We thus analyzed whether TCR-L/IFNα could increase the effect of HBV-specific CD8T cell recognition. We utilized HepG2 cells as target cells and HBV-specific CD8T cells as effectors and tested the effect of TCR-L/IFNα on CD8T activation (IFNγ production) as well as the effect on target cells (secretion IFNγ inducible chemokines CXCL-9 and CXCL-10). To avoid competition between

TCR-L/IFNα and HBV-specific CD8T cells for the identical HLA-class I/HBV peptide complexes, we tested the effect of cTCR-L/IFNα (specific for HBc18-27/A*02:01) on HBs183-91-specific CD8T cells by using HepG2 cells pulsed with both HBs183-91 SP600125 nmr and HBc18-27 peptides. Figure 6C shows that the CD8T cell function was neither affected by the presence of cTCR-L/IFNα nor by an IgG1/IFNα control (Fig. 6C, CD8) This is consistent with the minor effect of cTCR-L/IFNα on HLA-class I expression in HepG2. However, by measuring the concentration of chemokines Ribociclib molecular weight present in the supernatants under different experimental conditions, we could demonstrate that TCR-L/IFNα induces specific alteration of target cell responsiveness. Despite identical HBV-specific CD8T activation, chemokine production by target was increased specifically by cTCR-L/IFNα but not by control IgG1/IFNα (Fig. 6C). Importantly, the fusion proteins did not activate chemokine production without concomitant CD8T cell activation.

The ability of TCR-L/IFNα RVX-208 to increase chemokine production on specific target cells was further investigated by incubating IFNγ-treated HepG2 cells with sTCR-L/IFNα and analyzing their CXCL-10 production. Only HBs183-91 pulsed cells incubated with sTCR-L/IFNα displayed an increase in CXCL-10 production (Supporting Fig. 3). In this work we demonstrate that TCR-L antibodies can be used to deliver a cytokine selectively to HBV-infected cells. IFNα was chosen as a proof-of-concept

therapeutic molecule for a number of reasons. IFNα has been used for many years for the treatment of patients with various cancers or viral diseases. In addition, IFNα has demonstrated efficacy in clearing HBV infection with evidence for both direct antiviral and immunomodulatory effects. We found that genetic fusion of IFNα to TCR-L altered the biological activity of IFNα, resulting in a molecule that maintains its full IFNα activity only on cells expressing the correct HBV-peptide HLA-complex. Linking IFNα to other molecules (like Peg or albumin) has been previously described to reduce its biological activity substantially, which might be due to steric hindrance that prevents the binding of the cytokine to its receptor.18, 19 Our data are consistent with previous reports of conjugation impact on intrinsic IFNα activity but also show that specific binding of TCR-L/IFNα to target cells through recognition of the cognate HBV peptide/HLA complex can unmask the full biological activity of the IFNα on the target cells.