| Abstract |
HER-2/neu oncoprotein is overexpressed on several types of cancer, comprising an ideal tumor-associated antigen for immunotherapy protocols. Several HER-2/neu peptides recognized in the context of MHC class I and class II molecules have been identified. However, Th cell responses against tumor antigens are still ambiguous and therefore, the aim of this study is to establish a reliable and successful protocol for the efficient generation of helper Th1 clones, with specificity for HER-2/neu oncoprotein, with wide applications to other tumor antigens as well. Herein, we present the successful generation of CD4+ T cell clones and their expansion in vitro in great numbers, whilst maintaining their function after long time in culture. The production of great numbers of functional cells is prerequisite for their use in adoptive immunotherapy. Using repetitive in vitro stimulations of CD4+ T cells with HER-2 (884-899) peptide, we generated Th cell lines, from which we isolated CD4+ T cell clones. The specific for HER-2 (884-899) CD4+ T cell clones could specifically recognize HER-2/neu+ HLA-DR4+ cancer cell lines, as well as tumor cells from breast, colorectal and pancreatic carcinomas, indicating that HER-2 (884-899) is naturally expressed on the surface of these cells. This finding suggests the potential usage of HER-2 (884-899) for the successful designing of peptide vaccines targeting breast, colorectal and pancreatic cancer. In an attempt to design peptide cancer vaccines that could stimulate both Th and CTL responses, capable therefore of eliciting effective immune responses against tumor burden, we have identified two novel peptides derived from HER-2/neu oncoprotein, using reverse immunology. These peptides, namely HER-2 (80-96) and HER-2 (166-180) can be recognized in the context of MHC class II, while they encompass MHC class I epitopes, named HER-2 (85-93) and HER-2 (171-179) respectively. Applying the protocol of clone generation we established, we isolated from healthy donors peripheral blood, CD4+ Th1 cell clones, as well as functional CD8+ T cell clones. Using the peptides HER-2 (80-96) and HER-2 (166-180), we showed that Th cell help is indeed essential for CTL priming and maintenance of their effector function. The helper role of Th concerns the number and the viability of the generated CTL, as well as their cytotoxic potential. Finally, using the ELISPOT-assay we determined the frequencies of pre-existing CTL specific for HER-2 (9369), HER-2 (9435), HER-2 (9665), HER-2 (9689), HER-2 (9777) and HER-2 (9952) peptides in the peripheral blood of patients with breast, ovarian, lung, colorectal and prostate cancers, as well as in healthy individuals. For HER-2 (9369), which is the most extensively studied epitope of HER-2/neu oncoprotein, we found increased percentages of CTL in HLA-A2+ (25%) and HLA-A26+ (30%) patients, which were significantly higher (60%) in HLA-A3+ patients. The recognition of this peptide in the context of HLA-A3 and HLA-A26 alleles, besides HLA-A2, increases the applicability of HER-2 (9369)-based vaccinations in a considerably broader patients population. Regarding the rest of the peptides we studied, from the HLA-A2+ patients examined, 31% had increased CTL frequencies to HER-2 (9952), 19% to HER-2 (9665), 16% to HER-2 (9689) and 12.5% HER-2 (9435), whereas only the 6% responded to HER-2 (9777). None of the HER-2/neu- patients or healthy donors exhibited increased CTL frequencies to any of these peptides. Besides IFN-γ production, assessed with ELISPOT-assay, pre-existing CTL immunity to all six HER-2/neu peptides was also confirmed in cytotoxicity assays where patients PBMC with increased CTL frequencies specifically lysed autologous tumor targets and autologous peptide-pulsed DC. The estimation of increased frequencies of CTL specific for HER-2/neu-derived peptides in the peripheral blood of patients with HER-2/neu+ tumors, could be proven useful for a better evaluation of patients to be included in HER-2/neu-based immunotherapeutical protocols, due to the increases possibility that these patients could react to the vaccination and give better clinical responses. The findings of the present study contribute successfully to the design of cancer vaccines (i) identifying novel peptides capable of stimulating complete immune reactions against several types of cancer (peptide vaccines), (ii) establishing a novel protocol for the generation of great numbers of functional cells, which can be crucial for adoptive immunotherapeutical treatments and lastly, (iii) establishing a system for the evaluation of the appropriate patients and peptides that could be included in those immunotherapeutical protocols.
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Παραγωγή αντιγονοειδικών Τ λεμφοκυτταρικών κλώνων ενάντια σε πεπτίδια ογκοπρωτεϊνών με μεγάλη συχνότητα εμφάνισης σε ποικίλες μορφές καρκίνου
