Bioassay Development for Bispecific Antibodies-Challenges and Opportunities

Abstract

Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules.

Keywords: binding assays; bioassays; bispecific antibodies; mechanisms of action; potency assays.

Figure 1

Examples of BsAb formats and structural diversity: (a–f) IgG-like BsAbs and (g–l) fragment-based BsAbs.

Figure 2

Mechanisms of actions of BsAb: (a) Schematic diagram of cell-bridging BsAb MoA (e.g., TDB or NK-recruiting BsAb); (b) Schematic diagram of receptor activating/inhibiting MoA (e.g., receptor dimerization inhibitor or activator); (c) Schematic diagram of cofactor mimicking MoA (e.g., emicizumab); and (d) Schematic diagram of “homing” BsAb MoA (e.g., blood brain barrier crosser).

Figure 3

Representative bioassays for BsAb: (a) Reporter gene T-cell activation assay; (b) Single-arm binding SPR assay; (c) Cell proliferation assay; (d) Bridging ELISA. MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide.