Similarly, genotypes AZD6244 A, D, E, and F have a one amino acid difference in the HBc18-27 peptide as compared to genotypes B and C (Fig. 1D). The high specificity of both TCR-Ls for the cognate HLA-peptide complex was evident. The presence of a lysine (K) instead of an arginine (R)

at position 187 of the HBs183-91 peptide (HBV genotypes B, E, F) abolished sTCR-L recognition. Note that, in contrast to sTCR-L antibodies, we have shown that HBs183-91-specific CD8T cells are able to recognize the 187K s183-91 peptide,11 demonstrating that the 187K s183-91 peptide is indeed bound to the HLA-A2* class I molecules on the surface of the cells. In addition, sTCR-L recognized the HBs183-91 HBV genotypes A, C, and D peptides only in the context of A0201, A0202, and A02011. The cTCR-L showed a broader HBV genotype recognition profile, because it also recognized the HBc18-27 peptide sequence with isoleucine (I) instead of valine (V) at position 27 (characteristic of HBV genotypes B and C) when presented by A*02:01, A*02:02, A*02:11. Such promiscuity of recognition may be explained by the fact that the V27I polymorphism affects an epitope anchor residue which resulted in weaker MHC-binding

ability, but does not effect TCR-recognition.12 AZD6738 Specific recognition was also detected when A*02:07 molecules presented either HBc18-27V or HBc18-27I peptides. However, this specific binding was weaker than the ones obtained with HLA- A*02:01, A*02:02, and A*02:11 as presenting molecules, and cTCR-L showed crossreactivity to nonpeptide pulsed target HLA- A*02:07 + cells. (Fig. 1D). The exquisite binding specificity of the two murine TCR-Ls suggests that they could be used to target IFNα to HBV-infected cells. For this purpose, we generated antibody fusion proteins ifoxetine in which mature human IFNα (without signal sequence) was linked to the C-termini of the two heavy chains of chimeric mouse-human TCR-L antibodies. The fusion proteins called TCR-L/IFNα were constructed by joining the

murine TCR-like antibody light and heavy chain variable domains (VL or VH) to the human kappa light chain constant region (CK) and the human γ-1 heavy chain constant region (CH1-Hinge-CH2-CH3), respectively. A flexible glycine-serine linker consisting of two Gly4Ser repeats was used for joining human mature IFNα2a to the penultimate C-terminal amino acid of the antibody heavy chain (heavy chain —LSPGGGGSGGGGS— IFNα2a). The last basic amino acid of the antibody heavy chain, a Lys, was removed in order to eliminate a potential cleavage site for endoproteinases with trypsin-like activity. This C-terminal Lys of an antibody heavy chain can be deleted without influencing antibody functions because this amino acid is cleaved off during secretion by cellular carboxypeptidases to a variable extent, as observed in many antibody preparations.