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

IntroductionWe 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.MethodsTreatments with RT (12Gy) and/or CY (100 mg/kg i.p.) started when tumors reached 100-300 mm3 (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.ResultsSimilar 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.ConclusionTaken 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

A combined radio-immunotherapy regimen eradicates late-stage melanoma in mice.

Advanced cancers are typically resistant to treatment, which leads to most experimental cancer immunotherapy approaches being tested against smaller tumors. In this study, we show that even late-stage, weakly immunogenic mouse B78 melanomas, with tumor volumes of 1500-2000 mm3, can be eradicated by a combined radio-immunotherapy regimen (CRIR) which includes local radiotherapy, intratumoral interleukin 12, slow-release systemic interleukin 2 and checkpoint blockade with anti-CTLA-4 antibody. Flow analysis of the tumors revealed a reduction in T regulatory (Treg) cells and an increase in CD8/Treg ratios following CRIR. T cell depletion did not prevent the rapid shrinkage of treated tumors, but suppressed regression of distant tumors. Cured mice developed immunological memory. These findings highlight the largely underappreciated efficacy of certain cancer immunotherapy regimens against large, late-stage tumors