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Enhancement of polymeric immunoglobulin receptor transcytosis by biparatopic VHH.

ABSTRACT: The polymeric immunoglobulin receptor (pIgR) ensures the transport of dimeric immunoglobulin A (dIgA) and pentameric immunoglobulin M (pIgM) across epithelia to the mucosal layer of for example the intestines and the lungs via transcytosis. Per day the human pIgR mediates the excretion of 2 to 5 grams of dIgA into the mucosa of luminal organs. This system could prove useful for therapies aiming at excretion of compounds into the mucosa. Here we investigated the use of the variable domain of camelid derived heavy chain only antibodies, also known as VHHs or Nanobodies®, targeting the human pIgR, as a transport system across epithelial cells. We show that VHHs directed against the human pIgR are able to bind the receptor with high affinity (?1 nM) and that they compete with the natural ligand, dIgA. In a transcytosis assay both native and phage-bound VHH were only able to get across polarized MDCK cells that express the human pIgR gene in a basolateral to apical fashion. Indicating that the VHHs are able to translocate across epithelia and to take along large particles of cargo. Furthermore, by making multivalent VHHs we were able to enhance the transport of the compounds both in a MDCK-hpIgR and Caco-2 cell system, probably by inducing receptor clustering. These results show that VHHs can be used as a carrier system to exploit the human pIgR transcytotic system and that multivalent compounds are able to significantly enhance the transport across epithelial monolayers.

SUBMITTER: Emmerson CD

PROVIDER: S-EPMC3194793 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:High expression of the polymeric immunoglobulin receptor (PIGR) has previously been associated with a favourable prognosis in a few cancer forms, but its expression and relationship with clinical outcome in epithelial ovarian cancer (EOC) has not yet been reported. The aim of this study was therefore to examine the clinicopathological correlates and prognostic significance of PIGR expression in EOC. METHODS:After an initial screening in the Human Protein Atlas portal, a validated antibody was selected for extended analysis of immunohistochemical PIGR expression in tissue microarrays with tumours from 154 incident cases of EOC from two pooled prospective population-based cohorts. Subsets of corresponding benign-appearing fallopian tubes (n?=?38) and omental metastases (n?=?33) were also analysed. Kaplan-Meier analysis and Cox regression analysis were applied to examine the impact of PIGR expression on overall survival (OS) and ovarian cancer-specific survival (OCSS). RESULTS:PIGR expression was significantly higher in fallopian tubes compared to primary tumours and metastases (p?<?0.001) and lower in carcinoma of the serous subtype compared to other carcinomas (p?<?0.001). PIGR expression was significantly associated with lower grade (p?=?0.001), mucinous histological subtype (p?=?0.002), positive progesterone receptor expression (p?=?0.009) and negative or low Ki-67 expression (p?=?0.003). Kaplan-Meier analysis revealed a significantly improved OS (p?=?0.013) and OCSS (p?=?0.009) for patients with tumours displaying high expression of PIGR. These associations were confirmed in unadjusted Cox regression analysis (HR?=?0.48; 95% CI 0.26-0.87; p?=?0.015 for OS and HR?=?0.43, 95% CI 0.22-0.82; p?=?0.011 for OCSS) but did not remain significant after adjustment for age, grade and clinical stage. CONCLUSIONS:This study provides a first demonstration of PIGR expression in human fallopian tubes, primary EOC tumours and metastases. High tumour-specific expression of PIGR was found to be associated with a favourable prognosis in unadjusted, but not in adjusted, analysis. These findings are novel and merit further investigation.

Project description:1. A gamma camera was used to monitor continuously the uptake of radiolabelled polymeric immunoglobulin A (pIgA) into the rat body after intravenous injection. Uptake into liver was fast but, since the peak of liver labelling occurred only after 9-15 min, it was not sufficiently rapid to constitute a pulse dose. A perfused, isolated rat liver system was therefore established which could be given a single pass dose of pIgA; a variety of tests showed such livers remained viable for at least 3 h and could be subsequently fractionated on Ficoll and Nycodenz gradients with normal distributions of marker enzymes. 2. Subcellular fractionation at different times after a single pass dose of pIgA showed that whilst pIgA appeared sequentially in sinusoidal plasma membrane, light endosomes, dense endosomes, very dense endosomes and lysosomes as in vivo, the predominance of pIgA in the light endosome compartment disappeared much earlier than after injection in vivo of pIgA, presumably because this compartment was not being continuously loaded over the first 10-15 min. The time course of appearance of label in bile was unchanged. A large excess of unlabelled asialofetuin did not change these patterns, indicating that the asialoglycoprotein receptor was not involved. 3. Low doses of the microtubule agent colchicine reduced the proportion of pIgA reaching the bile, but subcellular fractionation of treated liver showed that distribution of label amongst liver fractions was little changed, although the overall liver pIgA content had increased. This would suggest that pIgA did not remain in the common compartment which could have supplied bile or lysosomes but rather flowed out of it as rapidly as in untreated liver but towards those compartments supplying the lysosomes. 4. Experiments with nocodazole, which reversibly disrupts microtubules, showed that very little of the pIgA taken into an inhibited liver appeared in the bile after nocodazole was removed 30 min later, even though a second dose of pIgA, given after nocodazole removal, appeared in bile with a normal time course. The first dose of pIgA must therefore have passed beyond the compartments competent to supply the bile before nocodazole was removed. Such compartments were undamaged since the second dose of pIgA appeared in bile normally. We therefore conclude that the bulk of pIgA must be supplied to the bile from light or dense endosomes rather than from very dense endosomes and lysosomes.

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Enhancement of polymeric immunoglobulin receptor transcytosis by biparatopic VHH.

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