Characterization of recombinant human monoclonal antibodies against the Bile Salt Export Pump

Abstract

Background: Inhibitory, Bile Salt Export Pump (BSEP)-reactive IgG-class antibodies cause phenotypic recurrence of BSEP deficiency in some patients after liver transplantation for Progressive Familial Intrahepatic Cholestasis Type 2 (PFIC-2). A detailed characterization of these disease-causing antibodies is key to elucidate the molecular mechanism of this emerging disease. Methods: BSEP-reactive B cells were isolated from peripheral blood of a patient with a high anti-BSEP titer by fluorescence-activated single cell sorting. Corresponding full-length immunoglobulin heavy and light chain variable region gene transcripts were amplified by single cell RT-PCR and cloned into eukaryotic expression vectors to enable the in vitro production of the respective monoclonal antibodies. These monoclonal antibodies were characterized by immunofluorescence studies of rat liver slices and transiently transfected cells, Western blot, FACS analysis, in situ rat liver perfusion and in vitro BSEP transport assays. Results: Immunofluorescent staining of fixed, transiently transfected human embryonal kidney (HEK293) cells identified several BSEP-reactive antibodies that also stained canalicular patterns on fixed rat liver slices. In contrast, only a few recognized denatured rat BSEP on Western blots, while all antibodies detected its denatured human homologue. FACS analysis of antibody-treated, unpermeabilized cells transiently expressing BSEP revealed BSEP-specific staining that was absent on empty control cells. BSEP-reactive antibodies reached the canalicular space of in situ perfused rat livers within two hours. Finally, in vitro vesicular transport assays showed clone-dependent influence on transport activity. Conclusion: We provide characterization of a novel set of patient-derived, monoclonal antibodies directed against extracellular features of BSEP. These include species that are BSEP-inhibitory, -neutral, and, strikingly, -stimulatory in vitro. The antibodies and their monovalent Fab derivatives may have great potential in applied research as well as in future clinical applications.