A homogeneous bioluminescent immunoassay for parallel characterization of binding between a panel of antibodies and a family of Fcγ receptors.

Fc engineering efforts are increasingly being employed to modulate interaction of antibodies with variety of Fc receptors in an effort to improve the efficacy and safety of the therapeutic antibodies. Among the various Fc receptors, Fc gamma receptors (FcγRs) present on variety of immune cells are especially relevant since they can activate multiple effector functions including antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Depending on the desired mechanism of action (MOA) of the antibody, interactions between Fc domain of the antibody and FcγR (denoted as Fc/FcγR) may need to be enhanced or abolished. Therefore, during the antibody discovery process, biochemical methods are routinely used to measure the affinities of Fc/FcγR interactions. To enable such screening, we developed a plate based, simple to use, homogeneous immunoassays for six FcγRs by leveraging a luminescent protein complementation technology (NanoBiT). An added advantage of the NanoBiT immunoassays is their solution-based format, which minimizes well known surface related artifacts associated with traditional biosensor platforms (e.g., surface plasmon resonance and biolayer interferometry). With NanoBiT FcγRs assays, we demonstrate that assays are specific, report IgG subclass specific affinities and detect modulation in Fc/FcγR interactions in response to the changes in the Fc domain. We subsequently screen a panel of therapeutic antibodies including seven monoclonal antibodies (mAbs) and four polyclonal intravenous immunoglobulin (IVIg) products and highlight the advantages of parallel screening method for developing new antibody therapies.