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. 2014 Apr;31(4):908-22.
doi: 10.1007/s11095-013-1212-3. Epub 2013 Sep 26.

Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis

Pamela J Hornby  1 , Philip R Cooper, Connie Kliwinski, Edwin Ragwan, John R Mabus, Benjamin Harman, Suzanne Thompson, Amanda L Kauffman, Zhengyin Yan, Susan H Tam, Haimanti Dorai, Gordon D Powers, Jill Giles-Komar
Affiliations

Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis

Pamela J Hornby et al. Pharm Res. 2014 Apr.

Abstract

Purpose: To 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).

Methods: Relative 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.

Results: In 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).

Conclusions: In 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.

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Figures

Fig. 1
Fig. 1
Human donor muscle-stripped intestinal segments (three segments in each of four regions per donor for a total of n = 9/region, mean ± SEM) assessed for transcytosis and FcRn protein and mRNA expression. Segments mounted in Ussing-type flux chambers were incubated on the mucosal-side with M15 (20 and 200 μg/mL) and concentration-related low levels of full-length mAb were detected on the serosal side after180 mins (a). In adjacent intestinal mucosal scrapings (b), protein levels of FcRn (by ELISA) in proximal colon were higher than in jejunum (P < 0.05 by ANOVA and Newman-Kreuls post-test). When FcRn mRNA/total mRNA was plotted against M15 mAb uptake for each donor (c), there was an apparent correlation.
Fig. 2
Fig. 2
FcRn immunoreactivity in fixed and paraffin-imbedded 5 μm sections of donor ileum (a) and colon (c) confirms the ELISA data shown in Fig. 1b. FcRn-immunostaining in intestinal enterocytes (arrowheads) was above background staining observed in adjacent control rat IgG stained sections (b, d). Unexpectedly, intense FcRn immunostaining was visualized in a few cells within the small intestinal crypts and proximal colon, which were not observed in controls (small arrows).
Fig. 3
Fig. 3
FcRn mRNA expression quantified to total mRNA in human donor mucosa (a) where n = 3; adult rat mucosa (b) where n = 5, and suckling rat pup full-thickness sections (c) where n = 4 (raw data were log transformed since underlying assumptions of equal variance and normal distribution shape were more tenable). (d) In suckling rat pups (n = 6), mAb serum levels were greater after administration of M15 into the proximal than to the distal small intestine and showed a functional proximal- distal gradient paralleling FcRn mRNA expression. P < 0.05 by 1-way ANOVA and Bonferroni’s post-tests.
Fig. 4
Fig. 4
Cynomolgus monkey intestine FcRn immunostaining and mAb levels after dosing. Duodenal/jejunal mucosal biopsy illustrated patchy FcRn immunostaining in villous enterocytes (a, arrowheads), which was not observed in adjacent control rat IgG section (b). In the same monkey, full-thickness proximal colon has FcRn-immunostaining evenly distributed throughout (c, arrowheads). Artifactual and non-specific staining (d) is due to post-mortem tissue handling. In 3 out of 4 isoflurane-anesthetized cynomolgus anti-growth factor mAb dosed into the proximal small intestine (1–2 mg/kg), resulted in low femoral vein serum levels post-dosing at 90 mins and up to 48 h (e). Dosing mAb acutely into the ileum and proximal colon (2 mg/kg anti-RSV H435A, n = 2; and WT, n = 3) resulted in much higher femoral vein serum levels at 90 mins in 3 out of 5 monkeys (f). In these 3 monkeys, mAb concentrations were slightly higher from jugular vein (taken near thoracic duct). Very low intestinal fluid mAb concentrations in two monkeys were associated with very low serum mAb levels. ND = Not Done.
Fig. 5
Fig. 5
FcRn expression and mAb localization in caco-2 cells. (a) Immunofluorescence of FcRn and nuclear DAPI illustrates intense intracellular FcRn in heterogeneous cells. (b) By flow cytometry, ~ 60% of dissociated caco-2 cells had cell surface FcRn expression that was competitively abolished using an equal concentration of soluble unlabeled FcRn. (c) A detectable signal for surface binding of an FcRn-binding affinity mAb (N434A 0.0001 – 5.0 μg/ml) in dissociated caco-2 cell resulted in an IC50 = 1.5 μg/ml (0.01 μM). A non-FcRn binding mAb (H435A) had no measurable surface binding (n = 3 per group) (d) In polarized caco-2 cell monolayers apical to basolateral mAb transcytosis did not occur after incubation at apical concentrations close to the surface FcRn-binding IC50 (1.5 μg/ml) for either WT or H435A (n = 3 per group). (e) In polarized caco-2 cell monolayers grown in transwells, apical incubation of mAb concentrations well above the surface FcRn-binding IC50 resulted in intracellular detection of more WT than non-FcRn binding variant in lysed cells at the highest concentration (n = 4 per group; P < 0.05 by 2-way ANOVA and Bonferroni’s post-test). (f) Similar apical-to-basolateral transcytosis of WT by caco-2 cell monolayers grown in transwells incubated at higher WT concentrations at either apical pH 6.0 or pH 7.4 (n = 6 per group).
Fig. 6
Fig. 6
Crypt cell FcRn immunostaining in human small intestine using in house anti-FcRn (top row) was colocalized (arrows) with commercially available anti-FcRn (b), chromagranin A (d), GLP-1 (f), and GLP-2 (h) immunostaining in the adjacent section that were 5 μm apart. A high background-signal using anti-FcRn (H-274, Santa Cruz; b) hampered detection of specific FcRn immunostained cells, though two apparent matches to in-house anti-FcRn stained cells are indicated (a, b arrows). FcRn-immunoreactive cells (c) were less prevalent than chromagranin A stained cells (e), with some matches that suggest a colocalized sub-population (arrows). FcRn-immunostaining (e, g) overlaps extensively in GLP-1 (f) and GLP-2 (h) immunoreactive cells.
Fig. 7
Fig. 7
Crypt cell FcRn immunostaining and adjacent control rat IgG in banked colon from stillborn (a, b), cynomolgus monkey (c,d) and C57b6 mice (e,f). The intensity numbers of FcRn immunostained cells were the highest in neonatal human. Adult cynomolgus was similar to adult human. No FcRn reactivity was apparent in C57b6 mice throughout the intestine compared to rat IgG negative control and illustrated in proximal colon.
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