Enhancement of Polymeric Immunoglobulin Receptor Transcytosis by Biparatopic VHH

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

The European Convention for the Protection of Pet Animals and tail docking in dogs

The European Convention for the Protection of Pet Animals was opened for signature in Strasbourg on 13 November 1987 and entered into force on I May 1992. This Convention states that: 'Surgical operations for the purpose of modifying the appearance of a pet animal or for other non-curative purposes shall be prohibited and, in particular: the docking of tails'. At present, 15 of the 27 States in the European Union have ratified this Convention (with or without reserving their position on tail docking) and have prohibited cosmetic surgical operations. In addition, four European States have prohibited these operations, even though they did not ratify the Convention. These policy positions agree with both the current knowledge on tail amputations in dogs and the opinions of official veterinary associations in Europe and North America

Polymeric IgA binding to the human pIgR elicits intracellular signalling, but fails to stimulate pIgR-transcytosis.

The intracellular pathway of polymeric immunoglobulin receptor (pIgR) is governed by multiple signals that lead to constitutive transcytosis. In addition, in transfected polarized MDCK cells, polymeric immunoglobulin A (pIgA) binding stimulates rabbit pIgR-transcytosis, owing to phospholipase-C gamma 1 activation and increase of intracellular calcium. Transcytosis of rat pIgR across hepatocytes is similarly accelerated by pIgA injection. In contrast we show here that human Madrin-Darby Canine Kidney (pIgR)-transcytosis, in human Calu-3 and human pIgR-transfected MDCK cells, is not promoted by pIgA, as monitored by a continuous apical release of its secreted ectodomain. However, the incubation of cells expressing human or rabbit pIgR with pIgA induces a comparable IP3 production, and pIgR-transcytosis of either species is accelerated by the protein kinase C (PKC)-activator phorbol myristate acetate. Without pIgA, mimicking phospholipase-C activation by combining low concentrations of phorbol myristate acetate with ionomycin, or high concentrations of ionomycin alone, stimulates the rabbit, but not the human, pIgR transcytosis. These data suggest that the species difference in pIgA-induced pIgR-transcytosis does not stem from the defective production of second messengers, but from a different sensitivity of pIgR to intracellular calcium. Our results outline the danger of extrapolating to humans the abundant data obtained from mucosal vaccination of laboratory animals

Crucial role of J chain in receptor-mediated epithelial transport of polymeric IgA: in vivo and in vitro data

The role of J chain in epithelial polymeric IgA (pIgA)-transport, shown by Brandtzaeg in 1975, is evidenced by lack of binding of J chain-deficient pIgA to free secretory component (SC) and to surface membrane SC on HT-29 colon carcinoma cells. Also, antibodies (Abs) to J chain inhibit such binding of J chain-containing pIgA. These in vitro data needed confirmation by functional experimental test systems. Here, we show inhibition of receptor-media ted epithelial transport of pIgA By anti-J chain Abs, and lack of transport of J chain-deficient pIgA, by two functional tests: in vivo rat hepatobiliary transport of intravenously-injected human pIgA, and apical transport of pIgA by cultured SC-expressing Madin-Darby canine kidney (SC-MDCK) cells grown as confluent monolayers on permeable filters separating the apical from the basolateral medium. Rabbit Abs to J chain, but not normal rabbit IgG, incubated with purified human pIgA inhibited its rat biliary transport in a dose- and time-dependent manner: F(ab')2 and Fab' fragments of anti-J chain Abs also inhibited, excluding Fc gamma-dependent clearance and excessive size of the immune complexes. Abs to J chain also inhibited pIgA transport by SC-MDCK cells. In addition, of three purified tetrameric IgA (tIgA) myeloma proteins only the two that contained J chain by several criteria, readily combined with SC by various methods In the functional transport tests, only the same two pIgAs were actively transported. Altogether our functional data convincingly confirmed the crucial role of the J chain in the epithelial transport of pIgA

Lack of SC/pIgR-mediated epithelial transport of a human polymeric IgA devoid of J chain: in vitro and in vivo studies.

Three human polymeric IgA (pIgA) myeloma proteins of tetrameric size were compared for their J-chain content, their in vitro secretory component (SC)-binding ability, and their capacity to be transcytosed by polymeric immunoglobulin receptor (pIgR)-expressing epithelial cells in vitro and rat hepatocytes in vivo. One of the three pIgA preparations, pIgA-L, was shown to lack J chain and was unable to combine with purified free human and rat SC, whereas pIgA-G and pIgA-C contained J chain and combined readily with SC. Furthermore, pIgA-L was not transferred into rat bile after intravenous injection, and was hardly transported apically by polarized Madin-Darbey canine kidney cell monolayers expressing the human pIgR, whereas pIgA-G and pIgA-C were efficiently transported in both test systems. Together with our recent demonstration that antibodies to human J chain block the SC/pIgR-mediated epithelial transport of pIgA, these data unanimously confirm the proposed key role of J chain in the epithelial transport of polymeric immunoglobulins into exocrine secretions