Project #2 of this project will focus on pre-clinical development of total tumor RNA transfected dendritic cell (DC) immunotherapy. The objective of this project is to develop optimal methods for using DC pulsed with mRNA isolated from tumor cells in cancer immunotherapy. Vaccination with the total antigenic content of tumor cells will induce immunity against a broad repertoire of tumor antigens and will reducer the emergence of antigen-loss variant tumor cells. Since the mRNA content of single cells can be amplified by PCR technology, the use of RNA as a source of tumor antigen will expand the patient population eligible for treatment to include (the majority of) cancer patients which have minimal, often undetectable, disease. Moreover, use of tumor antigen in the form of RNA provides a mechanism to reduce autoimmune manifestations, should they arise. Thus, the primary objective of this research proposal is to develop optimal methods for using DC pulsed with tumor RNA as tumor vaccines in CEA-expressing cancer patients with low volume metastatic cancer. The specific objectives of the proposed studies are: (1). To develop microdissection techniques to isolate tumor cells from pathology slides, and amplify biologically active mRNA. (2). To characterize the T cell response generated in vitro with tumor RNA transfected DC and optimize loading of tumor RNA on DC. (3). To develop methods to reduce the contribution of non-tumor specific, """"""""self"""""""" antigens by subtractive hybridization. Antigen presenting function and vaccines potency of tumor RNA pulsed DC will be first evaluated in a murine post-surgical metastasis model, and promising strategies will be further evaluated in human systems by measuring the ability of RNA transfected DC to initiate primary CEA- specific CTL responses in vitro.
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