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

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

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

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

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