613. Challenging an Old Drug in a New Context: Low-Dose Cyclophosphamide in Immunogenetherapy of Cancer
Authors: Sebastian Tuve, Shaoheng Ni, Andre Lieber
Journal: Molecular Therapy (2006)

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Breaking down immunological tolerance towards cancer cells is a major hurdle towards the aim of effective immunotherapy. Naturally occurring CD4+CD25+ T regulatory cells (Tregs) have an important role in peripheral self-tolerance and recent reports indicate that Tregs also mediate immunotolerance towards tumors. Cyclophoshamide (Cy) has been approved as a therapeutic drug since the 1970s. High- dose cyclophosphamide application is used for immunosuppression and cancer therapy due to its cytostatic and anti-angiogenetic effects. In contrast low-dose Cy application has been shown to trigger immune responses against cutaneous contact allergens and tumor- antigens. Here, we investigate the effect of low-dose Cy treatment on immune and cancer cells in a mouse model. MATERIAL AND METHODS: Immunocompetent and immunodeficient (SCID) C57Bl/6 mice received a single i.p. injection of Cy ranging from high-dose (200mg/kg) to low-dose (50mg/kg) and were sacrificed at different time-points for analysis of normal, malignant and immunecells. Syngenic TC-1 and EL-4 cells served as tumor models. To assess the effect of Cy on immunogenetherapy, adenoviral vectors expressing anti-CD3 and fetal liver tyrosine kinase 3 (Flt3 ligand) were constructed and applied intratumorally. RESULTS: High-dose Cy application led to a general immunosuppression in immunocompetent mice and delayed tumor growth in both immunocompetent and immunodeficient mice. In contrast, low-dose Cy strongly delayed tumor growth in immunocompetent, but not in immunodeficient mice. When Cy treatment was combined with immunotherapy, using the vectors described above, tumors were eliminated, whereas application of the vectors alone had no curative effect. Re-challenging with tumor cells led to delayed tumor growth when compared to naive mice. Immunohistochemistry analysis showed infiltration of immune-effector cells into the tumors upon low-dose Cy injection. Intracellular cytokine staining revealed activation of tumor-specific cytotoxic CD8+ T-cells. Our data suggest that low-dose Cy has a preferential effect against Tregs, whereas immune effector cells (e.g. CD4+ and CD8+ T-cells) are less Cy sensitive. Low-dose Cy depletes Tregs at the tumor site and in secondary lymphoid organs. As a serious side effect low-dose Cy downregulates CD19+ B-cells. Both, the number of splenic Tregs and CD19+ cells declined by day 4 after low-dose Cy injection and returned to normal levels by day 7. No signs of autoimmune diseases in low-dose Cy treated mice were observed. CONCLUSIONS: We show, that a single application of low-dose Cy is capable of inducing a strong immune response against autologous tumor cells. Moreover the susceptibility of tumors towards adenoviral immunogene- transfer was significantly increased leading to immune-mediated eradication of large tumors. Our data provide a rationale for using low-dose Cy in immunotherapy of cancer, either as a single agent or in combination with immunogenetherapy approaches.