Use of the TLR2 ligand polysaccharide krestin to maximize conventional cancer therapy

KEYWORDS:

• HER2
• polysaccharide kinase
• toll-like receptors
• natural killer cells
• CD8+
• T cells

The HER2+ breast cancer subtype accounts for 20–30% of cases and represents a particularly aggressive form of breast cancer. As this type of breast cancer is dependent on the HER2 receptor for proliferation, the introduction of the anti-HER2 monoclonal antibody (mAb) trastuzumab revolutionized the management of HER2+ disease and remains the foundation of anti-HER2 treatment [1]. Despite this improvement in treatment, patients with advanced HER2+ disease will progress on trastuzumab. The primary antitumor activity of trastuzumab is mediated by the immune process of antibody (Ab)-dependent cellular cytotoxicity (ADCC) [2]. Functionally, ADCC occurs when the Fc portion of an Ab binds to the Fcg receptor of a natural killer (NK) cell, inducing NK cell cytokine release (interferon gamma (IFN-γ) and cytolysis of the Ab-bound cell. In HER2+ breast cancer, studies have shown that the functional activity of NK cells impacts the antitumor effects of trastuzumab [3]. In addition, treatment with trastuzumab affects the localization of NK cells as was shown in patients undergoing neoadjuvant chemo and trastuzumab therapy, where trastuzumab treatment was associated with a significant increase in the numbers of tumor-associated NK cells [4]. Despite these observations, NK cell function is inhibited in cancer patients by multiple mechanisms, including the secretion of the immunosuppressive transforming growth factor-beta cytokine release by tumors [5] and the downregulation of the activating NK cell CD16 receptor [6] (a critical receptor for ADCC).

The presence of HER2-specific Type I or Th1 immunity is critical to antitumor efficacy in HER2+ disease as it represents an adaptive immune response that mediates a direct cytotoxic effect on tumor cells [7]. A recent study has documented that there is a progressive loss of a HER2-specific Th1 immune response through growth of HER2+ breast cancer [8]. It has been demonstrated that trastuzumab induces HER2-specific Th1 immunity in a minority of patients (30%), and that levels of the induced HER2-specific T cell immunity are variable [9]. We have shown that HER2 vaccination can induce additional immune response above what is generated with trastuzumab such that 70% of patients develop HER2-specific immunity following trastuzumab and vaccination [9]. Clinically, this is an important observation as measurable HER2-specific immunity has been linked to improved survival in HER2+ breast cancer [10]. This finding has been shown in several studies, including a recent investigation that revealed in patients who received chemo + trastuzumab, Th1-nonresponsive patients had a worse disease-free survival (median, 47 vs. 113 months; P < .001) compared with Th1-responsive patients [10].

Our investigation into interventions to augment trastuzumab-mediated ADCC and HER2-specific Th1 immunity led us to polysaccharide krestin (PSK), an extract from the mycelium of the mushroom Trametes versicolor. First approved in the 1970s, PSK has been used for decades in Japan as an anticancer therapy. Its clinical cancer use was supported by human trials that suggested improved survival when PSK was administered in gastric [11], colorectal [12], and lung cancers [13]. Despite this survival benefit, the exact mechanism of PSK’s antitumor activity has been unclear. Prior publications have suggested that PSK’s activity may be immune mediated, with studies reporting that PSK induces the gene expression of IL-8 in peripheral blood mononuclear cells (PBMCs) after oral administration [14], stimulates T-cell proliferation [15], and improves the function of CD4+ T cells in gut-associated lymphoid tissue [16].

Using knockout mouse models, our investigations of PSK have established that PSK selectively binds to the toll-like receptor 2 (TLR2) [17]. Toll-like receptors are elements of the innate immune system tasked with the detection of foreign microbes and viruses and the activation of an immune response against these foreign bodies. When bound by ligands and activated, TLR2 induces the activation of multiple immune cell subtypes. Specifically, TLR2 is primarily found on dendritic cells (DCs), and to a lesser extent on T cells and NK cells. In our preclinical models, PSK induces the maturation and activation of DCs, resulting in an increased percentage of CD86+ MHCH^high DCs. These PSK-activated DCs in turn release the Th1-linked cytokine, IL-12. Secretion of IL-12 induces the antitumor activities of multiple other immune cells including CD8+ T cells and NK cells. PSK administration also induced the secretion of other Th1-related cytokines including IFN-γ, TNF-alpha, and IL-2. We have demonstrated that PSK administration inhibits the growth of implanted or spontaneous breast tumors in neu-transgenic mice. To investigate which immune cells were responsible for the antitumor activity of PSK, we performed selective immune cell depletion in our mouse models. These depletion studies revealed that the antitumor activity of PSK is primarily due to CD8+ T cells and NK cells.

As we had established that PSK could induce NK cell maturation, we next investigated if PSK could also augment the functional activity of NK cells and enhance the ADCC of tumor antigen-specific mAbs. In these evaluations, we revealed that the addition of PSK to PBMCs augments the activation of NK cells, as determined by increased CD25 and CD69 expression, and induces the NK cell secretion of IFN-γ [18]. We confirmed that these effects on NK cells are mediated through both direct TLR2 binding of PSK and indirectly through the induction of DC secretion of IL-12, as noted earlier. We also showed that when added to PBMCs, PSK augments the cytolytic function of NK cells against cancer cell lines. In order to evaluate if PSK could augment trastuzumab-mediated ADCC, we exposed HER2+ breast cancer cell lines (SKBR3 and MDA-MB-231) to PSK-treated PBMCs, and verified that indeed PSK significantly enhanced trastuzumab-mediated ADCC against these cells. Lastly, in neu-transgenic mice bearing neu+ tumors, the combination of PSK and an anti-HER2 mAb exhibited enhanced antitumor effects compared to either therapy alone.

Based on these preclinical investigations, we have initiated a Phase II randomized clinical trial to investigate the immune and therapeutic effects of the addition of PSK to anti-HER2 therapy in advanced HER2+ breast cancer. Patients on this randomized study receive trastuzumab and a HER2-directed vaccine with or without PSK. To date, we have enrolled 24 patients of a planned 30 patients. In the treated patients, there have been no grade 3 or higher adverse events on the study. This trial will complete enrollment and treatment in 2017. The immune analysis from this study will investigate changes in NK cell functional activity and HER2-specific immunity from baseline and between patients who received PSK vs. those who did not. The ability of PSK to augment NK cell functional activity will be measured by NK cell secretion of IFN-γ and a CD107a NK cell degranulation assay (to measure NK cell cytotoxicity). We will also investigate changes in HER2-specific Th1 immunity with HER2 IFN-γ ELISPOT assays. The clinical and correlative science results from this trial will guide future development and application of PSK and similar therapies. Our long-term clinical plan is to integrate PSK into neoadjuvant anti-HER2 therapy to significantly improve upon the current pathologic complete response rate of 40% [19].

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

Funding received from the National Institutes of Health, National Cancer Institute 1U19AT006028-04.