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Assay Tutorials : Oct 15, 2011 ( )
Identifying Promising Drug Candidates
Screening and Characterizing Therapeutic Antibodies Targeting Receptor Tyrosine Kinase!--h2>
The development of potent and selective monoclonal antibodies (mAbs) is a significant challenge. More than 25 antibodies have been approved for human therapy and over 240 antibodies are being clinically developed worldwide for a wide range of diseases.
The quest for better therapeutic antibodies has gained great momentum in recent years, driven by the development of new technologies to better understand the mechanisms of action of antibody-based drugs.
Cisbio Bioassays has developed new tools for the screening and characterization of biotherapeutics using Tag-lite®, its recently launched cell-based technology based on HTRF® (Cisbio’s homogeneous fluorescence technology).
Due to its critical involvement in tumor progression, the receptor tyrosine kinase (RTK) family represents a validated target class for anticancer therapy.
How Tag-lite enables the screening of therapeutic antibodies for their binding to receptor tyrosine kinase and determination of their affinities is explained in this article. In addition, how the functional activity of the antibodies can be assessed through the measurement of ERK phosphorylation is shown.
Tag-lite starts with the cloning of the RTK sequence in frame with the SNAP-Tag® fragment in a mammalian expression vector. HEK293 cells are transfected with the SNAP tagged-RTK vector following the lipofectamine 2000 procedure, either in microplates or in T175 flasks. These cells are specifically labeled with the SNAP-Lumi4®-Terbium substrate and used for the antibody binding study in the presence of a d2-labeled antispecies antibody (mouse or human) or a d2-labeled anti-Tag antibody to capture the primary antibody (Figure1A).
In parallel, wild-type RTK or SNAP-RTK expressing cells enabled the antibody functional test to be performed by measuring the ERK phosphorylation level using a cell-based sandwich immunoassay (Figure1B).
Materials & Methods
Reagents: SNAP-Lumi4-Tb, anti-mouse Fc-d2, anti-human Fc-d2, anti-cMyc-d2, anti-His-d2, SNAP-RTK plasmids, and the labeling medium were from Cisbio Bioassays. Lipofectamin 2000 was purchased from Invitrogen, and the cell-dissociation buffer was from Sigma-Aldrich. Anti-RTK antibodies were obtained from different sources.
Covalent labeling of SNAP-RTK cells: A solution of Tag-lite SNAP-Lumi4-Tb substrate at 100 nM was prepared in Tag-lite labeling medium. After aspiration of the cell culture medium, the SNAP-Lumi4-Tb solution was added to the cells expressing the SNAP-RTK in the T175 flask, followed by one hour incubation at 37ºC. The cells were then washed four times to remove the excess of SNAP-Lumi4-Tb and detached using the cell-dissociation buffer. The cells were frozen under 1 million cells/vial in culture medium and 10% DMSO.
Antibody binding assay: The SNAP-Tb labeled cells were thawed and washed with PBS, then re-suspended in labeling buffer. Binding assays were performed in a total volume of 20 μL in 384 small volume white plates. 10,000 cells/well were incubated for two hours at room temperature with two different concentrations of primary antibodies (10 and 40 nM). Red labeled antispecies antibodies or Red-labeled antitag antibodies were added at a fixed and saturating concentration (100 nM) for the detection.
Erk phosphorylation assay: SNAP-EGFR1 expressing cells were dispensed in 384 sv plates and pretreated or not with Cetuximab or erlotinib (tyrosine kinase inhibitor) for two hours, at 37°C, then stimulated with EGF for 10 minutes. After cell lysis, phosphorylated ERK was quantified by HTRF using the Cellul’erk kit.
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.
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