Unlocking binding properties of single-domain antibodies targeting the polymeric immunoglobulin receptor to enhance mucosal enrichment of IgG against respiratory syncytial virus

A major driver of mucosal immunity is immunoglobulin A (IgA) that can translocate across mucosal epithelial barriers to protect against various pathogens in luminal spaces of the human body. The transcytosis of IgA is primarily mediated by the polymeric Ig receptor (pIgR), which is highly expressed in mucosal tissues and selectively transports polymeric IgA, but not IgG. IgG has been the preferred immunoglobulin isotype for therapeutic development because of its well-characterized biological functions and established manufacturing processes. Efficient transport of IgG across the epithelium into mucosal spaces is a highly desirable feature in the development of IgG-based neutralizing antibodies targeting respiratory infections. To address this challenge, we report discovery and characterization of anti-pIgR variable single-domain antibodies (V_HH) that facilitate pIgR-mediated transcytosis with efficiency comparable to dimeric IgA in epithelial cell models. Screening a panel of anti-pIgR V_HH-Fc molecules targeting the same epitope bin revealed correlations between binding parameters (K_D and <i>k_off</i> ) and transcytosis activity. Notably, several antibodies with highly efficient transcytosis exhibited faster dissociation rates at acidic pH relative to neutral pH, suggesting the potential of pH-dependent binding as a factor to influence the transcytosis pathway of pIgR-bound antibodies. Building on these insights, we engineered a bispecific antibody (bsAb) by fusing an anti-pIgR V_HH to the C-terminus of an IgG heavy chain targeting respiratory syncytial virus (RSV). This bsAb efficiently transcytosed across pIgR-expressing Madin-Darby canine kidney (MDCK) cells and a human airway mucosal barrier model, while fully retaining RSV binding and neutralization capabilities. Our study introduces a novel strategy to enhance the mucosal distribution of systemically administered biologics, with significant implications for the development of improved antibody therapeutics against mucosal pathogens.