Application Notes

Studying the interaction of the antibody-drug conjugate SYD985 with an anti-toxin antibody

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2 Results We first analyzed the binding of seco-DUBA, conjugated to the antibody (SYD985), to the anti- toxin monoclonal antibody. The murine anti-toxin monoclonal antibody was labeled using NanoTemper Technologies Protein Labeling Kit RED-NHS. The concentrations of labeled anti- toxin antibody were kept constant in all samples at 1.5 nM, whilst the concentration of ADC (SYD985) or of the non-conjugated antibody (SYD977) was varied. The samples were then loaded into MST Premium Coated capillaries. The MST measurements were performed at room temperature. For the interaction of the ADC (SYD985) with the NT647-labeled anti-toxin monoclonal antibody, an (observed) K d of 1.12 +/- 0.2 nM was found (Figure 2). As expected, no binding was observed between NT647-labeled anti-toxin monoclonal antibody and the non-conjugated antibody (SYD977), which does not contain the cytotoxic drug (Figure 2). Figure 2: Binding of the NT-647 labeled anti-toxin monoclonal antibody to the ADC and the native antibody. The fluorescently labeled anti-toxin monoclonal antibody was used at a constant concentration of 1.5 nM with varying concentrations of the ADC (SYD985) or the non-conjugated antibody (SYD977). The fluorescently labeled anti-toxin antibody binds with a K d of 1.12 +/- 0.2 nM to the ADC (SYD985) (squares), while no binding of the fluorescently labeled anti- toxin antibody (triangles) to the non conjugated antibody was observed. The error bars reflect standard deviation (SD) from 3 measurements. In a second experiment, we analyzed the binding of the ADC (SYD985) to NT647-labeled anti-toxin monoclonal antibody in 50 % human plasma. As expected, SYD985 binds with a high affinity to the NT647-labeled anti-toxin monoclonal antibody (observed K d = 1.5 +/- 0.23 nM). Figure 3: Binding of the NT-647 labeled anti-toxin monoclonal antibody to the ADC in human plasma. The fluorescently labeled anti-toxin monoclonal antibody was used at a constant concentration of 1.5 nM with varying concentrations of the ADC (SYD985). This experiment was performed at 50 % final concentration of human plasma. The fluorescently labeled anti-toxin antibody binds with a K d of 1.5 +/- 0.2 nM to the ADC (SYD985). Conclusions Our experiments show that MicroScale Thermophoresis (MST) is a suitable and powerful tool for the analysis of interactions between antibodies and their antigens. Moreover, MST is a suitable tool to investigate specificity of the anti-toxin antibody, i.e. it recognizes the drug conjugate whereas the non- conjugated antibody is not interacting at all. Performing binding experiments in bioliquids is very challenging using orthogonal biophysical methods. Therefore, this application note underlines the advantages in applying MicroScale Thermophoresis for analyzing antibody-antigen interactions even in complex biological liquids such as human plasma.

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