Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125 I-HA at 37°C by rat liver sinusoidal endothelial cells.125 I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 g/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues.125 I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.After Meyer and Palmer (1) discovered hyaluronan (HA), 1 it was found to be a component of essentially all vertebrate extracellular matrices (ECMs). Fibroblasts, keratinocytes, chondrocytes, and other cells continuously synthesize and secrete HA, which is a linear polymer with a native molecular mass that may exceed 10 7 Da and is composed of the repeating disaccharide 2-deoxy,2-acetamido-D-glucopyranosyl-␤ (1, 4)-D-glucuronopyranosyl-␤ (1, 3). Despite its simple structure, HA is involved in many cell functions including migration, differentiation, and phagocytosis (2-6). HA is important in development (4, 7), wound healing (8, 9), angiogenesis (10, 11), and tumor growth and metastasis (12, 13). Although previously believed to be only a structural component in the ECM, HA is now also recognized as an active cell-signaling molecule. Some cell types show distinct physiological responses to HA of different sizes. In particular, some cell types respond physiologically to very small, but not large, HA. Small HA oligosaccharides containing 14 -20 sugars stimulate angiogenesis by endothelial cells (10,11,14), induce gene expression in activated macrophages (15), and induce NO synthase expression in sinusoidal LECs and Kupffer cells, but not hepatocytes or stellate cells (16). Cell surface HA receptors identified to date include CD44, RHAMM (CD168), ICAM-1 (CD54), LYVE-1 (5), and an endocytic receptor that is specific for HA and chondroitin sulfate. This latte...

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Six new placental alkaline phosphatase monoclonal antibodies (MAbs) have been evaluated in order to select a potentially clinically useful antibody fragment for use in immunoxintigraphy or therapy. Initially, 3 antibodies were identified by trial pepsin digestion as likely to give satisfactory F(ab')2 yield. The corresponding intact antibodies were then compared for ability to localise human xenograft tumours in athymic mice. Of the best of these, designated 3F6, F(ab')2 and Fab fragments were then evaluated in similar xenograft experiments. In intact antibody biodistribution comparisons, 3F6 showed good tumour retention and satisfactory specificlnon-specific ratios at 8 days. In similar fragment biodistribution experiments 3F6 F(ab')2 gave the highest tumourlblood ratio (10) and tumourlorgan ratios (19) and the best specificlnon-specific localisation. This fragment also showed higher absolute uptake in the tumour than intact antibody, 18.9% and 14.40/, respectively of the injected dose. As expected, fragments showed much faster blood clearance rates than whole antibody. For Fab the in vivo instability by 6 hr was also demonstrated.