In VitroGeneration of Cytotoxic T Lymphocytes Against HLA-A2.1 -Restricted Peptides Derived from Human Thymidylate Synthase

Abstract

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.

Dendritic Cell-Mediated Cross-Presentation of Antigens Derived from Colon Carcinoma Cells Exposed to a Highly Cytotoxic Multidrug Regimen with Gemcitabine, Oxaliplatin, 5-Fluorouracil, and Leucovorin, Elicits a Powerful Human Antigen-Specific CTL Response with Antitumor Activity in Vitro

“…CIR-A2 cell line (13) was provided by Dr. Jeffrey Schlom (Experimental Oncology Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD). All of the tumor cell lines were cultured as previously described (3,4). PBMCs for the in vitro generation of CTL lines (see below) were obtained by Ficoll-Hypaque gradient centrifugation of heparinized blood collected from normal donors or cancer patients carrying HLA-A(*)02.01 haplotype.…”

“…, and TS-3 (TSTTSLELD)] peptide epitopes with HLA-A(*)02.01 binding motifs were synthesized and characterized as previously described (3,4). Another CEA-derived epitope peptide [(CAP)-1 (YLSGANLNL)] (14) was kindly provided by Dr. Jeffrey Schlom.…”

“…These cells were used to stimulate autologous PBMCs used for CTL line generation as described in previous studies (3,4,19) with the only exception that DCs used for CTL stimulation were not pulsed with epitope peptides but loaded with tumor cell lysates. Before being used as APC for CTL stimulation, DCs were routinely evaluated for the immune phenotype and had to show at least a 90% purity level.…”

“…n the hope to improve the efficacy of anti-cancer treatments, attempts have been made to combine cancer vaccines with biologic agents or cytotoxic drugs and to test new strategies aimed at inducing a simultaneous multi-antigen specific immune response (1)(2)(3)(4)(5). In previous studies, we have tested the activity in vitro against human colon cancer cells of a chemotherapy regimen with multiple cytotoxic drugs, such as gemcitabine (GEM), 3 oxaliplatin (OXA), leucovorin (LV), and 5-fluorouracil (5-FU) (i.e., GOLF regimen).…”

Phase Ib study of poly-epitope peptide vaccination to thymidylate synthase (TSPP) and GOLFIG chemo-immunotherapy for treatment of metastatic colorectal cancer patients

“…Radio-and chemoimmunotherapy have produced promising results in both preclinical and clinical studies. [6][7][8][9][10][11] We generated and characterized new vaccine constructs able to direct an effective immune-response to molecular structures which are over-expressed in cancer cells and, at the same time, are critical for their growth, survival, and diffusion, as the Epidermal growth Factor Receptor [EGFR], the parathyroid hormone related peptide [PTHrP] and TS. [6][7][8][9] TS, in particular, is a cancer-associated target enzyme, inhibited by several anticancer drugs (antimetabolites) such as 5 0 -FU, a cytotoxic drug which is a basic component of different regimens for the treatment of mCRC and other common malignancies.…”