The allogeneic, HER-2-expressing GM-CSF-secreting breast tumor vaccine was first tested as a single agent, and then in sequence with a range of low doses of cyclophosphamide (given the day prior to vaccination) and doxorubicin (given 7 days after vaccination) in 28 patients with stable metastatic breast cancer [19
]. This clinical study utilized an innovative three by three factorial matrix design to efficiently determine the best predicted dose combination of cyclophosphamide and doxorubicin when given with a fixed dose of vaccine cells (5 × 108
cells). Cyclophosphamide was tested at doses of 0, 200, 250, and 350 mg/m2
, and doxorubicin was tested at doses of 0, 15, 25, and 35 mg/m2
. These doses were chosen in order to encompass the human equivalent of the chemotherapy drug doses found to be effective in the murine model, where the most effective doses were 100 mg/kg cyclophosphamide (equivalent to a human dose of 225 mg/m2
), and 5 mg/kg doxorubicin (equivalent to a human dose of 12 mg/m2
]. Eligible patients received three monthly cycles of chemotherapy-modulated vaccination, followed by a fourth boost cycle of the same intervention provided disease was stable or better [19
]. The study demonstrated that chemotherapy-modulated vaccination is safe, and associated with the induction of HER-2-specific CD4+
T cell-dependent HER-2-specific immunity as measured by delayed type hypersensitivity (DTH) and antibody levels. Optimal chemotherapy doses (cyclophosphamide 200 mg/m2
and doxorubicin 35 mg/m2
) significantly augmented the relatively low levels of HER-2-specific antibody induced by vaccine alone. Cyclophosphamide doses greater than 200 mg/m2
completely inhibited both DTH and antibody responses specific for HER-2, illustrating the importance of studies defining cancer vaccine-drug interactions in patients. Interestingly, the effective dose of cyclophosphamide was similar in the murine and human settings, whereas the best doxorubicin dose was quite different for the murine model as compared to breast cancer patients.
The mechanism by which cyclophosphamide and doxorubicin augment vaccine activity is under investigation. Initial analysis of peripheral Treg levels with time after each vaccination cycle suggests that they do not change significantly (Emens, unpublished data). Importantly, the addition of chemotherapy to vaccination does appear to impact immune priming [19
]. With each vaccine cycle, serum GM-CSF levels peaked at day 2, and declined thereafter. When the vaccine was given as a single agent, peak levels were highest with the first vaccine, and declined with each subsequent vaccine cycle. When cyclophosphamide and doxorubicin were sequenced with the vaccine, peak levels of GM-CSF were maintained across all 4 cycles of vaccination. Further investigation of this observation, as well as analysis of the impact of chemotherapy dose on HER-2-specific T cell responses is ongoing.
This study has at least two broader implications. Importantly, this study defined a cyclophosphamide dose of 200 mg/m2
within the dose range tested as most optimal for enhancing vaccine-induced immunity compared with doses of 250 or 350 mg/m2
, which were ineffective [19
]. Historically, cyclophosphamide doses of 300 mg/m2
have been used for enhancing immunotherapies in Phase II and III cancer vaccine trials. This study provides one possible explanation for the lack of vaccine efficacy observed in those trials. As a result, we are taking a cyclophosphamide dose of 200 mg/m2
forward as the best dose of cyclophosphamide for immune modulation in current and future breast cancer vaccine trials. In addition, these results suggest that analysis of vaccine-induced DTH and/or antibody responses may be one straightforward measure of antigen-specific immunity in optimization trials designed to define the best immunotherapy drug doses and schedules. This study was initially designed to test a range of cyclophosphamide doses that included 0, 250, 350, and 450 mg/m2
. In conducting the study, we observed the induction of de novo HER-2-specific DTH with vaccine alone in the first six patients. Once chemotherapy was added to the vaccination regimen, we noticed in real-time that the induction of HER-2-specific immunity by DTH skin testing disappeared and some chemotherapy-related side effects appeared (hair loss, mild nausea). Therefore, we modified the study design to drop the highest dose of cyclophosphamide planned for testing (450 mg/m2
) and add a lower dose than originally planned (200 mg/m2
). Thus, these simple measures can be used for realtime analysis of immune responses to guide dose and schedule selection in proof of principle studies. They may also have an important role in studies that involve multiple sites where the T cell analysis might be more likely compromised by variations in sample collection, storage, and transport.