Peptides Derived From Mutated BCR-ABL Elicit T Cell Immunity In CML Patients

Abstract 887 Over 20 BCR-ABL mutations have been identified that result in imatinib resistance and relapse of chronic myelogenous leukemia (CML). CML is highly responsive to immunological manipulations and we therefore hypothesized that mutated BCR-ABL-derived peptides could serve as immunogenic tumor-specific targets. Herein, we present a multi-step strategy for identifying tumor-specific T cell epitopes generated from gene mutation. We first investigated whether peptides derived from 24 frequent BCR-ABL mutations could potentially bind 8 common class I MHC molecules by applying the well-validated prediction servers IEDB and NetMHC to tiled 9- and 10-mers around each mutation. More than 60 peptides were predicted to bind to one or more of the following alleles with IC50 IC50>500). From IEDB, 9 of 61 (15%) were predicted as high, 35 (57%) as intermediate and 17 (38%) as weak binders. 24 of 84 mutated peptides (29%) and 24 of 61 mutated peptides (39%) were predicted as binding with at least two-fold higher affinity compared to parental peptides, using NetMHC and IEDB, respectively. These predictions indicated that cells from 7 of 9 imatinib-resistant CML patients had the potential to present at least one mutated BCR-ABL derived peptide by binding autologous HLA alleles (with IC50<1000). CML cells from 3 of the 5 patients had an E255K mutation and expressed HLA-A3, and were predicted to generate two promising candidate peptides: E255K-A247-255 (KLGGGQYGK, IEDB IC50=113; NetMHC IC50=192) and E255K-B255-263 (KVYEGVWKK, IEDB IC50=29; NetMHC IC50=28). Both peptides were predicted to bind HLA-A*0301 at least ten-fold more tightly than parental peptides. Using a competitive MHC binding assay, we confirmed that E255K-A and –B were high binders with IC50 scores of 208nM and 17nM, respectively and that they both had at least ten-fold fold greater affinity than parental peptides. In addition, E255K-B also bound to the other HLA-A3 superfamily members HLA-A*1101, HLA-A*3001, HLA-A*3101, HLA-A*6801 (IC50: 39–603nM). We next successfully generated T cell lines against E255K-B but not E255 K-A from a normal HLA-A3+ donor that demonstrated greater specificity against the mutated peptide (2330±325 SFC/million cells, by IFNγ-ELISPOT) than the parental peptide (1270±42 SFC/million cells). E255K-B is endogenously processed and presented since E255K-B reactive T cells also responded to HLA-A3+ antigen-presenting cells (APCs) that were transfected with a minigene encompassing 227 base pairs surrounding the E255K mutation (1900±85 SFC/million cells). Finally, we assessed the potential for E255K-B to stimulate T cell responses in CML patients. E255K-B elicits T cell immunity in vivo in that we could detect antigen-specific CD8+ T cell reactivity from two HLA-A3+ CML patients bearing the E255K mutation by IFNγ-ELISPOT and by antigen-specific tetramer staining. T cell responses could be abrogated in the presence of class I blocking antibody w6/32. For both patients, reactive T cells were polyfunctional, expressing GM-CSF, IP10 and TNFα in response to APCs pulsed with mutated peptide or expressing the E255K minigene. For Patient 2, E255K reactivity developed only in the setting of donor-derived engraftment following curative allogeneic stem cell transplantation. Further analysis to explore the kinetics of mutated peptide-specific immunity in relationship to burden of mutation-expressing leukemia cells is in progress. Our studies have demonstrated that leukemia-driven genetic alterations can provide immunogenic tumor-specific antigen targets associated with clinical response in vivo . Importantly, even though BCR-ABL mutations generate resistance to imatinib, they also create novel epitopes that can be effectively recognized by cytotoxic CD8+ T cells. Our studies support the development of specific, nontoxic and personalized vaccination strategies for eradication of drug-resistant CML. Disclosures: No relevant conflicts of interest to declare.