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Purpose: High levels of type I T cells are needed for tumor eradication. We evaluated whether the HER2-specific vaccine–primed T cells are readily expanded ex vivo to achieve levels needed for therapeutic infusion. Patients and Methods: Phase I/II nonrandomized trial of escalating doses of ex vivo–expanded HER2-specific T cells after in vivo priming with a multiple peptide–based HER2 intracellular domain (ICD) vaccine. Vaccines were given weekly for a total of three immunizations. Two weeks after the third vaccine, patients underwent leukapheresis for T-cell expansion, then received three escalating cell doses over 7- to 10-day intervals. Booster vaccines were administered after the T-cell infusions. The primary objective was safety. The secondary objectives included extent and persistence of HER2-specific T cells, development of epitope spreading, and clinical response. Patients received a CT scan prior to enrollment and 1 month after the last T-cell infusion. Results: Nineteen patients received T-cell infusions. Treatment was well tolerated. One month after the last T-cell infusion, 82% of patients had significantly augmented T cells to at least one of the immunizing epitopes and 81% of patients demonstrated enhanced intramolecular epitope spreading compared with baseline (P < 0.05). There were no complete responses, one partial response (6%), and eight patients with stable disease (47%), for a disease control rate of 53%. The median survival for those with progressive disease was 20.5 months and for responders (PR+SD) was 45.0 months. Conclusions: Adoptive transfer of HER2 vaccine–primed T cells was feasible, was associated with minimal toxicity, and resulted in an increased overall survival in responding patients.

Multiple animal models have been developed to investigate the pathogenesis of colorectal cancer and to evaluate potential treatments. One model system uses azoxymethane, a metabolite of cycasin, alone and in conjunction with dextran sodium sulfate to induce colon cancer in rodents. Azoxymethane is metabolized by hepatic P450 enzymes and can also be eliminated through the kidneys. In this study, C57BL/6J mice were fed either standard or high-fat diet and then all mice received azoxymethane at 10 mg/kg body weight twice a week for 6 wk. Shortly after the end of treatment, high mortality occurred in mice in the high-fat diet group. Postmortem examination revealed hepatic and renal pathology in mice on both diets. Histologic changes in liver included hepatocytomegaly with nuclear pleomorphism and bile duct hyperplasia accompanied by mixed inflammatory-cell infiltrates. Changes in the kidneys ranged from basophilia of tubular epithelium to tubular atrophy. The results indicate that further optimization of this model is needed when feeding a high-fat diet and giving multiple azoxymethane doses to induce colon cancer in C57BL/6J mice.

Flow chart of participants.

Detailed adverse events.

Purpose:High levels of type I T cells are needed for tumor eradication. We evaluated whether the HER2-specific vaccine–primed T cells are readily expanded ex vivo to achieve levels needed for therapeutic infusion.Patients and Methods:Phase I/II nonrandomized trial of escalating doses of ex vivo–expanded HER2-specific T cells after in vivo priming with a multiple peptide–based HER2 intracellular domain (ICD) vaccine. Vaccines were given weekly for a total of three immunizations. Two weeks after the third vaccine, patients underwent leukapheresis for T-cell expansion, then received three escalating cell doses over 7- to 10-day intervals. Booster vaccines were administered after the T-cell infusions. The primary objective was safety. The secondary objectives included extent and persistence of HER2-specific T cells, development of epitope spreading, and clinical response. Patients received a CT scan prior to enrollment and 1 month after the last T-cell infusion.Results:Nineteen patients received T-cell infusions. Treatment was well tolerated. One month after the last T-cell infusion, 82% of patients had significantly augmented T cells to at least one of the immunizing epitopes and 81% of patients demonstrated enhanced intramolecular epitope spreading compared with baseline (P < 0.05). There were no complete responses, one partial response (6%), and eight patients with stable disease (47%), for a disease control rate of 53%. The median survival for those with progressive disease was 20.5 months and for responders (PR+SD) was 45.0 months.Conclusions:Adoptive transfer of HER2 vaccine–primed T cells was feasible, was associated with minimal toxicity, and resulted in an increased overall survival in responding patients.

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.

Supplementary Methods

A multiantigen multipeptide vaccine, targeting proteins expressed in preinvasive breast lesions, can stimulate type I CD4+ T cells which have been shown to be deficient in both patients with breast cancer and mice that develop mammary tumors. Transgenic mice (TgMMTV-neu) were immunized with a multiantigen peptide vaccine specific for neu, insulin—like growth factor-binding protein 2 and insulin-like growth factor receptor-I at a time when some of the animals already had preinvasive lesions (18 weeks of age). Although immunization with each individual antigen was partially effective in inhibiting tumor growth, immunization with the multiantigen vaccine was highly effective, blocking development of palpable lesions in 65% of mice and slowing tumor growth in the infrequent palpable tumors, which did arise. Protection was mediated by CD4+ T cells, and the few slow-growing tumors that did develop demonstrated a significant increase in intratumoral CD8+ T cells as compared with controls (P = 0.0007). We also combined the vaccine with agents that were, by themselves, partially effective inhibitors of tumor progression in this model; lapatinib and the RXR agonist bexarotene. Although the combination of lapatinib and vaccination performed similarly to vaccination alone (P = 0.735), bexarotene and vaccination significantly enhanced disease-free survival (P < 0.0001), and approximately 90% of the mice showed no pathologic evidence of carcinomas at one year. The vaccine also demonstrated significant clinical efficacy in an additional transgenic model of breast cancer (TgC3(I)-Tag). Chemoimmunoprevention combinations may be an effective approach to breast cancer prevention even when the vaccine is administered in the presence of subclinical disease. Cancer Prev Res; 6(12); 1273–82. ©2013 AACR.

Splenocytes from mice provided normal water (open circle, n=8), 20mM 2FF (closed circle, n=8), or 50mM 2FF (triangle, n=7) for tumor prophylaxis showed a mean {plus minus} SEM IFN-ɣ response toward MMC lysate of 41.7 {plus minus} 6.35 (20mM group, p

Representative flow cytometry panels are shown for a single CD4 (A, B), CD8 (C, D), NK (E, F), Untreated (G) or CD22 (H) treated animal before treatments began (A, C, E, G) or after treatment with respective antibodies (B, D, F, H).

Mean {plus minus} SEM of peripheral cytokines detected in terminal sera collected from untreated (shaded) and 2FF-treated (unshaded) TgMMTV-neu mice. N=6 to 8 per group, *p

Supplementary Figure from 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

Supplementary Table S1. Weight changes in different treatment groups for each mouse strain. Supplementary Table S2. Weight change in NTCU treated mice is sex independent. Supplementary Table S3. Survival in NTCU treated mice is sex independent. Supplementary Table S4. Immune phenotype of splenocytes from FVB and NIH Swiss mice treated 4 weeks with 40mM NTCU either subsequent or concomitant to vaccination with IGF1R. Supplementary Table S5. Immune phenotype of splenocytes from NIH Swiss and FVB mice treated with NTCU for 24-32 weeks.

Weights were collected from n=7 to 20 TgMMTV-neu mice per group at various times throughout the study; average weight is represented over age of untreated (circle), 20mM 2FF (square) and 50mM 2FF (triangle) treated mice. The solid gray arrow represents end of 50mM 2FF treatment, dashed black arrow represents end of 20mM 2FF treatment.

Shown is the effect of 2FF treatment on (A) Amount of IgG (Mean {plus minus} SEM) isolated from 75uL of sera collected, and (B) AAL lectin-based measurement of fucosylation (Mean {plus minus} SEM) of these samples from untreated (circle) or 2FF-treated (square) TgMMTV-neu mice (n=5 each).

Allred proportion (ranging from 0-5) and intensity (ranging from 0-3) scores for each tumor. The median score among triplicate TMA cores and reviewers was utilized. Most MCC tumors with an Allred score of 7 continued to express MHC class I in all tumor cells, however the intensity of expression was significantly reduced as compared to those with an Allred score of 8.

Related Article from The Ultimate in Cancer Chemoprevention: Cancer Vaccines

Supplementary Figure S1. Gene expression in mouse SCC is consistent with human SCC data. Supplementary Figure S2. Lung non-neoplastic lesions found in animals treated with NTCU. Supplementary Figure S3. Lymphoid aggregates and PD-L1 expression in the lung of mice treated with NTCU.

Effect of 2FF-treatment on (A) Mean {plus minus} SEM tumor cell binding of antibody from TgMMTV-neu mice as detected by FACS analysis using (A) untreated (circle) and 2FF-treated (square) purified serum IgG, or untreated (triangle) and 2FF-treated (inverted triangle) serum (n=5 each), (B) geometric mean of untreated (black, MMC; dark gray, M6) or 2FF-treated (unshaded, MMC; light gray, M6) cell surface fucose using LCA, AAL, and AOL lectins, and (C) in vitro growth of untreated cells (circle, MMC; triangle, M6) or cells pretreated with 2FF (>7 days, square, MMC; inverted triangle, M6) and continuously treated with 2FF during the growth analysis (Mean {plus minus} SEM, n=3 each).

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (P < 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC. Cancer Immunol Res; 2(11); 1071–9. ©2014 AACR.

PDF file - 3266K, Supplemental Figure 1. Protein expression of vaccine component antigens in preinvasive lesions derived from TgMMTV-neu mice. Supplemental Figure 2. Multi-antigen peptide vaccine controls tumor growth in a TgC3(I)-Tag implant model. Supplemental Table 1. Vaccine component peptides.

Outline of study design for Figures 1, 2, 4, and 5, as indicated. Dashed gray line represents treatment with 2FF. Lower line represents general time point in weeks or average mouse age.

Effect of 2FF-treatment on (A) Tumor volume (Mean {plus minus} SEM) over days since implant for mice treated with IgG2b (open circle), anti-CD8 (diamond), 20mM 2FF+IgG2b (closed circle), or 20mM 2FF+anti-CD8 (triangle), n=5 per group; and (B) Tumor volume (Mean {plus minus} SEM) over days since implant for mice treated with IgG2b (open circle), anti-NK (diamond), 20mM 2FF+IgG2b (closed circle), or 20mM 2FF+anti-NK (triangle), n=5 per group ****p

The development of therapeutic cancer vaccines is impacted by immunosuppressive elements in the tumor microenvironment. Most immunogenic cancer proteins are “self,” therefore, peripheral tolerance contributes substantially to tumor immune escape. Transforming growth factor β (TGFβ) actively modulates both inflammation and tolerance induction. Combining vaccination with agents that disarm TGFβ will enhance vaccine efficacy. (Clin Cancer Res 2009;15(21):1–3) (Clin Cancer Res 2009;15(21):6476–8)

Detailed Adverse Events and Flow diagram of patients progression through the trial .

Purpose:Cancer vaccines targeting nonmutated proteins elicit limited type I T-cell responses and can generate regulatory and type II T cells. Class II epitopes that selectively elicit type I or type II cytokines can be identified in nonmutated cancer-associated proteins. In mice, a T-helper I (Th1) selective insulin-like growth factor binding protein-2 (IGFBP-2) N-terminus vaccine generated high levels of IFNγ secreting T cells, no regulatory T cells, and significant antitumor activity. We conducted a phase I trial of T-helper 1 selective IGFBP-2 vaccination in patients with advanced ovarian cancer.Patients and Methods:Twenty-five patients were enrolled. The IGFBP-2 N-terminus plasmid-based vaccine was administered monthly for 3 months. Toxicity was graded by NCI criteria and antigen-specific T cells measured by IFNγ/IL10 ELISPOT. T-cell diversity and phenotype were assessed.Results:The vaccine was well tolerated, with 99% of adverse events graded 1 or 2, and generated high levels of IGFBP-2 IFNγ secreting T cells in 50% of patients. Both Tbet+ CD4 (P = 0.04) and CD8 (P = 0.007) T cells were significantly increased in immunized patients. There was no increase in GATA3+ CD4 or CD8, IGFBP-2 IL10 secreting T cells, or regulatory T cells. A significant increase in T-cell clonality occurred in immunized patients (P = 0.03, pre- vs. post-vaccine) and studies showed the majority of patients developed epitope spreading within IGFBP-2 and/or to other antigens. Vaccine nonresponders were more likely to have preexistent IGFBP-2 specific immunity and demonstrated defects in CD4 T cells, upregulation of PD-1, and downregulation of genes associated with T-cell activation, after immunization.Conclusions:IGFBP-2 N-terminus Th1 selective vaccination safely induces type I T cells without evidence of regulatory responses.

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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.

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Supplementary Table 1. Th1/Th2 for eleven epitopes derived from the HIF-1α protein; Supplementary Table 2. Number of tumorigenic implants in the Sca-1positive or Sca-1negative Population; Supplementary Table 3. Complete blood count (median and range) for the indicted immunization. n=4 mice/group; Supplementary Table 4. Serum chemistries (median and range) for the indicted immunization; Supplementary Figure 1. Sca-1positive cells exhibit several stem cell/EMT markers; Supplementary Figure 2. HIF-1α epitope-specific Th respond to naturally processed HIF-1α protein; Supplementary Figure 3. Sca-1positive cells have increased estrogen receptor-alpha (ER-α) gene expression; Supplementary Figure 4. The HIF-1α vaccine does not induce tissue toxicities.

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Purpose: Polysaccharide krestin (PSK) is a mushroom extract that has been long used in Asia and recently in Western countries as a treatment for cancer due to its presumed immune potentiating effects. Although there have been reports of clinical responses after patients have ingested PSK, the mechanism of action of the agent remains undefined. The current study was undertaken to investigate the mechanism of the antitumor actions of PSK.Experimental Design: The immunostimulatory effect of PSK was first evaluated in vitro using splenocytes from neu transgenic mice and Toll-like receptor (TLR) 2 knockout (TLR2−/−) mice. Then the immunostimualtory and antitumor effect of PSK was determined using tumor-bearing neu transgenic mice, TLR2−/−, and wild-type C57BL/6 mice.Results: We demonstrate that PSK is a selective TLR2 agonist, and the activation of dendritic cells (DC) and T cells by PSK is dependent on TLR2. Oral administration of PSK in neu transgenic mice significantly inhibits breast cancer growth. Selective depletion of specific cell populations suggests that the antitumor effect of PSK is dependent on both CD8+ T cell and NK cells, but not CD4+ T cells. PSK does not inhibit tumor growth in TLR2−/− mice suggesting that the antitumor effect is mediated by TLR2.Conclusion: These results demonstrate that PSK, a natural product commonly used for the treatment of cancer, is a specific TLR2 agonist and has potent antitumor effects via stimulation of both innate and adaptive immune pathways. Clin Cancer Res; 17(1); 67–76. ©2010 AACR.

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Related Articles from Enhancing Cancer Vaccine Efficacy via Modulation of the Tumor Microenvironment

Purpose: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody–based immunotherapy that includes the activation of natural killer (NK) cells.Experimental Design: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP).Results: VTX-2337 selectively activates TLR8 with an EC50 of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158).Conclusions: VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy. Clin Cancer Res; 18(2); 499–509. ©2011 AACR.