Intana Bioscience

Intana’s innovative FCCS technology has been used extensively for characterising drug-target interaction in physiological environments. Intana has broadened the scope of FCCS from affinity measurements to comprehensive interaction studies, including kinetics, multi component complex formation, stoichiometry assessment, aggregation quantification, PK (pharmacokinetic) studies, and target engagement.

Intana Bioscience has successfully applied FCCS to advance the pipelines of pharmaceutical organisations, ranging from preclinical studies and early stages of drug discovery to supporting clinical trials by measuring drug target occupancies in patient samples.

FCCS-Based Assays

Intana Bioscience has achieved this by developing and optimising various FCCS-based assays, such as:

Target Occupancy assay: This assay allows the measurement of the percentage of targets that are bound by a drug molecule, which is an important parameter to evaluate the efficacy and safety of a drug. The assay uses a fluorescently labeled tracer that competes with the drug for the same target. FCCS can also measure the total target concentration by using two fluorescently labeled antibodies against different epitopes of the target protein. This allows the absolute quantification of the fraction of occupied targets within a sample.

The assay can measure the target occupancy in various biological samples, such as cell lysates, tumour tissues, blood cells, and liquid biopsies. The assay can also measure the total target concentration by using two fluorescently labeled antibodies against different epitopes of the target protein. The approach facilitates dose finding, monitors drug response, identifies species barriers, helps to stratify patients, and to correlate the target occupancy with the therapeutic success.

PROTAC degradation assay: This assay measures the degradation of target proteins by proteolysis-targeting chimeras (PROTACs), a new drug class that recruits the ubiquitin-proteasome system. The assay uses a fluorescent tracer that binds to the target protein. It detects the degradation by the decrease of the tracer fluorescence. It also measures the target occupancy by PROTACs by using a tracer that competes with the PROTAC. The assay can help to quantify the activity of PROTACs, correlate the target occupancy with the protein degradation, support dosing, and select suitable cell models.

GPCR-ligand interaction assay: GPCRs are integral membrane proteins that are difficult to assay, due to their physico-chemical properties. The FCCS approach allows solubilization of GPCR containing membrane preparation and setting up biophysical affinity measurements by direct interaction measurements or competition assays for binding strength and kinetics.

Measuring Drug Target Occupancies in Cells, Animals and Patients

Target occupancy is a key parameter to evaluate the efficacy and safety of a drug. FCCS can be applied to quantify the target occupancy by detecting the fluorescence signal of a labeled tracer that competes with the drug for the same target. FCCS can also measure the total target concentration by using two labeled antibodies against different epitopes of the target protein. FCCS can be applied to various biological samples, such as cell lysates, tumour tissues, blood cells, and liquid biopsies.

Identifying the Species Barriers

Identifying species barriers is crucial for drug discovery as it aids in selecting suitable animal models for preclinical testing, predicting human pharmacokinetics and pharmacodynamics, and preventing potential adverse effects or toxicity. FCCS can be utilised to monitor the drug response in different species, and identify the species barriers, such as the difference in the drug absorption, distribution, metabolism, or target expression.

A unique advantage of using FCCS to identify the species barriers is that it can provide a direct and quantitative measurement of the drug/target interaction in vivo, without the need for invasive procedures or radioisotopes.

Optimising Drug Dosage and Predicting Clinical Outcomes

Target occupancy is an important parameter to evaluate the efficacy and safety of a drug. A drug that has a high target occupancy is expected to have a high pharmacological effect, while a drug that has a low target occupancy may have a low or no effect, or even adverse effects. Therefore, measuring the target occupancy is essential to optimise the drug dosage, monitor the drug response, and predict the clinical outcomes.

However, measuring the target occupancy is not easy, especially in living cells and organisms. The conventional methods such as radioligand binding assays and Surface Plasmon Resonance (SPR) have limitations which may lead to inaccurate or incomplete measurements of the target occupancy. This could result in failures in the later stages of drug development, such as Phase II clinical trials, where the drug efficacy and safety are tested in human subjects.

Conventional methods are often performed in vitro, which may not reflect the physiological environment of the cells or the organisms, such as the presence of off-targets, aggregates, or complex formation. But FCCS enables label free assay development with the accuracy of in vitro assays under in vivo conditions.

While SPR requires the immobilisation of one of the molecules on a solid surface, FCCS can measure the interaction of molecules in solution or in living cells. Thereby the physiological environment of the molecules is preserved, and the potential effects of immobilisation, such as conformational changes, steric hindrance, or denaturation are avoided.

Monitoring of Drug-target Interactions

FCCS can also measure the target occupancy in a time-resolved manner, by performing repeated measurements at different time points after the drug administration. FCCS captures the dynamics of the drug-target interaction, such as the association and dissociation rates, the residence time, and the half-life. FCCS can also measure the concentration of the drug in the sample, by using a fluorescently labeled drug or a tracer that binds to the drug. Thereby FCCS can correlate the target occupancy with the drug concentration, and determine the dose-response relationship. FCCS can help to optimise the drug dosage, and guide animal and human dosing.

Measuring Target Occupancy in Liquid Biopsies of Patients

Target occupancy can be measured in liquid biopsies of patients using FCCS, which can detect the fluorescence signals from a tracer and an antibody that compete for binding to the target protein in the lysate of PBMCs. FCCS can provide information on the target occupancy in patients, which can be used to personalise the drug therapy, and to predict the clinical outcomes.