The contribution of cell surface FcRn in monoclonal antibody serum uptake from the intestine in suckling rat pups

Abstract

The neonatal Fc receptor (FcRn) in intestinal epithelium is the primary mechanism for transfer of maternal immunoglobulin G (IgG) from suckled milk to serum; but the factors contributing to the rapid uptake of IgG are poorly understood. These studies help to determine the contribution of cell surface FcRn in IgG uptake in 2-week-old rat pups by varying local pH and binding conditions. Variants of a human wild-type (WT) IgG monoclonal antibody (mAb WT) were assessed for binding affinity (KD) to rat (r)FcRn at pH 6.0 and subsequent off-rate at pH 7.4 (1/s) by surface plasmon resonance. Selected mAbs were administered intra-intestinally in isoflurane-anesthetized 2-week rat pups. Full length mAb in serum was quantified by immunoassay, (r)FcRn mRNA expression by reverse transcription polymerase chain reaction, and mAb epithelial localization was visualized by immunohistochemistry. After duodenal administration, serum levels of mAb variants correlated with their rFcRn off-rate at pH 7.4, but not their affinity at pH 6.0. The greatest serum levels of IgG were measured when mAb was administered in the duodenum where rFcRn mRNA expression is greatest, and was increased further by duodenal administration in pH 6.0 buffer. More intense human IgG immunostaining was detected in epithelium than the same variant administered at higher pH. These data suggest an increased contribution for cell surface receptor. We conclude that, in the neonate duodenum, receptor off-rates are as important as affinities for FcRn mediated uptake, and cell surface binding of IgG to rFcRn plays contributes to IgG uptake alongside pinocytosis; both of which responsible for increased IgG uptake.

Keywords: FcRn; IgG; bioavailability; cell surface receptor; uptake.

FIGURE 1

Surface plasmon resonance (SPR) analysis using the ProteOn XPR36 in co-injection mode IgG1 variants, (A) wild-type (WT); (B) N434A or (C) N434Y were diluted 1:5 in a concentration series from 1000 to 1.6 nM, injected over rat FcRn for 1 min in PBS-TE (pH 6.0) and followed immediately by a co-injection of PBS-TE (pH 7.4) without mAb for 3 min.

FIGURE 2

Serum levels of IgG correlate with rat FcRn pH 6.0 to 7.4 off-rates: (A) Serum levels (ng/mL) of IgG1 variants (1 mg/kg) 90 min after intra-duodenal administration in sucking rat pups (n = 5) were highest for N434A andWT and low as expected for H435A, with N434Y similar to H435A. One-way Analysis of variance (ANOVA) statistics were carried out followed by a Bonferroni’s multiple comparison test versus WT. Serum levels were plotted against (B) Kd affinity constants (nM) to rat FcRn at pH 6.0 (r 2 = 0.43; P = 0.54); and (C) dissociation from FcRn (1/s) at pH 7.4. after pre-binding at pH 6.0 (r 2 = 0.99; P = 0.07). Key: □WT; ▪ N434Y; × N434A; and ▼ H435A. The H435A has low > 1000 nM binding affinity to FcRn, and its points are only added to (B) and (C) to show the trend. All statistics carried out excluded the H435A points to avoid bias.

FIGURE 3

Greatest serum levels of IgG measured when administered at site with greatest rat FcRn mRNA expression: (A) Serum levels of the WT mAb were measured after 90 min following administration at the proximal small intestine (S.I; duodenal region) and at the distal S.I (ileum region) in suckling rat pups (n = 6). A one-way ANOVA followed by Bonferroni’s multiple comparison test was carried out versus respective WT. (B) mRNA expression of rat FcRn, assessed using reverse transcription polymerase chain reaction (RT-PCR), from mucosal scrapings of proximal and distal small intestine, and proximal and distal colon. Data shown is fold change over proximal colon which had the lowest level of FcRn mRNA).

FIGURE 4

The effect of pH (pH 6.0, 7.4, and 8.0.) of high and low FcRn-affinity IgG variants on serum uptake: Serum levels (ng/mL) at 90 min after administration of N434A tended to be higher following intra-duodenal administration (0.01 mg/kg) in suckling rat pups (n = 6). There was no difference in the serum levels of H435A administered in different pH conditions. One-way ANOVA Statistical tests carried out compared to N434A pH 6.0.

FIGURE 5

Increased levels of IgG bound to enterocytes at pH 6.0 compared to pH 8.0: Representative examples of IgG –immuno-reactivity in enterocytes of suckling rat pup proximal small intestine 90 min after administration of (A) PBS, (B) IgG1 N434A at pH 6.0, (C) IgG1 N434A at pH 8.0, and (D) IgG1 H435A at pH 8.0. Images on the left-hand side are using rabbit anti-human IgG, and the images on the right are using the anti-rabbit IgG to show specificity. Images are at 10× magnification with the exception of the inserted image in (B) which is 60× magnification.