GENERATION OF ANTIBODIES BY GENETIC IMMUNIZATION

Genetic immunization provides major advantages over classic protein/peptide immunization for the generation of antigen-specific antibodies. In vivo expression of the antigen following gene transfer results in proper modification and folding, and thus presentation of natural epitopes. For many antigen candidates, no purified protein is available and peptide synthesis is not always feasible. To efficiently enable the generation of useful antibodies via genetic immunization methods, two technical hurdles need to be overcome, First, one needs to be able to screen for the presence of the antibodies in the sera of genetically immunized mice, without the availability of antigen. This problem can be solved by expressing the antigen from plasmid DNA expression vectors after in vitro or in vivo high-efficiency gene transfer. Cells expressing the antigen can be used in immunohistochemistry, flow cytometry, or Western blotting analysis to identify antigen-specific antibodies. Second, efficient, simple, and cost effective in vivo gene transfer is required to induce a humoral immune response. Mirus' intravascular plasmid DNA gene transfer methods fulfill these requirements and can efficiently induce antibody responses against expressed transgenes. In this Phase I application, we propose to determine which intravascular gene transfer method result in the most efficient humoral immune response against different classes of expressed antigens. We will also optimize Western blotting antibody screening methods by expressing antigens in different cell lines and in liver cells. During the Phase II studies, we will further optimize genetic immunization methods by including cytokine genes, refining the immunization schedule, and testing different expression vectors and mouse strains. The screening methods will be improved by testing pooling of sera (to limit the initial number of assays), and testing other assays (immunohistochemistry, flow cytometry, ELISA). This technology will allow Mirus to provide a comprehensive antibody generation service, identify novel antibodies for the research market as well as for therapeutic targets, and introduce genetic immunization and antibody screening kits.