Generation of a monoclonal human single chain antibody fragment to hepatic stellate cells--a potential mechanism for targeting liver anti-fibrotic therapeutics.
ABSTRACT Hepatic stellate cells are pivotal to fibrogenesis in the liver and many potential anti-fibrotic therapeutics are required to act on targets within hepatic stellate cells. The aim of this study was to generate a human antibody fragment to hepatic stellate cells.
Phage display was used to generate a human monoclonal antibody fragment to a peptide sequence present on an extracellular domain of synaptophysin, a protein expressed on the surface of hepatic stellate cells.
An antibody fragment was isolated (termed C1-3), expressed in bacteria and purified. Fluorescently-labelled C1-3 antibody associated with human hepatic stellate cells but not hepatocytes in culture. Binding of fluorescently labelled C1-3 to hepatic stellate cells was blocked by the extracellular synaptophysin peptide sequence and uptake of the antibody intracellularly was inhibited by monensin. The toxin tributyl tin-when conjugated to C1-3-retained the ability to kill hepatic stellate cells confirming that C1-3 is sequestered intracellularly.
This antibody fragment may be an effective means to target therapeutics to human hepatic stellate cells.
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ABSTRACT: Chronic liver disease results in a liver-scarring response termed fibrosis. Excessive scarring leads to cirrhosis, which is associated with high morbidity and mortality. The only treatment for liver cirrhosis is liver transplantation; therefore, much attention has been directed toward therapies that will slow or reverse fibrosis. Although anti-fibrogenic therapies have been shown to be effective in experimental animal models, licensed therapies have yet to emerge. A potential problem for any anti-fibrogenic therapy in the liver is the existence of the body's major drug metabolising cell (the hepatocyte) adjacent to the primary fibrosis-causing cell, the myofibroblast. This article reviews the development of a human recombinant single-chain antibody (scAb) that binds to the surface of myofibroblasts. This antibody binds specifically to myofibroblasts in fibrotic mouse livers. When conjugated with a compound that stimulates myofibroblast apoptosis, the antibody directs the specific apoptosis of myofibroblasts with greater specificity and efficacy than the free compound. The antibody also reduces the adverse effect of liver macrophage apoptosis and-in contrast to the free compound-reversed fibrosis in the sustained injury model used. These data suggest that specifically stimulating the apoptosis of liver myofibroblasts using a targeting antibody has potential in the treatment of liver fibrosis.Hepatology International 01/2009; 2(4):405-15. · 2.64 Impact Factor
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ABSTRACT: Pregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16alpha carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) - which may be identical or associated with the progesterone receptor membrane component 1 (PGRMC1) - in mediating this anti-fibrogenic effect has been examined, since binding of dexamethasone to the LAGS in liver microsomal membranes has previously been shown to be inhibited by PCN. Quiescent rat and human hepatic stellate cells (HSC) were isolated from livers and cultured to generate liver myofibroblasts. HSC and myofibroblasts expressed PGRMC1 as determined by RT-PCR and Western blotting. Quiescent rat HSC also expressed the truncated HC5 variant of rPGRMC1. Rat PGRMC1 was cloned and expression in COS-7 cells gave rise to specific binding of radiolabelled dexamethasone in cell extracts that was inhibited by PCN, suggesting that PGRMC1 may be identical to LAGS or activates LAGS binding activity. Liver microsomes were used to screen a range of structurally related compounds for their ability to inhibit radiolabelled dexamethasone binding to rat LAGS. These compounds were also screened for their ability to activate rat and human PXR and to inhibit rat HSC-to-myofibroblast trans-differentiation/proliferation. A compound (4 androstene-3-one 17beta-carboxylic acid methyl ester) was identified which bound rat LAGS with high affinity and inhibited both rat and human HSC trans-differentiation/proliferation to fibrogenic myofibroblasts without showing evidence of rat or human PXR agonism. However, despite potent anti-fibrogenic effects in vitro, this compound did not modulate liver fibrosis severity in a rat model of liver fibrosis. Immunohistochemical analysis showed that rat liver myofibroblasts in vivo did not express rPGRMC1. LAGS ligands inhibit HSC trans-differentiation and proliferation in vitro but show little efficacy in inhibiting liver fibrosis, in vivo. The reason(s) for this disparity is/are likely associated with an altered myofibroblast phenotype, in vitro, with expression of rPGMRC1 in vitro but not in vivo. These data emphasize the limitations of in vitro-derived myofibroblasts for predicting their activity in vivo, in studies of fibrogenesis. The data also demonstrate that the anti-fibrogenic effects of PCN in vivo are likely mediated entirely via the PXR.Comparative Hepatology 06/2009; 8:1. · 1.88 Impact Factor