Inducible HSP70 (HSP70i) is implicated in the development of autoimmune vitiligo and is overexpressed in melanoma. We generated an expression construct encoding a single amino acid modification to the dendritic cell (DC) activating moiety (HSP70i Q435A ). A topical application of the DNA construct has now demonstrated efficacy in reversing autoimmunity in Sinclair swine without obstructing anti-melanoma responses. We postulate that treatment with the HSP70i Q435A construct elicits a humoral response to stress protein selectively expressed on the surface of melanoma cells, providing measurable anti-tumor protection. To test this, we evaluated humoral responses to the protein in mice to measure their specificity and anti-tumor activity. Biolistic delivery of HSP70i Q435A or empty vector DNA to C57BL/6 mice was provided 5 times, every 6 days before a subcutaneous B16F10 tumor challenge. Melanoma growth was monitored and serum was collected at end point to determine antibody isotype/subclass titers by ELISA, measure anti-HSP70-mediated ADCC via an ELISAbased natural kill cell (NK) degranulation assay and perform epitope scanning analysis to detect binding epitopes to anti-HSP70 serum antibodies. Tumor growth was delayed in HSP70i Q435A -treated mice with measurable anti-HSP70i IgG and IgM titers found exclusively in HSP70i Q435A -treated mice. A consensus C-terminal peptide sequence was identified via epitope mapping, and significant degranulation of NK cells was observed in response to anti-HSP70 serum antibodies. Thus, topical application of HSP70i Q435A DNA might offer a safe treatment for the autoimmune side effects of immunotherapy for melanoma.
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