Fusogenic activity of vesicular stomatitis virus glycoprotein plasmid in tumors as an enhancer of IL-12 gene therapy.

We have characterized the fusogenic activity of a plasmid expression system encoding vesicular stomatitis virus G protein (VSVG) in vitro and in vivo. Over 70% of murine colon and renal carcinoma cells (MC38 and Renca, respectively) transfected with VSVG plasmid in vitro fused and formed polykaryons upon incubation with pH 5.5 media. Using a plasmid expression system encoding VSVG and bacterial green fluorescent protein (GFP) formulated in a polyvinyl pyrrolidone (PVP) delivery system, diffusion of GFP throughout the VSVG-induced syncytia was shown in vivo in MC38 and Renca tumors. Moreover, tumor-bearing mice showed tumor growth inhibition following in vivo transfection with VSVG plasmid at an optimal dose of 48 microg. We have previously shown that direct injection of interleukin -12 (IL -12) plasmid complexed with PVP into tumors induces a strong immune response. In the current study, we assessed the ability of VSVG to elicit an antitumor response by enhancing cytokine gene delivery within the tumor mass. Tumor-bearing mice treated intratumorally with both VSVG/PVP and IL-12/PVP (48 and 24 microg, respectively) showed increase in tumor rejection when compared to IL- 12 plasmid alone (75% vs. 50%, respectively). These data suggest that VSVG gene therapy can be used in combination with other therapeutic genes to induce an antitumor response in vivo by enhancing the expression of the gene of interest.

Therapeutic tumor immunity induced by polyimmunization with melanoma antigens gp100 and TRP-2.

To improve the immunogenicity of melanoma self-antigens, we used a novel strategy of nonviral genetic vaccination coupled with muscle electroporation. Electroporation-enhanced immunization with plasmids encoding either human gp100 or mouse TRP-2 antigens induced only partial rejection of B16 melanoma challenge. However, immunization with a combination of these two antigens caused tumor rejection in 100% of the immunized mice. Splenocytes from combination-immunized animals killed syngeneic targets loaded with peptides derived from both gp100 and TRP-2. Immune cell depletion experiments identified CD8+ T lymphocytes as the primary effectors of antitumor immunity. Most importantly, polyimmunization led to the generation of a therapeutic immune response that significantly improved the mean survival time of mice bearing established lung metastases. These results validated the usefulness of electroporation-enhanced, nonviral genetic immunization for the active immunotherapy of cancer and indicated that using a combination of different tumor antigens may be a decisive strategy for a successful therapeutic vaccination.

Antitumoral effect of a nonviral interleukin-2 gene therapy is enhanced by combination with 5-fluorouracil.

Using a novel cationic lipid delivery system consisting of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride and cholesterol, we delivered murine interleukin-2 (IL-2) cDNA directly into an established murine renal cell carcinoma (Renca). Production of IL-2 within the tumor induced rejection of established tumors (62% on average), whereas control plasmid had little or no effect (17% on average). Surviving animals treated with IL-2:lipid were highly resistant to Renca rechallenge, but not to cross-challenge with a syngeneic mammary adenocarcinoma. Experiments on selectively immunosuppressed animals indicated a requirement for CD8+ T, natural killer, and polymorphonuclear cells. By contrast, depletion of CD4+ T cells did not disrupt the ability of IL-2:lipid to induce tumor rejection. A combination of IL-2 gene therapy with 5-fluorouracil treatment increased the antitumoral efficacy and survival of mice bearing primary and metastatic Renca tumors (42% survival with IL-2:lipid compared with 94% survival with IL-2:lipid plus 5-fluorouracil). These data indicate that rejection of primary and metastatic tumors can be achieved after intratumoral delivery of a nonviral IL-2 gene therapy, and is increased in combination with systemic delivery of a conventional chemotherapeutic agent.

Combination of interleukin 12 and interferon alpha gene therapy induces a synergistic antitumor response against colon and renal cell carcinoma.

The antitumor effect and mechanism of action of IL-12 gene therapy combined with IFN-alpha gene therapy were investigated in tumor-bearing mice using renal and colon carcinoma models, Renca and CT26, respectively. Tumors were treated with murine IL-12 plasmid alone or in combination with IFN-alpha plasmid formulated with a polymeric interactive noncondensing (PINC) gene delivery system. Intratumoral injection of IL-12 DNA/polyvinyl pyrrolidone (PVP) alone induced rejection of 58 and 17% of Renca and CT26 tumors, respectively, whereas 25% (Renca) and 0% (CT26) rejection was observed in mice treated with IFN-alpha plasmid/PVP. Combination gene therapy of formulated plasmids, IL-12 with IFN-alpha, synergistically increased the antitumor response against Renca (100% tumor rejection) and CT26 (50%). In vivo depletion of leukocyte subsets indicated that CD8(+) T and NK cells were the primary effectors of the antitumor response induced by the combined cytokine gene therapy. Moreover, mice that rejected the primary tumors after combined treatment with IL-12 and IFN-alpha plasmid formulation developed protective immunity against a subsequent tumor challenge. Analysis of tumor-infiltrating leukocytes from mice treated with the combined IL-12 and IFN-alpha gene therapy showed upregulation of CD40 molecules on antigen-presenting cells (Mac-1(hi) cells). Finally, levels of mRNA for the chemokines IP-10 and TCA-3 were higher in tumors treated with the combination of cytokine plasmids than in tumors treated with either cytokine gene alone. These data provide evidence that IL12 gene therapy combined with IFN-alpha gene therapy synergistically induces regression of established tumors and may represent a novel therapeutic strategy for cancer treatment.

Nonviral interferon alpha gene therapy inhibits growth of established tumors by eliciting a systemic immune response.

A plasmid expression system encoding murine IFN-alpha4 and complexed with a protective interactive noncondensing polymeric (PINC) delivery system was used for in vivo immunotherapy treatment of an immunogenic murine renal cell carcinoma, Renca, and a nonimmunogenic mammary adenocarcinoma, TS/A. Mice bearing established tumors were treated with IFN-alpha/polyvinylpyrrolidone (PVP) expression complexes via direct intratumoral injection. Up to 100% inhibition of tumor growth was observed in the treated mice. By using an optimal dose of 96 and 48 microg of formulated IFN-alpha plasmid for the treatment of Renca and TS/A, respectively, 30% (Renca) and 10% (TS/A) of the treated animals remained tumor free. Inhibition of tumor growth was dependent on activation of the immune system. The antitumor activity elicited by IFN-alpha gene therapy was abrogated when mice were selectively depleted of CD8+ T cells. By contrast, depletion of CD4+ T cells resulted in enhanced tumor rejection following IFN-alpha/PVP treatments. Finally, mice that remained tumor free following IFN-alpha gene therapy displayed immune resistance to a subsequent tumor challenge. These data provide evidence that IFN-alpha gene therapy can be used to induce an efficient antitumor response in vivo.