Injecting Hope-A Review of Breast Cancer Vaccines

There is significant interest in investigating immunotherapeutic strategies to be used for the treatment of breast cancer patients. One form of immunotherapy under active investigation is the cancer vaccine. Vaccines are a form of active immune therapy designed to stimulate the immune system to recognize tumor cells as foreign. Vaccines include an antigen that serves as the target for the immune response, and an immunoadjuvant, which is a nonspecific stimulator of the immune response that promotes an environment conducive to immune stimulation. Vaccines are an appealing therapeutic strategy because they are specific and are associated with minimal toxicity. In addition, they stimulate the adaptive immune system, thereby producing a memory response allowing for sustained effect without repeated therapy. Currently, there are no US Food and Drug Administration–approved breast cancer vaccines; however, there are multiple vaccines and treatment strategies employing these vaccines that are being actively investigated in clinical trials.

Introduction

Recent successes with drugs targeting T-cell inhibitory molecules has led to a significant increase in enthusiasm for immunotherapy. Broadly speaking, immunotherapy is a treatment designed to enhance a patient’s immune response against their tumors. There are multiple forms of immunotherapy, which include: 1) checkpoint blockade, the class of drugs targeting T-cell inhibitory molecules; 2) adoptive cellular therapy, which involves direct infusion of T cells into patients; and 3) vaccines, which are a form of active immune therapy designed to stimulate the patient’s immune system to recognize tumor cells as foreign. The majority of cancer vaccines include an antigen that serves as the target for the immune response, as well as an immunoadjuvant, which is a nonspecific stimulator of the immune response that promotes an environment conducive to immune stimulation. The appeal of vaccines as a therapeutic strategy is that they are target-specific and associated with minimal toxicity. In addition, they stimulate the adaptive immune system, thereby producing a memory response allowing for sustained effect without repeated therapy. For these reasons, physicians and patients alike are enthusiastic about the possibility of a breast cancer vaccine that could stimulate the patient’s immune system to recognize, attack, and eliminate tumor cells. Although there are currently no US Food and Drug Administration (FDA)-approved breast cancer vaccines, there are multiple vaccines and vaccine treatment strategies that are being actively investigated in clinical trials.

NeuVax-Vaccination for Secondary Prevention

The breast cancer vaccine farthest along in development is NeuVax, which is comprised of the human epidermal growth factor receptor 2 (HER2)-derived peptide E75 (nelipepimut-S) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) as an immunoadjuvant. E75 is a major histocompatibility complex (MHC) class I epitope that stimulates a CD8+ cytotoxic T-lymphocyte (CTL) response. After preclinical studies confirmed the immunogenicity of the peptide,[1-4] phase I trials enrolling patients with metastatic breast cancer showed the administration of E75 + GM-CSF to be safe and capable of stimulating an antigen-specific immune response.[5] Subsequent to that, our group conducted two early-phase trials evaluating the vaccine in the adjuvant setting. The first trial, which was designed as a standard dose-escalation study, enrolled node-positive breast cancer patients. The second trial, designed as a dose and inoculation schedule optimization study, enrolled high-risk node-negative patients. Both trials enrolled patients with tumors that expressed any degree of HER2 (1–3+ by immunohistochemistry [IHC]). All patients had completed standard-of-care therapy and were disease free at the time of enrollment. Those with hormone receptor–positive tumors that had been prescribed endocrine therapy continued on that treatment. Because E75 is human leukocyte antigen (HLA)-restricted, patients that were determined to be HLA-A2– or HLA-A3–positive were vaccinated, while HLA-A2/A3–negative patients were followed prospectively as unvaccinated controls. Patients were administered escalating doses of E75 + GM-CSF monthly for 4 or 6 months. The vaccination series was well tolerated, with minimal toxicity at all dose levels. The trials also confirmed that the vaccine could stimulate an antigen-specific immune response.[6] Because there were encouraging early efficacy data,[7] the trials transitioned into phase II studies that were analyzed jointly.

Combined, the trials enrolled 187 evaluable patients; 108 were vaccinated and 79 were followed as controls. The two groups were well matched for all demographic and prognostic factors except hormone receptor status, with vaccinated patients being more likely to be hormone receptor–negative.[8] It is widely acknowledged that endocrine therapy administered to hormone receptor–positive patients improves survival; therefore, this difference in hormone receptor status between vaccinated and control patients represents a potential bias against the vaccine. The vaccine was again shown to be safe and well tolerated, with generally mild toxicities. The majority of patients experienced low-grade local toxicity, including erythema and pruritus at the injection site, and low-grade systemic toxicity, most often mild influenza-like symptoms, fatigue, and bone pain. All patients completed 5 years of follow-up, and the 5-year disease-free survival (DFS) rates were 90% in vaccinated patients vs 80% in controls (P = .08). Because the trials began with a dose-escalation phase, not all patients received the optimal dose of E75 peptide. For those who were optimally dosed, the 5-year DFS rate was 95%, a difference that was statistically significant when compared with unvaccinated controls (P = .05).[8]

We have postulated that this simple vaccine strategy, which employs only a single immunogenic epitope, may be effective because of its ability to initiate a response against the immunizing peptide with subsequent epitope spreading, reflecting the initiation of a broader immune response. Patients vaccinated with E75 + GM-CSF have shown an increase not only in levels of E75-specific CTL, but also in the levels of CTL specific for GP2, a second MHC class I epitope derived from the HER2 protein, as well as CTL specific for epitopes derived from folate-binding protein, a second tumor antigen.[9,10] Another important consideration for this vaccination strategy is that we have administered the vaccine in the adjuvant setting to patients with minimal residual disease. Previous trials investigating single-epitope peptide vaccines had enrolled patients with diffusely metastatic cancer. In a report from the National Cancer Institute on their initial experience evaluating peptide vaccines in patients with metastatic melanoma and ovarian cancer, investigators reported objective response rates less than 3%.[11] In a previous phase III trial evaluating Theratope, a breast cancer vaccine that targets a carbohydrate epitope found on cancer-associated mucins, there was no improvement in time to progression or overall survival.[12] The trial enrolled patients with metastatic breast cancer, and when reporting the results, the investigators suggested that vaccination may have been more effective if administered to patients with earlier-stage disease. The failure of vaccines in these trials may be attributable in part to the immunosuppressive microenvironment of metastatic cancer.

At the time of this review, NeuVax is the only breast cancer vaccine being evaluated in a phase III trial. The PRESENT trial (Prevention of Recurrence in Early-Stage, Node-Positive Breast Cancer With Low to Intermediate HER2 Expression With NeuVax Treatment; ClinicalTrials.gov identifier: NCT01479244), which completed randomization in April 2015, enrolled over 750 HLA-A2–positive patients with node-positive, low to intermediate (IHC 1+ or 2+) HER2-expressing breast cancer. The trial targeted these low and intermediate HER2-expressing patients for several reasons. First, correlative studies from the early-phase trials demonstrated robust immune responses in patients with HER2 1+ or 2+ tumors.[13] Second, patients with low to intermediate HER2-expressing tumors represent an unmet medical need, as they currently have no targeted therapy (with the caveat that those with hormone receptor–positive tumors are advised to take endocrine therapy). Finally, the drug development space for what is defined clinically as HER2-positive breast cancer (IHC 3+ or gene amplification demonstrated by fluorescence in situ hybridization) is “crowded” with multiple agents under investigation, including pertuzumab and trastuzumab emtansine. However, as will be discussed further below, there are data to suggest that the combination of a CTL-eliciting vaccine and trastuzumab could be synergistic, and ongoing phase II trials are evaluating this strategy. Patients enrolled on the PRESENT trial were randomized to receive NeuVax or GM-CSF alone for a primary vaccination series consisting of six inoculations administered monthly, followed by booster inoculations every 6 months, through 3 years. The study’s primary endpoint is DFS.

Strategies to Enhance Response to Vaccination

Stimulation of a CD4+ helper T-cell response

Because CD8+ T cells do not sustain a prolonged memory response in the absence of antigen exposure and stimulation by antigen-presenting cells,[7] there is concern regarding the durability of the immune response stimulated by vaccination with an MHC class I peptide. In fact, in the early E75 trials, we noted waning antigen-specific immunity over time, which we addressed by adding a series of booster inoculations that were well tolerated and effective in maintaining immunity.[14] Another strategy to both enhance the immune response and maintain long-term immunity is to also stimulate a CD4+ helper T-cell response by vaccinating in combination with an MHC class II epitope.[15,16]

In an early-phase clinical trial that enrolled patients with stage IV breast, ovarian, or non–small-cell lung cancer, Disis and colleagues evaluated multi-epitope vaccines that incorporated MHC class II epitopes.[17] One vaccine consisted of three MHC class II epitopes derived from the HER2 protein’s extracellular domain (ECD). These epitopes included HLA-A2–restricted MHC class I epitopes within their natural sequence; therefore, this vaccine was designed to stimulate a CTL response. Thirteen patients received this vaccine, with the majority developing T-cell–mediated immunity. A second cohort of 11 patients received a vaccine comprised of MHC class II epitopes derived from the intracellular domain (ICD) of the HER2 protein. All of these patients developed T-cell responses against a component of the HER2 protein. Additional correlative studies identified an antibody response in the serum of vaccinated patients, providing evidence that these vaccines, which stimulated CD4+ helper T cells, supported a B-cell response.[18]

One of the epitopes included in the ICD vaccine investigated by Disis et al[17] is AE36. Our group has investigated a modified form of this MHC class II epitope. One weakness of class II epitopes is that they have lower binding affinity than class I epitopes. To address this, the Ii-Key peptide LRMK, which improves antigen presentation by enhancing epitope charging, was covalently linked to the amino terminus of the AE36 peptide to generate AE37.[19-21] Preclinical studies demonstrated the hybrid peptide to be more effective than the native peptide in stimulating peptide-specific CD4+ T cells, which provided stronger helper effect to HER2-specific CD8+ T cells.[22] We then conducted a phase I trial of AE37 + GM-CSF in breast cancer patients, which confirmed the vaccine to be safe and capable of inducing peptide-specific immune responses.[23] Subsequently, we completed a prospective multicenter phase II trial that enrolled 298 patients in the adjuvant setting and randomized them to AE37 + GM-CSF (n = 153) or GM-CSF alone (n = 145).[24] At the time of the planned primary analysis, there was no difference in estimated 5-year DFS rates for vaccinated (80.8%) vs control (79.5%) patients. Similar to previous studies, this trial enrolled patients with tumors expressing any degree of HER2; therefore, there were planned subset analyses based on the extent of HER2 expression as well as hormone receptor expression. For patients with HER2 1+/2+ tumors, regardless of hormone receptor status, those receiving the vaccine (n = 76) had a 5-year DFS rate of 77.2% compared with 65.7% in control patients (n = 78; P = .21). In patients with HER2 1+/2+ tumors that were also hormone receptor–negative (ie, triple-negative breast cancer), the DFS rate was 77.7% in vaccinated patients (n = 25) vs 49.0% in control patients (n = 25; P = .12). Although the overall intent-to-treat analysis showed no benefit to vaccination, the subset analyses suggest that further evaluation in patients with triple-negative breast cancer may be warranted. Alternatively, consideration could be given to combining AE37 with an MHC class I epitope in a multi-epitope vaccine, or perhaps using the vaccine to stimulate a T-cell response prior to treatment with either an agonistic antibody targeting T-cell stimulatory molecules, or an antagonistic antibody targeting T-cell inhibitory molecules.

In a study presented at the 2015 San Antonio Breast Cancer Symposium (SABCS), Knutson and colleagues reported early results from a phase I trial investigating a multi-epitope vaccine designed to elicit helper T-cell immunity.[25] The vaccine consists of a pool of four HLA-DR (antigen D related)–restricted epitopes that are naturally processed, combined with the immunoadjuvant GM-CSF. The approach is novel in that the epitopes are all subdominant, which could circumvent the issue of tolerance, potentially limiting the efficacy of a vaccine employing dominant epitopes. The study enrolled 22 patients with stage II to III HER2-positive breast cancer who were disease free after completing standard therapy. All patients were vaccinated monthly for 6 months. The vaccine was well tolerated, with grade 1 and 2 toxicities attributable to inoculation, including injection site reactions and fatigue. Importantly, T-cell immunity to HER2 occurred in 94% of patients.

Combining Vaccination With Chemotherapy

It has long been thought that chemotherapy suppresses the immune response. However, recent data have indicated that certain chemotherapeutic agents can stimulate an immunogenic cell death,[26] suggesting a potential opportunity for synergy with immunotherapeutic agents. A recently reported phase II trial investigated the combination of the poxviral vaccine PANVAC with docetaxel in metastatic breast cancer patients.[27] The PANVAC vaccine administers a priming dose with a recombinant vaccinia vector followed by boosting doses with recombinant fowlpox vectors. Transgenes for the tumor antigens CEA and MUC1, as well as for the T-cell costimulatory molecules B7.1, ICAM-1, and LFA-3, are encoded in each vector.[28] The chemotherapy partner, docetaxel, was chosen in part because preclinical data showed it improved the immune response to recombinant viral vaccines, with enhanced antigen-specific T-cell responses.[29] The study enrolled 48 patients with metastatic breast cancer and randomized them to docetaxel plus PANVAC or docetaxel alone. The median progression-free survival for vaccinated patients was 8 months vs 4 months for those receiving docetaxel alone (hazard ratio, 0.56 [95% CI, 0.34–1.14]; P = .09). The study therefore achieved its primary endpoint of providing data to inform the design of a larger definitive randomized trial.[27]

Combining Vaccination With Targeted Therapy: Vaccination Plus Trastuzumab

Preclinical data have shown synergistic activity when active immunotherapy (a cancer vaccine) is combined with passive immunotherapy (a monoclonal antibody). Studies were performed employing a HER2/neu transgenic mouse model and showed that cellular and humoral anti-neu immune responses are both required to eliminate HER2/neu-expressing tumors.[30-32] Using a second murine model, Park et al confirmed that anti-HER2 antibody–induced tumor regression is dependent on T cells.[33] Additionally, because trastuzumab is an immunoglobulin G antibody, antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer cells is one mechanism of action. ADCC induces tumor cell lysis, with subsequent release of antibody-coated tumor antigens that can be taken up by dendritic cells and presented on MHC class I molecules.[34] In effect, trastuzumab treatment functions in part to turn the tumor into a vaccine. Furthermore, there is clinical evidence supporting an immunologic response to trastuzumab that could promote synergy with a CD8+ T-cell–eliciting vaccine. Taylor et al showed generation of HER2-specific CD4+ T-cell responses and endogenous anti-HER2 antibody responses in a small study of HER2-positive metastatic breast cancer patients receiving trastuzumab.[35] In a study of a subset of patients from whom serum was available, treated on the trastuzumab arm of the North Central Cancer Treatment Group N9831 adjuvant therapy trial, Knutson et al demonstrated generation of endogenous HER2-specific antibody responses that correlated with survival outcomes.[36]

A recent report by Chen et al detailed a single-arm feasibility study evaluating a vaccine combined with cyclophosphamide and trastuzumab in HER2-positive metastatic breast cancer patients.[37] The vaccine was allogeneic, targeting the HER2 antigen that consisted of the SKBR3 and T47D cell lines genetically modified to secrete GM-CSF. This strategy addresses the issue of the hostile metastatic microenvironment by administering: 1) a cytokine-modified vaccine; 2) an immune-modulating cyclophosphamide dose that can relieve the suppressive effects of regulatory T cells[38]; and 3) the monoclonal antibody trastuzumab, which, as previously detailed, has immunomodulatory activity. The trial confirmed the safety of this combination approach and demonstrated antigen-specific immune responses. The clinical benefit-defined as complete response + partial response + stable disease-at 1 year was 40%. A randomized phase II trial further evaluating this strategy has completed accrual; the results are anticipated within a year (personal communication with Leisha Emens, MD).

Our group has also been interested in evaluating the synergy between trastuzumab and single-epitope CD8+ T-cell–eliciting vaccines. The early-phase trials evaluating E75 + GM-CSF began accruing before trastuzumab became part of the standard adjuvant therapy regimen used to treat patients with HER2-positive breast cancer. During the course of those trials, data showing the benefit of trastuzumab led to a change in practice. Therefore, 12 patients with HER2-positive breast cancer, who were enrolled later in the study, received trastuzumab as part of their standard therapy followed by vaccination with E75 + GM-CSF. After 5 years, none of these 12 patients had disease recurrence.[39] Our group also completed a phase II trial evaluating a second MHC class I peptide derived from the HER2 protein, GP2, combined with GM-CSF and administered in the adjuvant setting.[10] The trial randomized HLA-A2–positive patients to receive GP2 + GM-CSF vs GM-CSF alone. In a per-treatment analysis at a median follow-up of 34 months, HER2-overexpressing patients vaccinated with GP2 + GM-CSF after receiving trastuzumab (n = 48) had a DFS rate of 100% compared with 89% in control patients inoculated with only GM-CSF after trastuzumab (n = 50; P = .08).[40] These studies administered the vaccine following completion of trastuzumab. To evaluate the safety of concurrent administration, we completed a phase I study that demonstrated the combination to be safe, without cardiac toxicity.[41] These data are consistent with a report from Disis et al that showed concurrent treatment with trastuzumab and a HER2-specific helper T-cell vaccine was safe for metastatic HER2-positive breast cancer patients.[42] Our group is currently conducting two phase II trials investigating trastuzumab with NeuVax. One study is accruing high-risk HER2-positive breast cancer patients and randomizing them to vaccine plus trastuzumab or trastuzumab alone (ClinicalTrials.gov identifier: NCT02297698). The second study is enrolling patients with HER2 low- and intermediate-expressing (IHC 1+ or 2+) tumors that are node-positive-or if node-negative, also hormone receptor–negative-and randomizing them to vaccine plus trastuzumab or trastuzumab alone (ClinicalTrials.gov identifier: NCT01570036).

Immunoprevention

There is great interest in investigating cancer vaccines for primary prevention. Through a process known as immune surveillance, tumor antigen–specific T cells can identify and eliminate nascent tumor cells. Tumor cells that escape detection and elimination can progress to become clinical disease.[43] This process of escape can be mediated by many processes to include MHC class I expression loss, increased expression of T-cell inhibitory receptors, and enhanced immunosuppressive mechanisms within the tumor microenvironment.[44-50] It has therefore been hypothesized that strengthening immunosurveillance by vaccination would favor elimination of tumor cells before they could become established cancers,[44-50] which would be comparable to the infectious disease model. For virally induced tumors, such as cervical cancer, the FDA has approved vaccines that may guard against the development of these cancers.

The majority of malignancies are not virally induced. For these cancers, vaccines against tumor antigens that the immune system targets could be developed. One strategy to begin investigating such an approach is to initially evaluate vaccines in patients with premalignant lesions to boost or prime the immune response, thereby preventing recurrence or progression to cancer. As an example, Kimura et al has completed a study evaluating an MUC1 vaccine in patients who recently had advanced colonic adenomas removed.[51] In that study, immune responses were stimulated in approximately 50% of patients.

Ductal carcinoma in situ (DCIS) represents a premalignant lesion in breast cancer that could be targeted with vaccination. In a trial conducted by Sharma et al, patients diagnosed with DCIS were vaccinated with an autologous dendritic cell vaccine before surgical resection of their tumor. The dendritic cells were pulsed with a combination of six MHC class II HER2-derived peptides. For HLA-A2–positive patients, E75 and a second MHC class I peptide were added. The study enrolled 27 patients and demonstrated the vaccine to be safe and effective at stimulating an immune response. Five patients had no evidence of residual disease in their surgical specimen. In 11 of the remaining patients, HER2 expression was eradicated following vaccination.[52] Our group has designed a similar trial evaluating the E75 + GM-CSF vaccine in DCIS. The study will enroll 48 patients randomized 2:1 to receive vaccine or GM-CSF alone. Three inoculations will be administered prior to surgery, and patients will then complete the six-shot vaccination series postoperatively. The primary objective is to evaluate E75-specific CTL levels in patients receiving E75 + GM-CSF vs GM-CSF alone. Secondary objectives include evaluating for epitope spreading by looking for the presence of CTL in the peripheral blood specific for other tumor antigens, as well as the degree of lymphocytic infiltrate and extent of HER2 expression in the surgically resected specimen. The trial will begin accrual in the spring of 2016.

Future Directions

Ipilimumab is a monoclonal antibody targeting the T-cell inhibitory molecule, which has been approved for use in patients with unresectable or metastatic melanoma. Based largely on the success of ipilimumab for melanoma in 2013, Science magazine named immunotherapy as the breakthrough of the year.[53] In the intervening years, immunotherapeutic agents targeting another T-cell inhibitory molecule (programmed death 1 [PD-1]) or its ligand (PD-L1) have been approved for use in the treatment of melanoma and advanced lung cancer. Importantly, there are emerging data to suggest that the benefit of these drugs will extend beyond just a few tumor types. For this reason, the American Society of Clinical Oncology recently named immunotherapy as its 2016 Advance of the Year.[54] There is significant interest in employing these agents in breast cancer, particularly in triple-negative breast cancer. At the 2014 SABCS, Nanda et al reported an overall response rate of 18.5% in a phase Ib trial investigating the anti–PD-1 antibody in patients with PD-L1–expressing recurrent or metastatic triple-negative breast cancer.[55] At the 2015 American Association for Cancer Research Annual Meeting, Emens et al reported an overall response rate of 24% in a phase Ia trial investigating the anti–PD-L1 antibody atezolizumab in metastatic triple-negative breast cancer.[56] At the 2015 SABCS, data were reported on early-phase studies evaluating these agents in patients with metastatic hormone receptor–positive breast cancer. In a trial evaluating pembrolizumab, the overall response rate was 12%,[57] while in a trial evaluating avelumab, an anti–PD-L1 antibody, the overall response rate was less than 5%.[58] These data suggest an opportunity for incorporating vaccines into a strategy of targeting these tumors with checkpoint blockade. The majority of breast tumors do not have a significant lymphocytic infiltrate; the median percentage of stromal area infiltrated with tumor-infiltrating lymphocytes in hormone receptor–positive breast cancer is 10%; in HER2-positive breast cancer, 15%; and in triple-negative breast cancer, 20%.[59] It is therefore possible that stimulating a T-cell response by vaccination will enhance the efficacy of agents that target T-cell inhibitory molecules.

Conclusion

There is great enthusiasm for stimulating the immune system to treat or prevent breast cancer. It is likely that vaccines will play a role in this effort; however, the rational development of vaccines and treatment strategies incorporating these vaccines will be critical. As previously highlighted, in order for breast cancer vaccines to be efficacious, they will need to be administered to the correct patient in the correct setting (ie, prevention vs adjuvant vs metastatic) as a component of the correct strategy. A simple strategy such as NeuVax, which combines a single immunogenic peptide with an immunoadjuvant, may prevent disease recurrence when administered in the adjuvant setting. For patients with metastatic disease, it is likely that vaccines will need to be a component of combination therapy that includes chemotherapy, trastuzumab, or checkpoint blockade as previously described. Thoughtful consideration of how to incorporate vaccines into our therapeutic armamentarium will be required to accomplish our goal of “injecting hope” into breast cancer patients.

Financial Disclosure:Dr. Mittendorf is the principal investigator on the phase III clinical trial evaluating nelipepimut-S, as well as two phase II trials evaluating nelipepimut-S in combination with trastuzumab; she is also the principal investigator on a phase II clinical trial evaluating the AE37 vaccine. The University of Texas MD Anderson Cancer Center receives funding from Galena Biopharma to support the conduct of these studies; it also receives funding from Antigen Express to support the conduct of the phase II trial evaluating AE37. Dr. Peoples has inventor rights to the E75 vaccine. This vaccine has been licensed for commercial development and he is entitled to financial proceeds associated with this license per federal policy; he also has partial inventor rights to the AE37 and GP2 vaccines. If licensed, he would be entitled to financial proceeds associated with these licenses per federal policy. Dr. Peoples consults in the development of the E75 and AE37 vaccines.

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