Viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells.

Expression of viral fusogenic membrane glycoproteins (FMGs) is a potent strategy for antitumor cytotoxic gene therapy in which tumor cells are fused into large multinucleated syncytia. To understand how local cell killing can potentiate activation of antitumor immune responses, we characterized the mechanism of FMG-mediated cell killing. Here, we show that syncytia are highly ordered structures over 24-48 h but then die through processes that, by multiple morphological and biochemical criteria, bear very little resemblance to classical apoptosis. Death of syncytia is associated with nuclear fusion and premature chromosome condensation as well as severe ATP depletion and autophagic degeneration, accompanied by release of vesicles reminiscent of exosomes (syncytiosomes). Dying syncytia produce significantly more syncytiosomes than normal cells or cells killed by irradiation, freeze thaw, or osmotic shock. These syncytiosomes also load dendritic cells (DCs) more effectively than exosomes from cells dying by other mechanisms. Finally, we demonstrate that syncytiosomes from either autologous or allogeneic fusing melanoma cells lead to cross-presentation of a defined tumor antigen, gp100, by DCs to a gp100-specific CTL clone. Cross-presentation was significantly more efficient than that with exosomes from normal, irradiated, or herpes simplex virus thymidine kinase/ganciclovir-killed tumor cells. Therefore, FMG-mediated cell killing combines very effective local tumor cell killing with the potential to be a highly immunogenic method of cytotoxic gene therapy. In addition, these data open the way for novel methods of loading DCs with relevant tumor-associated antigens for vaccine development.

Enhancing the efficacy of a weak allogeneic melanoma vaccine by viral fusogenic membrane glycoprotein-mediated tumor cell-tumor cell fusion.

We have investigated how to make K1735 cells, a poor allogeneic melanoma vaccine, more effective for protection against B16 in vivo. To promote antigen release in an immunologically effective manner, tumor cells were transfected with a viral fusogenic membrane glycoprotein (vesicular stomatitis virus G glycoprotein), which kills cells through the formation, and degeneration, of large multinucleated syncytia. Vaccines consisting of a 1:1 mix of fusing allogeneic and autologous cells led to dramatic increases in survival of mice in both prophylactic and therapy models, dependent upon T cells, the mechanism of tumor-tumor cell fusion, and the nature of the fusion partner. Syncytia activate macrophages and fusogenic membrane glycoprotein-mediated cell killing very efficiently promotes cross-priming of immature dendritic cells with a model tumor antigen. Our data suggest that the unique manner in which syncytia develop and die provides a highly effective pathway for tumor antigen release and presentation to the immune system and offers a novel mechanism by which cancer cell vaccines may be prepared for clinical use.