Widening the Applicability of Human Neutrophil Elastase and Proteinase 3 Peptide Vaccines by Elucidating Immunogenic Non-HLA-A2 MHC Class I Restricted Epitopes.

Human Neutrophil Elastase (HNE) and proteinase 3 (Pr3) belong to a group of myeloid tissue restricted serine proteases, aberrantly expressed by myeloid leukemia cells of interest as a source of myeloid-restricted antigens applicable to immunization strategies. HNE shares with Pr3 the antigenic PR1 peptide sequence inducing HLA-A*0201 restricted cytotoxic T cells (CTL) against leukemic cells. Our previous studies with full length HNE protein have demonstrated its potential for inducing both CD4+ and CD8+ T cell responses in healthy individuals including HLA-A2 negative individuals, suggesting an immunogenic potential of HNE beyond the PR1 peptide (Fujiwara H et al Blood 2004). To find epitopes other than PR1 in HNE and Pr3, peripheral blood mononuclear cells (PBMC) from 15 AML patients were screened at diagnosis or relapse with a peptide matrix consisting of 15mers overlapping by 11 amino acids spanning HNE and Pr3.The corresponding Pr3 and HNE 15mers were also used to generate and test short term T cell lines derived from patient samples. Responses to the peptide matrix pools were measured by a CFSE flow cytometry-based proliferation assay where the proliferating fraction in response to peptide pools was compared to the background co-stimulatory antibody only control for each assay. A positive response was defined as a proliferating fraction minimum 0.1% of the total CD8/CD3 population after subtraction of background proliferation and at least one and a half times background for each assay. These steps helped to control for variability between samples. Using this strategy we identified PR1 and 6 new candidate epitopes contained within HNE and Pr3. Corresponding immunogenic Pr3 sequences in 2 epitopes differed by only 3 amino acids from the immunogenic HNE sequence. CD8 short term T cell lines generated to these epitopes were restricted by HLA-A2, B35 and A68. Optimal peptide length was predicted using RANKPEP (http://www.mifoundation.org/Tools/rankpep.html) and confirmed with flow cytometric assays measuring responses to candidate epitopes, by interferon-gamma, tumor necrosis factor-alpha, IL-2 production and degranulation in response to HLA-matched or mismatched EBV-transformed B cell antigen-presenting cells pulsed with relevant and irrelevant peptides. This study confirms the presence of multiple immunogenic epitopes in HNE and Pr3 and represents the first step in developing a multi-peptide leukemia vaccine broadly applicable to individuals of diverse HLA type.