HLA-A2–Restricted Cytotoxic T Lymphocyte Epitopes from Human Heparanase as Novel Targets for Broad-Spectrum Tumor Immunotherapy

Abstract

Peptide vaccination for cancer immunotherapy requires identification of peptide epitopes derived from antigenic proteins associated with tumors. Heparanase (Hpa) is broadly expressed in various advanced tumors and seems to be an attractive new tumor-associated antigen. The present study was designed to predict and identify HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in the protein of human Hpa. For this purpose, HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: 1) a computer-based epitope prediction from the amino acid sequence of human Hpa, 2) a peptide-binding assay to determine the affinity of the predicted protein with the HLA-A2 molecule, 3) stimulation of the primary T-cell response against the predicted peptides in vitro, and 4) testing of the induced CTLs toward different kinds of carcinoma cells expressing Hpa antigens and/or HLA-A2. The results demonstrated that, of the tested peptides, effectors induced by peptides of human Hpa containing residues 525-533 (PAFSYSFFV, Hpa525), 277-285 (KMLKSFLKA, Hpa277), and 405-413 (WLSLLFKKL, Hpa405) could effectively lyse various tumor cell lines that were Hpa-positive and HLA-A2-matched. We also found that these peptide-specific CTLs could not lyse autologous lymphocytes with low Hpa activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-gamma-producing T cells compared to a negative peptide. Our results suggest that Hpa525, Hpa277, and Hpa405 peptides are new HLA-A2-restricted CTL epitopes capable of inducing Hpa-specific CTLs in vitro. Because Hpa is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy for patients with advanced tumors.

Multiple Antigenic Peptides of Human Heparanase Elicit a Much More Potent Immune Response against Tumors

“…Since then, several other epitopes have been reported for both murine and human heparanase and tested for their antitumor activity on various tumor cells in vitro and in vivo (13)(14)(15). Despite the fact that peptide vaccines are easy to produce, free from pathogen contamination, and have minimal oncogenic potential, peptide vaccines usually cannot elicit an ideal immune response in the body due to small molecular weight and single structure, which limit their clinical application.…”

“…This assay was conducted as described previously (13)(14)(15). Briefly, DCs from PBMCs were loaded with different heparanase peptides and a negative control peptide at a final concentration of 30 mmol/L for 4 hours and then irradiated with 20 Gy, which prevented all outgrowths in the control cultures.…”

“…To evaluate the levels of CTL activity, a 4-hour 51 Cr release assay was used as previously described (13)(14)(15). Briefly, target cells were incubated with 51 Cr (100 mCi per 1 Â 10 6 cells) for 2 hours in a 37 C water bath.…”

“…By using HLA-A2-restricted epitope prediction algorithms based on supermotif and quantitative motif methods, we successfully predicted and identified another 3 HLA-A2-restricted CTL epitopes, Hpa277 (277-285, KMLKSFLKA), Hpa405 (405-413, WLSLLFKKL) and hHpa525 (525-533, PAF-SYSFFV), in human heparanase and 2 H-2Kb-restricted CTL epitopes, mHpa398 (398-405, LSLLFKKL) and mHpa519 (519-526, FSYGFFVI), in mice. In vitro and in vivo experiments showed that these heparanase epitopes could induce heparanase-specific CTLs to lyse various tumor cells that were heparanase positive and MHC matched (13)(14)(15).…”

“…Using the strategy outlined above, we designed three 4-branched MAP vaccines on the basis of HLA-A2-restricted CTL epitopes from human heparanase that we have identified previously (13,15). To investigate the immune responses against various tumors, CTL activity induced by these heparanase MAP vaccines was determined by a 4-hour 51 Cr release assay in vitro and ex vivo.…”