Early phase clinical trials evaluating CD8+ T cell-eliciting, HER2-derived peptide vaccines administered to HER2-positive breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the monoclonal antibody targeting the HER2 protein. Among 60 patients enrolled on clinical trials evaluating the E75+GM-CSF and GP2+GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein; an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. Based on these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy.

Background CD8+ T cell-eliciting vaccines are being investigated in breast cancer patients. Preclinical data showed that trastuzumab increases the susceptibility of tumor cells to lysis by vaccine-generated CD8+ T cells suggesting potential benefit of a combination immunotherapy strategy. The current trial was undertaken to demonstrate the safety of this approach. Methods This study was designed as a dose-escalation trial enrolling clinically disease-free, HLA-A2+ or A3+, HER2-positive breast cancer patients. Patients received six monthly inoculations of GP2+ granulocyte-macrophage colony-stimulating factor (GM-CSF) administered concurrently with standard of care trastuzumab. Local and systemic toxicity as well as left ventricular ejection fraction (LVEF) were monitored. Immunologic responses were assessed in vivo by measuring the local reaction and in vitro using an IFN-γ ELISPOT assay. Results Seventeen disease-free breast cancer patients were vaccinated. There were no dose-limiting or grade 3-5 local or systemic toxicities. The median LVEF was unchanged from baseline after vaccination. Mean local reaction at initial inoculation was 28±10 mm and increased to 68±8 mm at the final inoculation (p<0.01). Mean ELISPOT response to GP2 increased from 47±19 at baseline to 144±60 (p=13) after vaccination. Based on safety and immunologic data, the appropriate dose was determined to be 1000 μg of GP2 + 250 μg of GM-CSF. Conclusion The GP2+GM-CSF vaccine is safe and stimulates an immunologic response when given concurrently with trastuzumab. An ongoing phase II trial is evaluating the efficacy of combining a CD8 T cell-eliciting vaccine with trastuzumab in HER2-positive breast cancer patients. Synopsis Combining trastuzumab with CD8+ T cell-eliciting HER peptide vaccines may provide synergistic immunotherapeutic benefit in breast cancer. This phase I trial demonstrates the GP2 + GM-CSF vaccine given concurrently with trastuzumab is safe and stimulates an immune response.

E75 is an immunogenic peptide derived from the human epidermal growth factor receptor 2 (HER2) protein. A large amount of preclinical work evaluated the immunogenicity of E75, after which phase I trials investigated using E75 mixed with an immunoadjuvant as a vaccine. Those studies showed the vaccine to be safe and capable of stimulating an antigen-specific immune response. Subsequent to that, our group conducted trials evaluating E75 + granulocyte macrophage colony-stimulating factor (GM-CSF) in the adjuvant setting. The studies enrolled node-positive and high-risk node-negative breast cancer patients, with the goal being to determine if vaccination could decrease the recurrence risk. The studies included 187 evaluable patients: 108 vaccinated ones and 79 controls. The 5-year disease-free survival for the vaccinated patients was 89.7% compared to 80.2% for the control patients, a 48% reduction in relative risk of recurrence. Based on these data, E75 + GM-CSF, now known as NeuVax™, is being evaluated in a phase III trial. In this article, we review preclinical data and results of the early-phase trials and provide an update on the ongoing phase III study. We also present additional strategies for employing the vaccine to be included as a component of combination immunotherapy as well as in the setting of ductal carcinoma in situ as an initial step towards primary prevention.

Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen processing machinery proteins TAP1, LMP2, and calnexin do not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I, and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

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