Epidermal growth factor receptor derived peptide vaccination to prevent lung adenocarcinoma formation: An in vivo study in a murine model of EGFR mutant lung cancer.

The ability to prevent disease is the holy grail of medicine. For decades, efforts have been made to extend the successes seen with vaccination against infectious diseases to cancer. In some instances, preventive vaccination against viruses (prototypically HPV) has successfully prevented tumorigenesis and will make a major impact on public health in the decades to come. However, the majority of cancers that arise are a result of genetic mutation within the host, or non-viral environmental exposures. We present compelling evidence that vaccination against an overexpressed self-tumor oncoprotein has the potential to prevent tumor development. Vaccination against the Epidermal Growth Factor Receptor (EGFR) using a multipeptide vaccine in a preventive setting decreased EGFR-driven lung carcinogenesis by 76.4% in a mouse model of EGFR-driven lung cancer. We also demonstrate that anti-EGFR vaccination primes the development of a robust immune response in vivo. This study provides proof of concept for the first time that targeting tumor drivers in a preventive setting in lung cancer using peptide vaccination can inhibit tumorigenesis and may provide useful clinical insights into the development of strategies to vaccinate against EGFR in populations where EGFR-mutant disease is highly prevalent. © 2015 Wiley Periodicals, Inc.

Natural history of tumor growth and immune modulation in common spontaneous murine mammary tumor models.

Recent studies in patients with breast cancer suggest the immune microenvironment influences response to therapy. We aimed to evaluate the relationship between growth rates of tumors in common spontaneous mammary tumor models and immune biomarkers evaluated in the tumor and blood. TgMMTV-neu and C3(1)-Tag transgenic mice were followed longitudinally from birth, and MPA-DMBA-treated mice from the time of carcinogen administration, for the development of mammary tumors. Tumor-infiltrating CD4(+) and CD8(+) T-cells, FOXP3(+) T-regulatory cells, and myeloid-derived suppressor cells were assessed by flow cytometry. Serum cytokines were evaluated in subsets of mice. Fine needle aspirates of tumors were collected and RNA was isolated to determine levels of immune and proliferation markers. Age of tumor onset and kinetics of tumor growth were significantly different among the models. Mammary tumors from TgMMTV-neu contained a lower CD8/CD4 ratio than that of other models (p < 0.05). MPA-DMBA-induced tumors contained a higher percentage of FOXP3(+) CD4(+) T-cells (p < 0.01) and MDSC (p < 0.001) compared with the other models. Individuals with significantly slower tumor growth demonstrated higher levels of Type I serum cytokines prior to the development of lesions compared to those with rapid tumor growth. Moreover, the tumors of animals with more rapid tumor growth demonstrated a significant increase in the expression of genes associated with Type II immunity than those with slower-progressing tumors. These data provide a foundation for the development of in vivo models to explore the relationship between endogenous immunity and response to standard therapies for breast cancer.

Mining the pre-diagnostic antibody repertoire of TgMMTV-neu mice to identify autoantibodies useful for the early detection of human breast cancer.

BACKGROUND: The use of autoantibodies for the early detection of breast cancer has generated much interest as antibodies can be readily assayed in serum when antigen levels are low. Ideally, diagnostic autoantibodies would be identified in individuals who harbored pre-invasive disease/high risk lesions leading to malignancy. Prospectively collected human serum samples from these individuals are rare and not often available for biomarker discovery. We questioned whether transgenic animals could be used to identify cancer-associated autoantibodies present at the earliest stages of the malignant transformation of breast cancer. METHODS: We collected sera from transgenic mice (TgMMTV-neu) from the time of birth to death by spontaneous mammary tumors. Using sera from a time point prior to the development of tumor, i.e. "pre-diagnostic", we probed cDNA libraries derived from syngeneic tumors to identify proteins recognized by IgG antibodies. Once antigens were identified, selected proteins were evaluated via protein arrays, for autoantibody responses using plasma from women obtained prior to the development of breast cancer and matched controls. The ability of the antigens to discriminate cases from controls was assessed using receiver-operating-characteristic curve analyses and estimates of the area under the curve. RESULTS: We identified 6 autoantibodies that were present in mice prior to the development of mammary cancer: Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35. In rodent validation cohorts, detecting both IgM and IgG antibody responses against a subset of the identified proteins could discriminate pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. IgG and IgM autoantibodies, specific for a subset of the identified antigens, could discriminate the samples of women who eventually developed breast cancer from case-matched controls who did not develop disease. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if plasma samples were collected greater than 5 months prior to a breast cancer diagnosis (AUC 0.68; CI 0.565-0.787, p=0.0025). CONCLUSION: Genetically engineered mouse models of cancer may provide a facile discovery tool for identifying autoantibodies useful for human cancer diagnostics.

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity.

BACKGROUND: The paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor receptor 2 (HER2) is an attractive candidate for CAR T-cell therapy in humans but has the potential for eliciting on-target off-tumor toxicity. We developed an immunocompetent tumor model of CAR T-cell therapy targeting murine HER2 (mHER2) and examined the effect of CAR affinity, T-cell dose, and lymphodepletion on safety and efficacy. METHODS: Antibodies specific for mHER2 were generated, screened for affinity and specificity, tested for immunohistochemical staining of HER2 on normal tissues, and used for HER2-targeted CAR design. CAR candidates were evaluated for T-cell surface expression and the ability to induce T-cell proliferation, cytokine production, and cytotoxicity when transduced T cells were co-cultured with mHER2+ tumor cells in vitro. Safety and efficacy of various HER2 CARs was evaluated in two tumor models and normal non-tumor-bearing mice. RESULTS: Mice express HER2 in the same epithelial tissues as humans, rendering these tissues vulnerable to recognition by systemically administered HER2 CAR T cells. CAR T cells designed with single-chain variable fragment (scFvs) that have high-affinity for HER2 infiltrated and caused toxicity to normal HER2-positive tissues but exhibited poor infiltration into tumors and antitumor activity. In contrast, CAR T cells designed with an scFv with low-affinity for HER2 infiltrated HER2-positive tumors and controlled tumor growth without toxicity. Toxicity mediated by high-affinity CAR T cells was independent of tumor burden and correlated with proliferation of CAR T cells post infusion. CONCLUSIONS: Our findings illustrate the disadvantage of high-affinity CARs for targets such as HER2 that are expressed on normal tissues. The use of low-affinity HER2 CARs can safely regress tumors identifying a potential path for therapy of solid tumors that exhibit high levels of HER2.

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.

T-helper I immunity, specific for the breast cancer antigen insulin-like growth factor-I receptor (IGF-IR), is associated with increased adiposity.

Numerous lines of evidence demonstrate that breast cancer is immunogenic; yet, there are few biologically relevant immune targets under investigation restricting the exploration of vaccines to limited breast cancer subtypes. Insulin-like growth factor-I receptor (IGF-IR) is a promising vaccine candidate since it is overexpressed in most breast cancer subtypes, is part of a dominant cancer growth pathway, and has been validated as a therapeutic target. We questioned whether IGF-IR was immunogenic in cancer patients. IGF-IR-specific IgG antibodies were significantly elevated in early-stage breast cancer patients at the time of diagnosis as compared to volunteer donors (p = 0.04). Predicted T-helper epitopes, derived from the IGF-IR extracellular and transmembrane domains, elicited a significantly higher incidence of Th2 immunity in breast cancer patients as compared to controls (p = 0.01). Moreover, the magnitude of Th2 immunity was greater in breast cancer patients compared to controls (p = 0.02). In contrast, both breast cancer patients and volunteer donors demonstrated a similar incidence of Th1 immunity to IGF-IR domains with the predominant response directed against epitopes in the intracellular domain of the protein. As the incidence of IGF-IR type I immunity was not associated with a breast cancer diagnosis, we questioned whether other factors were contributing to the presence of IGF-IR-specific T-cells in both populations. While age was not associated with Th1 immunity, we observed a significantly greater magnitude of IGF-IR IFN-γ-secreting T-cells in obese subjects as compared to overweight (p < 0.001) or healthy-weight (p = 0.006) subjects, regardless of breast cancer diagnosis. No significant difference was observed for Th2 incidence or magnitude when stratified by age (p = 0.174, p = 0.966, respectively) or body mass index (p = 0.137, p = 0.174, respectively). Our data demonstrate that IGF-IR is a tumor antigen and IGF-IR-specific Th1 immunity may be associated with obesity rather than malignancy.

Three-dimensional scaffolds to evaluate tumor associated fibroblast-mediated suppression of breast tumor specific T cells.

In the tumor microenvironment, the signals from tumor-associated fibroblasts (TAF) that suppress antitumor immunity remain unclear. Here, we develop and investigate an in vitro three-dimensional (3D) scaffold model for the novel evaluation of TAF interaction with breast tumor cells and breast specific, neu antigen (p98) reactive T cells. Breast cancer cells seeded on 3D chitosan-alginate (CA) scaffolds showed productive growth and formed distinct tumor spheroids. Antigen specific p98 T cells, but not naïve T cells, bound significantly better to tumor cells on scaffolds. The p98 T cells induced potent tumor cell killing but T helper cell cytokine function was impaired in the presence of TAF coseeding on scaffolds. We found that the immunosuppression was mediated, in part, by transforming growth factor beta (TGF-b) and interleukin-10 (IL-10). Therefore, TAF appear capable of inducing potent T cell suppression. CA scaffolds can provide clinically relevant findings prior to preclinical testing of novel immunotherapies.

Treatment failure of a TLR-7 agonist occurs due to self-regulation of acute inflammation and can be overcome by IL-10 blockade.

Multiple TLR agonists have been shown to have antitumor effects in animal models. However, the therapeutic efficacy of TLR agonist monotherapy in cancer treatment has been limited, and the mechanisms of failure remain unknown. We demonstrate that topical treatment with a TLR-7 agonist, imiquimod, can elicit significant regression of spontaneous breast cancers in neu transgenic mice, a model of human HER-2/neu(+) breast cancer. However, tumor growth progressed once imiquimod therapy was ended. Gene expression analysis using tumor-derived RNA demonstrated that imiquimod induced high levels of IL-10 in addition to TNF-alpha and IFN-gamma. Elevated levels of circulating IL-10 were also detected in sera from imiquimod-treated mice. Elevated serum IL-10 appeared to be derived from IL-10 and dual cytokine secreting (IFN-gamma(+) and IL-10(+)) CD4(+) T cells rather than CD4(+)CD25(+)Foxp3(+) T regulatory cells, which were also induced by imiquimod treatment. Blockade of IL-10, but not TGF-beta, enhanced the antitumor effect of imiquimod by significantly prolonging survival in treated mice. These data suggest that the excessive inflammation induced by TLR agonists may result in a self-regulatory immunosuppression via IL-10 induction and that blocking IL-10 could enhance the therapeutic efficacy of these agents.

COX-2 Inhibitors Decrease Expression of PD-L1 in Colon Tumors and Increase the Influx of Type I Tumor-infiltrating Lymphocytes.

Colon cancer is initiated under inflammatory conditions associated with upregulation of immune checkpoint proteins. We evaluated immune modulation induced by nonsteroidal anti-inflammatory agents used for colon cancer prevention. Both celecoxib and naproxen inhibited polyp growth in APC Min mice. Treatment of mice with either drug significantly decreased PD-L1 expression on polyps in a dose-dependent manner (P < 0.0001 for both). The decrease in PD-L1 was associated with an influx of CD8+ T cells into polyps (P < 0.0001, celecoxib; P = 0.048, naproxen) compared with lesions from untreated animals and correlated with disease control. Naproxen is a nonselective inhibitor of both COX-1 and COX-2, and we questioned the role of the different cyclooxygenases in PD-L1 regulation. Silencing either COX-2 or COX-1 RNA in the murine colon cancer cell line MC38, reduced PD-L1 expression by 86% in COX-2-silenced cells (P < 0.0001) while there was little effect with COX-1 siRNA compared with control. Naproxen could inhibit the growth of MC38 in vivo. Naproxen-treated mice demonstrated a significant reduction in MC38 growth as compared with control (P < 0001). Both Tbet+ CD4 and CD8 tumor-infiltrating lymphocytes (TIL) were significantly increased (P = 0.04 and P = 0.038, respectively) without a concurrent increase in GATA3+ TIL (P > 0.05). CD8+ TIL highly expressed the activation marker, CD69. Not only was PD-L1 expression decreased on tumors, but LAG3+CD8+ T cells and PD-1 and LAG3 expression on regulatory T cells was also reduced (P = 0.008 and P = 0.002, respectively). These data demonstrate COX-2 inhibitors significantly decrease PD-L1 in colonic lesions and favorably impact the phenotype of tumor-infiltrating lymphocytes to control tumor growth. PREVENTION RELEVANCE: Nonsteroidal anti-inflammatories (NSAID) are an essential component of any combination chemoprevention of colon cancer. We show NSAID treatment reduces PD-L1 expression on intestinal tumor cells. NSAID regulation of PD-L1 is dependent on COX-2 expression. These data underscore an important immunologic mechanism of action for NSAID in colon cancer prevention. See related Spotlight, p. 209.

Anti-tumor activity of a T-helper 1 multiantigen vaccine in a murine model of prostate cancer.

Prostate cancer is one of the few malignancies that includes vaccination as a treatment modality. Elements of an effective cancer vaccine should include the ability to elicit a Type I T-cell response and target multiple antigenic proteins expressed early in the disease. Using existing gene datasets encompassing normal prostate tissue and tumors with Gleason Score ≤ 6 and ≥ 8, 10 genes were identified that were upregulated and conserved in prostate cancer regardless of the aggressiveness of disease. These genes encoded proteins also expressed in prostatic intraepithelial neoplasia. Putative Class II epitopes derived from these proteins were predicted by a combination of algorithms and, using human peripheral blood, epitopes which selectively elicited IFN-γ or IL-10 dominant antigen specific cytokine secretion were determined. Th1 selective epitopes were identified for eight antigens. Epitopes from three antigens elicited Th1 dominant immunity in mice; PSMA, HPN, and AMACR. Each single antigen vaccine demonstrated significant anti-tumor activity inhibiting growth of implanted Myc-Cap cells after immunization as compared to control. Immunization with the combination of antigens, however, was superior to each alone in controlling tumor growth. When vaccination occurred simultaneously to tumor implant, multiantigen immunized mice had significantly smaller tumors than controls (p = 0.002) and a significantly improved overall survival (p = 0.0006). This multiantigen vaccine shows anti-tumor activity in a murine model of prostate cancer.

Multi-Epitope-Based Vaccines for Colon Cancer Treatment and Prevention.

Background: Overexpression of nonmutated proteins involved in oncogenesis is a mechanism by which such proteins become immunogenic. We questioned whether overexpressed colorectal cancer associated proteins found at higher incidence and associated with poor prognosis could be effective vaccine antigens. We explored whether vaccines targeting these proteins could inhibit the development of intestinal tumors in the azoxymethane (AOM)-induced colon model and APC Min mice. Methods: Humoral immunity was evaluated by ELISA. Web-based algorithms identified putative Class II binding epitopes of the antigens. Peptide and protein specific T-cells were identified from human peripheral blood mononuclear cells using IFN-gamma ELISPOT. Peptides highly homologous between mouse and man were formulated into vaccines and tested for immunogenicity in mice and in vivo tumor challenge. Mice treated with AOM and APC Min transgenic mice were vaccinated and monitored for tumors. Results: Serum IgG for CDC25B, COX2, RCAS1, and FASCIN1 was significantly elevated in colorectal cancer patient sera compared to volunteers (CDC25B p=0.002, COX-2 p=0.001, FASCIN1 and RCAS1 p<0.0001). Epitopes predicted to bind to human class II MHC were identified for each protein and T-cells specific for both the peptides and corresponding recombinant protein were generated from human lymphocytes validating these proteins as human antigens. Some peptides were highly homologous between mouse and humans and after immunization, mice developed both peptide and protein specific IFN-γ-secreting cell responses to CDC25B, COX2 and RCAS1, but not FASCIN1. FVB/nJ mice immunized with CDC25B or COX2 peptides showed significant inhibition of growth of the syngeneic MC38 tumor compared to control (p<0.0001). RCAS1 peptide vaccination showed no anti-tumor effect. In the prophylactic setting, after immunization with CDC25B or COX2 peptides mice treated with AOM developed significantly fewer tumors as compared to controls (p<0.0002) with 50% of mice remaining tumor free in each antigen group. APC Min mice immunized with CDC25B or COX2 peptides developed fewer small bowel tumors as compared to controls (p=0.01 and p=0.02 respectively). Conclusions: Immunization with CDC25B and COX2 epitopes consistently suppressed tumor development in each model evaluated. These data lay the foundation for the development of multi-antigen vaccines for the treatment and prevention of colorectal cancer.

Tumor-associated autoantibodies from mouse breast cancer models are found in serum of breast cancer patients.

B cell responses to tumor antigens occur early in breast tumors and may identify immunogenic drivers of tumorigenesis. Sixty-two candidate antigens were identified prior to palpable tumor development in TgMMTV-neu and C3(1)Tag transgenic mouse mammary tumor models. Five antigens (VPS35, ARPC2, SERBP1, KRT8, and PDIA6) were selected because their decreased expression decreased survival in human HER2 positive and triple negative cell lines in a siRNA screen. Vaccination with antigen-specific epitopes, conserved between mouse and human, inhibited tumor growth in both transgenic mouse models. Increased IgG autoantibodies to the antigens were elevated in serum from women with ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC). The autoantibodies differentiated women with DCIS from control with AUC 0.93 (95% CI 0.88-0.98, p < 0.0001). The tumor antigens identified early in the development of breast cancer in mouse mammary tumor models were conserved in human disease, and potentially identify early diagnostic markers in human breast tumors.

Therapeutic and Prophylactic Antitumor Activity of an Oral Inhibitor of Fucosylation in Spontaneous Mammary Cancers.

2-fluorofucose (2FF) inhibits protein and cellular fucosylation. Afucosylation of IgG antibodies enhances antibody-dependent cell-mediated cytotoxicity by modulating antibody affinity for FcγRIIIa, which can impact secondary T-cell activation. Immune responses toward most common solid tumors are dominated by a humoral immune response rather than the presence of tumor-infiltrating cytotoxic T cells. IgG antibodies directed against numerous tumor-associated proteins are found in the sera of both patients with breast cancer and transgenic mice bearing mammary cancer. We questioned whether 2FF would have antitumor activity in two genetically distinct transgenic models; TgMMTV-neu (luminal B) and C3(1)-Tag (basal) mammary cancer. 2FF treatment significantly improved overall survival. The TgMMTV-neu doubled survival time compared with controls [P < 0.0001; HR, 7.04; 95% confidence interval (CI), 3.31-15.0], and survival was significantly improved in C3(1)-Tag (P = 0.0013; HR, 3.36; 95% CI, 1.58-7.14). 2FF treated mice, not controls, developed delayed-type hypersensitivity and T-cell responses specific for syngeneic tumor lysates (P < 0.0001). Serum IgG from 2FF-treated mice enhanced tumor lysis more efficiently than control sera (P = 0.004). Administration of 2FF for prophylaxis, at two different doses, significantly delayed tumor onset in both TgMMTV-neu; 20 mmol/L (P = 0.0004; HR, 3.55; 95% CI, 1.60-7.88) and 50 mmol/L (P = 0.0002; HR: 3.89; 95% CI, 1.71-8.86) and C3(1)-Tag; 20 mmol/L (P = 0.0020; HR, 2.51; 95% CI, 1.22-5.18), and 50 mmol/L (P = 0.0012; HR, 3.36; 95% CI, 1.57-7.18). Mammary cancer was prevented in 33% of TgMMTV-neu and 26% of C3(1)-Tag. 2FF has potent antitumor effects in mammary cancer models. The agent shows preclinical efficacy for both cancer treatment and prevention.

Tumor-associated antigens identified early in mouse mammary tumor development can be effective vaccine targets.

Breast cancer vaccines composed of antigens identified by serological analysis of cDNA expression libraries (SEREX) induce antigen specific immune responses in patients but have had disappointing clinical benefits. While many attempts to modify the adjuvants and vaccine method have been tried, one issue not addressed was whether the SEREX tumor-associated antigens identified from late stages of disease were ideal targets. We questioned in the transgenic TgMMTV-neu mouse model whether the antigen repertoire is distinct between early and late stage breast cancer and whether the antigens identified via SEREX from transgenic mice with early or late stage tumors would elicit differential anti-tumor effects to address this question. Three early stage antigens, Pdhx, Stk39, and Otud6B, were identified from a SEREX screen of mice prior to development of palpable lesions. Formulated into a vaccine, each early antigen inhibited tumor growth (p < 0.0001). The antigens identified from mice with late stage tumors (Swap70, Gsn, and Arhgef2) were unable to inhibit tumor growth when used as vaccines (for example Gsn p = 0.26). Each of the three early stage antigens were essential for tumor survival in syngeneic mouse tumor cells and in human breast cancer cell lines across breast cancer subtypes. Silencing protein expression of the early antigens increased apoptosis (p < 0.0001 for all antigens in mouse and p < 0.05 for all antigens in human triple negative breast cancer) and decreased survival (p < 0.0001 for all antigens in mouse and human triple negative and HER2 positive breast cancer). Overexpression of the early stage antigens in women with breast cancer predicted worse prognosis (p = 0.03) while overexpression of late stage antigens did not impact prognosis (p = 0.09). These data suggest that antigens expressed earlier in breast tumor development and functionally relevant to breast tumor growth may be more effective targets for therapeutic breast cancer vaccines than antigens identified in later disease.

The Effect of Mouse Strain, Sex, and Carcinogen Dose on Toxicity and the Development of Lung Dysplasia and Squamous Cell Carcinomas in Mice.

In order to translate new treatments to the clinic, it is necessary to use animal models that closely recapitulate human disease. Lung cancer develops after extended exposure to carcinogens. It has one of the highest mutation rates of all cancer and is highly heterogenic. Topical treatment with N-nitrosotris-(2-chloroethyl)urea (NTCU) induces lung squamous cell carcinoma (SCC) with nonsynonymous mutation rates similar to those reported for human non-small cell lung cancer. However, NTCU induces lung cancer with variable efficacy and toxicity depending on the mouse strain. A detailed characterization of the NTCU model is needed. We have compared the effect of three different NTCU doses (20, 30, and 40 mmol/L) in female and male of NIH Swiss, Black Swiss, and FVB mice on tumor incidence, survival, and toxicity. The main findings in this study are (1) NIH Swiss mice present with a higher incidence of SCC and lower mortality compared with Black Swiss and FVB mice; (2) 30 mmol/L NTCU dose induces SCC at the same rate and incidence as the 40 mmol/L dose with lower mortality; (3) female mice present higher grade and incidence of preinvasive lesions and SCC compared with males; (4) NTCU-induced transformation is principally within the respiratory system; and (5) NTCU treatment does not affect the ability to elicit a specific adaptive immune response. This study provides a reference point for experimental designs to evaluate either preventive or therapeutic treatments for lung SCC, including immunotherapies, before initiating human clinical trials.

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.

The Antitumor Efficacy of IL2/IL21-Cultured Polyfunctional Neu-Specific T Cells Is TNFα/IL17 Dependent.

PURPOSE: Infusion of HER2-specific T cells, derived from vaccine-primed patients and expanded with IL2/IL12, has induced tumor regression in a minority of patients with metastatic treatment-refractory HER2(+) breast cancer. We questioned whether alteration of cytokine growth factors used to culture vaccine-primed T cells could improve antitumor activity. EXPERIMENTAL DESIGN: Using the TgMMTV-neu murine mammary tumor model, we cultured T cells derived from mice immunized with a previously defined neu class II peptide, p98-114 (neu p98), and evaluated different cytokine combinations for expansion. RESULTS: Infusion of neu p98-specific T-cell lines derived from all cytokine conditions evaluated resulted in significant antitumor activity compared with infused naïve splenocytes (P < 0.05). T cells cultured with IL2/IL21 could uniquely mediate complete regression of spontaneous mammary tumors. IL2/IL21 cultured neu-specific T cells demonstrated a different cytokine secretion pattern as compared with other cultured T cells; secreting high levels of TNFα and IL17 (P < 0.05). Moreover, tumor-infiltrating CD8(+) cells were significantly increased after the infusion of IL2/IL21 cultured T cells as compared with tumors treated with T cells expanded under other cytokine conditions (P < 0.001). The antitumor effect of the infusion of IL2/IL21 cultured cells was mediated by CD8 T cells. Depletion of TNFα or IL17, but not IFNγ, abrogated the tumor growth inhibition induced by the IL2/IL21 T cells and markedly decreased the influx of CD8 into tumors. Finally, IL2/IL21-cultured human antigen specific T cells also displayed a similar polyfunctional Th1/Th17 phenotype. CONCLUSIONS: Expansion of HER2 vaccine-primed T cells with IL2/IL21 may have the potential to effectively mediate tumor regression when used in adoptive transfer. Clin Cancer Res; 22(9); 2207-16. ©2015 AACR.

Preservation of tumor-host immune interactions with luciferase-tagged imaging in a murine model of ovarian cancer.

BACKGROUND: Ovarian cancer is immunogenic and residual tumor volume after surgery is known to be prognostic. Ovarian cancer often follows a recurring-remitting course and microscopic disease states may present ideal opportunities for immune therapies. We sought to establish the immune profile of a murine model of ovarian cancer that allows in vivo tumor imaging and the quantitation of microscopic disease. RESULTS AND DISCUSSION: Baseline imaging and weight measurements were taken within 1 and 2 weeks after intraperitoneal tumor injection, respectively. Significantly higher photons per second from baseline imaging were first observed 5 weeks after tumor cell injection (p < 0.05) and continued to be significant through 8 weeks after injection (p < 0.01), whereas a significant increase in weight above baseline was not observed until day 56 (p < 0.0001). Expression of luc2 in ID8 cells did not alter the cellular immune microenvironment of the tumor. FOXP3+ T cells were more likely to be detected in the intraepithelial compartment and CD4+ T cells in the stroma as compared to CD3+ T cells, which were found equally in stroma and intraepithelial compartments. CONCLUSIONS: Use of an intraperitoneal tumor expressing a codon-optimized firefly luciferase in an immunocompetent mouse model allows tumor quantitation in vivo and detection of microscopic tumor burdens. Expression of this foreign protein does not significantly effect tumor engraftment or the immune microenvironment of the ID8 cells in vivo and may allow novel immunotherapies to be assessed in a murine model for their translational potential to ovarian cancers in remission or minimal disease after primary cytoreductive surgery or chemotherapy. METHODS: Mouse ovarian surface epithelial cells from C57BL6 mice transformed after serial passage in vitro were transduced with a lentiviral vector expressing a codon optimized firefly luciferase (luc2). Cell lines were selected and luc2 expression functionally confirmed in vitro. Cell lines were intraperitoneally (IP) implanted in albino C57BL/6/BrdCrHsd-Tyrc mice and albino B6(Cg)-Tyrc-2 J/J mice for serial imaging. D-luciferin substrate was injected IP and tumors were serially imaged in vivo using a Xenogen IVIS. Tumor take, weights, and luminescent intensities were measured. Immunohistochemistry was performed on tumors and assessed for immune infiltrates in stromal and intraepithelial compartments.

Protein-bound polysaccharide-K induces IL-1ß via TLR2 and NLRP3 inflammasome activation.

Inflammasome activation has been shown to regulate both innate and adaptive immune responses. It is important to investigate whether immune-enhancing natural products can also activate inflammasome. The current study examined the potential of protein-bound polysaccharide-K (PSK), a hot water extract from Trametes versicolor, to activate inflammasome. Using THP-1 cells, we have demonstrated that PSK induces both pro-IL-1ß and mature IL-1ß in THP-1 cells in a caspase 1- and NLRP3-dependent manner. PSK also induces IL-1ß and IL-18 in human PBMC. Cathepsin B is required for PSK-induced inflammasome activation as CA-074-Me, a cathepsin B inhibitor, significantly decreased PSK-induced IL-1ß. PSK induces NLRP3 at both mRNA and protein level. Comparison of PSK-induced IL-1ß in bone marrow-derived macrophages from wild type C57BL/6 mice, TLR2(-/-), P2X7R(-/-) and NLRP3(-/-) mice demonstrated that PSK-induced IL-1ß is dependent on both TLR2 and NLRP3. P2X7R is not required for PSK-induced inflammasome activation, but enhances PSK-induced caspase-1 activation and IL-1ß induction. Altogether, these results demonstrated that PSK induces inflammasome activation and production of IL-1ß in a TLR2- and NLRP3-dependent mechanism. These results provide novel insights into the mechanisms of the immune modulatory effects of PSK.

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.