Cyclophosphamide mechanism of action in preclinical tecemotide studies

Abstract

Background: Tecemotide is a MUC1 antigen-specific therapeutic cancer vaccine. In phase III clinical studies, delivery of tecemotide is preceded by a single low dose of cyclophosphamide (CPA) to inhibit regulatory T cells (Tregs) and enhance the response to the tumor-associated antigen. Here, we investigated effects of CPA on the immune environment and tumor growth in preclinical murine models. Methods: The effect of CPA was investigated in tumor-free human MUC1 transgenic mice and anti-tumor responses were evaluated in the mice engrafted with syngeneic colorectal and ovarian cancer cells expressing human MUC1. The mice received either vehicle control, CPA (100 mg/kg), tecemotide (100 μg) or CPA + tecemotide. The immune cell phenotype and function in the spleen were assessed on Days 1, 3 and 7 postadministration. Results: The single administration of CPA led to a reduction of the absolute numbers of splenocytes, including CD8+ cells and Tregs (p<0.05 on Days 1, 3 and 7 vs. control). But while by Day 7 post-administration the total number of splenocytes and CD8+ T cells partially recovered, the number of Tregs stayed low. This resulted in a higher CD8+/Treg ratio. Further, Tregs exhibited significantly reduced suppressor activity on a per cell basis as observed on Day 3. In the MC38/MUC1 colorectal cancer model, CPA alone reduced the growth of tumors, which was enhanced by the combination of CPA + tecemotide (2-way ANOVA, p<0.05). Tecemotide monotherapy and vehicle control did not show an effect. Likewise, mice treated with the combination survived longer (median survival 31 d vs. 19 d with tecemotide; 24.5 d, CPA; 19.5 d, control; PTREND=0.04). The combination therapy also significantly increased the precursor frequency of endogenous P15E-antigen-specific CD8+ T cells and IFN-γ production indicating antigen spreading. In the MUC1+ orthotopic ovarian cancer model MOSEC/MUC1, tecemotide inhibited tumor growth (2-way ANOVA, p<0.05) and the development of abdominal ascites. Tecemotide significantly elevated BP25-specific CD4+ T cell IFN-γ production as compared to the control or CPA, a response that was significantly enhanced by the combination of tecemotide with CPA. Conclusions: Besides its direct cytotoxic effects on tumor cells, CPA can reduce immunosuppressive mechanisms in the tumor and thereby create a favorable immune environment for the combination with tecemotide to exert its activity. These findings provide mechanistic evidence to support the use of low-dose CPA as a preconditioning agent designed to potentiate the immune and antitumor efficacy of tecemotide.