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Feb 15, 2012 (Vol. 32, No. 4)

Membrane-Binding Studies with Living Cells

Investigating the Real-Time Binding Kinetics of Therapeutic Antibodies to Cell Surface Antigens

  • Click Image To Enlarge +
    Figure 1. The samX biosensor for performing kinetic binding analysis (left). TandAb (bispecific) antibodies bind to tumor cell antigens, mediating effector and target cell interaction (right).

    A large proportion of pharmacologically relevant targets are located on the surface of cell membranes. In addition, many compounds capable of modulating cellular responses do so by directly binding to native cellular proteins on the surface of the cell. The higher the affinity that a compound or antibody has for its target, the more effective it will be at triggering the desired response at a physiologically relevant concentration.

    It is, therefore, not surprising that assays investigating the interaction of potential drugs with their targets are of particular interest to the pharmaceutical industry.

    Several approaches exist to measure binding between a drug compound and its target. These include ELISA, Western blot, isothermal titration calorimetry, KinExa, and flow cytometry. However, these techniques often involve radioisotope or fluorescent labeling. Such labels may inhibit the antibody-target interaction in vitro and would not be used in vivo, limiting the biological relevance of results produced using this sort of approach.

    In addition, many of these equilibrium techniques lack the temporal resolution required to facilitate accurate kinetic binding analysis, while another option, surface plasmon resonance (SPR), has only been able to work with purified receptors and membrane fractions.

    To address these limitations, SAW Instruments has developed the sam family of biosensor solutions for detecting and displaying biomolecular interactions in real-time. The systems use an innovative approach based on surface acoustic wave (SAW) technology, whereby an aqueous sample is guided over a proprietary sensor chip that converts a high-frequency signal into a SAW via the inverse piezoelectric effect.

    The phase and amplitude of the resulting SAWs are sensitive to changes in mass loading and liquid viscosity respectively, allowing biomolecular interactions at the chip’s surface to be detected with a high level of sensitivity.

    The great advantage of the SAW method is that it provides real-time data rather than relying on equilibrium-based models. This allows rapid analysis and accurate kinetic information to be captured. The system can also be used to study living cells, vesicles, antibodies, enzymes, and even LMW compounds (< 200Da in size) without the need for labeling. Molecular conformational changes, such as those that occur when a ligand binds to its target active site, can also be detected using SAW technology.

    Recently, researchers at SAW Instruments and Affimed Therapeutics collaborated to investigate the interaction between a therapeutic (bispecific) antibody fragment and its specific tumor cell surface target in a live cell-based assay (Figure 1). This is an early example where real-time kinetics data has been generated to describe the binding of a label-free therapeutic antibody to cancer cells.

  • Methods and Results

    Click Image To Enlarge +
    Figure 2. Binding curve of 15 nM TandAb to antigen-positive tumor cells (10, 000 cells/mL). (A) Overlay plot illustrating TandAb binding data at different ligand concentrations, with fits based on a 1:1 binding model (B).

    In order to study the binding of the Affimed TandAb (bispecific antibody) ligand to its target antigen on tumor cells, the ligand was first incubated in 10 mM acetate buffer (pH 5) and immobilized on the surface of a sam short-chain CMDextran (2-D surface) sensor chip to form the cell capture surface. Human IgG mix, at a concentration of 150 nM, was also immobilized on the same chip and used as a reference position. The sensor chips and fluidic cells use state-of-the-art microfluidics, allowing one chip to house multiple sensor elements. Using this approach, a concentration analysis can be performed and an accurate affinity constant (KD) calculated.

    Freshly cultured tumor cells in suspension, positive for the antigen of interest, were injected directly from the autosampler at a concentration of 10,000 cells per mL in phosphate buffered saline solution (PBS, pH 7.4) and immobilized on the capture surfaces. The interactions between the TandAb ligand (at different concentrations between 5–40 nM) and the antigens on the surface of the immobilized cells were measured via repeated cycles of injections. SAW phase changes were recorded and used to generate binding data based on a 1:1 binding model (Figure 2).

    As the data shows, increases in binding were observed as the concentration of the ligand was increased, as would be expected. Figure 2 also includes the raw association and dissociation kinetics of ligand binding, as well as the binding curve observed when using the ligand at the median concentration of 15 nM.

    Using the data generated by the concentration analysis, a plot of observed rate constant (kobs) versus concentration (nM) was constructed (Figure 3). The data exhibited a linear trend, allowing the researchers to determine the association rate constant (kon) and dissociation rate constant (koff) for the ligand/target interaction.

    These figures were then used to calculate the KD value for the on-cell interaction, which in this case was KD = 2 nM. This result was independently confirmed using laborious flow cytometric analysis that can only resolve steady-state KD. This work, along with other customer projects conducted by SAW Instruments, shows that the sam biosensor can accurately measure on-cell affinity constants over a large spread of concentrations, including those in the low nM to high pM ranges.

    After each concentration measurement experiment, the surface of the chip was regenerated using a 10 mM glycine regeneration buffer (pH 2.0). This strips the bound cells from the immobilized ligands on the chip’s surface without affecting the capture ligands themselves, thereby allowing the chip to be reused for another round of analysis using a fresh cell suspension sample.

  • Summary and Outlook

    Click Image To Enlarge +
    Figure 3. Evaluation of TandAb antibody binding to antigen-positive tumor cells using FitMaster data analysis software (Origin8.5-based), which comes on the sam biosensor workstation.

    The data presented here illustrate that real-time, label-free kinetic binding analysis on whole viable mammalian cells produces reliable KD values. The rapid speed (minutes rather than hours) of the assay is key, as the use of freshly cultured, unfixed cells allows the analysis to be performed before any internalization of receptors can take place.

    The results obtained during this study have also been confirmed using more traditional methods (e.g., FACS analysis), suggesting the system is robust and accurate enough for widespread adoption. Such studies can be conducted using suspension cells, such as in the capture assay described, or using adherent cells, which can be cultured and grown directly on the sensor chip surfaces.

    The SAW approach using cells could now facilitate new studies exploring the following:

    • Target molecule accessibility,
    • Bispecific antibody functionality,
    • Receptor co-localization,
    • Antibodies binding to GPCR receptors on the surface of cells.

    The latest addition to the sam family, the samX, advances the cell-based workflow a step further by having two independent sensor chip positions, instead of one, providing eight sensor elements that can be individually addressed via the fluidics and which facilitates on-chip immobilization. This expands the workflow options available for increased power, flexibility, and throughput.

    The new product also makes it possible to inject a different concentration of ligand into each sensor element position of the chip. This allows cells to be captured or adhered in all positions, meaning that each sensor element position can represent a unique ligand concentration. By increasing the flexibility offered, these factors make samX the ideal platform for performing rapid, real-time, cell-based binding analyses.



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