HER2-specific Immunity Research Articles

A phase II study of concurrent WOKVAC vaccination with neoadjuvant chemotherapy and HER2-targeted monoclonal antibody therapy.

TPS636 Background: HER2+ breast cancer (BCa) is an aggressive breast cancer subtype. The treatment of Stage I-III HER2+ BCa with neoadjuvant chemotherapy and HER2 monoclonal antibodies (trastuzumab and pertuzumab) induces a pathologic complete response in 43-67% of patients. Residual HER2+ disease (i.e. residual cancer burden) after neoadjuvant therapy is associated with a significantly higher risk of recurrence. Evidence supports that HER2 immunity is progressively lost in HER2 oncogenesis and that restoration of HER2 specific CD4+ Th1 immunity is associated with a lower risk of recurrence. Trastuzumab can partially restore HER2 specific immunity, but only in a minority of patients. We have demonstrated that HER2 specific vaccination can augment HER2 specific immunity. While taxane chemotherapy can deplete immunosuppressive cells (MDSCs) 13 – 17 days after treatment creating an optimal time for vaccination. The WOKVAC vaccine is a plasmid vaccine encoding Th1 selective epitopes from HER2 and two other high-risk cancer antigens IGFBP-2 and IGF-1R. The Phase I trial of this vaccine reported that it is safe and all doses induced antigen specific Type I immune responses. We hypothesize that addition of WOKVAC to neoadjuvant chemo/HER2 targeted therapy will induce a significant increase in the number of patients with Th1 CD4+ and CD8+ T cells after neoadjuvant vaccine/chemo/HER2 therapy. Methods: Trial Design: Phase II single arm trial of WOKVAC vaccination with concurrent neoadjuvant taxane based chemotherapy, trastuzumab, and pertuzumab in patients with Stage I-III HER2+ (HR+/-) breast cancer. Enrolled patients will receive WOKVAC vaccine 150mcg on day 13 of cycles 1-3. WOKVAC vaccination may be given with either TCHP or THP. A maximum of 16 patients will be enrolled in this study. Objectives: The primary objective is to evaluate whether concurrent WOKVAC vaccine with chemotherapy and HER2 antibody therapy can significantly increase T-bet+ CD4+ and CD8+ TIL. Secondary objectives are to determine: (1) whether concurrent WOKVAC with neoadjuvant chemo/HER2 therapy is safe, (2) if the magnitude of vaccine induced antigen specific Th1 immunity after neoadjuvant therapy is associated with pathologic response. Statistical Methods: (1) In order to detect a 30% increase in the number of patients with Tbet+ CD4+ and CD8+ TIL in the enrolled patients with 80% power and a one-side alpha of 0.05, our enrollment goal for this study will be 16 patients. (2) Safety will be assessed per CTCAE v5.0, Benchmarks for safety needed to advance this trial concept to a Phase III study will be a grade 3 toxicity rate of ≤13% and a grade 4 toxicity rate of ≤ 7%. (3) The induction of Th1 immunity will be compared against the presence or absence of a complete pathologic response to determine if there is a significant correlation using Pearson r correlation analysis. Targeted Accrual: The study will accrue up to 16 patients. Clinical trial information: NCT04329065 .

Abstract 3564: Vaccination using different platforms encoding HER2-LAMP with heterologous boosting enhances adaptive HER2-specific immunity to enable potent anti-tumor responses

Abstract Background: Immune checkpoint blockade (ICB) antibodies have demonstrated that effective induction of T cell responses can elicit tumor regression of different metastatic cancers, although these responses are restricted to certain individuals with specific types of cancer. To enhance these responses, there has been renewed emphasis in developing different types of cancer-specific vaccines to stimulate and direct T cell and B cell immunity to important oncologic targets, such as the oncogene human epidermal growth factor receptor 2 (HER2), which is expressed in ~20% of breast cancers (BC). In our previous work we used a DNA plasmid vaccine targeting HER2, which utilized a lysosome-associated membrane protein 1 (LAMP) domain to traffic HER2 antigen to endolysosomal compartments. We demonstrated that this approach was more effective at eliciting antigen specific CD4+ and CD8+ T cell responses, and provided a significant but limited survival benefit in an endogenous mouse model of HER2+ BC compared to a HER2-wild type vaccine. Methods: In our current study, we explored the use of this modified antigen construct using different vaccine modalities, including DNA plasmid, adenoviral (Ad), modified vaccinia ankara (MVA), and self-replicating RNA vaccines to identify effective vaccine platforms to elicit HER2-specific immunity and anti-tumor immunity. Additionally, we have tested the combination of these modalities in homologous and heterologous vaccine boosting regimens to determine an optimal strategy to elicit anti-tumor immunity in vivo. Results: In our studies, plasmid and MVA vectors elicited suboptimal immune priming responses, in comparison to Ad and self-replicating RNA vectors that elicit significant HER2-specific T and B cell responses upon vaccination. While timing of boosting is critical, our results reflect that certain platforms (e.g., plasmid DNA vectors) are capable of homologous boosting capacity, while others (e.g., Ad vectors) elicit more robust T and B cell responses to capsid epitopes, limiting homologous boosting capacity for HER2-specific immunity. Significantly, using an endogenous model of metastatic HER2+ BC, we have found that heterologous vaccination with different HER2-LAMP targeted vectors can elicit significantly augmented HER2-specific T and B cell responses that translated into more effective anti-tumor immunity. This immunity proved capable of eliciting tumor regression in ~80% of vaccinated mice, compared to 0% using homologous HER2-WT plasmid vaccination, 30% using homologous HER2-LAMP plasmid vaccination, or 50% with HER2-WT heterologous vaccination. Conclusions: These data demonstrate the potential of utilizing heterologous vaccine platforms encoding LAMP-based endolysosomal trafficking vaccines to elicit HER2-specific immunity and effect anti-tumor responses. Citation Format: Robert D. Marek, Alan Chen, Renhuan Xu, Junping Wei, Tao Wang, Xiao Yang, Gangjun Lei, Teri Heiland, Zachary C. Hartman. Vaccination using different platforms encoding HER2-LAMP with heterologous boosting enhances adaptive HER2-specific immunity to enable potent anti-tumor responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3564.

Phase II trial to evaluate immune-related biomarkers for pathological response in stage II-III HER2-positive breast cancer receiving neoadjuvant chemotherapy with subsequent randomization to multi-epitope HER2 vaccine versus placebo in patients with residual disease post-neoadjuvant chemotherapy.

TPS610 Background: Several studies demonstrated worsening disease-free survival in patients who failed to achieve complete pathological response after neoadjuvant chemotherapy (NAC), particularly in HER2-positive (HER2+) breast cancer. Despite the recent approval of trastuzumab emtansine (T-DM1) in patients with residual disease after NAC approximately 12% of patients still develop recurrent and metastatic disease. TPIV100 is a multi-epitope vaccine that includes a pool of 4 degenerate HER2-derived HLA-DR epitopes, which activate CD4 helper T cells, admixed with GM-CSF. In our previous phase I trial this vaccine was shown to be safe in combination with trastuzumab in stage II-III HER2+ breast cancer after completion of standard of care chemotherapy. Furthermore, this vaccine also generated robust long-lasting T-cell immune responses and antibody immunity against HER2. Methods: This trial is a multi-center, randomized, placebo-controlled, phase II trial of TPIV100 in combination with T-DM1 in stage II-III HER2+ breast cancer patients with residual disease after NAC. This trial is currently opened through the ACCRU consortium. Eligible patients include those with stage II-III HER2+ with residual disease, in the breast and/or lymph nodes, after trastuzumab ± pertuzumab-based NAC, with ECOG PS ≤ 2 and adequate organ function. Patients with baseline left ventricular ejection fraction < 50%, history of trastuzumab-related cardiac toxicity, myocardial infarction or stroke < 6 months, history of congestive heart failure, autoimmune disease, immunocompromised patients with known HIV or those on chronic steroid, hypersensitivity to GM-CSF, and other active malignancy < 3 years are excluded. TPIV100 or placebo, in combination with GM-CSF, will be given concurrently with T-DM1. To ensure that TPIV100 in combination with T-DM1 is safe, there is a run-in phase with 20 patients treated with the combination. If there is no significant dose-limiting toxicity observed in the run-in phase, the trial will be expanded to the randomized phase II portion, which will include 240 patients. Eligible patients will be randomized in a 2:1 fashion with ER/PR as a stratification factor. The primary endpoint is invasive disease-free survival, and the secondary endpoint includes immunogenicity of TPIV100 as assessed by IFN-g ELIspot analysis. Correlative studies include assessment of helper T-cell response distribution (including Th1, Th2, Th17, Tfh), HER2-specific antibody immunity, and HLA genotype. Currently, 20 patients in the run-in phase have been enrolled. Enrollment to the randomized phase II portion is expected to begin in March 2022. Clinical trial information: NCT04197687.

High frequency of HER2-specific immunity observed in patients (pts) with HER2+ cancers treated with margetuximab (M), an Fc-enhanced anti-HER2 monoclonal antibody (mAb).

1030 Background: Previous studies have shown that 44-71% of trastuzumab (T)-treated pts develop HER2-specific immunity (Clin Cancer Res 2007, 13:5133; Cancer Res 2016, 76:3702; Breast Cancer Res 2018, 20:52). M is an Fc-engineered mAb that shares similar HER2 binding and antiproliferative activity as T. The Fc region of M has been engineered for increased affinity to the activating FcγRIIIA (CD16A) and lower binding to the inhibitory FcγRIIB (CD32B), attributes that may enhance the mAb’s immune function, such as antigen presentation. Methods: HER2+ cancer pts who progressed on prior therapy received M (0.1-6 mg/kg QW for 3 of every 4 weeks [N = 34]; or 10-18 mg/kg Q3W [N = 32]) in phase 1 trial NCT01148849. PBMC and plasma were collected pre-dose and 50 days post-dose for 46 pts and > 4 years for 3 pts on long-term treatment. Response to HER2 or control antigens (Ag) was assessed by IFNγ ELISpot and antibody (Ab) ELISA. In 14 pts, T-cell antigen receptor (TCR) repertoire was assessed by immunosequencing. Results: Following M treatment, mean frequencies of IFNγ-producing T cells specific for intra- or extracellular fragments of HER2 increased by 2.5 to 6-fold (p < 0.0027, paired t test). Most (95%) of subjects responded to ≥2 of 6 (median = 5) HER2 Ag. Mean HER2-specific Ab concentration increased by 19-54% (p < 0.0001), with all subjects responding to ≥2 (median = 5) of the 6 Ag. A small 1.6-fold increase in IFNγ response to control CMV/EBV/Flu (but not tetanus or cyclin D1) peptides was observed; no increase in Ab response to control Ag was noted. Subsets of HER2-specific T-cell and Ab responses persisted during long-term treatment. Median TCR clonality increased by 54% (p = 0.003), with an average of 125 unique clones expanding, while overall TCR diversity remained unchanged (p = 0.19). Conclusions: Treatment of HER2+ cancer with M was associated with enhanced HER2-specific T-cell and Ab responses together with increased TCR clonality, indicative of a more focused T-cell repertoire. The high frequency of HER2-specific immunity in M-treated patients ( > 95%) is consistent with its enhanced Fc region contributing to linkage of innate and adaptive immune responses.

What is the role of HER2-specific antibody immunity in patients with HER2-positive early breast cancer receiving chemotherapy plus trastuzumab?

Breast cancer is the most common malignancy among women worldwide and the leading cause of cancer death in many countries (1). In around 20% to 25% of breast cancer cases, there is an overexpression of HER2 or an amplification of its gene (HER2-positive subtype); HER2 is a transmembrane receptor implicated in tumor replication, invasion and dissemination (2). HER2-positive breast cancer is characterized by a worse prognosis, aggressive disease, higher mitotic index, worse nuclear grade and lymph nodes positivity (3).

Generation of HER2-specific antibody immunity during trastuzumab adjuvant therapy associates with reduced relapse in resected HER2 breast cancer

BackgroundResected HER2 breast cancer patients treated with adjuvant trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induce HER2-specific antibodies which correlate with improved survival in HER2 metastatic breast cancer patients. It remains unclear whether the generation of immunity required trastuzumab and whether endogenous antibody immunity is associated with improved disease-free survival in the adjuvant setting. In this study, we addressed this question by analyzing serum anti-HER2 antibodies from a subset of patients enrolled in the NCCTG trial N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively.MethodsPre-and post-treatment initiation sera were obtained from 50 women enrolled in N9831. Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and compared between arms and with disease-free survival.ResultsPrior to therapy, across all three arms, N9831 patients had similar mean anti-HER2 IgG levels. Following treatment, the mean levels of antibodies increased in the trastuzumab arms but not the chemotherapy-only arm. The proportion of patients who demonstrated antibodies increased by 4% in Arm A and by 43% in the Arms B and C combined (p = 0.003). Cox modeling demonstrated that larger increases in antibodies were associated with improved disease-free survival in all patients (HR = 0.23; p = 0.04).ConclusionsThese results show that the increased endogenous antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant, in view of its correlation with improved disease-free survival. The findings may have important implications for predicting treatment outcomes in patients treated with trastuzumab in the adjuvant setting.Trial registrationClinicalTrials.gov, NCT00005970. Registered on July 5, 2000.

Nitrated T helper cell epitopes enhance the immunogenicity of HER2 vaccine and induce anti-tumor immunity

Human epidermal growth factor receptor 2 (HER2) is an attractive target for cancer vaccine. However, autoimmune tolerance prevents vaccines based on HER2 protein from inducing long-lasting, highly effective anti-tumor immunity. In this study, we proved that the introduction of p-nitrophenylalanine in the universal T cell epitope (named NitraTh) enhances humoral immunity induced by B cell epitope and cellular immunity induced by CTL epitope. Moreover, this NitraTh epitope can work in both mouse and human immune system. When fused with extracellular domain 23-83 of HER2, NitraTh epitope help to break the self-tolerance of HER2 and induced strong HER2 specific humoral immunity and cellular immunity. Vaccination with HER2-NitraTh can significantly inhibit the growth of HER2+B16F10 tumor cells. These findings have important implications for developing therapeutic cancer vaccines.

Abstract LB-113: Recombinant HER2/Neu expressing Listeria combined with radiation safely delays tumor progression and prolongs overall survival in a phase I clinical study in canine osteosarcoma

Abstract The purpose of this study is to determine the safety and efficacy of a highly attenuated recombinant Listeria monocytogenes expressing a chimeric human HER2/neu (Lm-LLO-HER2/neu) in combination with palliative radiation (RT) to induce HER2 specific immunity and delay tumor progression in dogs with spontaneous osteosarcoma (OSA). Dogs develop OSA that recapitulates many aspects of human OSA, and are recognized as a clinically relevant, large animal model in which to evaluate novel therapies. Previously we have shown that Lm-LLO-HER2/neu administration to dogs with HER2+ OSA following amputation and chemotherapy is safe, breaks tolerance to HER2 and prolongs overall survival. Ten systemically healthy dogs with histopathologically confirmed, treatment naïve, HER2+ appendicular OSA, and no evidence of cardiac or metastatic disease were enrolled. All dogs received 2 × 8Gy fractions of RT on consecutive days, followed by the first of 8 intravenous doses of Lm-LLO-HER2/neu given once every 3 weeks. Dogs were monitored at each treatment and every 2 months after for systemic and cardiac toxicity, lameness and quality of life (QOL) (using a validated owner questionnaire). Radiographs were performed at baseline, week 10, week 22 and every 2 months thereafter to determine the effect on the primary tumor and development of pulmonary metastases. PBMCs were collected every 3 weeks to evaluate HER2/neu specific T cell responses. The primary endpoint was time to progression; secondary end points were safety and overall survival. Results are compared to historical canine controls that received 2 × 8Gy RT alone. Repeat Lm-LLO-HER2/neu administration was well tolerated with no systemic or cardiac toxicity. Lameness and QOL showed near uniform improvement over the study period. To date, limb radiographs of 7 dogs have been taken 10 weeks post enrollment and showed no evidence of tumor progression. 4 dogs have currently completed the scheduled 8 vaccinations, and limb radiographs of these dogs taken at 22 weeks, showed no progression of the primary lesion. At the time of writing, 4/10 dogs have died; 2 dogs were euthanized due to pathological fracture, and 2 dogs were euthanized due to metastatic disease. One dog, still alive, has developed pulmonary metastatic disease. At the time of writing, median time to progression is 243 days and median survival time (MST) is 285 days. Historically, MST of dogs treated with RT alone is 136 days. Results of IFN-γ ELISpot assays are pending. To conclude, our preliminary results show repeat Lm-LLO-HER2/neu administration is safe and well tolerated with no cardiac toxicity. Our results also suggest that combination RT and Lm-LLO-HER2/neu immunotherapy improves QOL, delays primary tumor progression and prolongs overall survival in canine OSA. These findings may have important translational relevance for human patients with OSA and other HER2/neu+ cancers. Citation Format: Nicola Mason, Josephine Gnanandarajah, Danielle Laughlin, Julie Engiles, Anu Wallecha, Yvonne Paterson. Recombinant HER2/Neu expressing Listeria combined with radiation safely delays tumor progression and prolongs overall survival in a phase I clinical study in canine osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2015-LB-113

Associations of HER2-specific immunity with survival during treatment with trastuzumab and chemotherapy in breast cancer.

587 Background: The addition of trastuzumab to chemotherapy improves response to therapy and extends survival among patients with HER2-positive (HER2+) breast cancer. Prior work showed that trastuz...

A phase 1 study of a heterologous prime-boost vaccination involving a truncated HER2 sequence in patients with HER2-expressing breast cancer

A phase 1 clinical trial was conducted to assess the safety, tolerability, and preliminary efficacy of a heterologous prime-boost strategy involving plasmid DNA (pHM-GM-CSF, expressing truncated human epidermal growth factor receptor 2 (HER2) and granulocyte macrophage colony-stimulation factor (GM-CSF) as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in patients with stage III–IV metastatic breast cancer expressing HER2. Nine eligible subjects were divided into three cohorts based on the dosages (2, 4, and 8 mg/patient/visit) of pHM-GM-CSF used as the primer, which was intramuscularly injected three times at weeks 0, 2, and 4. It was followed by a single injection of Ad-HM (3 × 109 virus particles), used as a booster, at week 6. During the 6-month follow-up period, adverse events (AEs), pharmacokinetics and pharmacodynamics, and HER2-specific cellular and humoral immune responses were evaluated. Seven cases of minor grade 1 toxicities in four of nine subjects and no serious drug-related AEs were reported. HER2-specific cell-mediated or humoral immunity was produced in all (100%) or three subjects (33%), respectively. One subject showed a partial response, and seven subjects had stable diseases. However, there were no differences in clinical tumor response and HER2-specific immune responses among the cohorts. These results showed that intramuscular injections of pHM-GM-CSF and Ad-HM were well tolerated and safe.

Exosomal pMHC-I complex targets T cell-based vaccine to directly stimulate CTL responses leading to antitumor immunity in transgenic FVBneuN and HLA-A2/HER2 mice and eradicating trastuzumab-resistant tumor in athymic nude mice

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.

Generation of adaptive HER2-specific immunity in HER2 breast cancer patients by addition of trastuzumab to chemotherapy in the adjuvant setting: NCCTG (Alliance) study N9831.

522 Background: In the adjuvant setting, patients with HER2 breast cancer treated with trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induces HER2-specific antibodies which correlate with response to therapy in patients with HER2+ metastatic breast cancer. It remained unclear from those studies, however, whether the HER2-specific immunity played a role and if antibody immunity was associated with improved disease free survival in the adjuvant setting. In the present study, we addressed these questions by analyzing sera samples from a subset of patients enrolled in the NCCTG adjuvant trial, N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively. Methods: Pre-and post-treatment initiation sera were obtained from 50 women enrolled in N9831 (22 Arm A; 14 Arm B, and 14 Arm C). Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and presented as an index (>0.2 was considered a positive response). Results: Prior to therapy, across all three arms, N9831 patients had similar mean HER2 IgG levels (0.19 units in Arm A, 0.14 in Arm B, and 0.23 in Arm C, P=0.85). Following treatment, the mean levels of antibodies increased in Arm B to 0.35 units and in Arm C to 0.56 units and were higher (p<0.001) than in Arm A where levels did not increase. The proportion of patients who demonstrated antibody immunity increased by 9% in Arm A, 50% in Arm B and 28% in Arm C (P=0.026). Although the event rate was low in this cohort, Cox modeling suggested that larger increases in antibodies were associated with improved disease free survival (HR=0.23; p=0.04). Conclusions: These results show that the increased antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant and results from the inclusion of trastuzumab. The findings may have important implications for improving treatment outcomes in patients treated with trastuzumab.

Abstract OT3-1-02: Phase II randomized study of combination immunotherapy with or without Polysaccharide Krestin (PSK®) concurrently with a HER2 ICD peptide-based vaccine and trastuzumab in patients with stage IV breast cancer

Abstract Background: Endogenous immunity in patients with HER2+ metastatic breast cancer (MBC) is likely dampened by an immune-suppressive tumor microenvironment and not sufficient to control tumor growth. Thus, most patients have disease relapse after achieving complete remission with standard therapies. Immunomodulation directed at enhanced stimulation of tumor specific immunity could result in immunologic eradication of residual HER2+ tumor cells and prevent BC relapse. We have shown PSK to be a potent TLR-2 agonist that stimulates both innate and adaptive immunity in a BC mouse model. Additionally, we have shown combination immunotherapy with HER2 peptide vaccines and trastuzumab (TRAZ) to be safe and able to elicit HER2 specific Th1 immunity and epitope spreading (ES) which has been associated with survival in vaccinated patients. Lastly, decreased serum TGF-β elicited by HER2 vaccination correlates with Th1 ES and may serve as a biomarker to predict cancer vaccine efficacy. We hypothesize that PSK, when given with TRAZ can augment vaccine induced HER2 specific TH1 immunity and prevent disease relapse in patients with optimally treated HER2+ MBC. Trial design: Phase II randomized two-arm clinical trial. Patients will be enrolled and randomly assigned in equal numbers to 1 of 2 arms (15 patients/arm) as follows: Arm 1:HER2 ICD vaccine, TRAZ and placebo or Arm 2:HER2 ICD vaccine, TRAZ and PSK. All patients will receive 3 monthly HER2 ICD vaccines plus TRAZ and 4 months of concomitant PSK or placebo. Serial blood draws for immunologic monitoring will be done. Eligibility criteria: Patients with Stage IV HER2+ BC who have been treated with definitive therapy and are: (1) without evidence of disease or have stable-bone only disease, (2) receiving TRAZ monotherapy, and (3) without clinically significant autoimmune disease. Patients must have normal LVEF per MUGA scan or echocardiogram. Aims: (1) Evaluate safety of PSK when given with a HER2 vaccine and TRAZ (2) Evaluate the effect of PSK on serum TGF-β levels when given with a HER2 vaccine and TRAZ and (3) Evaluate the effect of PSK on intermolecular ES when given with a HER2 vaccine and TRAZ. A secondary objective is to evaluate progression free survival (PFS) and overall survival (OS). Statistical methods: (1) Toxicity will be determined by clinical and chemical parameters and grading will be done per CTEP CTCAE 4.0.; (2) Evaluation of TGF-β levels, pre and post-PSK treatment will be assessed with linear regression models; and analysis of multiple post-baseline measurements will be performed using generalized estimating equations; (3) A positive antigen-specific immune response will be defined as a precursor frequency >1:20,000 antigen-specific peripheral blood mononuclear cells. Differences in the levels of HER2 immunity will be evaluated between arms using a two-tailed T test. The degree of ES in each arm will be evaluated with generalized linear modeling; (4) Large differences in PFS and OS observed between groups will be noted and described. Target accrual: 30 patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-1-02.

Abstract P5-18-07: Completed SN33 Trial: 60 month follow-up of Early Stage Node Positive HER2 Negative patients with NeuVax ™(E75) and sargramostim.

Abstract Background: Despite recent advances in adjuvant treatment, the prognosis for HER2 negative patients has not improved. Unlike HER2 positive patients, the benefit of Herceptin has not been shown. Here we present the final 60-month results of a phase 1/I2 clinical trial vaccinating early stage node positive breast cancer patients with NeuVax (E75) and sargramostim following adjuvant chemotherapy and radiation. Previously, data reported from this trial was combined with a node negative trial that used the same treatment schema. Those reports showed the vaccine was safe, and effective at stimulating HER2-specific immunity. We report safety and efficacy (Disease Free Survival, DFS) data through 60 months. Methods: Eligible patients had excised early stage node-positive breast cancer with any level of HER2 expression. After completion of standard of care chemotherapy and radiation, patients were enrolled but not randomized: patients were assigned to the vaccine group (VG) only if they were HLA A2/A3+. The non-treated patients were followed as a control group (CG). Hormone receptor positive patients received appropriate adjuvant therapy concomitant with vaccine. The phase I/II trial was performed with phase 1 as a dose escalation/schedule optimization where VG was given 3–6 monthly inoculations of NeuVax (E75) with the immunoadjuvant, sargramostim. The phase 2 portion utilized the optimal dose and schedule carried forward. Herceptin became commercially available during the conduct of the trial, and HER2 positive patients were offered this treatment but remained on trial. DFS was summarized by Kaplan-Meier lifetables and analyzed by the log-rank test. Due to waning immunity after the scheduled monthly vaccinations, a voluntary booster program was initiated to be integrated into the trial process. Results: 97 patients were enrolled; 53 in the VG and 44 in the CG. The VG and CG were well-matched with the only difference being trastuzumab therapy (18.5% in VG vs. 4.8% in CG, p = 0.03). There were no HER2 0 patients in the VG. Vaccination was well tolerated with primarily grade 1 and grade 2 toxicity. No VG patients relapsed during the vaccination period. ITT patients in this phase 1/2 trial favored VG in DFS at 24 months (VG 90.6%, CG 79.5%, p = 0.1194); at 60 months, the VG continues to have fewer recurrences than the CG (VG 84.5% vs. 77.1%, p = 0.3046). In the patients who were treated at the optimum (Phase 2) dose, the difference in the DFS at 24 months observed was (VG 96.2%, CG 79.5%, p = 0.0597); at 60 months, the VG continues to have fewer recurrences than the CG (92.3% vs. 77.1%). When the HER2 positive patients were removed from the population, the Phase 2 HER2 Negative VG had a significantly improved DFS at 24 months (100% vs. 80.6%, p = 0.045); at 60 months, VG retains a 17% difference (VG 94.7% vs. CG 77.4%). Conclusions: NeuVax (E75) and sargramostim vaccine is safe and well–tolerated in early stage breast cancer patients. With follow-up at 60 months, the vaccine used as adjuvant therapy continues to reduce relapses in node positive HER2 negative patients treated at an optimal dose. A phase 3 multicenter, multinational double-blind trial is underway. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-18-07.

Abstract P2-20-03: Combination Immunotherapy for Breast Cancer Patients: Safety and Immunologic Data from a Phase II Trial Administering a HER2/neu-Derived Peptide vaccine (AE37+GM-CSF) Sequentially or Concurrently with Trastuzumab in the Adjuvant Setting

Abstract Background. AE37 is a HER2-derived peptide (AA:776-790) modified to enhance antigen-specific stimulation of CD4+ T cells. A phase I trial has shown that AE37 mixed with GM-CSF (GM) is safe and effective in raising HER2-specific immunity. Our group is conducting a prospective, randomized, single-blinded phase II clinical trial of AE37+GM vs GM alone. The goal of the current study is to determine the effects on cardiac safety and immunologic response of vaccinating sequentially or concurrently with administration of trastuzumab (Tz). Methods. The trial is enrolling high-risk breast cancer patients with tumors having any degree of HER2 expression (IHC 1-3+). At the time of enrollment, all patients are disease-free having completed surgery, chemotherapy and when indicated, XRT. Patients receiving Tz as part of their standard therapy were administered the vaccine sequentially after completion of Tz or concurrently with Tz. Cardiac toxicity was assessed by determining ejection fraction (EF). Immunologic response was monitored by delayed-type hypersensitivity (DTH) responses and [3H]thymidine proliferative assays for the hybrid AE37 and native AE36 (unmodified AA:776-790) peptides. Results. To date the trial has enrolled 102 patients at our international site, including 51 receiving Tz as part of standard therapy. Of these, 30 were randomized to receive AE37+GM; 13 sequentially after Tz (group 1) and 17 concurrently (group 2). 21 patients were randomized to receive GM alone. There was no change in EF pre-and post completion of vaccination series for patients in group 1 (mean 63±1.7% vs 65±2.9%, p=.32) or group 2 (mean 68±3.7% vs 63±%, p=.14). There was a statistically significant difference in DTH response pre-vs post-vaccination with AE37+GM in both group 1 (mean 1 mm vs 20 mm, p<.001) and group 2 (2 mm vs 29 mm, p<.001); however, there was no significant difference in the post-vaccination DTH response when comparing group 1 to group 2 (p=.16). There was no significant difference in the DTH response pre vs post inoculation for patients receiving GM alone. There was a significant proliferative response to AE36 and AE37 in both group 1 and 2 with the response greater in group 2. There was no proliferative response in patients receiving GM alone. Conclusions. Administration of the AE37+GM peptide vaccine with Tz administered either concurrently or sequentially is safe without any increase in the incidence of cardiotoxicity. Vaccination with AE37+GM concurrently with Tz results in enhanced proliferation suggesting that there may be clinical benefit to such a combination immunotherapy strategy. Patients will continue to be followed to determine the effects of vaccination with sequential vs concurrent Tz administration on clinical outcome. Figure available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-20-03.

Vaccination with a plasmid DNA encoding HER-2/neu together with low doses of GM-CSF and IL-2 in patients with metastatic breast carcinoma: a pilot clinical trial

BackgroundAdjuvant trastuzumab (Herceptin) treatment of breast cancer patients significantly improves their clinical outcome. Vaccination is an attractive alternative approach to provide HER-2/neu (Her2)-specific antibodies and may in addition concomitantly stimulate Her2-reactive T-cells. Here we report the first administration of a Her2-plasmid DNA (pDNA) vaccine in humans.Patients and MethodsThe vaccine, encoding a full-length signaling-deficient version of the oncogene Her2, was administered together with low doses of GM-CSF and IL-2 to patients with metastatic Her2-expressing breast carcinoma who were also treated with trastuzumab. Six of eight enrolled patients completed all three vaccine cycles. In the remaining two patients treatment was discontinued after one vaccine cycle due to rapid tumor progression or disease-related complications. The primary objective was the evaluation of safety and tolerability of the vaccine regimen. As a secondary objective, treatment-induced Her2-specific immunity was monitored by measuring antibody production as well as T-cell proliferation and cytokine production in response to Her2-derived antigens.ResultsNo clinical manifestations of acute toxicity, autoimmunity or cardiotoxicity were observed after administration of Her2-pDNA in combination with GM-CSF, IL-2 and trastuzumab. No specific T-cell proliferation following in vitro stimulation of freshly isolated PBMC with recombinant human Her2 protein was induced by the vaccination. Immediately after all three cycles of vaccination no or even decreased CD4+ T-cell responses towards Her2-derived peptide epitopes were observed, but a significant increase of MHC class II restricted T-cell responses to Her2 was detected at long term follow-up. Since concurrent trastuzumab therapy was permitted, λ-subclass specific ELISAs were performed to specifically measure endogenous antibody production without interference by trastuzumab. Her2-pDNA vaccination induced and boosted Her2-specific antibodies that could be detected for several years after the last vaccine administration in a subgroup of patients.ConclusionThis pilot clinical trial demonstrates that Her2-pDNA vaccination in conjunction with GM-CSF and IL-2 administration is safe, well tolerated and can induce long-lasting cellular and humoral immune responses against Her2 in patients with advanced breast cancer.Trial registrationThe trial registration number at the Swedish Medical Products Agency for this trial is Dnr151:785/2001.

Timed Sequential Treatment With Cyclophosphamide, Doxorubicin, and an Allogeneic Granulocyte-Macrophage Colony-Stimulating Factor–Secreting Breast Tumor Vaccine: A Chemotherapy Dose-Ranging Factorial Study of Safety and Immune Activation

Granulocyte-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. We conducted a 3 x 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. Twenty-eight patients received at least one immunization, and 16 patients received four immunizations. No dose-limiting toxicities were observed. HER2-specific delayed-type hypersensitivity developed in most patients who received vaccine alone or with 200 mg/m(2) CY. HER2-specific antibody responses were enhanced by 200 mg/m(2) CY and 35 mg/m(2) DOX, but higher CY doses suppressed immunity. Analyses revealed that CY at 200 mg/m(2) and DOX at 35 mg/m(2) is the combination that produced the highest antibody responses. First, 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.

HER2/neu (HER2) specific T-cell immunity in patients with HER2+ inflammatory breast cancer (IBC) and prognosis

3057 Background: IBC is rare, highly aggressive, and associated with worse prognosis when compared to non-IBC tumors. Moreover, multimodality treatment has had little impact on overall prognosis. HER2 is overexpressed in about 40% of IBC tumors and is associated with worse overall survival (OS). We have developed vaccines that elicit both HER2-specific CD4+ and CD8+ T-cell immunity in HER2+ cancer patients. Generation of HER2-specific T-cell immunity could (1) target immunogenic and biologically relevant proteins such as HER2 in IBC, (2) result in immunogenic eradication of HER2+ tumor cells, and (3) potentially prevent disease relapse when used in the adjuvant setting after standard therapy. A retrospective analysis of IBC patients immunized with HER2 vaccines was conducted to better understand the development of HER2-specific T-cell immunity and its possible impact on overall prognosis in IBC. Methods: Clinical and immunological data of IBC patients enrolled in University of Washington IRB approved HER2 vaccine trials was collected and reviewed. 27 patients immunized between 1996–2008 were identified; and 24/27 subjects who received vaccines designed to elicit both CD4+/CD8+ immunity were included in immunologic and survival analysis. The 24 subjects received either a HER2 DNA or HER2 peptide-based vaccine that were admixed with GM-CSF and given intradermally monthly for a total of 3 DNA or 6 peptide vaccines. Immune responses were assessed via IFN-γ ELISPOT at baseline and post-vaccination. Results: All 24 subjects had stage III IBC and median age was 48 (range 34–77). 10/24 (42%) patients had ER/PR+ tumors, 9/24 (37%) had received trastuzumab, and 15/24 (62%) had received multimodality treatment (chemotherapy, mastectomy, radiotherapy). 12/18 subjects (66%) evaluable for immunologic response developed HER2-specific T-cell immunity post-vaccination. Median OS for patients (n=6) not generating HER2-specific immunity was 31 months and median OS for the 12 patients who developed HER2-specific immunity has not been reached at median follow-up of 46 months, (p=0.026). Conclusions: Patients with IBC are able to generate HER2-specific T-cell immunity after HER2 vaccination, and development of HER2-specific immunity may impact survival. No significant financial relationships to disclose.

Association of the level of HER2/neu (HER2) gene amplification in breast cancer and the magnitude of antigen specific T-cell immunity achieved after HER2 vaccination

3059 Background: Studies have demonstrated that the level of HER2 gene amplification in breast cancer, assessed by fluorescence in situ hybridization (FISH), correlates with favorable clinical response after treatment with trastuzumab. We questioned whether HER2 gene amplification impacted the development of HER2-specific T-cell immunity following immunization with a HER2 vaccine. Methods: Patients with HER2+ stage III or IV breast cancer, treated to complete remission or stable bone only disease, were enrolled in one of two concurrent clinical trials of HER2-specific vaccines. Eligibility criteria between the two studies were similar. Patients received either a plasmid DNA-based vaccine encoding the HER2 intracellular domain or a peptide-based vaccine composed of 3 HER2 class II epitopes. Peripheral blood was assessed for HER2-specific T-cell responses by interferon gamma (IFN-g) ELISPOT prior to, immediately after, and 6 months to 1 year after the end of vaccinations. Both immune response and FISH data were available on 31 patients. Results: Correlation of FISH levels to IFN-g spots/well in evaluable patients revealed the level of HER2 gene amplification was not related to the presence of pre-existent HER2-specific T-cell immunity prior to vaccination (p=0.43), the generation of a HER2-specific immune response after vaccination (p=0.35), or the persistence of the HER2-specific T-cell response (p=0.33). However, the magnitude of the T-cell response achieved was less as HER2 gene amplification increased (p=0.05). Conclusions: The level of HER2 gene amplification in the primary tumor can adversely impact the magnitude of HER2-specific T-cell immunity achieved after vaccination. No significant financial relationships to disclose.

Persistent immunity and survival after immunization with a HER2/neu (HER2) vaccine

3010 Background: Our initial vaccine studies showed that optimally treated breast cancer patients can be immunized against HER2 during active immunization. The majority of patients developed T-cell immunity to HER2 peptides and protein and also epitope spreading (ES). The goal of this study was to determine if patients previously immunized with a HER2 vaccine had persistent immunity years after active immunization and to assess their clinical outcome in terms of overall survival (OS). Methods: Subjects eligible for this IRB-approved long term follow-up (LTFU) study: (1) had HER2+ breast cancer and were immunized between 1996–1999 in a phase I HER2 peptide vaccine trial, (2) were at least 1 year out from their last vaccine, and (3) if donating blood samples could not be receiving chemotherapy. 52 patients (37 stage IV, 15 stage III) were identified and 21/52 patients (12 stage IV, 9 stage III) were determined to be living. All 21 subjects were contacted by letter and sent a LTFU Questionnaire. OS was defined as the time between date of vaccine study entry and death or last follow-up and was estimated using the Kaplan-Meier method. Cox proportional hazards were used to determine associations between OS and known clinical and vaccine-related immunologic factors; analyses included all 52 subjects. Long-term T-cell immunity was evaluated using IFN-γ ELISPOT assay. Results: Median follow-up time for the 21 patients still alive was 112 months (range, 104–126 months). Blood samples were collected in 10/21 subjects and 6/8 (75%) evaluble patients had persistent T-cell immunity to immunizing HER2 peptides; and 7/8 patients (88%) had T-cell immunity specific for HER2 protein and peptides not contained in their immunizing mix (defined as ES). In multivariate analysis, number of chemotherapy regimens prior to vaccination (HR=5.7 (CI 95%, 1.5–23; p=<0.001)), and development of ES after HER2 vaccination (HR=0.34 (CI 95%, 0.12–1.0; p=0.05)) were independent predictors of OS. Median OS for 33 subjects who developed ES was 84 months vs 25 months for 16 subjects who did not develop ES. Conclusions: HER2-specific T-cell immunity elicited with active immunization is durable years after vaccination has ended and the generation of ES is an independent predictor of OS. No significant financial relationships to disclose.