285 (PB065) - Using Fluorescence Cross Correlation Spectroscopy to Monitor and Quantify Drug Target Occupancy in Cells, Tissues and Patient Biopsies

Abstract

Fluorescence Cross Correlation Spectroscopy (FCCS) is a singly molecule sensitive method to analyze molecular interactions. The approach has been employed in the past by Intana to set up high throughput screens, based on affinity or enzymatic activities. Similarly, we have investigated the formation and assembly of large protein complexes and studied complex targets as channels, transporters and even GPCRs. Among many other advantages, FCCS allows measurements in cellular lysates, bypassing protein purification and retaining the physiological environment. Consequently, assay development is straight forward and allows tackling also demanding targets. With this study we aimed to quantify the absolut drug target occupancy in cell in order to correlate the ratio of inhibited target molecules to the physiological, i.e. therapeutic effect. In order to measure unlabeled drugs and targets without purification of either binding partner for subsequent analysis Intana has set up an FCCS based assay format making use of fluorescent antibodies to label targets without interfering with drug target interactions and fluorescent tracer to quantify the unoccupied fraction of targets. For this samples are processed to cellular lysates using proprietary protocols and co-incubated with 2 different antibodies, carrying 2 different fluorescent labels and binding independently on 2 epitopes on the target. By this the total concentration of targets in the lysate can be precisely quantified. In a second step a fluorescent tracer molecule, which binds into the same pocket as the drug of interest is added to the lysate to quantify all unoccupied targets. In addition, a fluorescent antibody directed against the target is added for increase sensitivity. Knowing the exact concentration of targets in the sample and the concentration of targets that is bound to the tracer yields a precise quantification of target occupancy in the sample. Our approach was tested in cells, tissues, xenograft tumors and clinical patient samples. Data can help to guide cellular and animal experiments and to correlate or stratify patient responses to drug treatments. Conflict of interest: Ownership: Intana Bioscince GmbH, siTOOLs Biotech GmbH, Alpine Antiviral GmbH.