Cyclophosphamide augments the efficacy of in situ vaccination in a mouse melanoma model

INTRODUCTION: We have previously shown that an intratumoral (IT) injection of the hu14.18-IL2 immunocytokine (IC), an anti-GD2 antibody linked to interleukin 2, can serve as an in situ vaccine and synergize with local radiotherapy (RT) to induce T cell-mediated antitumor effects. We hypothesized that cyclophosphamide (CY), a chemotherapeutic agent capable of depleting T regulatory cells (Tregs), would augment in situ vaccination. GD2(+) B78 mouse melanoma cells were injected intradermally in syngeneic C57BL/6 mice. METHODS: Treatments with RT (12Gy) and/or CY (100 mg/kg i.p.) started when tumors reached 100-300 mm(3) (day 0 of treatment), followed by five daily injections of IT-IC (25 mcg) on days 5-9. Tumor growth and survival were followed. In addition, tumors were analyzed by flow cytometry. RESULTS: Similar to RT, CY enhanced the antitumor effect of IC. The strongest antitumor effect was achieved when CY, RT and IC were combined, as compared to combinations of IC+RT or IC+CY. Flow cytometric analyses showed that the combined treatment with CY, RT and IC decreased Tregs and increased the ratio of CD8+ cells/Tregs within the tumors. Moreover, in mice bearing two separate tumors, the combination of RT and IT-IC delivered to one tumor, together with systemic CY, led to a systemic antitumor effect detected as shrinkage of the tumor not treated directly with RT and IT-IC. Cured mice developed immunological memory as they were able to reject B78 tumor rechallenge. CONCLUSION: Taken together, these preclinical results show that CY can augment the antitumor efficacy of IT- IC, given alone or in combination with local RT, suggesting potential benefit in clinical testing of these combinations.