Discovery and characterization of single-domain antibodies for polymeric Ig receptor-mediated mucosal delivery of biologics

Abstract

Mucosal immunity is dominated by secretory IgA and IgM, although these are less favorable compared to IgG molecules for therapeutic development. Polymeric IgA and IgM are actively transported across the epithelial barrier via engagement of the polymeric Ig receptor (pIgR), but IgG molecules lack a lumen-targeted active transport mechanism, resulting in poor biodistribution of IgG therapeutics in mucosal tissues. In this work, we describe the discovery and characterization of single-domain antibodies (VHH) that engage pIgR and undergo transepithelial transport across the mucosal epithelium. The anti-pIgR VHH panel displayed a broad range of biophysical characteristics, epitope diversity, IgA competition profiles and transcytosis activity in cell and human primary lung tissue models. Making use of this diverse VHH panel, we studied the relationship between biophysical and functional properties of anti-pIgR binders targeting different domains and epitopes of pIgR. These VHH molecules will serve as excellent tools for studying pIgR-mediated transport of biologics and for delivering multispecific IgG antibodies into mucosal lumen, where they can target and neutralize mucosal antigens.

Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment

“…We previously identified a panel of heavy chain only (VHH) antibodies that could bind human pIgR with affinities ranging from ~4 to 500 nM. 41 Of these, VHH2 and VHH6 were included in this analysis since VHH2 displayed cross-reactivity to mouse pIgR and both displayed strong transcytosis in an MDCK monolayer-based assay and in a human epithelial airway model. To mediate binding to the SARS-CoV-2 spike glycoprotein, we identified 2 antibodies that were reported to display neutralization activity – D001 (Sino Biological cat.…”

“…Bifunctional molecules containing VHH2 (CV19B283, CV19B307, and CV19B308) bound to pIgR with Kd values ~5 nM, consistent with our previous findings. 41 Bifunctional molecules containing VHH6 (CV19B277 and CV19B301) bound with Kd ~ 0.2 mM or 265 nM, respectively. The significantly weaker binding by CV19B277 may have been due to its being fused in tandem on the N-terminus of ACE2, although the mechanism for its reduced binding was unknown.…”

“…The anti-pIgR VHH modules could mediate transcytosis across both MDCK cell bilayers and in a human epithelial microtissue model, and we thus asked whether this pIgR-mediated transcytosis could occur in vivo . 41 We selected VHH2, and VHH6 for pharmacokinetic analysis in either monovalent or bivalent format in C57BL/6 mice ( Supplementary Fig. S4, Supplementary Table S1 ).…”

“… 40 We and others have developed anti-pIgR single-domain antibodies (VHH) that can exploit pIgR-mediated transcytosis to deliver IgG across the endothelium into the lung mucosa. 41 , 42 We have shown that molecules harboring the anti-pIgR domains displayed increased level of transport across Madin-Darby canine kidney (MDCK) cell-derived monolayers and across human-derived lung epithelial layers by >30-fold compared to conventional IgG. 41 This platform, which we call BRING-IT ( B i-di r ect i o n al polymeric I g receptor med i ated t ransport of biologics), has the potential to deliver IgG into the mucosal lumen, where it can target and neutralize mucosal antigens or pathogens such as SARS-CoV-2.…”

Improvements in drug delivery and monitoring of diseases in biotherapeutics

“…[1][2][3] The ability of nanobodies to specifically target unique epitopes with subnanomolar affinity, and to penetrate dense solid tumours make them a powerful tool in medical therapeutic procedures and in tumour pathobiology. 2,[5][6][7] Improvements in drug delivery and monitoring of diseases in biotherapeutics…”