Highly sensitive detection of antibody nonspecific interactions using flow cytometry

Abstract

The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery. These candidates must be rapidly pared down to identify the most drug-like candidates for in-depth analysis of their safety and efficacy, which can only be performed on a limited number of antibodies due to time and resource requirements. One key biophysical property of successful antibody therapeutics is high specificity, defined as low levels of nonspecific binding or polyspecificity. Although there has been some progress in developing assays for detecting antibody polyspecificity, most of these assays are limited by poor sensitivity or assay formats that require proprietary antibody surface display methods, and some of these assays use complex and poorly defined polyspecificity reagents. Here we report the PolySpecificity Particle (PSP) assay, a sensitive flow cytometry assay for evaluating antibody nonspecific interactions that overcomes previous limitations and can be used for evaluating diverse types of IgGs, multispecific antibodies and Fc-fusion proteins. Our approach uses micron-sized magnetic beads coated with Protein A to capture antibodies at extremely dilute concentrations (<0.02 mg/mL). Flow cytometry analysis of polyspecificity reagent binding to these conjugates results in sensitive detection of differences in nonspecific interactions for clinical-stage antibodies. Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures. Moreover, we also find that a unique reagent, namely ovalbumin, results in the best assay sensitivity and specificity. Importantly, our assay is much more sensitive than standard assays such as ELISAs. We expect that our simple, sensitive, and high-throughput PSP assay will accelerate the development of safe and effective antibody therapeutics.

Keywords: Developability; affinity; bispecific; multispecific; mAb; IgG; Fc; Fab; non-specific; nonspecific; off-target binding; polyreactivity; specificity.

Figure 1.

Overview of the PolySpecificity Particle (PSP) assay for evaluating antibody nonspecific interactions. In the PSP assay, mAbs are immobilized on micron-sized magnetic beads coated with Protein A and then the conjugates are incubated with different types of polyspecificity reagents. The levels of antibody nonspecific interactions are evaluated via fluorescence detection using standard flow cytometry methods. The primary advantages of the PSP assay relative to the conventional PolySpecificity Reagent (PSR) assay are that the PSP assay does not require proprietary technologies for displaying full-length IgGs on yeast and it is compatible with the soluble IgGs and other soluble Fc-fusion proteins (including bispecific and multispecific antibodies)

Figure 2.

Impact of mAb loading on flow cytometry detection of antibody nonspecific interactions with soluble membrane proteins. mAb immobilization on Protein A beads was detected using a F(ab’)₂ antibody specific for human Fc (Alexa Fluor 488) and mAb binding to biotinylated soluble membrane proteins was detected using streptavidin (Alexa Fluor 647). The measurements were evaluated as a function of the mAb concentration relative to the IgG binding capacity of the Protein A beads. The experiments were performed three times, and a representative example is shown

Figure 3.

Detection of antibody nonspecific interactions for mAbs reported to display high and low levels of polyspecificity. The measurements were performed using biotinylated soluble membrane proteins. The PSP scores are average values for each mAb normalized between a polyreactive control mAb (ixekizumab) with a score of 1 and a specific mAb (elotuzumab) with a score of 0. The experiments were repeated three times, the data are average values, and the error bars are standard deviations

Figure 4.

Novel flow cytometry method detects mAb nonspecific interactions in a manner that is strongly correlated with previous measurements using a proprietary method. Measurements of antibody interactions with soluble membrane proteins using Protein A beads in this work are shown on the y-axis, while the corresponding published measurements using a proprietary yeast display technology are reported on the x-axis. The PSP scores were calculated as described in Figure 3. The flow cytometry measurements performed using Protein A beads are averages of three experiments, and the error bars are standard deviations

Figure 5.

Comparison of ELISA and previously published flow cytometry measurements of antibody nonspecific interactions. (a, b) ELISA measurements of antibody nonspecific interactions using two assay formats: (a) ELISA #1 format in which Protein A is immobilized, mAbs are captured and mAb interactions with soluble membrane proteins are detected; and (b) ELISA #2 format in which soluble membrane proteins are immobilized and interactions with soluble (non-immobilized) mAbs are detected. In (A) and (B), the ELISA measurements are compared to previously reported flow cytometry measurements (soluble membrane proteins). The ELISA measurements are averages of three experiments and the error bars are standard deviations

Figure 6.

Comparison of the ability of the PSP flow cytometry and ELISA methods to differentiate between mAbs with low and high levels of nonspecific interactions. Raw binding signals for two control antibodies with low (elotuzumab) and high (ixekizumab) levels of polyreactivity measured using ELISAs and flow cytometry. ELISA #1 and #2 are defined in Figure 5. The measurements are averages of three experiments and the error bars are standard deviations

Figure 7.

Evaluation of complex and defined protein reagents for detecting antibody nonspecific interactions using the PSP assay. (a) Soluble cytosolic proteins (SCP), (b) Hsp90, (c) HSA and (d) ovalbumin were evaluated for detecting antibody nonspecific interactions relative to previous flow cytometry measurements using yeast surface display (soluble membrane proteins).^, The PSP scores (y-axes) were calculated as described in Figure 3, and are averages of three experiments and the error bars are standard deviations

Figure 8.

Accuracies of the PSP flow cytometry measurements for classifying mAbs with low and high polyspecificity. mAbs were defined as polyreactive if their published PSR values (soluble membrane proteins) were >0.27¹⁵ and the classification accuracies and cutoff values for the PSP measurements in this work are reported for each reagent. The PSP scores were calculated as described in Fig. 3. The abbreviated reagents are soluble membrane proteins (SMP), soluble cytosolic proteins (SCP), human serum albumin (HSA), and ovalbumin (OVA)

Figure 9.

Impact of Fv isoelectric point on antibody nonspecific interactions. Ovalbumin, soluble cytosolic proteins and soluble membrane proteins preferentially detect a subset of mAbs with high Fv isoelectric points, while soluble cytosolic proteins and soluble membrane proteins also detect a subset of mAbs with low Fv isoelectric points. The PSP scores were calculated as described in Fig. 3, and the isoelectric points were calculated based on the Fv sequences