Ratio (DAR)

The site of conjugation is crucial to control the DAR and ensure that the toxin-linker payload does not interfere with the antigen-binding region for negative impacts on the targeting properties. An increased DAR may enhance the overall toxicity when using less potent payloads, but it is also associated with off-target cytotoxicity and plasma clearance. The drug-load distribution (DLD) may in turn impact the structure, stability and performance of the final ADC. The site-specific material obtained using GlyCLICK was evaluated by trastuzumab conjugated to MMAE (Fig. 2), the spectra showing mass shifts corresponding to complete deglycosylation (top), azide-activation (middle) and incorporation of a toxic payload (bottom).

Site-specific ADCs using GlyCLICK


 
Figure 2. LC/MS analysis of trastuzumab conjugated with MMAE (monomethyl auristatin E) using GlyCLICK, showing a) native, b) deglycosylated, c) conjugated trastuzumab.  

Enhanced Efficacy and Serum Stability

The pharmacokinetic properties (PK) of ADC design include the aspects of dose-response cytotoxic effect against targeted cells and longer half-life for optimal efficacy in vivo. In the early ADC discovery process, such properties are vital for optimal combination of antibody, linker and toxic payload. Instability in serum and biotransformation events can further cause premature drug loss in circulation and affect the ADC distribution in tissue, potentially limiting dose-response toxicity.

 

 

Figure 3.  Qualitative HIC analysis after serum incubation. T-GlyCLICK-DM1 was affinity purified by CaptureSelectTM IgG CH1 (ThermoFisher) after rat serum incubation for 144 hours. Samples were analyzed as described in Fig. 2.

 

To demonstrate the stability and cytotoxicity of GlyCLICK generated ADCs, we labeled a monoclonal antibody site-specifically at the Fc glycan sites using the GlyCLICK Azide Activation kit. The resulting conjugate carrying DM1 (Mertansine) toxin payload (DAR 2.0) was used to evaluate both serum stability and cytotoxic effect on HER2 positive cells.  The results show robust conjugates with minimal drug loss in serum (Fig. 3) and expected dose-response cytotoxicity (Fig. 4).

 

 

Figure 4. Cytotoxicity analysis of HER2(+) SK-BR-3 by DBCO-PEG4-Ahx-DM1 GlyCLICK-conjugated trastuzumab. Viability measured with CellTiter-Glo® Luminescent assay, 5000 cell/well incubated with 0-10 ug/ml conjugate at 37°C, 5% CO2 for 72 h prior to addition of reagent.

 

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