Scientific Report 2005

Immunology

Legumain as a Target for a Genetic Vaccine Against Breast Cancer

R. Xiang, C. Dolman, C. Liu, D. Markowitz, Y. Luo

In our efforts to develop cancer immunotherapies, we are using legumain as a target to induce a strong, cell-mediated immunity against breast cancer cells. Legumain, an asparaginyl endopeptidase, is a newly discovered stress protein that is an almost ideal target for our DNA-based vaccines. First, positional gene expression profiling of tumor tissues in a search for genes that are upregulated in tumors and tumor vasculature indicated that legumain is highly expressed in many murine breast tumor tissues. In contrast, expression of legumain is very low or absent in all normal tissues from which breast tumors arise. In addition, the protein is overexpressed by endothelial cells in the breast tumor vasculature and by tumor-associated macrophages in the breast tumor microenvironment. It is well known that tumor-associated macrophages are recruited by chemokine gradients established by tumor cells into the tumor stroma, where the macrophages mediate immunosuppression by making T cells ineffective and by promoting angiogenesis, leading to increased tumor cell growth and metastases. Importantly, cell-surface expression of legumain occurs under stress, such as tumor growth in vivo, but not in tissue culture.

We obtained the following evidence in our initial experiments. First, we found that legumain was overexpressed in vivo by most of the solid tumors we tested, especially on neoplastic cells, neovasculature, and tumor-associated macrophages, but not by the corresponding cultured tumor cell lines in tissue culture. Most normal tissues had either no or essentially undetectable levels of legumain expression. Second, we found that legumain is a stress-responsive protein induced under certain conditions such as heat shock, drug treatment, and hypoxia and is associated with tumor invasion, dissemination of metastases, and tumor angiogenesis. Third, in a prophylactic setting, the legumain-based DNA vaccine induced suppression of both growth of 4T1 primary breast tumors and dissemination of spontaneous pulmonary metastases. Finally, this antitumor effect could be achieved by suppression of angiogenesis in the tumor vasculature combined with tumor-cell killing mediated by cytotoxic T lymphocytes and/or antibodies. Taken together, the results indicate that legumain can be used as an effective target for a DNA vaccine against breast cancer. This approach may lead to the rational design of such vaccines for future clinical application.