The monoclonal antibody 7.16.4 sequenced with cyclophosphamide-modulated vaccination maximizes tumor immunity and tumor-free survival in tumor-bearing tolerant neu-N mice

4995 HER-2/neu (HER-2) is overexpressed in up to 25% of human breast cancers, and portends a poor prognosis. HER-2 is also a target of the antitumor immune response in breast cancer patients. Notably, the humanized monoclonal antibody Trastuzumab can result in the clinical remission of treatment-refractory metastatic breast tumors that overexpress HER-2, and improves clinical outcomes for early breast cancer as well. Neu-N mice transgenic for the rat protooncogene neu spontaneously develop mammary tumors, and are profoundly immune-tolerant to HER-2. We previously showed that a low dose of Cyclophosphamide (CY) given one day prior to vaccination can eliminate the negative influence of regulatory T cells in neu-N mice, enabling the cure of up to 25% of tumor-bearing mice. We also showed that HER-2-specific monoclonal antibodies combined with vaccine could augment HER-2-specific CD8+ T cell immunity and tumor-free survival in neu-N mice. We hypothesized that both abrogating the influence of regulatory T cells with CY, and augmenting antigen processing and presentation with the HER-2-specific MAb would maximize the tumor rejection response in tumor-bearing animals. As a model for clinical translation, we used the Trastuzumab-like murine MAb 7.16.4. The administration a low dose of CY with weekly 7.16.4 MAb to neu-N mice with pre-established tumors resulted in tumor-free survival rates of about 10% at 50 days after tumor challenge. Treating tumor-bearing neu-N mice with HER-2+, GM-CSF-secreting vaccination added to CY + 7.16.4 MAb therapy increased the tumor-free survival rate to 55%. The effect was associated with an increase in HER-2-specific CD8+ T cells, anti-tumor antibodies, and tumor cell apoptosis. These data support the testing of CY-modulated vaccination in the setting of weekly Trastuzumab therapy in patients with advanced HER-2+ breast cancers.