A novel DNA methyltransferase I–derived peptide eluted from soluble HLA‐A*0201 induces peptide‐specific, tumor‐directed cytotoxic T cells

Abstract

Key words: soluble HLA; DNMT-1; cancer vaccine; tumor antigen; immunotherapyThe preferred approach for the elimination of tumor cells is to elicit a specific antitumor CTL response that will select tumor cells presenting the appropriate T-cell epitopes. 1-4 Human clinical trials for a variety of malignant diseases showed that T-cell therapy was effective and curative for some individuals. 5,6 A number of TAAs have been identified, mainly melanoma antigens, such as Melan-A and MART-1. Studies were initiated to vaccinate tumor patients against these antigens, and their ability to induce antitumor T-cell immunity has been shown in clinical studies. 7 Many of the recently identified antigens are nonmutated differentiation antigens overexpressed in tumors, e.g., a peptide derived from protein tyrosinase that was shown to successfully induce the attack and lysis of melanoma cells from cancer patients. 8 Several strategies have evolved to identify novel TAAs, e.g., cloning of epitopes recognized by CTLs 9 and SEREX technology, 10 where a patient's antibodies are used to identify tumor antigens from a cDNA expression library made from mRNA of tumor specimens.Conventionally, endogenous peptides have been eluted from affinity-purified HLA, such as HLA-A*0201, followed by analysis and sequencing using HPLC combined with tandem mass spectrometry. 11,12 HLA class I molecules are integral membrane glycoproteins; however, the presence of a soluble form of these molecules was demonstrated decades ago. 13,14 Similar to membrane-bound MHC, sMHC class I molecules bind peptides which are 8 -10 amino acids long, with a few exceptions having a length of 6, 11 and 12 amino acids. 15 Purification of MHC molecules is complicated due to their unavoidable contamination with cell debris and detergents after cell lysis. We recently identified HLA-restricted, tumor-specific antigens presented by the sMHC class I molecule by transfecting human tumor cell lines with truncated genes of HLA-A2. After purification of the secreted sHLA by affinity chromatography, peptides were eluted and sequenced. 15 Novel peptides unique to the sMHC variants were identified and screened for tumor-relevant precursor proteins. Detection of these peptides on sMHC indicates that these peptides are properly processed and displayed on transfected sMHC molecules similarly to those displayed on membrane-bound MHC molecules. Peptides recovered from sHLA molecules form similar patterns to those of membrane-bound HLA and include known MHC peptides. 15 Therefore, the repertoire of MHC peptides recovered from sMHC molecules is a good representation of the naturally processed and displayed peptides of transfected cells. Peptides detected this way should be presented at many copy numbers per cell and are likely to be bound with relatively high affinity to HLA.Use of sHLA as a source of peptides enabled us to identify large numbers of MHC peptides presented by human cancer cells and to screen these for peptides possibly derived from TAAs as candidates for tumor vaccination. 15 ...

Identification of Breast Cancer Peptide Epitopes Presented by HLA-A*0201

“…We sequenced over 150 peptides presented by the HLA A*0201 from the 3 tumorigenic cell lines and the nontumorigenic line. Most of these peptides were shared by all 4 cell lines and were used to ensure proper alignment of the fractions, including the bona fide CTL epitope, GLIEKNIEL from DNA methyl transferase 1 . A few peptides were unique to an individual cell line, such as LLQEVEHQL from the E3 ubiquitin ligase TRIM37 found only in the MCF-7 peptide pool.…”

“…Most of these peptides were shared by all 4 cell lines and were used to ensure proper alignment of the fractions, including the bona fide CTL epitope, GLIEKNIEL from DNA methyl transferase 1. 18 A few peptides were unique to an individual cell line, such as LLQEVEHQL from the E3 ubiquitin ligase TRIM37 found only in the MCF-7 peptide pool. The peptides corresponding to ODC1, Cdk2, EXOSC6, and KNTC2 were presented by the HLA A*0201 of all three tumorigenic lines and missing from the MCF10A pool.…”