Immobilization-Free Binding and Affinity Characterization of Higher Order Bispecific Antibody Complexes Using Size-Based Microfluidics

Abstract

Simultaneous targeting of different antigens by bispecific antibodies (bsAbs) is permitting synergistic binding functionalities with high therapeutic potential, but is also rendering their analysis challenging. We introduce flow-induced dispersion analysis (FIDA) for the in-depth characterization of bsAbs with diverse molecular architectures and valencies under near-native conditions without potentially obstructive surface immobilization. Individual equilibrium dissociation constants are determined in solution, even in higher-order complexes with both antigens involved, hereby allowing the analysis of binding cooperativity and elucidation of a potential interference between the interactions. We further illustrate bispecific binding functionality as incremental increases in complex sizes when the bsAbs are exposed to one or two antigens. The possibility for comprehensive binding analysis with low material consumption and high matrix tolerability irrespective of molecular format and with little optimization renders FIDA a versatile tool for format selection and characterization of complex bi/multispecific protein therapeutics throughout the drug development and biomanufacturing pipeline.

Figure 1

FIDA principle enables size-based detection of bispecific binding behavior. (A) Schematic illustration of the FIDA principle for bispecific antigen binding. Complexation of the fluorescent antigen with binding partners causes signal dispersion, which translates to a change in the apparent hydrodynamic radius of the antigen. The red circle indicates the fluorescently labeled (yellow stars) primary antigen used for detection, and the green circle indicates the unlabeled secondary antigen that is affecting the signal indirectly through ternary complex formation. (B) Various bsAbs were constructed through genetic fusion of anti-PD-L1 IgG1 and anti-HER2 single-domain antibodies through a flexible (GGGGS)×2 linker. Symmetric bsAb formats included sdAb C-terminal coupling on HC (CHC-IgG), sdAb C-terminal coupling on LC (CLC-IgG), and sdAb C-terminal coupling on both LC and HC (CHC-CLC-IgG). An asymmetric bsAb monovalent for each antigen (sdAb-Fc/IgG) was constructed by controlled pairing of sdAb-Fc and IgG HC by introduction of KiH mutations in the CH3 domains (illustrated as the black circle between the two CH3 domains). (C) Example of Taylor dispersion of unbound fluorescent PD-L1-DY490 (blue), monospecific CHC-IgG binding to PD-L1-DY490 (orange), and bispecific binding of CHC-IgG to PD-L1-DY490 and unlabeled HER2 (green). (D, E) FIDA complex sizing. Apparent hydrodynamic radii of antibodies in complex with fluorescently labeled (DY-490) primary antigen ± an excess of unlabeled secondary antigen. The monospecific and bispecific antibodies have been separated by stippled squares similar to (B).

Figure 2

Quantitative characterization of bsAb binding. (A) Examples of antibody binding curves generated by plotting apparent hydrodynamic radius of the fluorescently labeled antigen (indicator) as a function of antibody concentration measured using FIDA. Increasing amounts of antibody increases the indicator apparent hydrodynamics radius through complexing. Inclusion of a secondary unlabeled antigen allows characterization of interference with indicator binding arising from the secondary binding event. (B) Equilibrium dissociation constants from fitting of binding curves and the related goodness of fit.

Figure 3

Complex Rh of asymmetric bsAbs correlate with heterodimerization. (A) CH3 steering platforms promote HC heterodimerization for assembly of asymmetric bsAbs. Without any HC heterodimerization, 50% heterodimer and 25% of each homodimer will form through random assembly. (B) Capillary electrophoresis of asymmetric sdAb-Fc/IgG bsAbs produced with (KiH) or without (WT CH3) HC steering. The electropherogram peaks represent the distribution of antibody species. (C) Overview of changes in apparent Rh of fluorescent PD-L1-DY490 upon binding of asymmetric sdAb-Fc/IgG bsAbs ± unlabeled HER2 antigen.