High throughput solution-based measurement of antibody-antigen affinity and epitope binning

Abstract

Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (k(a)) rate constant measurements suggest that this is mainly caused by inaccurate k(a) measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods.

Keywords: BLI; KD; MSD; affinity; antibody; epitope; high throughput; kinetic exclusion.

Figure 1. Octet RED384 Binning. Four antibodies were probed for competitive and non-competitive binding against the same antigen. In a typical binning assay, control antibodies were loaded onto AHQ sensors, blocked with a human IgG1 antibody, and the sensors were exposed to Antigen A (step 1) followed by an antibody of interest (step 2). Additional binding of the antibody of interest indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (self-competing antibodies shown by borders around graphs).

Figure 2. Octet RED384 Kinetic Measurements. (A) Octet RED384 K_D profile screening. Antigen A, with a human Fc, was loaded to 0.2 nm on AHQ sensors and exposed to solutions of 200 nM Fab in PBSF. Traces for Fabs 1 and 2 were fit using a truncated dissociation step; trace 3 was fit using the “Partial” fit function in the Octet Data Analysis software v. 7.0. (B) Octet RED384 loading density titration. The Fc-antigen was loaded onto AHQ sensors at densities ranging from 1.0 to 0.2 nm and exposed to 50 nM Fab solutions in PBSF. (C) Octet RED384 analyte titration. The Fc-antigen was loaded onto AHQ sensors at 0.2 nm and exposed to different concentrations of Fab solutions in PBSF (50–12.5 nM). (D) Optimized Octet RED384 K_D measurements of 48 antibodies. 48 antibodies were loaded onto AHQ sensors and then exposed to a solution of 25 nM monomeric antigen. The (□) symbols indicate that a 5E-05 sec⁻¹ limit has been reached for a 30 min dissociation step. The dotted line represents a 100 pM threshold.

Figure 3. Octet RED384, KinExA and MSD-SET K_D Measurements. (A) Octet RED384 K_D values plotted against KinExA K_D values. Octet RED384 experiments were performed using AHQ sensors. KinExA experiments were performed in the reverse orientation, and both KinExA and MSD experiments were designed to obtain K_D-controlled curves. (B) MSD-SET K_D measurements with varying contact times. MSD-SET experiments were performed by incubating equilibrated solutions on an antibody-coated MSD plate for 2, 10, 30 and 60 min. As plate contact times increased, the fitted K_D values also increased. Only fits are shown for clarity. (C) Reproducibility studies of 3 antibodies by KinExA. Each of the 16 points on the curve represents duplicate measurements from a 3-fold dilution series, with error bars representing standard deviation. The K_Ds of mAb F.1, mAb F.2 and mAb D.2 were 680 fM, 360 pM and 5 nM, respectively. (D) Reproducibility studies of 3 antibodies by MSD-SET. MSD-SET experiments were performed as outlined in Figure 4. Plate incubation times were fixed at 150 sec to minimize disruption of equilibrium. Each of the 12 points on the curve represents duplicate measurements from a 5-fold dilution series, with error bars representing standard deviation. The K_Ds of mAb F.1, mAb F.2 and mAb D.2 were 1 pM, 260 pM and 4.4 nM, respectively.

Figure 4. Solution Equilibrium Titrations (SET), KinExA and MSD experimental design. (A) SET. A solution equilibrium titration (SET) is prepared with antigen (blue) held constant and antibody (orange) titrated. The solution is allowed to come to equilibrium. (B) KinExA. A column is packed with antibody-conjugated beads. The SET sample is passed over the column at a fixed flow rate (typically 0.25 mL/minute) (C) Only free antigen is captured on the antibody-conjugated beads. (D) The captured antigen is detected with a fluorophore-labeled antibody (pink) that binds in a non-competitive manner on the antigen. (E) The fluorophore’s signal results in the trace shown, where the end points of a raw signal trace can be used to plot signal as a function of antibody concentration and fit to a quadratic equation to extract a K_D. (F) MSD. Antibody is coated to a standard-bind MSD plate. The SET sample is added by a liquid handling robot to the antibody-coated plate. (G) After 150 sec, free antigen is captured on the plate, while the remaining SET solution is washed off the plate. (H) The captured antigen is then detected by a sulfotag-conjugated antibody that binds in a non-competitive manner on the antigen.(I) The sulfotag is electrochemically activated to provide signal that is measured by a CCD camera. The raw signal can then be plotted as a function of antibody concentration and fit to a quadratic equation to extract a K_D.

Figure 5. MSD-SET K_D measurements. (A) KinExA K_D values plotted against MSD-SET K_D values. KinExA and MSD experiments were performed to provide K_D-controlled curves. (B) MSD-SET K_D of 12 different panels of antibodies. MSD-SET was performed to provide K_D-controlled curves. Antigens used included cytokines, cell-surface receptors, enzymes and peptides. The inset shows affinities of antibodies binding to Antigen H across four separate bins.