618. Hydrodynamic Limb Vein-Delivered Xenogeneic Plasmid DNA Cancer Vaccine Effectively Induces Antitumor Protection

We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids that results in high-level transgene expression in myofibers. This novel delivery method is a very effective means to induce a high-titer antibody response against expressed transgene antigens following genetic immunization. Here, we examine the utility of HLV as a method to deliver a genetic cancer vaccine in mice.Using the human gp100 (hgp100) xenogeneic DNA cancer vaccine in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV delivery was highly effective at breaking tolerance against the homologous murine gp100 tumor-associated-antigen and induced prophylactic antitumor protection against B16 tumor challenge. Mice were vaccinated by repeated (days 0, 14 and 28) injections of 50 μg of plasmid DNA encoding the hgp100 into a distal site of the hind limb saphenous vein in a volume of 1.0 ml at a rate of 8.0 ml/min. Tolerance was broken in hgp100-vaccinated mice as demonstrated by prophylactic antitumor protection and in vitro cytolytic activity against B16 and hgp100-transfected B16 (B16-hgp100) cells. Tetramer (hgp100 25-33 /H2Db) analysis of peripheral blood lymphocytes indicated that 3-5% of the CD8+ T cells were gp100-specific following HLV vaccination. Although HLV genetic vaccination elicited an anti-hgp100 antibody response able to recognize and bind appropriately (processed in B16 melanocytic cells) and aberrantly (processed in non-melanosomal COS-7 cells) processed hgp100, adoptive and passive transfer studies demonstrated that protection was cell-mediated. Furthermore, a durable antitumor memory response was evident in HLV vaccinated mice 6 months after tumor challenge. Survivor mice showed delayed tumor progression upon B16 rechallenge, with 3-4% of their CD8 + T cells still exhibiting gp100-specificity. Effective reactivation of antitumor protection was achieved in survivor mice by a single HLV hgp100 boost that resulted in expansion of gp100-specific CD8+ T cells (17%) and protection from tumor rechallenge.Our results indicate that genetic vaccination by HLV delivery is an effective method to break tolerance and induce T cell immunity capable of prophylactic antitumor protection. Most importantly, since HLV gene delivery is equally effective in small and large research animals (mice, rats, rabbits, dogs and monkeys), it represents a clinically promising alternative to existing genetic vaccinat