A B S T R A C T PurposeGranulocyte-macrophage colony-stimulating factor (GM-CSF) -secreting tumor vaccines have demonstrated bioactivity but may be limited by disease burdens and immune tolerance. We tested the hypothesis that cyclophosphamide (CY) and doxorubicin (DOX) can enhance vaccine-induced immunity in patients with breast cancer. Patients and MethodsWe conducted a 3 ϫ 3 factorial (response surface) dose-ranging study of CY, DOX, and an HER2-positive, allogeneic, GM-CSF-secreting tumor vaccine in 28 patients with metastatic breast cancer. Patients received three monthly immunizations, with a boost 6 to 8 months from study entry. Primary objectives included safety and determination of the chemotherapy doses that maximize HER2-specific immunity. Results Twenty ConclusionFirst, immunotherapy with an allogeneic, HER2-positive, GM-CSF-secreting breast tumor vaccine alone or with CY and DOX is safe and induces HER2-specific immunity in patients with metastatic breast cancer. Second, the immunomodulatory activity of low-dose CY has a narrow therapeutic window, with an optimal dose not exceeding 200 mg/m 2 . Third, factorial designs provide an opportunity to identify the most active combination of interacting drugs in patients. Further investigation of the impact of chemotherapy on vaccine-induced immunity is warranted.

Purpose: Given the complex tumor microenvironment, targeting multiple cellular components may be the most effective cancer treatment strategy. Therefore, we tested whether antiangiogenic and immune-based therapy might synergize by characterizing the activity of DC101, an antiangiogenic monoclonal antibody specific for vascular endothelial growth factor receptor-2 (VEGF-R2), alone and with HER-2/neu (neu)^targeted vaccination. Experimental Design: Neu-expressing breast tumors were measured in treated nontolerant FVB mice and immune-tolerant neu transgenic (neu-N) mice. Neu-specific and tumor cellŝ pecific immune responses were assessed by intracellular cytokine staining, ELISPOT, and CTL assays.Results: DC101 decreased angiogenesis and increased tumor cell apoptosis. Although DC101 increased serum levels of the immunosuppressive cytokine VEGF, no evidence of systemic immune inhibition was detected. Moreover, DC101 did not impede the influx of tumor-infiltrating lymphocytes. In FVB mice, DC101 inhibited tumor growth in part through a T cell^dependent mechanism, resulting in both increased tumor-specific CD8 + T cells and tumor regression. Combining DC101 with neu-specific vaccination accelerated tumor regression, augmenting the lytic activity of CD8 + cytotoxicTcells. In tolerant neu-N mice, DC101only delayed tumor growth without inducing frank tumor regression or antigen-specific T-cell activation. Notably, mitigating immune tolerance by inhibiting regulatoryTcell activity with cyclophosphamide revealed DC101-mediated augmentation of antitumor responses in vaccinated neu-N mice. Conclusions: This is the first report of DC101-induced antitumor immune responses. It establishes the induction of tumor-specificT-cell responses as one consequence of VEGF-R2 targeting with DC101. These data support the development of multitargeted cancer therapy combining immune-based and antiangiogenic agents for clinical translation.

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