989. Induction of Antitumor Immunity by Cell- Penetrating Peptide-Coupled Hsp70 Gene Transfer to Tumor Cells
Authors: Takayuki Otsuki, Makiya Nishikawa, Atsushi Ota, Seiji Takemoto, Yoshinobu Takakura
Journal: Molecular Therapy (2006)

Cancer immunotherapy is expected to be a safe and highly effective strategy that shows promise for treatment of cancer patients. Effective delivery of antigens to antigen presenting cells (APC) is important for inducing tumor-specific immune response. Heat shock protein 70 (Hsp70) can present a broad repertoire of tumor antigens to dendritic cells and elicit innate immunity. We and others have developed Hsp70-based antigen delivery systems to APC. Because tumor cells possess a variety of tumor-related antigens within the cells, their delivery outside the cells to APC may lead to an effective induction of tumor-specific immune response without administration of antigens. In recent years, cell-penetrating peptides (CPP), such as HIV Tat protein, HSV VP22 and oligo-arginines, have been used for the delivery of a variety of large cargoes, such as proteins, DNA and liposomes, into cells through a mechanism that has not fully been elucidated. VP22 has been reported to be secreted from cells and enters surrounding other cells. We hypothesized that VP22-coupled Hsp70 expressed in tumor cells can bind endogenous tumor-related antigens and leave the cells, then encounters APC to induce antigen-specific immune response. To prove this, we constructed plasmid DNA by which a fusion protein of mouse Hsp70 and the C-terminal 34 amino acids of VP22 (|[Delta]|VP22) is expressed under the control of CMV promoter (pHsp70-|[Delta]|VP22). When expressed in B16-F10 melanoma cells, Hsp70-|[Delta]|VP22 showed a distribution to the plasma membrane, suggesting its high affinity to lipid bilayer. When Hsp70-|[Delta]|VP22-expressing cells and GFP-expressing cells were cultured together, Hsp70-|[Delta]|VP22 was detected in GFP-expressing cells using an anti-VP22 antibody. These results suggest the secretion of Hsp70-|[Delta]|VP22 out of cells after transgene expression. Then, plasmid DNA was injected into tumors and its antitumor potency was evaluated in tumor-bearing mice. A lymphoma cell line, E.G7-OVA, which had been established by transfection of EL4 cells with ovalbumin cDNA, was selected and inoculated into the dorsal skin of C57BL/6 mice. When the volume of tumor tissue approached around 40 mm3, 100 |[mu]|g plasmid DNA was injected directly into the tumor, followed by electroporation. This treatment was repeated three times with each 4 days-interval. In a CTL assay using E.G7 as target cells, splenocytes from mice receiving pHsp70-|[Delta]|VP22 showed a significantly higher cytolytic activity than those with pHsp70. In addition, low, but detectable cytolytic activity was observed against EL4 cells, but not against B16-F10 cells, suggesting that not only OVA-derived antigens but also other antigens common to EL4 and E.G7 are presented to APC after intratumoral injection of pHsp70-|[Delta]|VP22. Furthermore, pHsp70-|[Delta]|VP22 inhibited the tumor growth in mice and increased the survival of tumor-bearing mice. These results indicate that tumor-specific antitumor immunity can be induced by intratumoral gene transfer of Hsp70 coupled with the VP22 peptide, in which Hsp70 may act as a binder of tumor antigens as well as a delivery vehicle to APC, and the VP22 peptide may facilitate the extracellular transport of Hsp70-antigen complex out of tumor cells.