Antibody Drug Conjugate Development

GlyCLICK is a site-specific conjugation technology for IgG using enzymatic remodeling of the Fc-glycan sites and click-chemistry. The key characteristics of GlyCLICK facilitate the generation of tailored and robust conjugates for reproducible and reliable results independent of the label or payload.

GlyCLICK workflow ADC

Reliable payload quantitation using GlyCLICK

Traditional conjugation strategies for cytotoxic drug development based on lysine or reduced cysteine chemistry result in heterogeneous conjugation and may impact the in vivo performance of the ADC. Using the GlyCLICK technology, site-specific conjugation preserves the affinity and increases tumor uptake in vivo, compared to randomly conjugated antibodies.

The quality and efficacy of an antibody conjugation method is a crucial aspect of ADC development. To evaluate the antibody-drug-ratio (DAR) of conjugates produced with GlyCLICK, trastuzumab was site-specifically conjugated with DM1, DBCO-PEG4-Ahx-DM1 (T-GlyCLICK-DM1), and compared to randomly conjugated trastuzumab-DM1 (T-DM1). Trastuzumab and the conjugates were analyzed by HIC separation and LC-MS using brentuximab vedotin as a control (Fig. 1). Homogenous conjugation with DAR=2.0 was observed for T-GlyCLICK-DM1 compared to the heterogenous T-DM1 and brentuximab-vedotin.

GlyCLICK Technology ADC fig 1

Figure 1. Characterization of T-GlyCLICK-DM1 and T-DM1. Samples were analyzed by HIC on a TSKgel®Butyl-NPR column (Tosoh Bioscinece) in 25 mM NaP, 1.5 M ammonium sulfate and eluted with a decreasing salt gradient with 20 % isopropanol. The DAR variants of T-DM1 were not resolved due to the heterogeneity in hydrophobicity of the material.

 

GlyCLICK conjugates show dose-response cytotoxicity

Cytotoxicity analysis by titration is a standard method for assessing the potency and safety of the ADC. The GlyCLICK conjugated antibody (T-GlyCLICK-DM1) was tested for cytotoxicity using SK-BR-3 cells which yielded a dose-response curve (Fig. 2). The curvature is similar to that of T-DM1, however, as 2.0 instead of 3.53 drugs are conjugated using the GlyCLICK kit, these conjugates were less cytotoxic.

GlyCLICK Technology ADC fig 2

Figure 2. Cytotoxicity analysis of T-GlyCLICK-DM1. Cell viability was measured using CellTiter-Glo® Luminescent assay (Promega). HER2(+) cells (SK-BR-3), 5000 cells/well were incubated with 0-10 ug/ ml T-GlyCLICK-DM1 in medium at 37°C, 5% CO2 for 72 hours prior to addition of reagent.

Minimal drug loss in serum using GlyCLICK

The loss of payload from the conjugated antibody resulting in changes in antibody-drug-ratio (DAR) during circulation in serum may compromise the efficacy and safety of the ADC. In addition, increased hydrophobicity due to the payload contributes to decreased stability and complex behavior of the ADC. To evaluate the in vivocharacteristics, serum stability was evaluated for affinity to HER2 using ELISA and loss of drug using HIC analysis of T- GlyCLICK-DM1 and T-DM1. The antigen binding assay showed similar relative binding after 168 hours of incubation (Fig. 3a). The HIC profile indicates that GlyCLICK material is less hydrophobic compared to T-DM1 and retains binding to its antigen in serum (Fig. 3b).

GlyCLICK Technology ADC fig 3

Figure 3. Serum stability analysis. a) Antigen binding after serum incubation. T-GlyCLICK-DM1 and T-DM1 (0.2 mg/ml) were incubated in rat serum, 85% in TBS for 168 hours at 37°C. Diluted samples (1:2000) were analyzed against HER2(+) using ELISA with binding visualized using a secondary antibody of goat anti-human IgG HRP using ABTS as substrate. b) Qualitative HIC analysis after serum incubation. T-GlyCLICK-DM1 was affinity purified by CaptureSelectTMIgG CH1 (ThermoFisher) after rat serum incubation for 144 hours. Samples were analyzed as described in Fig. 1.

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GlyCLICK Poster

GlyCLICK® generates homogenous antibody conjugates with preserved affinity. Genovis Poster, World ADC 2018.
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