Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (KD values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent KD and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens

DKK1/DS4 interaction studied in the fixed antibody and fixed antigen assay orientations on the KinExA.

<p>(A) In the fixed antibody orientation, a series of samples is prepared by titrating DKK into a fixed concentration of antibody binding sites. After sample equilibration, free antibody binding sites are captured on beads and detected by a fluorescently labeled anti-species antibody. In our modified KinExA method, the beads are coated with a murine anti-idiotypic mAb instead of antigen. (B) In the fixed antigen orientation, a series of samples is prepared by titrating the antibody into a fixed DKK concentration. Free DKK in equilibrated samples is captured on antibody-coated beads and detected with a customized sandwiching mAb that is fluorescently-labeled (one step detection) or unlabeled and followed by a fluorescently-labeled reagent (two step detection); see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036261#s4" target="_blank">Materials and Methods</a>. (C) Global analysis of DS4's interaction with human DKK1 in the fixed antibody orientation. The “unknown ligand” model in the KinExA software automatically corrects the concentration of the titrated component with the best fit for its apparent activity, so that the x-axis shows the antigen's active concentration, rather than its nominal concentration. (D) Global analysis of DS4's interaction with human DKK1 in the fixed antigen orientation. For both panels C and D, the nominal concentration of the fixed binding partner is indicated per titration curve; in panel D, the best fit binding site concentration is indicated in parentheses. The apparent K_D values for panels C and D were 0.49 pM and 0.42 pM, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036261#pone-0036261-t001" target="_blank">Table 1</a>).</p

Determination of active antigen concentrations using complementary label-free methods.

<p>(A) Titration-based Octet measurement obtained over immobilized anti-Id mAb for samples containing 1 nM DS4 binding sites titrated with a purified preparation of carrier-free human DKK4. (B) Sharp inhibition curve obtained from the data shown in panel A showing that a nominal concentration of 50 nM human DKK4 was needed to exactly titrate out 1 nM DS4 (corresponding to an antigen activity of 2%). (C) CFCA data collected on the Biacore for a nominal concentration of 0.1 µg/mL human DKK1 flowed at 100 µL/min (blue) and 5 µL/min (red) over a high capacity of immobilized DS4. The curve fit is shown in black.</p

Affinity determinations for crude antigens from conditioned media.

<p>(A) Dual-curve analysis in the fixed antibody assay orientation of recombinant rhesus DKK4 in conditioned media of 293F cells. (B) Overlay plot of the curves obtained in the fixed antigen assay orientation using recombinant human DKK2 (black squares) and cyno DKK2 (red circles) from conditioned media of 293F cells, each diluted threefold with running buffer. The blue curve shows the cyno DKK2 assay performed at a fourfold higher flow rate and the green curve represents a control titration of purified mouse DKK1 spiked into a threefold dilution of media from untransfected cells. (C) Dual-curve analysis in the fixed antigen assay orientation of native human DKK1 in the media of a Hepatocarcinoma cell line, diluted 1000-fold (red) and 20-fold (black) with running buffer. The apparent K_D values were (A) 2.1 pM – rhesus DKK4, (B) 110 nM – human DKK2; 120 nM (red) and 170 nM (blue) – cyno DKK2, and (C) 0.16 pM – human DKK1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036261#pone-0036261-t001" target="_blank">Table 1</a>).</p

Association rate determination by KinExA.

<p>“Kinetics direct” measurements using (A) 50 pM DS4 and 30 pM human DKK1 and (B) 100 pM DS4 and 90 pM human DKK4. The apparent k_a values were 1.05×10⁷ M⁻¹s⁻¹ for human DKK1 and 1.4×10⁷ M⁻¹s⁻¹ for human DKK4.</p

Kinetics and affinity determinations by Biacore.

<p>Binding kinetics of (A) human DKK1, (B) mouse DKK4, and (C) human DKK2. Measured data in color contrast the global fits in black. Panels A and B each show a simultaneous fit of the data obtained from three different capacity surfaces (red – low, green – medium, and blue – high; in panel A, the data are separated out by surface for clarity but were fit simultaneously). Panel C shows data from a single surface. In each case, DKK was flowed as a threefold dilution series with nominal top concentrations of (A) 13 nM, (B) 40 nM, and (C) 500 nM over immobilized DS4. Only the association phase was monitored for DKK1 because the dissociation was too slow to resolve by our capture-based assay. Panel D shows an alternate analysis of the DKK2 data, using the equilibrium binding responses. Panel E shows the inhibition of DS4 Fab with titrating levels of human DKK4 over immobilized anti-Id. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036261#pone-0036261-t002" target="_blank">Table 2</a> for the extracted kinetic rate constants and affinities. The upper cartoon depicts the assay orientation used in panels A–D while the lower cartoon refers to panel E.</p