PurposeTo evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs).MethodsRelative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys.ResultsIn human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1–5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing).ConclusionsIn adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.
Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.
Background: Relatively little has been reported comparing the ability of different induced pluripotent stem cells (iPSCs) and protocols to derive human intestinal organoids (HIO), although there is potential to supply HIO for translational research and regenerative medicine. In view of the time and effort required to differentiate HIO, protocols for differentiation were compared and five iPSC lines, produced by retroviral or non-viral reprogramming methods, were concurrently differentiated to HIO to evaluate the robustness and repeatability of using cellular markers by flow cytometry and immunohistochemistry. Methods: iPSCs were differentiated to definitive endoderm by Activin A treatment (Protocol I) or Myostatin (GDF8), GSK-3β inhibitor (CHIR99021) and B27 supplement (Protocol II) and then differentiated to hindgut using FGF4 and WNT3A (Protocol I), or KGF and Retinoic Acid (Protocol II) resulting in spheroids that were differentiated to HIO in 3-dimensional culture in matrigel containing EGF, Noggin, and R-Spondin1. Results: Definitive endoderm markers (CXCR4 and SOX17) were similar using both protocols and in all cell lines, but in their absence, spheroids to not develop. HIO derived from excised hindgut spheroids (Protocol II) showed more consistent expression of epithelial markers E-Cadherin, CDX2, villin, and chromogranin A. Morphological characteristics such as budding are predictive of robust enterocyte differentiation (villin positive) in HIO. Conclusions: Substituting different members of the growth factor families (e.g., TGFβ) is equally or more effective for producing epithelial lineages in HIO. Although there are no early quantitative predictors of level of success, all reprogrammed somatic cells could be differentiated to produce HIO. Several morphological characteristics (excisable spheroids and budding in HIO) were associated with HIO success. This could improve cost-effectiveness and ease of differentiation as HIO become more commonly available as translational 3D models of the gut.
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