A B S T R A C T PurposeThe 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 MethodsTwenty-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. ResultsConcurrent 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. ConclusionCombination 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.

Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of ∼10–25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.

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 À/À ) 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 CD8þ T cell and NK cells, but not CD4 þ T cells. PSK does not inhibit tumor growth in TLR2 À/À 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.