Human Fc CH2 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 CH2 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 CH2-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 CH2-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 CH2 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.