Antibody binding assay to screen mouse and human antibodies:
As shown in Figure 2, the antibody screening procedure was successfully applied to 12 different RTK, using either red-labeled antispecies conjugates or red-labeled antitag antibodies. The assay window ranges from 3 to 34 depending on the concentration and antibody affinity.
The same assay format was used to determine the affinity constant (KD) of the previously screened antibodies by increasing the concentration of primary antibody in the presence of a saturating concentration of Red-labeled secondary antibody. The KD values obtained were in the nanomolar range and in good agreement with the antibody specifications.
Moreover, taking advantage of the nondestructive HTRF measurement, we were able to record the fluorescence signal evolution at different time points during one hour and therefore calculate the Kon constant. In the same way, we also measured the Koff rate by adding a large excess of unlabeled antibody at the equilibrium.
Functional characterization of Cetuximab measured by ERK phosphorylation:
The mechanism of action underlying a therapeutic effect was then investigated. We addressed MAPKinase activation induced after ligand stimulation through the measurement of the ERK phosphorylation level. The results presented in Figure 3 demonstrate how Cetuximab and erlotinib can efficiently inhibit ERK phosphorylation. They confirm that Cetuximab antibody targets the receptor binding site of EGF and therefore prevents the activation of the ERK signalling pathway.
This could contribute, along with some other cellular events, to the positive therapeutic effect of Cetuximab.
The same kind of experiment has been performed on endogenous EGF receptor expressing cells with the same type of inhibition curves.
Tag-lite technology is both powerful and efficient in the screening and functional characterization of biotherapeutics on RTKs. As this technology is based on fluorescent probes, no radioisotope- labeled components are required.
Tag-lite is a flexible method: a number of assay formats are possible (direct antibody screening with labelled probe or indirect using secondary antibodies). Labeled frozen or fresh cells can be employed. As a truly homogeneous method, Tag-lite requires no washing steps, making it user-friendly. Tag-lite also generates high signal to noise windows and is amenable to miniaturizable from 96- to 1,536-well plates.
Perfectly adaptable from the screening stage to maturation studies, through production and quality control phases, this new technological platform helps in the understanding of biotherapeutic mechanisms of action, and opens new opportunities to develop biologic drugs with improved patient clinical benefits.