The ability to manipulate genes encoding potential immunomodulators such as cytokines and tumor antigens has opened a new era of research attempts. Poorly immunogenic murine tumor model systems demonstrate that vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony stimulating (GM-CSF) generates potent, specific, and long-lasting immunity. To test this strategy in patients with advanced melanoma, the laboratory of the applicants performed a clinical trial of vaccination with lethally irradiated, autologous melanoma cells expressing human GM-CSF. Vaccination elicits a striking infiltrate of pre-existing metastatic lesions with large numbers of lymphocytes, plasma cells, macrophages, and eosinophils, resulting in tumor destruction, fibrosis, and edema. Consistent production of humoral and cellular immune responses is exemplified by tumor specific cytotoxicity and cytokine production characterizing tumor-infiltrating lymphocytes, and post-vaccination sera containing IgG antibodies that recognize intracellular and cell surface melanoma determinants. These results provide a solid foundation for undertaking a molecular analysis of the melanoma antigens stimulating specific T and B cell responses in vaccinated patients. A number of candidate antigens have been identified from initial screening with autologous sera of a cDNA expression library constructed from a patient's tumor cell line. The first revealed a novel product with structural similarities to MUC-1, a member of a family of immunogenic cell surface molecules expressed in a variety of cancers. This antibody-based strategy will likely be productive for identifying a number of melanoma antigens. The applicants have designed several approaches to characterize immune responses against each candidate antigen. Humoral responses will be evaluated by immunoblotting lysates of CRIP packaging cell lines transfected with MFG based retroviral vectors expressing each candidate. Autologous EBV transformed lymphoblasts transfected with MFG retroviruses will be used to determine T cell cytotoxicity, proliferation, and cytokine production in response to each candidate antigen. Expression patterns will be determined by Northern analyses and RT-PCR from a variety of tumors and their normal tissue counterparts. Attempts by numerous investigators to utilize previously identified antigens in vaccine strategies demonstrate an ability to develop significant immune responses against these antigens. The analysis of the applicants should contribute to the understanding of the relative importance of common versus unique targets in anti-tumor immunity. Further identification of immunogenic antigens in melanoma offers promise for future therapies.
