The selective detection of IFNα biological activity on cells expressing the cognate HBV peptide/HLA-complex is thus the direct consequence of the exclusive targeting of IFNα to cells expressing the cognate HBV-peptide/HLA-complexes, whereas IFNα biological activity on cells not expressing the cognate complexes is dramatically reduced by the covalent attachment of IFNα to the antibodies. The therapeutic efficacy of IFNα treatment is linked to its antiviral and immunomodulatory effects. We tested whether our TCR-L/IFNα selleck kinase inhibitor could specifically induce antiviral and immunomodulatory

effects. The antiviral effector function of TCR-L/IFNα was measured using a system in which HepG2 cells were transfected with the entire HBV genome. These HBV-transfected HepG2 cells secrete HBsAg that is quantifiable in the supernatants of the cells Nutlin-3 mw 4-5 days after transfection. In the experiment shown in Fig. 6A, HepG2 cells were treated with the indicated concentrations of either cTCR-L/IFNα ± HBc18-27 peptide, or IFNα 6 hours after transfection with the HBV construct. On day 4 supernatants were collected and the respective concentrations of HBsAg were quantified by enzyme-linked immunosorbent assay (ELISA). Although IFNα significantly reduced the amount of HBsAg secreted into the cell culture medium, the addition of cTCR-L/IFNα fusion protein did not have any effect

on HBsAg production by the transfected cells. However, if the same cells were pulsed with HBc18-27 peptide at the time of cTCR-L/IFNα treatment, significant inhibition of HBsAg secretion was observed, similar to the levels detected

with the control IFNα molecule (Fig. 6A). Under these experimental conditions (HBV transfection) therefore, the expression of endogenous HBV peptide may have not been sufficient Methocarbamol to allow adequate levels of TCR-L binding. Nevertheless, these results show that the cTCR-L/IFNα fusion molecule possesses an HBV-specific antiviral effect, dependent on the level of HBV peptide/HLA-complexes on the surface of target cells. We also analyzed whether immunomodulatory functions directly mediated by IFNα could be directed toward the specific target cells. First, we tested whether TCR-L/IFNα was able to up-regulate HLA-class I molecules or NK-cell costimulatory molecules MICA and MICB.3 Experiments using HBV-transfected or parental HepG2 cell lines showed only a minimal increase of HLA-class I, MICA and MICB expression, and only in the presence of very high concentrations of TCR-L/IFNα (1 nM, data not shown). Using a different cell line of hepatic origin (PLC), an HBsAg+ hepatocellular carcinoma line that can be recognized by HBV-specific CD8T cells,23 we observed the ability of TCR-L/IFNα to slightly up-regulate HLA-class I expression in an antigen-specific fashion and at low concentrations (50 pM). No changes of MICA and MICB expression were observed in these cell lines at all concentrations tested (Fig. 6B).