Disparities Between Antibody Occupancy, Orientation, and Cytotoxicity in Immunotherapy.

We report fusion proteins designed to bind spatially distinct epitopes on the extracellular portion of HER2, a breast cancer biomarker and established therapeutic target, and recruit IgG (either anti-His6 or serum IgG) to the cell surface. When incubated with anti-His6 antibody and varied concentrations of a single HER2-binding protein His6 fusion, we observe interference and a decrease in antibody recruitment at HER2-binding protein concentrations exceeding ~30 nM. In contrast, concomitant treatment with two or three distinct HER2-binding protein His6 fusions, and anti-His6, results in increased antibody recruitment, even at relatively high HER2-binding protein concentration. In some instances, increased antibody recruitment leads to increased Antibody-Dependent Cellular Cytotoxicity (ADCC) activity. While a fusion protein consisting of a HER2-binding nanobody and Sac7d, a protein evolved to recognize the Fc domain of IgG, binds IgG from serum, antibody recruitment does not lead to ADCC activity. Rationales for these disparities are provided. Collectively, our findings have implications for the design of efficacious targeted immunotherapeutic biologics, and ensembles thereof.