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

Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

<div><p>Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their <i>in vitro</i> characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling. </p> </div

Influence of pH on the apparent affinity (top) and apparent activity (bottom) of different mAbs towards their purified recombinant antigens.

<p>The K_D values and mAb activities for each interaction were obtained from a single curve KinExA analysis performed at different pH values that spanned the pH range encountered during serum experiments. The bars represent the best fit values and the error bars represent the 95% confidence interval. The arrows indicate the trend observed with increasing pH and the range of best fit values for K_D and activity. Only sweet spot experiments enabled a determination of both the K_D and the mAb activity (no mAb activity is reported for 19F7 and 33B12 because those curves were mostly K_D-controlled). The antigen concentrations used were 128 pM rhPCSK9, 42 pM rhPGRN, 21 pM rhPGRN, 100 pM rhFABP4, and 1nM rhFABP4 (from left to right).</p

KinExA-determined affinities of three mAbs (J17, 2B2, and 33B12) towards their purified recombinant antigens (left) and native antigens in human serum (right).

<p>The data are presented in the same way as in Figure 2.</p

KinExA-determined apparent affinities for four mAbs (J16, J17, 19F7, and 21B8) towards their purified recombinant antigens (left) and their serum antigens (right) at stable, non-neutral pH values.

<p>Titration curves are presented as described in Figure 2.</p

Kinetic analysis of anti-PGRN mAbs 2B2 (left) and 19F7 (right) in TBST buffers at different pH values.

<p>The data were collected on a ProteOn XPR36 biosensor by injecting a dilution series of rhPGRN (0.8, 2.4, 7.1, 21.3, and 64 nM) over amine-coupled mAbs. Double-referenced sensorgrams (colored lines) obtained from two ligand channels per mAb were fit globally to a 1:1 binding model with mass transport limitation (fit shown in black); the results from one channel per mAb is shown along with the global best fit K_D.</p

Human serum titrated with anti-PCSK9 mAb J16.

<p>(A) Raw data trace of fluorescence (in Volts) as a function of time recorded by the KinExA instrument for a typical experiment: (I) packing of mAb-coated beads inside the flow cell; (II) baseline signal; (III) auto-fluorescence signal obtained from serum components (presumably porphyrins); (IV) buffer wash; (V) detection of bead-captured PCSK9 with a Dylight-labeled mAb; and (VI) buffer wash, after which the final fluorescence signal for bead-captured PCSK9 is recorded (relative to the baseline signal). (B) Global fit of normalized data obtained from titrating J16 into different dilutions of serum prepared in PBS. (C) Error plots for K_D and PCSK9 concentration for the global analysis in panel B with best fit values (solid line) and 95% confidence interval (dotted lines). (D) Comparison of the fits obtained for single-curve and multi-curve (global) analysis of the data in panel B. The PCSK9 concentration is back-calculated for undiluted serum. Open and closed symbols indicate independent experiments performed with the same dilution factor. </p

Expression of human lambda expands the repertoire of OmniChickens.

Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken®, a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads

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

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