Topoisomerase inhibitors modulate expression of melanocytic antigens and enhance T cell recognition of tumor cells

Abstract

While there are many obstacles to immune destruction of autologous tumors, there is mounting evidence that tumor antigen recognition does occur. Unfortunately, immune recognition rarely controls clinically significant tumors. Even the most effective immune response will fail if tumors fail to express target antigens. Importantly, reduced tumor antigen expression often results from changes in gene regulation rather than irrevocable loss of genetic information. Such perturbations are often reversible by specific compounds or biological mediators, prompting a search for agents with improved antigen-enhancing properties. Some recent findings have suggested that certain conventional chemotherapeutic agents may have beneficial properties for cancer treatment beyond their direct cytotoxicities against tumor cells. Accordingly, we screened an important subset of these agents, topoisomerase inhibitors, for their effects on antigen levels in tumor cells. Our analyses demonstrate up-regulation of antigen expression in a variety of melanoma cell lines and gliomas in response to nanomolar levels of certain specific topoisomerase inhibitors. To demonstrate the ability of CD8+T cells to recognize tumors, we assayed cytokine secretion in T cells transfected with T cell receptors directed against Melan-A/MART-1 antigen. Three days of daunorubicin treatment resulted in enhanced antigen expression by tumor cells, in turn inducing co-cultured antigen-specific T cells to secrete Interluekin-2 and Interferon-γ. These results demonstrate that specific topoisomerase inhibitors can augment melanoma antigen production, suggesting that a combination of chemotherapy and immunotherapy may be of potential value in the treatment of otherwise insensitive cancers.

A Screening Assay to Identify Agents That Enhance T-Cell Recognition of Human Melanomas

“…Likewise, chemotherapeutic agents such as topoisomerase inhibitors 27 enhance antigen expression in both melanomas and gliomas. The plastic nature of antigen expression is further indicated by the fact that cytokines such as Oncostatin M, 19 interleukin 1a (IL-1a), and IL-1b 28 can diminish antigen expression in melanomas.…”

“…The protocol for evaluation of the cytokine IL-2 was performed as previously described 27 by using the BD OptEAI kit, human IL-2 ELISA set from BD Biosciences (San Diego, CA) following the manufacturer's recommendations. The absorbance at 450 nm was read in a BioRad 3550 plate reader.…”

“…27 The 480 compounds were tested in six parallel 96-well plates. The first two columns (16 wells) of each plate were used for standards (8 wells), untreated controls (4 wells), and positive controls (4 wells).…”

“…27 For transduction of the Melan-A/MART-1 specific TCR, 1·10 5 J.RT3-T3.5 cells were incubated with lentiviral vector at a MOI of 10. The surface expression and peptide specificity of the transduced TCR was established by using tetramer staining.…”

“…27 Briefly, a TCR specific for the MART-1 peptide EAAGIGILTV presented in HLA-A2 MHC Class I molecules (1D3 38,39 ) was synthesized by GeneArt (Burlingame, CA) and placed into a third-generation lentiviral transfer vector. 40 The full-length insert (1,820 base pair) containing both chains of the TCR was then subcloned into the lentiviral vector using the restriction sites NheI and SalI.…”