XBP-1 a Selective and Specific Target for Immunotherapy in Myeloma.

X-box binding protein 1 (XBP-1) is a transcription factor is essential for the differentiation of plasma cells and the unfolded protein response. XBP-1 is significantly up-regulated in myeloma cells compared to both normal plasma cells and B-cells. Selective and specific requirement of XBP-1 for differentiation of B cells to plasma cells and its further up-regulation in multiple myeloma (MM) cells makes it a promising target for immunotherapy directed at MM. We have evaluated XBP-1 as a target antigen to develop MM-specific immunotherapy. In order to generate XBP-1 antigen-specific cytotoxic T lymphocytes (CTLs), we have identified HLA-A2-specific peptides derived from non-spliced (short form) and spliced (long form) XBP-1 proteins. We have further modified the peptide by altering one amino acid, which is critical for HLA-A2 affinity, to obtain epitope, which has higher stability to HLA-A2 clefts. The modified XBP-1 peptides were able to evoke higher levels of IFN-g release compared to native peptides and were able to induce CTLs highly specific to MM, demonstrated by Calcein-released cytotoxicity assay. Cytotoxic activity of these XBP1 peptide-specific-CTLs against U266, a HLA-A2⁺/XBP-1⁺ MM cell line was 60% and 79% by CTLs stimulated by non-spliced peptide and 69% and 88% by CTLs stimulated by spliced peptide at Effector:Target ratios of 20:1 and 60:1, respectively. The CTLs did not lyse HLA-A2⁻ or XBP-1⁻ cells. These results were further confirmed by the novel CD107 cytotoxicity assay, which detects MM-specific cytolytic CD8⁺ T cells by flow-cytometric analyses. The CTLs generated with XBP-1 peptide displayed distinct phenotypes, showing high CD69⁺CD45RO⁺ cell population (62% in non-spliced peptide-CTLs, 67% in spliced peptide-CTLs, 4% in unstimulated control-CTLs) and CD45RA⁺CCR7⁺ cell population (1% in non-spliced peptide-CTLs, 1% in spliced peptide-CTLs, 24% in unstimulated control-CTLs). In conclusion, we report the identification of highly immunogenic heteroclitic XBP-1 epitopes that have ability to generate MM-specific CTLs. Confirmation of in vivo activity of these CTLs in SCID mouse model is currently underway prior to its evaluation in clinical studies.