458?Antitumor mechanisms of local radiation and combination immunotherapy in an immunologically cold model of neuroblastoma

摘要

Background The standard treatment for high-risk neuroblastoma includes a combination immunotherapeutic approach consisting of IL-2, GM-CSF, and monoclonal antibodies directed against GD2, a disialoganglioside preferentially expressed in neuroblastoma and melanoma (1). We recently described an effective a preclinical in-situ vaccination strategy combining local radiation therapy (RT), IL-2-linked to anti-GD2 monoclonal antibody (intratumoral immunocytokine, IT-IC), checkpoint inhibition (anti-CTLA4), and drivers of innate immunity (anti-CD40 and CpG) (2). This strategy is effective in curing mice with immunologically-cold neuroblastoma. We sought to better characterize the anti-tumor mechanisms that mediate this effect. Methods Mice bearing GD2-expressing, immunologically-cold neuroblastoma tumors (9464D-GD2) were treated with 12Gy RT and combination immunotherapy (IT-IC, anti-CTLA-4, CpG, anti-CD40) over 12 days as previously described (2). Depletion of individual immune cell sets during treatment was achieved by depleting monoclonal antibodies and confirmed by flow cytometry. T-cell receptor deficient (TCR KO) mice were used to confirm findings in T-cell depletion experiments. 9464D-GD2 parental cells have low MHC-I expression; subclones with low and moderate MHC Class I expression were obtained by flow cytometry sorting and the impact of MHC class I expression on immune cell infiltrate and survival was assessed. Results The effectiveness of RT and combination immunotherapy was not significantly reduced by NK or T cell depletion, and TCR KO mice had similar tumor growth and survival to mice that underwent T-cell depletion. Moderate MHC class I expression did not slow tumor growth or improve survival in mice bearing 9464D-GD2 tumors (over those with low MHC-I) following treatment. Moderate MHC class I expression also did not alter individual immune cell subsets in treated tumors. Overall, increased infiltration of CD8 T-cells, CD4 T-cells, and depletion of T regulatory cells was observed in all treated tumors (p0.05). Abstract 458 Figure 1 Effect of MHC class I expression on response to RT and combination immunotherapy (IT-IC, anti-CTLA4, anti-CD40, CpG). A) Increased MHC class I expression in 9464D-GD2 derived tumors did not alter tumor growth or survival following treatment. B) Increased MHC class I expression did not alter immune subsets following treatment of 9464D-GD tumors with radiation and combination immunotherapy. Increased numbers of CD8 and CD4 T-cells was observed with both moderate and absent MHC class I expression. T regulatory cells were also effectively depleted in both treated groups Conclusions Treatment with RT and combination immunotherapy (IT-IC, anti-CTLA4, anti-CD40, CpG) may act through mechanisms that are MHC class I, NK-cell and T-cell independent. Further investigation of the role of innate immunity and myeloid subsets in this scenario is warranted. Acknowledgements Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number T32 CA090217.