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Targeting a Novel Breast Differentiation Antigen for Breast Cancer Immunotherapy
Background: Manipulation of T cell immunity could provide a highly specific cancer treatment modality. Efforts to develop vaccination or adoptive T-cell therapy for breast cancer have been hindered by a lack of well-characterized antigens expressed uniformly in tumor cells and recognized by T cells. A novel breast differentiation antigen, NY-BR-1, has been described to be a target of antibody (Ab) responses in a subset of breast cancer patients. NY-BR-1 is a putative transcription factor that is expressed in up to 80% of breast tumors by RNA analysis, but restricted in its expression in normal tissues to mammary epithelium and testes. We have demonstrated that high-avidity CD8+ cytotoxic T cells (CTL) specific for NY-BR-1 epitopes presented by HLA-A2 can be isolated from normal females, suggesting that T cell tolerance to this self-protein is incomplete. NY-BR-1-specific CTL clones efficiently lyse HLA-A2 positive breast tumor cells expressing NY-BR-1. The objective of this proposal is to develop NY-BR-1 as a target for active vaccination or T cell therapy for breast cancer. Specific aims: (1) To define epitopes of NY-BR-1 presented by common HLA alleles and recognized by CD8+ CTL; (2) To isolate a NY-BR-1-specific Ab from a yeast display library of single-chain variable fragments (scFv) and evaluate the frequency and homogeneity of NY-BR-1 protein expression in primary and metastatic breast tumors; (3) To determine whether CD8+ CTL specific for NY-BR-1 can be isolated from patients with NY-BR-1+ breast tumors. Study design: We will use a capsid-modified gutless adenovirus vector to introduce NY-BR-1 into autologous dendritic cells, which will be used as antigen-presenting cells to generate NY-BR-1 specific CD8+ CTL from normal females. Epitopes of NY-BR-1 presented by common HLA alleles will be mapped using deletion constructs of NY-BR-1 and synthetic peptides. NY-BR-1-specific monoclonal Ab will be developed and used to examine the frequency and homogeneity of NY-BR-1 protein expression in breast tumors using a tissue micro-array at the FHCRC breast specimen repository. Peptide-MHC tetramers will be used to determine if NY-BR-1 specific T cells can be isolated and expanded from patients with NY-BR-1+ breast cancers and to assess the functional status of such T cells. Potential outcomes and benefits: The proposed studies will assist in validating NY-BR-1 as a target antigen for T cell-based immunotherapy and for monitoring immune responses to protein or whole-cell based vaccines, and in elucidating interactions between tumor and host immunity.
Despite advances in conventional treatment of primary breast cancer, patients frequently relapse many years after initial therapy because of residual microscopic disease. Advanced breast cancer is still not curable with currently available therapeutic options. Harnessing the body’s own immune system by vaccination or adoptive T cell therapy to eradicate residual or progressive tumor could provide a highly specific treatment modality without the toxicity of chemotherapy or radiation. A major obstacle in breast cancer immunotherapy is a lack of well-characterized antigens that can be recognized by cytotoxic T lymphocytes, one of the main effectors of the immune system. Most breast tumor-associated antigens are non-mutated self-proteins that are also widely expressed in normal tissues, and natural mechanisms of immune tolerance to prevent autoimmunity also limit potent antitumor activity. A novel breast differentiation antigen termed NY-BR-1 has been shown to be a target of antibody responses in a subset of breast cancer patients. NY-BR-1 is restricted in its expression in normal tissues to mammary epithelium and testes but is abundantly expressed in up to 80% of breast tumors. In preliminary studies, we have demonstrated that NY-BR-1 is recognized by cytotoxic T cells that can kill breast tumor cells expressing this antigen, suggesting that immune tolerance to this protein is incomplete. This proposal seeks to develop NY-BR-1 as a target for active vaccination or adoptive T cell therapy. To accomplish this goal, we will map the protein fragments of NY-BR-1 that can activate T cells in a majority of breast cancer patients. We will also develop a monoclonal antibody to examine the pattern of NY-BR-1 protein expression in breast cancer cells using a high-throughput breast tumor tissue micro-array. We will then determine if NY-BR-1 specific T cells can be isolated and expanded from breast cancer patients whose tumor express NY-BR-1, and assess the functional status of such T cells. The proposed studies will assist in elucidating the interactions between tumor and host immunity, and in designing a rational approach for targeting NY-BR-1 for breast cancer immunotherapy.