Newborn humans rely on maternally-derived antibodies for protection against pathogens. IgG is transported across the placenta to the fetus. After birth lgA in the milk and colostrum protects the GI tract, but these antibodies are not specifically absorbed into the infants circulation. The purpose of this proposal is to develop a general method by which ingested antibodies can be absorbed across the intestine and into the circulation of the infant. Intestinal epithelial cells express the polymeric immunoglobulin receptor (pIgR), which transport dimeric IgA from the basolateral to the apical surface of the epithelial cell. At the apical surface the pIgR is proteolytically cleaved and the extracellular domain, known as secretory component (SC), is released into the intestinal lumen. Cleavage is not very rapid, so there is a pool of uncleaved pIgR at the apical surface. We have shown that ligands can bind to this pIgR, and are internalized and transcytosed to the basolateral surface. We plan to optimize this process for absorption of ingested antibodies across the intestinal epithelium and into the circulation. Our specific goals will be: 1). We will prepare antibodies against the extracellular, membrane- proximal """"""""stalk"""""""" of the pIgR. These antibodies may prevent cleavage of the pIgR to SC and/or bind to the stalk that remains after cleavage. In either case, the antibodies should be efficiently taken up by the pIgR at the apical surface. Fab fragments of these antibodies will be used to study transcytosis from the apical to the basolateral surface. Fab fragments of antibodies of varying affinity will be used to optimize transcytosis. 2). These Fab-anti-stalk (Fab-AS) will be conjugated to Fab fragments of a model anti-pathogen antibody. Transcytosis of these conjugates will be analyzed. 3). These conjugates will also be linked to transferrin. The transferrin can bind to the transferrin receptor (TR) in endosomes and promote transcytosis to the basolateral surface. 4). For use in human recombinant antibodies against the stalk region of human pIgR will be selected by a phage display approach: Single chain Fy (scFv) fragments be genetically fused to scFv fragments of the anti-pathogen antibody and an anti-TR antibody.
| Low, S H; Roche, P A; Anderson, H A et al. (1998) Targeting of SNAP-23 and SNAP-25 in polarized epithelial cells. J Biol Chem 273:3422-30 |
| Low, S H; Chapin, S J; Wimmer, C et al. (1998) The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells. J Cell Biol 141:1503-13 |
| Altschuler, Y; Barbas, S M; Terlecky, L J et al. (1998) Redundant and distinct functions for dynamin-1 and dynamin-2 isoforms. J Cell Biol 143:1871-81 |
| Reich, V; Mostov, K; Aroeti, B (1996) The basolateral sorting signal of the polymeric immunoglobulin receptor contains two functional domains. J Cell Sci 109 ( Pt 8):2133-9 |
