Off-target testing assays

23rd January 2018

The label-free Attana Cell 200
Cell-based assays
Off-target interactions on cells

It is of high importance to understand and verify desired or unwanted off-target interactions of a drug molecule. Off-target interactions are all interactions not specific to the aimed binding site of the target molecule. The off-target interactions could be non-specific interactions to e.g. carbohydrates on a cell surface but also specific interactions to other molecules than the target itself.1 The drug distribution in the body could be highly affected in presence of off-target interactions, consequently affecting drug performance. In this paper, we discuss a proprietary advanced Quartz Crystal Microbalance (QCM) based biosensor offering applications to study the above mentioned off-target interactions, label free and in real time.

A biosensor solution designed to make a difference

The Attana Cell 200 biosensor (as shown in Fig.1.) is based on the piezoelectric effect of quartz. When applying an alternating electric potential over a quartz crystal, it will start to oscillate with a resonance frequency related to the mass of the crystal. Hence when any molecules are bound to the crystal, the frequency will change, and the interaction can be characterised, for example, as kinetic rate constants and affinity.

The Attana Cell 200 biosensor enables label-free characterisation of molecular interactions in biochemical-, sera- and cell-based assays.

Cell-based assays

To determine off-target interactions using the Attana Cell 200 biosensor, the cells will easily be captured to the sensor surface (blue and green spheres, Fig.2). Adherent cells are grown directly on the sensor surface, while suspension cells are captured onto the sensor. A common way is to use lectins specific for the cell of interest. To map the off-target properties of a drug molecule, a panel of cells is used for screening a variety of different cell membranes and biological relevant environments.

During the measurement, the drug molecule is flown over cells expressing the target (purple spheres, Fig. 2) and cells without the target, mimicking the fluidics in the human body, and off-target interactions can be studied.

Off-target interactions

The cell membrane with its variety of components, may lead to natural interactions between the protein product and other molecules than the target antigen. Measuring the protein binding in a cell-based assay provides an added value to the kinetic information and is more biologically relevant than to measure the protein-antigen binding in a purified biochemical assay having the target isolated from its natural environment.

Deglycosylation of the membrane

It has been reported by Peiris et al2, the glycosylation profile of the cells affects the interactions between a protein and its target in the cell membrane. They showed deglycosylation of the membrane proteins revealed an increase in the accessibility of the target. Deglycosylation of membrane proteins gives a tool for understanding the impact the membrane environment has on the aimed protein-target interaction.

Detecting off-target properties

The importance of detecting off-target properties has been of increasing interest and the Attana Cell 200 has been proven to be a useful tool. As an example, researchers screened different antibodies – some clearly displayed off-target interactions (bottom row, Fig.3), while other did not and had no interactions to the negative reference cells (top row, Fig.3). Off-target interactions have often fast association and dissociation, which results in a hill-shaped binding profile. Although the off-target interaction often is fast and weak, it clearly affects the binding profile of the drug. The off-target property could be identified to be specific parts of the antibody. The protein was refined and optimised for better drug distribution.

Longterm results

Discovering desired or unwanted off-target interaction properties of a drug can lead to refined, optimised drugs with enhanced distribution in the body and increase patient safety and lowering the side effects when decreasing the dose thanks to increased deliver efficiency.

Samuel Altun & Teodor Aastrup are with Attana.


1 Salanti et al., Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein, 2015, Cancer Cell 28, 500-514.

2 Peiris et al., Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors, 2017, Scientific reports, 7:43006.






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