Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor-specific antigens

Abstract

The Human MHC Project aims at comprehensive cataloging of peptides presented within the context of different human leukocyte antigens (HLA) expressed by cells of various tissue origins, both in health and in disease. Of major interest are peptides presented on cancer cells, which include peptides derived from tumor antigens that are of interest for immunotherapy. Here, HLA‐restricted tumor‐specific antigens were identified by transfecting human breast, ovarian and prostate tumor cell lines with truncated genes of HLA‐A2 and HLA‐B7. Soluble HLA secreted by these cell lines were purified by affinity chromatography and analyzed by nano‐capillary electrospray ionization‐tandem mass spectrometry. Typically, a large peptide pool was recovered and sequenced including peptides derived from MAGE‐B2 and mucin and other new tumor‐derived antigens that may serve as potential candidates for immunotherapy.

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

“…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.…”

“…Soluble HLA-A2-eluted peptides were sequenced, synthesized and precursor proteins identified. 15 Theoretical binding affinity for peptides was calculated in the database for MHC ligands and peptide motifs by Rammensee. 19 427 sHLA-A2-ELUTED, DNMT-1-DERIVED PEPTIDE…”

“…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.…”

“…15 We selected 4 peptides from the pool of sMHC-derived peptides identified on the basis of their relevance to tumors and demonstrate the immunogenicity of a DNMT-1-derived peptide that efficiently induces peptide-specific cytotoxic T cells.…”