Lauren Rastetter

Dendritic Cell–Activating Vaccine Adjuvants Differ in the Ability to Elicit Antitumor Immunity Due to an Adjuvant-Specific Induction of Immunosuppressive Cells

Purpose: We questioned whether the vaccine adjuvant combination of TLR-7 ligand agonist, imiquimod, with granulocyte macrophage colony-stimulating factor (GM-CSF) would result in enhanced dendritic cell recruitment and activation with increased antigen-specific immunity as compared with either adjuvant used alone. Experimental Design: The adjuvant effects of GM-CSF and imiquimod were studied in ovalbumin (OVA) and MMTVneu transgenic mice using peptide-based vaccines. Type I immunity, serum cytokines, myeloid-derived suppressive cells (MDSC), and regulatory T cells (Treg) levels were examined. Results: Both GM-CSF and imiquimod equally induced local accumulation and activation of dendritic cells. Both adjuvants effectively enhanced OVA-specific T-cell responses. We further evaluated the antitumor efficacy of adjuvant GM-CSF and imiquimod immunizing against murine insulin-like growth factor–binding protein-2 (IGFBP-2), a nonmutated oncoprotein overexpressed in the tumors of MMTVneu transgenic mice. Tumor growth was significantly inhibited in the mice receiving IGFBP-2 peptides with GM-CSF (P = 0.000), but not in imiquimod vaccine-treated groups (P = 0.141). Moreover, the addition of imiquimod to GM-CSF negated the antitumor activity of the vaccine when GM-CSF was used as the sole adjuvant. While GM-CSF stimulated significant levels of antigen-specific T-helper cell (TH)1, imiquimod induced elevated serum interleukin (IL)-10. Both MDSC and Tregs were increased in the imiquimod-treated but not GM-CSF–treated groups (P = 0.000 and 0.006, respectively). Depleting MDSC and Treg in animals immunized with imiquimod and IGFBP-2 peptides restored antitumor activity to the levels observed with vaccination using GM-CSF as the sole adjuvant. Conclusion: Adjuvants may induce regulatory responses in the context of a self-antigen vaccine. Adjuvant triggered immunosuppression may limit vaccine efficacy and should be evaluated in preclinical models especially when contemplating combination approaches. Clin Cancer Res; 18(11); 3122–31. ©2012 AACR.

Elimination of IL-10–Inducing T-Helper Epitopes from an IGFBP-2 Vaccine Ensures Potent Antitumor Activity

Immunization against self-tumor antigens can induce T-regulatory cells, which inhibit proliferation of type I CD4(+) T-helper (TH1) and CD8(+) cytotoxic T cells. Type I T cells are required for potent antitumor immunity. We questioned whether immunosuppressive epitopes could be identified and deleted from a cancer vaccine targeting insulin-like growth factor-binding protein (IGFBP-2) and enhance vaccine efficacy. Screening breast cancer patient lymphocytes with IFN-γ and interleukin (IL)-10 ELISPOT, we found epitopes in the N-terminus of IGFBP-2 that elicited predominantly TH1 whereas the C-terminus stimulated TH2 and mixed TH1/TH2 responses. Epitope-specific TH2 demonstrated a higher functional avidity for antigen than epitopes, which induced IFN-γ (P = 0.014). We immunized TgMMTV-neu mice with DNA constructs encoding IGFBP-2 N-and C-termini. T cell lines expanded from the C-terminus vaccinated animals secreted significantly more type II cytokines than those vaccinated with the N-terminus and could not control tumor growth when infused into tumor-bearing animals. In contrast, N-terminus epitope-specific T cells secreted TH1 cytokines and significantly inhibited tumor growth, as compared with naïve T cells, when adoptively transferred (P = 0.005). To determine whether removal of TH2-inducing epitopes had any effect on the vaccinated antitumor response, we immunized mice with the N-terminus, C-terminus, and a mix of equivalent concentrations of both vaccines. The N-terminus vaccine significantly inhibited tumor growth (P < 0.001) as compared with the C-terminus vaccine, which had no antitumor effect. Mixing the C-terminus with the N-terminus vaccine abrogated the antitumor response of the N-terminus vaccine alone. The clinical efficacy of cancer vaccines targeting self-tumor antigens may be greatly improved by identification and removal of immunosuppressive epitopes.

Protein-bound polysaccharide activates dendritic cells and enhances OVA-specific T cell response as vaccine adjuvant

Protein-bound polysaccharide-K (PSK) is a hot water extract from Trametes versicolor mushroom. It has been used traditionally in Asian countries for its immune stimulating and anti-cancer effects. We have recently found that PSK can activate Toll-like receptor 2 (TLR2). TLR2 is highly expressed on dendritic cells (DC), so the current study was undertaken to evaluate the effect of PSK on DC activation and the potential of using PSK as a vaccine adjuvant. In vitro experiments using mouse bone marrow-derived DC (BMDC) demonstrated that PSK induces DC maturation as shown by dose-dependent increase in the expression of CD80, CD86, MHCII, and CD40. PSK also induces the production of multiple inflammatory cytokines by DC, including IL-12, TNF-α, and IL-6, at both mRNA and protein levels. In vivo experiments using PSK as an adjuvant to OVAp323-339 vaccine showed that PSK as adjuvant leads to enlarged draining lymph nodes with higher number of activated DC. PSK also stimulates proliferation of OVA-specific T cells, and induces T cells that produce multiple cytokines, IFN-γ, IL-2, and TNF-α. Altogether, these results demonstrate the ability of PSK to activate DC in vitro and in vivo and the potential of using PSK as a novel vaccine adjuvant.

Gamma delta T cells are activated by polysaccharide K (PSK) and contribute to the anti-tumor effect of PSK

Polysaccharide K (PSK) is a widely used mushroom extract that has shown anti-tumor and immunomodulatory effects in both preclinical and clinical studies. Therefore, it is important to understand the mechanism of actions of PSK. We recently reported that PSK can activate toll-like receptor 2 and enhances the function of NK cells. The current study was undertaken to study the effect of PSK on gamma delta (γδ) T cells, another important arm of the innate immunity. In vitro experiments using mouse splenocytes showed that γδ T cells produce IFN-γ after treatment with PSK and have up-regulated expression of CD25, CD69, and CD107a. To investigate whether the effect of PSK on γδ T cells is direct or indirect, purified γδ T cells were cultured either alone or together with bone marrow-derived DC in a co-culture or trans-well system and then stimulated with PSK. Results showed that direct cell-to-cell contact between γδ T cells and DC is required for optimal activation of γδ T cells. There was also reciprocal activation of DC by PSK-activated γδ T cells, as demonstrated by higher expression of costimulatory molecules and enhanced production of IL-12 by DC in the presence of γδ T cells. PSK can also co-stimulate γδ T cells with anti-TCR and anti-CD3 stimulation, in the absence of DC. Finally, in vivo treatment with PSK activates γδ T cells among the tumor infiltrating lymphocytes, and depleting γδ T cells during PSK treatment attenuated the anti-tumor effect of PSK. All together, these results demonstrated that γδ T cells are activated by PSK and contribute to the anti-tumor effect of PSK.

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.

Abstract PD3-08: Modulation of immunity with 2-fluorofucose (2FF) for breast cancer treatment and prevention

The majority of patients with breast cancer have robust Type II immune responses directed against their tumors with little to no Type I immunity. The dominance of a Type II microenvironment is established early in breast tumorigenesis with a Type II immune signature prevalent even in pre-invasive lesions such as ductal carcinoma in situ. As a result, breast cancer is associated with abundant autoantibodies directed against tumor associated antigens and few infiltrating T-cells in the majority of patients. A recently reported inhibitor of protein and cellular fucosylation, 2-fluorofucose (2FF) has been shown to enhance immune cell function, in part through the generation of fucose-deficient antibodies which result in enhanced antibody dependent cell mediated cytotoxicity, as well as apparent modulation of T-cell dependent activity [Ca Res, Oct 1, 2014 74;2890]. For treatment studies, TgMMTV-neu (MMTVneu; luminal) and C31-Tag (C3T; triple negative) mice were treated orally with vehicle alone and 20mM 2FF when spontaneous tumor volume reached 75-100mm3 until sacrifice (n=20/grp). To assess the ability to prevent breast cancer development, mice were treated orally with vehicle alone, 20 or 50mM 2FF for up to 200 days starting at 6-8 weeks of age (n=20/grp). Tumor kinetics, disease free survival, and overall survival were calculated. Immune responses were evaluated by both DTH and IFN-gamma ELISPOT. To determine immune mechanism of action, NK, B, CD4, or CD8 cells were depleted concurrently with tumor implant. When tumor bearing mice were treated with 2FF, tumor growth was significantly inhibited in both groups over the course of therapy (MMTVneu p 2FF, a novel inhibitor of fucosylation has potent anti-tumor effects in 2 transgenic models of breast cancer; luminal and triple negative mammary tumors. The agent is active in these mouse models in both the treatment and prevention setting and thus may represent a rational therapeutic approach to evaluate in breast cancer patients. Citation Format: Disis ML, Rastetter L, Gad E, Koehnlein M, Senter PD, Gardai S, Okeley NM. Modulation of immunity with 2-fluorofucose (2FF) for breast cancer treatment and prevention. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-08.

Abstract P5-04-06: Oral immunomodulatory agents prevent tumor growth and increase tumor CD8 T cell infiltrate; bexarotene further improves tumor response to conventional chemotherapy in breast tumors

The tumor immune environment is important in breast cancer with greater than 50% immune infiltrate (LPBC) prior to neoadjuvant chemotherapy predicting improved pathologic complete response, and LPBC and CD8 infiltrate prior to adjuvant therapy predicting improved survival. Unfortunately, the majority of breast cancers do not have LPBC or robust CD8+ infiltrate. Evidence has emerged that conventional chemotherapy can increase CD8+ T cells therefore discovering ways to boost this response should further enhance the anti-tumor function. Three oral agents have modest anti-tumor function: metformin (oral biguanide), bexarotene (retinoic receptor agonist), and celecoxib (COX2 inhibitor). In vitro data suggest a role for these agents in increasing Th1 immunity: metformin was shown to increase MHC class I expression on tumor cells, bexarotene was shown to decrease CD8+ T cell apoptosis, and celecoxib has been shown to decrease MDSC cells. The goal of this study was to demonstrate whether addition of these oral agents to conventional chemotherapy enhanced the anti-tumor function of chemotherapy, possibly by modifying the immune environment in the transgenic mouse mammary tumor model TgMMTV-neu (genetically similar to luminal breast cancer). Two active chemotherapies in human breast cancer, doxorubicin and paclitaxel, inhibited tumor growth and increased CD8+ T cell tumor infiltrate in transgenic mice. Treatment of mice with 100 mm3 tumors with doxorubicin (5 mg/kg weekly for four weeks) showed a 32% increase in CD8+ T cells and 85% decrease in tumor growth as compared to control mice (p=0.0001) and treatment with paclitaxel (10 mg/kg weekly for four weeks) showed a 40% increase in CD8+ tumor infiltrate (p=0.0068) and 60% decrease in tumor growth as compared to control treated mice (p=0.0026). A third chemotherapy cyclophosphamide (100 mg/kg weekly for four weeks) increased CD8+ tumor infiltrate by 45% (p=0.011) but did not show a significant decrease in tumor volume (p=0.57). Metformin and bexarotene also demonstrated increased CD8+ tumor infiltration and decreased breast tumor growth but celecoxib did not. Metformin treated mice had a 46% increase in CD8+ tumor infiltrate (p=0.001) and a 52% decrease in mean tumor volume (p=0.011) as compared to controls (75 mg/m2 of metformin for four weeks). Bexarotene treated mice had 44% increase in CD8+ tumor infiltrate (p=0.05) and 60% decrease in mean tumor growth (p=0.03) compared to control (treated with 50 mg/m2 bexarotene for four weeks). Of all three oral therapies, bexarotene was most effective inhibiting spontaneous tumor growth in the mice. Furthermore, addition of bexarotene to chemotherapy was superior to chemotherapy alone. Adding bexarotene to weekly doxorubicin decreased tumor growth by 98% (p=0.008 compared to doxorubicin alone), adding bexarotene to weekly paclitaxel decreased tumor growth by 86% (p=0.02 compared to paclitaxel alone), and adding bexarotene to weekly cyclophosphamide inhibited tumor growth by 82% (p=0.04 compared to cyclophosphamide alone). These results suggest that addition of bexarotene, a well-tolerated oral agent, to chemotherapy may improve tumor response in breast cancer. Citation Format: Sasha E Stanton, Ekram Gad, Edmond Marzbani, Lauren Rastetter, Mary L Disis. Oral immunomodulatory agents prevent tumor growth and increase tumor CD8 T cell infiltrate; bexarotene further improves tumor response to conventional chemotherapy in breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-06.

Abstract 1352: Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Preventative vaccine therapy may benefit women at high risk for breast cancer but currently it is impossible to identify women who will develop breast cancer prior to tumor development to identify initiating cancer antigens. Immunocompetent transgenic mouse mammary tumor models that are genetically similar to human breast cancer subtypes may identify pre-invasive antigens because mice can be followed longitudinally. Comparing tumor growth characteristics in two mouse models (TgMMTV-neu is genetically similar to luminal B breast cancer and C3(1)tag is genetically similar to triple negative breast cancer) demonstrated that, similar to triple negative breast cancer, C3(1)Tag mice develop tumors earlier and tumors grow more rapidly than TgMMTV-neu tumors. Furthermore, C3(1)Tag tumors have increased CD8+/CD4+ ratio (p<0.05) similar to human triple negative tumors. Using these two mouse models, we identified putative pre-invasive breast cancer antigens present prior to tumor development by serologic analysis of recombinant cDNA expression libraries and serological analysis of chip-arrayed proteins. We identified 65 pre-invasive antigens that were present in mice that would develop cancer but not parental control mice. The goal of this study was to identify which of the 65 antigens were required for human breast cancer survival with the goal to develop a preventative multi-antigen polyepitope breast cancer vaccine. We chose the pre-invasive antigens necessary for human breast cancer tumor cell survival using a high throughput siRNA screen evaluating for increased apoptosis and decreased cell survival in either HER2 positive or triple negative human breast cancer cell lines with decreased expression of the target protein. Five of the antigens were essential for human breast cancer cell survival: VPS35, SERBP1, ARPC2, PDIA6, and KRT8. All of these genes have roles in human cancer progression, and KRT8, SERBP1, and PDIA6 have identified roles in breast cancer pathogenesis. After designing human MHC class II peptides for each of these targets that cover at least 25% of the protein, we evaluated implanted tumor inhibition in the mouse models. Vaccination VPS35 peptides inhibited tumor growth by 47% (p<0.0001), vaccination with ARPC2 peptides inhibited tumor growth by 54% (p<0.0001), and vaccination with SERBP1 peptides inhibited tumor growth by 61% (p<0.0001) in the TgMMTV-neu mice. In C3(1)Tag mice, vaccination with VPS35 peptides inhibited tumor growth by 39% (p<0.0001) and vaccination with SERBP1 peptides inhibited tumor growth by 59% (p<0.0001) but vaccination with ARPC2 peptides did not inhibit tumor growth. These studies have demonstrated that mouse models can be used to identify pre-invasive breast cancer antigens. Further studies will evaluate the use of vaccines containing epitopes from several of the antigens to prevent spontaneous tumors in these mouse models. Citation Format: Sasha E. Stanton, Ekram Gadd, Lauren Rastetter, James Annis, Jianning Mao, John Ladd, Samir Hanash, Mary L. Disis. Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development. [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 1352. doi:10.1158/1538-7445.AM2015-1352

Abstract 279: Bexarotene increases tumor CD8+ T cells and improves response to conventional breast chemotherapy in the transgenic mouse mammary tumor model TgMMTV-neu

In breast cancer, increased immune infiltrate prior to neoadjuvant chemotherapy predicts improved pathologic complete response (pCR) and improved survival, however almost half of breast tumors have no CD8+ T cell infiltrate. Evidence has emerged that conventional breast cancer chemotherapy can increase CD8+ T cells (doxorubicin) and decrease the immunosuppressive regulatory CD4+ T cells (paclitaxel and cyclophosphamide); therefore discovering well tolerated agents that further enhance the anti-tumor immune function of these chemotherapies may further improve response in breast cancer patients. The oral agent bexarotene (a retinoic receptor agonist) showed a 20% disease response as a single agent in metastatic breast cancer and has been shown to increase CD8+ T cell tumor infiltration and decrease CD8+ T cell apoptosis in vitro. The goal of this study is to demonstrate whether this well tolerated oral agent can enhance the anti-tumor response of conventional chemotherapy through increasing intratumoral CD8+ T cells in TgMMTV-neu transgenic mice that are immunologically competent and genetically similar to human luminal B breast cancer. Two of the most active chemotherapies in human breast cancer, doxorubicin and paclitaxel, each could inhibit tumor growth by 70-80% and increase CD8+ T cell tumor infiltrate by approximately 40% in tumor bearing TgMMTV-neu mice, a third breast cancer chemotherapy cyclophosphamide increased CD8+ tumor infiltrate by 45% (p = 0.011) but did not show a statistically significant decrease in tumor volume. When TgMMTV-neu mice with ∼100 mm3 tumors were treated with 50 mg/m2 bexarotene for four weeks they demonstrated a 44% increase in CD8+ tumor infiltrate (p = 0.05) and 60% decrease in mean tumor growth (p = 0.03) compared to control. However, the addition of bexarotene to chemotherapy was superior to chemotherapy alone, and significantly more effective than bexarotene alone. This enhanced anti-tumor function was even seen with cyclophosphamide that by itself had not inhibited tumor growth. Adding bexarotene to weekly doxorubicin decreased tumor growth by 98% (p = 0.008 compared to doxorubicin alone), adding bexarotene to weekly paclitaxel decreased tumor growth by 86% (p = 0.02 compared to paclitaxel alone), and adding bexarotene to weekly cyclophosphamide inhibited tumor growth by 82% (p = 0.04 compared to cyclophosphamide alone). Further studies are now ongoing to identify the anti-tumor role of bexarotene particularly its immune modulatory role. These results suggest that the addition of bexarotene, a relatively well-tolerated oral agent, may modify the immune environment and improve tumor response to chemotherapy in breast cancer possibly improving response to neoadjuvant chemotherapy. Citation Format: Sasha E. Stanton, Ekram Gadd, Edmond Marzbani, Lauren Rastetter, Mary L. Disis. Bexarotene increases tumor CD8+ T cells and improves response to conventional breast chemotherapy in the transgenic mouse mammary tumor model TgMMTV-neu. [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 279. doi:10.1158/1538-7445.AM2015-279