Meredith Slota

Concurrent Trastuzumab and HER2/neu-Specific Vaccination in Patients With Metastatic Breast Cancer

PURPOSE The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer. PATIENTS AND METHODS Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected. RESULTS Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. CONCLUSION Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.

ELISpot for measuring human immune responses to vaccines

The enzyme-linked immunosorbent spot (ELISpot) assay is one of the most commonly used methods to measure antigen-specific T cells in both mice and humans. Some of the primary reasons for the popularity of the method are that ELISpot is highly quantitative, can measure a broad range of magnitudes of response and is capable of assessing critical cellular immune-related activities such as IFN-γ secretion and granzyme B release. Furthermore, ELISpot is adaptable not only to the evaluation of a variety of T-cell functions, but also to B cells and innate immune cells. It is no wonder that ELISpot has evolved from a research tool to a clinical assay. Recent Phase I and II studies of cancer vaccines, tested in a variety of malignancies, have suggested that ELISpot may be a useful biomarker assay to predict clinical benefit after therapeutic immune modulation. This article will discuss the most common applications of ELISpot, overview the efforts that have been undertaken to standardize the assay and apply the method in the analysis of human clinical trials, and describe some important steps in the process of developing a clinical-grade ELISpot.

Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

BackgroundStandardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response.ResultsLinear regression analysis indicates that both stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a robust T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses.ConclusionThese data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

Phase II Study of a HER-2/Neu (HER2) Intracellular Domain (ICD) Vaccine Given Concurrently with Trastuzumab in Patients with Newly Diagnosed Advanced Stage Breast Cancer.

HER2 is a tumor antigen in breast cancer and several trials have demonstrated that breast cancer patients can be immunized against this protein. We have developed HER2 peptide based vaccines that are aimed at eliciting CD4+ Th1 tumor antigen specific T cell responses. Th1 effectors provide immunologic memory, enhance cross priming which will allow the elaboration of tumor specific CD8+ T cells, and stimulate epitope spreading which we have shown to be a potential biomarker of clinical response. 52 patients will be enrolled with the primary objective to determine relapse free survival after active immunization. Eligible patients are newly diagnosed with Stage III (B or C) or Stage IV breast cancer and begin vaccination within 6 months of starting maintenance trastuzumab. This interim report will present data on the first 25 patients enrolled; 21 stage IV and 4 locally advanced patients. The vaccine is well tolerated with all adverse events (AE) being Grade I or 2. The most common AE is injection site reaction. Moreover, the combination of HER2 vaccination with trastuzumab did not result in additive cardiac toxicity in these patients. Immune responses were evaluated by IFN-gamma ELISPOT. To date, 88% of patients immunized developed significant immunity to the components of the ICD vaccine. The majority, 75%, developed robust immunity to the HER2 protein. Our group has recently demonstrated that a broadening of immunity throughout the HER2 protein, to components of the protein that weren9t in the vaccine, i.e. epitope spreading, may be associated with improved survival in vaccinated patients. 63% of immunized patients demonstrated evidence of intramolecular epitope spreading. We questioned whether such high frequencies of homing Type 1 T cells might modulate the immunosuppressive tumor microenvironment, so we evaluated whether circulating serum immunosuppressive cytokines were impacted by immunization. TGF-beta is an immunosuppressive cytokine secreted by tumor stroma and regulatory T cells. We found that the levels of serum TGF-beta decreased significantly in the majority of patients after vaccination. We further analyzed the correlation between the change of serum levels of TGF-beta post vaccination and HER2 ICD vaccine-induced T cell responses. We found that the greater the magnitude of the HER2 specific T cell response, as demonstrated by IFN-gamma secretion, the greater the decrease in serum TGF-beta (p=0.0045, r=0.742). The correlation between the increased epitope spreading T cell response and decreased levels of TGF-beta was even more significant (p=0.0003). The median overall survival has not been reached with 100% of patients alive at this time. Relapse free survival data will be presented. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5102.

Immunization against HIF-1α Inhibits the Growth of Basal Mammary Tumors and Targets Mammary Stem Cells In Vivo

Purpose: Triple-negative breast cancer (TNBC) represents a cancer stem cell–enriched phenotype. Hypoxia-inducible factor-1α (HIF-1α) induces the expression of proteins associated with stemness and is highly upregulated in TNBC. We questioned whether HIF-1α was immunogenic and whether vaccination targeting HIF-1α would impact the growth of basal-like mammary tumors in transgenic mice. Experimental Design: We evaluated HIF-1α–specific IgG in sera from controls and patients with breast cancer. Class II epitopes derived from the HIF-1α protein sequence were validated by ELISPOT. To assess therapeutic efficacy, we immunized Tg-MMTVneu and C3(1)Tag mice with HIF-1α Th1-inducing peptides. Stem cells were isolated via magnetic bead separation. Levels of HIF-1α and stem cells in the tumor were quantitated by Western blotting and flow cytometry. Results: The magnitude (P < 0.001) and incidence (P < 0.001) of HIF-1α–specific IgG were elevated in TNBC patients compared with controls. Both breast cancer patients and donors showed evidence of HIF-1α–specific Th1 and Th2 immunity. Three HIF-1α–specific Th1 class II restricted epitopes that were highly homologous between species elicited type I immunity in mice. After HIF-1α vaccination, mammary tumor growth was significantly inhibited in only C3(1)Tag (basal-like/stem cellhigh; P < 0.001) not TgMMTV-neu (luminal/neu/stem celllow; P = 0.859) murine models. Vaccination increased type I T cells in the tumor (P = 0.001) and decreased cells expressing the stem cell marker, Sca-1, compared with controls (P = 0.004). Conclusions: An HIF-1α vaccine may be uniquely effective in limiting tumor growth in TNBC. Inhibiting outgrowth of breast cancer stem cells via active immunization in the adjuvant setting may impact disease recurrence. Clin Cancer Res; 23(13); 3396–404. ©2016 AACR.

Optimizing the cryopreservation of murine splenocytes for improved antigen-specific T cell function in ELISPOT

ELISPOT assays are routinely used to measure immune responses of T cells in fresh and frozen splenocytes preparations; however, standardized methods for murine ELISPOT are not widely available. Freezing cells can significantly impact the function of T cells. The aim of this study was to optimize cryopreservation protocols to retain antigen-specific T cell function at similar levels as freshly isolated T cells using murine splenocytes. We examined four factors that might have an impact on cell viability and function: freezing medium, resting cells prior to freezing, temperature of the medium for initial dilution of cells after thawing and resting of cells prior to ELISPOT. FVB/N mice were vaccinated with adjuvant only (CFA/IFA) or IGF-IR peptides, previously shown to be immunogenic by ELISPOT analysis. Mouse splenocytes were cryopreserved using five different media: Medium 1 (50% X-Vivo media, 40% FBS, 10% DMSO), Medium 2 (25% RMPI, 65% FBS, 10% DMSO), Medium 3 (90% FBS, 10% DMSO), Medium 4 (Amresco Media) and Medium 5 (EZ-CP2 Media). Prior to freezing, the cells were either rested on ice for 5 hours or immediately frozen and moved to liquid nitrogen. After four weeks, we used media at 37°C or 4°C for initial dilution of cells after thawing. The thawed cells were either rested overnight at 37°C or not rested. The recovery efficiency and T cell function were evaluated by determining cell viability, background levels, peptide responses, and mitogen responses in ELISPOT assays, respectively. We observed significantly lower cell viability when using freezing Medium 4 and 5, compared to the other media tested (Medium 5 vs Media 1-4: P<0.001; Medium 4 vs 1: P<0.001, Medium 4 vs 2 P<0.05). Resting cells for 5 hours prior to freezing resulted in higher viability, however this difference was not significant (P=0.072). Using media warmed to 37°C to dilute thawed samples resulted in significantly higher cell viability as compared to using media at 4°C (P<0.0001). Media 2 and 3 gave significantly lower background than Fresh cells (P<0.05). Medium 3 was the only freezing medium to result in similar levels of IGF1R peptide responses compared to Fresh. Overnight resting of cryopreserved cells before ELISPOT gave significantly lower T cells responses in both PHA and PMA/ionomycin controls compared to unrested cryopreserved cells (P<0.001).However, overnight resting of cells gave mitogen responses most similar to that of fresh cells when PHA or PMA/ionomycin controls were used. The method of cryopreservation can have a tremendous impact upon splenocytes viability and function. By using an optimized cryopreservation protocol, it is possible to obtain antigen-specific T cell function at levels similar to freshly isolated cells.

Vaccine targeting HIF1A in triple negative breast cancer

The high rates of relapse in triple negative breast cancer (TNBC) are thought to be due to the presence of increased levels of cancer stem cells (CSC), which have been shown to be resistant to standard therapies. It has been demonstrated that hypoxia-inducible factor 1 (HIF1A) can induce the expression of numerous gene products associated with stem-ness and epithelial-mesenchymal transition in breast cancer cells and has been shown to be hyperactivated in TNBC. In this study, we aimed to target HIF1A with a therapeutic immune response through active immunization. HIF1A is a tumor-associated antigen. We have determined that both the magnitude and incidence of HIF1A-specific IgG is significantly elevated in TNBC compared to volunteer donors. We identified epitopes derived from HIF1A that selectively elicited IFN-gamma secretion with little to no IL-10 secretion in human peripheral blood mononuclear cells and T cell lines generated with these epitopes responded to recombinant HIF1A protein. Furthermore, these epitopes are highly homologous between mouse and man. To evaluate therapeutic efficacy, we immunized MMTV-neu (HER2+ model) and C3(1)Tag (TNBC model) mice with a plasmid-based vaccine containing an extended sequence of the identified epitopes. Tumor growth was inhibited over 80% (p < 0.0001) in the TNBC model; however, growth was inhibited only by 40% (p < 0.01) in the HER2+ model. We determined the majority of the tumor cells from the TNBC model expressed the mouse stem cell marker, Sca-1, whereas only a minority of the cells derived from the HER2+ model expressed the marker. Finally, we detected a 52% decrease in tumor Sca-1 expression after HIF1A-specific vaccination in the TNBC model (p = 0.004). Targeting HIF1A via active immunization may be an effective way to prevent disease relapse in patients with TNBC.

Abstract P2-05-03: Role of tumor immune environment in tumor initiation and growth rates in mouse mammary tumor models

The role of the tumor immune environment is critical in human breast cancer tumorigenesis and response to therapy: tumors with a Th1 immune activating environment (high cytotoxic CD8+ T cells and low FoxP3 and myeloid derived suppressor cells (MDSC)) show improved response to chemotherapy and improved disease free survival and prognosis, where tumors with a Th2 immune suppressive environment (increased FoxP3, increased MDSC, and decreased CD8 T cells) shows poor response to chemotherapy and worse disease free survival and prognosis. There has not been good correlation with animal models of mammary tumorigenesis and human response to chemotherapy because in mouse models (1) tumor implant models do not show the tumor immune infiltration seen in human tumors (2) xenograph models are not immunocompetent (3) the tumor immune infiltration has not been evaluated the spontaneous mouse mammary models. We evaluated the tumor immune environment in relation to time to tumor development and rate of tumor growth in two commonly used transgenic mouse models: TgMMTV-neu and C3(1)Tag to better define role of the tumor immune environment in tumorigenesis in spontaneous mouse mammary tumor models. Spontaneous tumorigenesis was studied in 80 TgMMTV-neu and 58 C3(1)Tag mice who were observed for tumor development from 6 weeks until tumor ∼1000 mm3. Differences in (1) time to tumor development (2) rate of tumor growth (3) tumor immune environment were evaluated. Between the transgenic mouse models, TgMMTV-neu mice (n = 10) developed tumors later, around 35.0 weeks, and the tumors grew slower with an average growth rate of 47.9 mm3/week than C3(1)Tag mice which developed tumors around 18.2 weeks with an average growth rate of 88.1 (p = 0.006). The tumor infiltration of MDSC inhibitory cells were higher in the C3(1)Tag mice than the TgMMTV-neu mice and approaches significance (17.4% of all lymphocytes in C3(1)Tag and 4.1% in TgMMTV-neu p = 0.08). Furthermore, there are increased CD8+ T cells in TgMMTV-neu mouse tumors than in C3(1)Tag (34.08% CD3+ cells in TgMMTV-neu mice and 22.38% in C3(1)Tag mice, p = 0.07) with similar FoxP3 levels (6.4% in TgMMTV-neu and 10.2% in C3(1)Tag mice p = 0.2) therefore the CD8/FoxP3 ratio in TgMMTV-neu mice is 5.3 where the CD8/FoxP3 ratio in C3(1)Tag mice is 2.2. These data demonstrate that C3(1)Tag mice have more aggressive tumorigenesis than the TgMMTV-neu mice and a more immunosuppressive tumor environment (higher MDSC, lower CD8, and smaller CD8/FoxP3 ratio) supporting the tumor immune environment plays an important role in tumorigenesis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-03.

Abstract P5-16-04: A phase I study of a DNA plasmid based vaccine encoding the HER-2/neu intracellular domain in subjects with HER2+ breast cancer.

HER2+ breast cancer (BC) is associated with early disease relapse, usually to distant sites. This would suggest relapse is due to residual microscopic disease. Generation of vaccine-induced HER2-specific CD4+ T helper immunity (Th1) may result in immunologic eradication of residual HER2+ tumor cells and subsequent development of immunologic memory and epitope spreading (ES), which has been associated with a survival benefit in vaccinated BC patients. We have shown HER2 peptide-based vaccines can generate immunity in BC however, more recently we developed a plasmid DNA based vaccine (pNGVL3-hICD) which may have additional advantages over synthetic peptides. DNA vaccines offer a strategy to immunize against multiple tumor antigens and are able to elicit both CTL and Th1 immunity. Plasmid DNA can also remain at the vaccine site, providing a constant source of antigen. Intradermal (i.d.) delivery of DNA vaccines with GM-CSF as adjuvant may enhance immunogenicity due to local influx of dermal Langerhans cells. We have recently completed a phase I trial utilizing pNGVL3-hICD in optimally treated stage III and IV HER2+ BC patients and have defined vaccine safety profile, optimal dose and schedule; and demonstrated vaccine biologic activity. Methods: A total of 66 subjects with stage III and IV HER2+ BC in complete remission were enrolled sequentially into 1 of 3 pNGVL3-hICD dose arms (22 subjects/arm): Arm 1=10µg, Arm 2=100 µg, and Arm 3 = 500µg. All vaccines were admixed with 100µg GM-CSF and given i.d. monthly for a total of 3 vaccines. Toxicity was assessed at baseline, during vaccination and at follow-up. Immune responses to HER ICD and ECD were assessed with IFN-γ ELISPOT at baseline and serially through week 60 post-vaccination. Linear regression analysis was used to compare differences in immune responses from baseline over the whole study period between dose arms. Vaccine site skin biopsies and peripheral lymphocytes were serially analyzed for plasmid persistence via RT-PCR. Results: 64 subjects (20 in Arm 1; 22 in Arm 2; 22 in Arm 3) completed 3 vaccines. Age, stage/status, number of previous chemotherapy regimens, and use of bisphosphonate and trastuzumab therapies was similar across dose arms. Vaccine-related toxicity was primarily Grade 1/2 injection site reactions, myalgias, arthralgias and not significantly different between arms; no cardiac or grade IV toxicity was observed. Immune responses to HER2 ICD were significantly better in Arms 2 and 3 vs Arm 1 ( p = 0.001 and 0.002, respectively) but not statistically different between Arms 2 and 3. 38 patients had DNA plasmid persistence at the vaccination site with no difference between arms. There has been no detection of DNA plasmid in lymphocytes from patients in all arms. Analyses of survival and ES (HER ECD immune responses) are on-going and will be presented. Conclusions: pNGVL3-hICD was safe and effectively induced persistent HER2 ICD specific Th1 immunity without increased cardiac toxicity. Moreover, immunity was present more than 1 year after end of vaccination, indicative of vaccine-induced immunologic memory. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-16-04.

P1-13-04: Phase II Study of Topical Imiquimod and Abraxane for Treatment of Breast Cancer Cutaneous Metastases.

Background: Breast cancer (BC) cutaneous lesions can present as local chest wall recurrence or isolated sites of metastatic disease. Current treatments with full thickness chest wall resection, radiation therapy and chemotherapy are not curative; and have significant morbidity and poor overall response rates. Combining local immunomodulation and systemic chemotherapy may be more effective in treating cutaneous disease. Topical imiquimod (IMQ), a TLR-7 agonist, has shown clinical activity against cutaneous metastasis. Pre-clinical studies have shown IMQ to stimulate Th1 cytokine secretion and up-regulate immune co-stimulatory molecules at the tumor site; resulting in augmented tumor specific T cell immunity and tumor growth inhibition. Use of paclitaxel in BC, has demonstrated immunostimulatory effects of increased serum IFN-γ and enhanced NK/LAK cell activity. Abraxane (albumin-bound paclitaxel) may be used in conjunction with IMQ as steroid pre-treatment is not required. We hypothesize the immune effects of Abraxane may synergize and augment the IMQ anti-tumor effects, resulting in greater clinical response. A phase II single-arm study of chemoimmunotherapy with topical IMQ and Abraxane was initiated to determine its safety and therapeutic efficacy; and examine its effect on augmenting endogenous tumor specific immunity and inducing tumor molecular alterations associated with inhibition of tumor growth and/or common pathways of BC immune escape. Materials and Methods: Up to 15 BC patients with cutaneous lesions no longer amenable to standard therapy are enrolled and receive 3 treatment cycles. A treatment cycle consist of topical 5% IMQ to target lesions 4 days/week (wk.) and Abraxane 100 mg/m2 on Days 1, 8, 15 every 28 Days. Toxicity is evaluated per CTCAE v3.0 on Days 1, 8, 15 of each cycle and wks. 13, 16, 20, 24. Target lesion antitumor activity is assessed per modified WHO criteria (Complete response (CR); Partial response (PR); Stable disease (SD); Progressive disease (PD)) at baseline, wks. 4, 8, 12, 16, 20, 24. 2-mm target lesion skin biopsies are obtained pre-and post-treatment for histologic analysis and RT-PCR analysis of a 7 IFN-related gene signature associated with tumor inhibition. Immunity to HER2, IGFBP-2, TOPO-IIα, p53 and serum TGF-β levels are evaluated at baseline and wks. 12, 24 with IFN-γ ELISPOT and ELISA, respectively. Results: 10 patients have been enrolled. Median (range) values include: age, 54 years (48-92), time from metastatic diagnosis, 134 months (58-728), prior chemotherapy regimens, 5 (2-10). 5/10 patients had received prior local therapy, e.g., radiation. 5/10, 4/10, and 2/10 patients had triple negative, HER2+ and ER+/PR+ tumors, respectively. In 5 patients completing 3 treatment cycles, overall response rate (ORR) = 100% (3 CR, 2 PR). In the 5 patients who completed 1–2 treatment cycles, ORR = 80% (2 PR, 2 SD, 1 PD). Treatment related toxicity is primarily grade I/II neutropenia, anemia; grade I skin toxicity. Immunologic analyses are ongoing and will be presented with completed clinical data on all patients. Conclusions: Chemoimmunotherapy with topical IMQ and Abraxane is well-tolerated and shows excellent clinical efficacy in treating metastatic cutaneous lesions in heavily pretreated BC patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-13-04.