While progress has been made understanding the requirements for effective T-cell recognition of human malignant melanoma, this disease remains incurable once metastatic. The clinical imperative is to translate and apply T-cell recognition into an effective treatment for melanoma patients. This project is intended to provide a critical link between the basic science ofT-cell recognition and the initiation of a cancer gene therapy trial to stimulate anti-melanoma T-cell immunity in melanoma patients. The necessary components for effective T-cell recognition of human melanoma include peptide that is preferentially expressed in melanoma and presented by appropriate MHC molecules. Genes encoding shared human melanoma antigens that stimulate cytolytic T cells have been cloned and are available. These include the genes for tyrosinase and MART-1, both of which are presented in the context of HLA-A2. As approximately 50% of individuals are HLA-A2+, this is clinically quite relevant. In addition, sufficient MHC surface molecule expression needs to be present to effectively present antigen to T cells. The use of interferon gamma (IFN- gamma) to increase expression of MHC molecules, and other important components for antigen processing/ presentation, mad augment presentation of these immunogenic peptides. This presentation of peptides may still fall to stimulate T cells to lyse autologous melanoma unless given with necessary co-stimulation. The genes for the human B7-1 and B7-2 co- stimulatory molecules have been cloned and are available. We hypothesize that HLA-A2+ cells expressing high levels of MHC molecules, the co-stimulatory B7-1 and B7-2 molecules, and tyrosinase or MART-1 molecules, will better induce anti-melanoma T-cell immunity than melanoma cells expressing low or undetectable levels of these molecules. We will initially gene-modify HLA-A2+ melanoma cells, epithelial cells, and B-cell lines to make them potent stimulators of anti-melanoma T-cell immunity. We will then use peripheral blood lymphocytes (PBL) from healthy volunteers to determine the in vitro immunogenicity of these gene-modified cells. Lymphocytes from the peripheral blood, lymph bodes and distant metastases from melanoma patients will be evaluated to determine if these same gene- modified cells can stimulate T-cell reactivity to autologous unmodified melanoma cells. We will then gene-modify in vivo human HLA-A2+ melanoma cells that are growing as subcutaneous nodules in SCID mice, and will evaluate their ability to stimulate in vitro T-cell proliferative and cytotoxic responses to autologous unmodified melanoma cells. Gene transfer for these in vitro and in vivo studies will utilize the novel technology of particle-mediated gene transfer to deliver combinations of up to four genes to target cells to stimulate anti-melanoma T-cell immunity. This project should provide a foundation to initiate a clinical gene therapy trial for patients with metastatic melanoma.
| Rakhmilevich, A L; Imboden, M; Hao, Z et al. (2001) Effective particle-mediated vaccination against mouse melanoma by coadministration of plasmid DNA encoding Gp100 and granulocyte-macrophage colony-stimulating factor. Clin Cancer Res 7:952-61 |
