Potent Selection of Antigen Loss Variants of B16 Melanoma following Inflammatory Killing of MelanocytesIn vivo

Abstract

We have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8 + T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8 + T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy. (Cancer Res 2005; 65(5): 2009-17)

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

“…injection of the hsp70 plasmid is required for the priming of TRP-2-specific responses in the spleens of vaccinated mice (Fig. 1B), confirming our previous data that TRP-2 and tyrosinase, but not gp100, are targets of the curative T cell responses raised in vivo by inflammatory killing of melanocytes (13,23).…”

“…We have reported previously (13,23) that three rounds of TyrHSVtk/CMV-hsp70/GCV treatment (a total of 9 intradermal plasmid injections and 15 i.p. injections of GCV) cures 70 -100% of mice bearing 3-day established s.c. B16 tumors on the contralateral flank (Fig.…”

“…GCV was administered i.p. on days 4 -8, 11-15, and 18 -22 as described previously (13,23). Survival (tumor size Ͼ1.0 cm) is shown.…”

“…When we attempted to treat more established tumors seeded 9 days previously, we observed therapeutic delays in tumor progression with plasmid injections, but the percentage of cures decreased to between 0 and 10% (23). In those mice where tumor growth was delayed but not cured, there was a potent in vivo immunoselection by a CD8 ϩ T cell response of tumor cell variants that had lost expression of a specific subset of known melanocyte Ags (23).…”

CD27 stimulation unveils the efficacy of linked class I/II peptide vaccines in poorly immunogenic tumors by orchestrating a coordinated CD4/CD8 T cell response

“…Recently, a novel, fully human anti-human CD27 monoclonal antibody (αhCD27) was developed which binds with high affinity to induce potent human T cell responses in the context of T cell receptor stimulation. 22 Because CD27 stimulation on both CD4 + T cells and CD8 + T cells can lead to their enhanced effector function and concomitant vaccine-induced CD4 + T cell help strengthens CD8 + T cell vaccine responses, we hypothesized that αhCD27 could be leveraged as an adjuvant for peptide vaccines and that it would provide a therapeutic benefit preferentially in the setting of peptide vaccines comprised of class I and II epitopes.…”