Killing of Normal Melanocytes, Combined with Heat Shock Protein 70 and CD40L Expression, Cures Large Established Melanomas

Abstract

Previously, we showed that nine intradermal injections of a plasmid in which the HSVtk suicide gene is expressed from a melanocyte-specific promoter (Tyr-HSVtk), combined with a plasmid expressing heat shock protein 70 (CMV-hsp70), along with systemic ganciclovir, kills normal melanocytes and raises a CD8+ T cell response that is potent enough to eradicate small, 3-day established B16 tumors. We show in this study that, in that regimen, hsp70 acts as a potent immune adjuvant through TLR-4 signaling and local induction of TNF-α. hsp70 is required for migration of APC resident in the skin to the draining lymph nodes to present Ags, derived from the killing of normal melanocytes, to naive T cells. The addition of a plasmid expressing CD40L increased therapeutic efficacy, such that only six plasmid injections were now required to cure large, 9-day established tumors. Generation of potent immunological memory against rechallenge in cured mice accompanied these therapeutic gains, as did induction of aggressive autoimmune symptoms. Expression of CD40L, along with hsp70, increased both the frequency and activity of T cells activated against melanocyte-derived Ags. In this way, addition of CD40L to the hsp70-induced inflammatory killing of melanocytes can be used to cure large established tumors and to confer immunological memory against tumor cells, although a concomitant increase in autoimmune sequelae also is produced.

Lysosomal Rerouting of Hsp70 Trafficking as a Potential Immune Activating Tool for Targeting Melanoma

“…Furthermore, we showed that B16 melanoma cells chronically expressing excess Hsp70 lost their ability to release Hsp70 through active transport mechanisms. In line with this, constitutive excess of Hsp70 expressed in melanomas has been found less immunogenic [19,41,44], a phenomenon that has been attributed to cellular acclimation to non-needed excess of Hsp70 [41,51,52]. Accordingly, acute excess-mediated active release mechanisms of Hsp70 appear to contribute to a more efficient immune stimulation against melanoma cells.…”

“…In accordance, melanoma cells are usually low immunogenic irrespective of their basal level of Hsp70 [44,56]. Increasing immunogenicity of melanoma cells, frequently resistant to conventional therapies [57] would be therefore a desirable strategy.…”

“…An acute excess achieved by repeated Hsp70 gene transfer alone or in combination with hyperthermia proved to kill large established melanoma or to arrest tumor growth in mice, respectively [44,58]. Alternatively, an acute excess of Hsp70 induced by different therapies of human melanomas [1,43] may be utilized to stimulate the immune system against resistant tumor populations.…”

“…vivo through up-regulation of Hsp70 in the low immunogenic B16 cells [19,44] made this cell model a valuable experimental tool to study mechanism of Hsp70 trafficking and potential impact of elevated Hsp70 levels on its localization hence on its immune activating properties.…”

Inflammatory tumour cell killing by oncolytic reovirus for the treatment of melanoma

“…41 Indeed, the deliberate establishment of an autoimmune response against normal melanocytes has been proposed as a potential approach for promoting antimelanoma immunity. 42,43 Therefore, therapies that provoke immune activation in addition to direct tumour-specific cytotoxicity represent rational approaches to the treatment of MMM.…”