Abstract

Imatinib mesylate is now standard treatment for chronic myelogenous leukemia. In spite of its high efficacy in controlling the disease, residual bcr-abl cells measured by RT-PCR are still detectable in more than 95% of the patients treated with this drug. In addition, recent reports describe the emergence of resistance to Imatinib mesylate. A major mechanism for the appearance of resistance, are point mutations changing amino acids in the bcr-abl tyrosine kinase that make imatinib ineffective. We hypothesized that some bcr-abl kinase mutations selected by imatinib would generate new immunogenic peptide-epitopes that could be recognized by the human immune system. For this purpose, we selected with predictive computer algorithms potentially immunogenic mutated amino acid sequences and synthetic analogues of mutated bcr-abl kinase sequences to study for immunogenicity and anti-leukemia activity in an in vitro human system. Initially we designed a number of synthetic peptides derived from the most frequent bcr-abl kinase mutations induced by imatinib in which single amino acid substitution was introduced at different MHC-binding positions. We investigated the most common mutations: E255V, E255K, Y253H, Y253F, F311L, T315I, M351T and H396P. Specific single and combined mutations improved the predicted binding of peptides to HLA A0201. Further additional single amino-acid substitutions on mutated peptides generated high predicted binding. Selected sequences were evaluated for eliciting MHC-restricted, peptide-specific CTL responses in an in vitro model using T-cells from different HLA A0201 healthy donors after stimulation with monocyte-derived DC. CTL lines were assessed by either IFN gamma ELISPOT or by a chromium51 release assay. Mutated derived peptides from E255K, E255V, Y253F and T315I were shown to be immunogenic in vitro by the specific release of IFN gamma. Furthermore, single amino-acid substitutions in key residues improved the immunogenicity of these peptides. Finally, T cell lines generated with optimized peptides derived from E255K and T315I (the most common imatinib-induced mutations) were able to kill peptide pulsed targets; more important, these T cell lines cross-reacted with the mutated peptides (a heteroclitic response). In conclusion, Imatinib-selected mutations of peptides from bcr-abl kinase have a better predicted binding than native kinase sequences. Selected peptides from the most common mutations are immunogenic in vitro in a HLA A0201 restricted manner. T cells generated with optimized peptides from these mutations are able to elicit heteroclitic peptide-specific cytotoxicity.

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