Identification of immunotherapeutic targets in melanoma

Introduction Melanoma is a common malignant tumor of the skin, with a bad prognosis when disseminated disease is present. Conventional therapies are of little benefit because most tumor cells are relatively chemoresistant. Lymphocyte infiltrates in or around the melanoma are associated with prolonged survival, suggesting a cellular immune response against melanoma. Active immunotherapy has been shown to stimulate the immune response and has been subject of previous studies as therapeutic modality in malignant melanoma. Tumor antigenic peptides can stimulate the immune response. Modification of these peptides may result in a stronger binding to HLA molecules, resulting in an increased immune response. Previous studies showed that peptides spanning the BRAFV600E mutation, a mutation in the Map-kinase pathway that is present in 70% of melanoma cells, were immunogenic. In this study we determined whether modified BRAFV600E peptides that bind stronger to HLA molecules, may induce a stronger immune response than unmodified BRAFV600E peptides in melanoma. Material and methods Twenty-three melanoma patients were included in this study. The HLA-A2 status was determined by flow cytometry. Genomic DNA was isolated from tumor cells and used to determine the BRAFV600E mutation status by amplification in a Polymerase Chain Reaction (PCR), followed by sequencing of the amplified DNA fragment using the primer SP6 and reverse primer T7, and M13 forward and M13 reverse primer. A T2 peptide binding assay was performed to determine the binding ability of modified peptides to T2 cells, to select the peptides with a high binding ability. A standard lymphocyte proliferation assay (3HT-thymidine incorporation assay) was used to determine the extent of proliferation of T-cells cultured with various peptides. Proliferating lymphocytes were phenotyped to determine whether it were CD4 (Th) or CD8 (CTL) cells. The proliferating cells were expanded further in the presence of IL-2 and used in a cytotoxicity assay, to determine whether they were efficient in killing tumor cells. Results Lymphocytes of 14 out of 23 patients were tested for their HLA-A2 status. Four (29%) of those patients were HLA-A2 positive, and thus could be used for further experiments. Another nine patients were tested positive for HLA-A2 before and were therefore included in this study. Four of the 11 patients tested for their BRAF status, had the V600E mutation. The T2 binding assay showed a strong binding of modified peptide 4, which is altered in both anchor residues, compared to the unmodified peptides. The lymphocyte proliferation assay showed a low proliferation in all tested cells, except WM35. The phenotype of the proliferating lymphocytes was mostly of the CD8 subtype. The cytotoxicity assay gave inconsistent results, with no significant difference in cytotoxicity of the modified peptides compared to the unmodified peptides. Conclusion Modified melanoma peptide 4 bound stronger in a peptide binding assay than the unmodified peptides and may therefore be a good candidate for further studies with specific cellular immunotherapy in patients with malignant melanoma.