Identifying Leukemia-Specific Neoepitopes from Next-Generation Sequencing Data to Develop Targeted Immunotherapy for Pediatric Acute Myeloid Leukemiaig

Abstract

Although allogeneic hematopoietic stem cell transplantation (HCT) can cure patients with chemotherapy-refractory acute myeloid leukemia (AML), post-HCT relapse remains common and extremely challenging to resolve. T cell immunotherapy could augment HCT efficacy by inducing deeper remissions pre-HCT to prevent relapse, or to treat or prevent relapse post-HCT. However, development of T cell immunotherapy for AML has been limited by a paucity of antigens established to be safe and effective T cell targets. Recurrent AML-specific somatic variants, such as fusion genes and insertion-deletion mutations (indels), may produce novel amino acid sequences (neoepitopes) recognized by T cells. Because T cells recognize peptide epitopes presented in the context of HLA molecules, algorithms that predict peptide binding to HLA can be used to identify potential T cell epitopes from within a mutation-specific amino acid sequence. We propose to identify immunogenic AML-specific neoepitopes by merging next-generation sequencing (NGS) data of somatic leukemia-specific variants with established HLA-binding prediction algorithms, before performing a high-throughput immunogenicity screen. As proof-of-principle, we combined the output of 4 HLA-binding prediction algorithms to identify candidate neoepitopes with 12 prevalent HLA restrictions from 11 fusions and 8 indels identified by NGS from a cohort of w300 pediatric AML patients. We identified 96 peptide neoepitopes with 11 HLA-restrictions in 16 genomic anomalies. So far we have isolated CD8 + T cell clones specific for two HLA-A*02:01-restrictedneoepitopes, one from the inv(16) fusion, CBFB-MYH11, and one from the most prevalent NPM1 mutation. Titration of peptide concentration in a chromium-51 release assay revealed high functional avidity of the CBFB-MYH11 specific clone (Figure 1: CBFB-MYH11 peptide, solid line; irrelevant peptide, dashed line. Mean and SD of 3 experiments shown). Moreover, this clone specifically lysed primary inv(16)-positive AML blasts from an HLA-A*02:01 + patient, but did not significantly lyse blasts from an inv(16) + but HLA-A*0201patient (Figure 2; also shown are lysis of peptide-pulsed A2 + T2 cell line as controls). Targeted PCR sequencing of the CBFB-MYH11 fusion to determine the frequency of the fusion variant producing our candidate epitope in patients with inv(16) + AML is in progress. Ongoing studies will also determine the breadth and specificity of the CBFB-MYH11 specific clone's target recognition, and identify additional immunogenic neoepitopes using the methods described above. Our method detects naturally occurring AML-specific epitopes that may prove tractable T cell targets with which to develop novel immunotherapies for use before, during or after HCT.

Personal tumor antigens in blood malignancies: genomics-directed identification and targeting

“…Gene fusions often arise from chromosomal translocations and are a hallmark of hematological malignancies (51,(80)(81)(82)(83). Although immunogenic neoantigens arising from gene fusions have long been described in hematological neoplasms (45,(84)(85)(86), only BCR-ABL has been targeted therapeutically using vaccination approaches, which were able to induce specific T cell responses (87)(88)(89)(90). Efforts to develop a T cell-based therapy directed at a particular neoepitope arising from ETV6-RUNX1, the most common fusion gene in childhood B-ALL, were stopped because of a lack of endogenous processing (91).…”