4B). ISGs induction by cTCR-L/IFNα was similar to the one of IFNα in HLA-A*02-positive hepatocytes (1.9- and 1.6-fold lower) but was much lower (6.8- and 7.8-fold

lower) in HLA-A*02-negative HBV-infected hepatocytes. The addition of peptides did not have any significant effect (Fig. 4B). These results demonstrate the specific activity of TCR-L/IFNα on HBV-infected primary hepatocytes, confirming that expression of the correct HLA-HBV peptide complex is required for efficient induction of IFNα signaling and that this induction can be mediated by HLA-HBV peptide complexes generated in the context of natural infection. To further explore the mechanism of the targeted activation of IFNα-induced genes on cells

expressing R788 cell line the cognate HBV peptide/HLA complexes, we compared the binding of cTCR-L/IFNα and sTCR-L/IFNα, and of the native, unconjugated TCR-L antibodies, to cells expressing the respective ACP-196 clinical trial HBV-peptide/HLA-complexes. It was initially considered that the overall binding affinity of TCR-L/IFNα might be dominated by the IFNα part of the molecule because the affinities [as determined by surface plasmon resonance (SPR)] of the two TCR-Ls for their specific HBV-peptide/HLA complexes (cTCR-L Kd = 22 nM; sTCR-L Kd = 32 nM) were lower than the published affinity of the native unconjugated IFNα2a for its own high-affinity receptor (Kd = 5 nM).22 However, because it has been described that conjugation of IFNα to other molecules can substantially alter the affinity to its receptor,19 the binding of the conjugated molecules cTCR-L/IFNα and sTCR-L/IFNα to HepG2 cells stably transfected with HBV genotype D (HepG2-13) or untransfected HepG2 control cells was studied. Figure 5A shows that both cTCR-L/IFNα and sTCR-L/IFNα fusion proteins bound specifically to the HBV-transfected cell line this website similarly or better than the corresponding unconjugated TCR-L antibodies. Similar specific binding was also seen using HBV peptide-pulsed HepG2 cells or other HLA-A*02+ cell lines, whereas there was no staining observed using

a control IgG1/IFNα fusion protein or using cell lines not expressing HLA-A*02 (data not shown). To further assess the binding specificity of the cTCR-L/IFNα and sTCR-L/IFNα fusion proteins, we analyzed their ability to bind specifically to target cells expressing HBV epitopes in a mixed HBV-positive and HBV-negative cell population. For this purpose, HBV-producing HepG2-13 and the parental HepG2 cells were labeled with two different concentrations of CSFE, mixed in a 1:1 ratio, and then stained with cTCR-L/IFNα fusion proteins. Binding of fusion protein to the target was measured by flow cytometry using a fluorescent antihuman IgG1 secondary antibody. Indeed, cTCR-L/IFNα fusion proteins bound preferentially to HBV-producing target cells (HepG2-13), whereas binding to parental cells was negligible (Fig. 5B).