Human Fc C_H2 domain modifies cholix transcytosis pathway to facilitate efficient oral therapeutic protein delivery

The first domain of the cholix (Chx) exotoxin can rapidly cross small intestinal epithelium using a vesicular apical to basal (A → B) transcytosis mechanism that exploits interactions with specific host cell proteins. This non-toxic element of Chx can efficiently ferry a covalently attached therapeutic protein cargo that results in deposition of the carrier-cargo chimera within the lamina propria where it is retained due the presence of Chx receptors present on cells in this compartment. Systemic delivery of a cargo using this pathway requires separation from Chx at a late stage of epithelial A → B transcytosis. Here, we use a furin protease-specific cleavage sequence (FCS) to genetically conjoin a protein cargo, human growth hormone (hGH), to produce chimeras designed to separate a Chx-cargo chimera in a furin⁺ vesicular compartment in the basal region of intestinal epithelium cells to facilitate systemic hGH delivery. Our studies demonstrate that in response to the Chx carrier, there is a redistribution and augmentation of apical furin⁺ compartments where pre-mature carrier-cargo separation events would dramatically limit systemic hGH delivery using this FCS strategy. Apical application of soybean trypsin inhibitor blocked A → B transcytosis of the Chx-FCS-hGH chimera, likely by inhibiting early trafficking events associated with RME involving furin. We now show that combining the Chx carrier with the Fc domain C_H2 element of human IgG1 in a position-specific manner provides a mechanism to bypass the apical furin⁺ vesicular compartment to reach the basal furin⁺ vesicular compartment where FCS scission allows for efficient hGH release into systemic circulation. Colocalization of the C_H2-Chx carrier with the Fc receptor-like A protein in the apical region of enterocytes soon after RME demonstrates this deviation from the A → B transcytosis pathway normally accessed by Chx. Using a solution optimized for delivery to rat ileum we observed the amount of hGH reaching the systemic circulation to be ∼4 % of the material delivered using the C_H2-Chx carrier, demonstrating that oral delivery of a therapeutic protein can be achieved using targeted vesicular transcellular routing. ONE SENTENCE SUMMARY: Combining domain I of the Vibrio cholerae exotoxin protein cholix with the C_H2 element of the human IgG1 Fc domain results in a modification of the transcytosis pathway used by this toxin element that avoids the apical endosomal compartment of polarized intestinal epithelial cell where furin-like proteases can prematurely separate cholix-based chimera molecules.