Background

Engineered bispecific antibodies (BsAbs) that recognize two separate antigens or epitopes are an emerging class of next-generation biological therapeutics. Such antibodies, capable of engaging multiple targets, shed new light on clinical treatments with the prospect of additive or synergistic mechanisms of action (MoA) and/or superior potency compared to monoclonal antibody (mAb) or combination therapies (Kontermann, 2012; Register et al., 2021). Many BsAbs are currently marketed as therapeutics in several disease areas, and more than 100 BsAbs have progressed into clinical pipelines (Kaplon et al., 2022; Register et al., 2021).

The binding assays are necessary components of in vitro BsAb characterization (Saldanha et al., 2018). They must be readily conducted during initial product development phases for the candidate screening and stability assessments and can therefore potentially be used as MoA-reflective potency assays (Lee et al., 2017). The bioassay strategy is outlined to characterize the independent or simultaneous binding affinities of a BsAb to their dual-antigen targets and demonstrate the full biological binding activity, which is the prerequisite of the envisioned MoA (Register et al., 2021).

Bridging ELISA is a type of sandwich ELISA that has been widely used in bi-functional quantitative assays that cover both binding events simultaneously. This assay is based on a bridging format and utilizes an immobilized capture recombinant antigen 1 in a solid phase (usually polystyrene microplates), followed by the addition of a biotinylated version of the antigen 2, to form the antigen 1–BsAb–antigen 2 bridging complex. The horseradish peroxidase (HRP)–labeled streptavidin is used as the detector. Major advantages of this method are the rapid setup, easy preparation of reagents, and effortless handling when compared to other bridging bioassays [e.g., surface plasmon resonance (SPR) or cell-based flow cytometry]. By applying this bridging approach, several BsAbs, including PD-L1/TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domain), HER2/PD-1(programmed death protein 1), 4-1BB(CD137) (tumor necrosis factor receptor superfamily 9)/HER2, and OX40(CD134) (tumor necrosis factor receptor superfamily 4)/4-1BB (Hinner et al., 2019; Ljungars et al., 2020; Chu et al., 2022; Mu et al., 2022), have been determined to being capable of binding to both targets simultaneously. The bridging ELISA assay should be viewed as a potential new standard and well-established procedure for measuring dual-target binding. Here, we focus on HER2(human epidermal growth factor receptor 2)/PD-L1(programmed cell death ligand 1) BsAb and describe an effective dual-target binding ELISA protocol that allows the measurement of a bispecific drug binding to both targets in a single assay format.