Background A. Natural killer (NK) cell deficiency has been found in patients with breast cancer. NK cells are part of the innate immunity that plays a fundamental role in tumor immune surveillance. As surveillance cells in the tumor microenvironment, DCs and NK cells have been shown to infiltrate tumors and monitor the presence of different antigen derived from tumors.[10] Thus, NK cells limit not only the emergence of viral infections but also of cancers by surveillance of “missing- self” and “induced- self” ligands, and by direct r ecognition of pathogen -associated molecules. Unfortunately, NK cell maturation and cytotoxic function is frequently impaired in breast cancer patient s.[1-3] Moreover, NK cell activity of IFN -γ production has been shown to be significantly impaired in invasive breast tumors, especially in advanced stage diseas e. [3] The activation of NK cells is regulated by a balance of signals transduced by activating and inhibitory receptors expressed on the surface of NK cells. More recently, it has been shown that breast tumor cells can alter NK cell function through modulation of their surface recepto rs.3 Specifically, expression of activating NK cell receptors (i.e., NKp30, NKG2D) were significantly decreased while expression of inhibitory receptors (i.e., NGK2A) were increased in both peripheral blood NK cells (p- NK) and breast tumor infiltrating NK cells (Ti- NK); and this alteration was correlated with decreased NK cell function, especially cytotoxicit y.3 Moreover, impairment of Ti -NK cytotoxicity was more pronounced when compared to that of peripheral NK cells. Additionally, in situ levels of inhibitory factors such as TGF -β and 7866/135 08/23/2021 Page 10 of 39 regulatory T cell infiltrates correlated with the NK cell impaired phenotype and functions. These data support the notion that breast tumors are able to model their environment to evade NK cell antitumor immunity which likely contributes to tumorigenesis . Thus, further investigation of immunomodulators such as PSK to restore NK cell maturation and activity within an immunosuppressive tumor microenvironment and prevent breast cancer progression and/or recurrence is warranted. B. Mushroom extract PSK is a TLR2 agonist that activates human NK cells. Hot-water extracts of mushrooms have been used in traditional medicine in China, Japan, and other Asian countries for centuries. Among other properties, the anticancer and immunomodulatory effects of certain mushroom extracts have been described and studied in depth in the lab and in clinical trials, mainly in Asia. PSK was isolated in Japan from the Trametes Versicolor mushroom in the 1960s and has been studied in more than 48 clinical trials, mainly for colorectal, gastric, esophageal, lung, and breast cancer. Based on the findings of randomized PSK clinical trials which showed a low toxicity profile and improvements in patient survival, PSK is commonly used as adjuvant or neoadjuvant treatment with or without chemotherapy for patients with colorectal, breast, and gastric c ancer in Japan. [11-14] TLRs play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection or tissue injur y.[15] Specifically, TLRs are a group of pathogen recognition receptors that can recognize the pathogen associated molecular patterns (PAMPs) such as dsRNA or ssRNA, unmethylated DNA, or lipopeptide from virus, bacteria, and fungus. TLRs can induce strong anti -tumor activity by regulating the functions of the immune cells that infiltrate the tumor microenvironment. The potential of TLR agonists, such as polyI:C (TLR3), resiquimod (TLR7/8), and CpG (TLR9), to activate NK cells, either directly or indirectly via dendritic cells (DC), have been shown in both pre-clinical and clinical studies. [4-6] Our gr oup has been studying the immune modulatory and anti -tumor potential of PSK. Our research studies have demonstrated that PSK is a potent TLR2 agonist (Fig. 1). Pre-clinically, in vitro studies have shown that PSK can activate human NK cells to produce IFN -γ and lyse tumor cells; and induce secretion of pro- inflammatory cytokines and chemokines (e.g., IL- 12, TNF- α, MIP-1α, and MIP -1β). NK cells represent a highly specialized lymphoid population with potent cytolytic activity against tumor or virus infected cells. There are two subpopulations of NK cells, the CD56bright NK cells which are mainly responsible for cytokine production and the CD56dim NK cells that are mainly responsible for the cytotoxic effect of NK cells.[16] Our preclinical studies have shown that PSK can activate both subpopulations of NK cells and stimulates both the IFN -γ production and cytotoxicity of NK cells (Fig. 2 ).[9] In a recent Phase 1 study of Trametes versicolor ( Tv) in women with breast cancer conducted by Standish et al, NK cell activity increased to approximately 2- fold of baseline after 4 weeks of oral administration of Tv.[17] Thus, the immune measure of PSK -induced NK cell activity in this study will be assessed with functional NK flow cytometry assays used in our previously published papers and at similar time points as described in Section 7. C.2. (Outcome Measures).[18] 1 10 100 10000123 PSK (µg/mL)TLR4TLR2SEAP Activity (OD650 nm) A B PBS PSK K562 12.525 50100020406080 PBSPSK *** E:T ratio% Specific Lysis CD56bright CD56dim Figure 1. PSK selectively activates TLR2. HEK cells transfected TLR2 or 4 were stimulated with serial dilutions of PSK (0.5- 1000 µg/mL, 24 hr). PSK also had no effect on TLR3, 5, 7, 8, 9 ( data not shown). Figure 2. PSK stimulates IFN -γ production and augments the cytotoxicity of NK cells. (A) Intracellular staining showing that PSK induces IFN γ production by NK cells. (B) PSK- stimulated PBMC have enha nced lysis against K562. Data shown is representative of results from 3 donors. CD56 IFN-γ 7866/135 08/23/2021 Page 11 of 39 C. PSK enhances trastuzumab mediated ADCC and may improve its clinical efficacy. Trastuzumab is a humanized anti -HER2 monoclonal antibody (mAb), and is the first HER2- targeted therapy approved by FDA. Tr astuzumab has significantly advanced the clinical management of patients with HER2+ breast cancer by prolonging disease- free survival in patients with locally advanced breast cancer, and progression- free and overall survival in patients with metastatic breast cancer.[19, 20] Trastuzumab inhibits tumor cell growth through multiple mechanisms including signaling blockade and downregulating the HER2/ne u receptor. One of the major mechanisms is believed to be ADCC, in which the tumor cells are coated with trastuzumab and then lysed by immune cells via binding of Fc gamma recep tor (FcγR) to the Fc portion of the mAb.[21] Increase in tumor infiltrating NK cells after trastuzumab therapy has been found in human breast cancer biopsy samples[22, 23] and FcγR gene polymorphism can impact the clinical response to trastuzumab.[24] NK cells constitutively express FcγRIIIA (CD16) and are the major effectors of ADCC.[25] Therefore the function of NK cells may impact the efficacy of ADCC and clinical response to trastuzumab.[26] In our pre -clinical studies, we have found that PSK significantly enhanced trastuzumab- mediated ADCC against two breast cancer cell lines, SKBR3 and MDA - MB-231. Furthermore , we showed that combination of oral PSK and a HER2- targeted mAb therapy in mice resulted in enhanced anti -tumor effect .[18] Patients enrolled to this study will have metastatic breast cancer which has been optimally treated to a state of NED (no evidence of disease) and therefore off cytotoxic chemotherapy. However, our patient population will continue use of maintenance trastuzumab monotherapy or combination trastuzumab and pertuzumab therapy as dictated per standard of care. Although not a study outcome, combination of PSK with trastuzumab could potential ly enhance NK-cell-mediated ADCC of residual microscopic disease (i.e., HER2+ tumor cells) in our study patients. D. PSK can act ivate DC and augment antigen -specific T cells when used as a vaccine adjuvant. TLR2 is highly expressed on DC, and treatment with PSK can induce DC maturation and secretion of multiple inflammatory cytokines, including TNF- γ, IL-12p40, and IL -12p70. [8, 9] Furthermore, PSK -induced IFN-γ produced by NK cell s and gamma -delta T cells can reciprocally activate DC. Therefore, we hypothesize that PSK may work as vaccine adjuvant to augment antigen -specific T cells. Our pre -clinical studies in a mouse model of vaccination with ova peptide antigen p323 has shown that PSK as a vaccine adjuvant significantly enhanced ova- specific T cell immunity. Specifically, BALB/c mice were given a vaccine of OVA p323 -339 peptide at the dose 0.5 mcg per mouse concurrently with an intradermal (ID) adjuvant of either PSK (1000 mcg ), GM -CSF (5 mcg), or PBS. For two days following initial injection, adjuvant was given alone once per day and animals were sacrificed on Day 4. Draining lymph nodes (dLN) were harvested for evaluation of DC activation and antigen -specific T cells. dLNs from mice that received GM -CSF or PSK were enlarged compared to dLNs from mice in the PBS control group (Fig. 4A); and the percentage and number of CD11c + DC were significantly increased in GM -CSF and PSK treated mice per FACS analysis. The expression of activation markers, CD80, CD86, MHCII, and CD40 were also up- regulated in DC isolated from dLNs from PSK treated animals (Fig. 4B). The number of ova antigen -specific T cells, as evaluated in an IFN -γ ELISPOT assay was also significantly increased in mic e that received PSK as adjuvant (Fig. 4C). Although PSK was given ID in these experiments, we hypothesize that oral PSK may also enhance the effect of vaccination. Indeed, a recent publication by Tanaka et al has shown that combining peptide vaccine with oral ingestion of Lentinula endodes mycelia extract enhances anti -tumor activity in B16 melanoma- bearing mice. [27] SKBR3 12.525 50100020406080100 PSK PBS E:T ratio% Specific Lysis MDA-MB-231 12.525 50100010203040 PBSPSK E:T ratio% Specific Lysis Figure 3. PSK enhances trastuzumab -mediated ADCC in lysing of SKBR3 and MDA -MB-231 breast cancer cells . PSK or PBS- stimulated PBMCs were incubated with 3000 target cells at the indicated E:T ratio. Shown are specific lysis at each E:T ratio (mean±sem of triplicate wells). A PBS GM-CSF PSK B CD80 CD86 MHCII CD40 C 7866/135 08/23/2021 Page 12 of 39 E. PSK may augment vaccine- induced HER2 Th1 immunity in optimally treated metastatic breast cancer patients. Disease relapse in optimally treated HER2+ breast cancer patients who are in complete remission suggest the presence of persistent residual microscopic disease not eradicated by systemic therapy. One approach to the eradication of residual subclinical disease in this high- risk population is the development of tumor vaccines that target HER2, an immunogenic biologically relevant protein. Generation of vaccine- induced HER2- specific Type I inflammatory CD4+ T helper immunity (Th1) could result in immunolo gic eradication of residual HER2+ tumor cells and potentially prevent disease relapse or tumor spread. Recent evidence indicates that Type I immunity which is associated with the production of high levels of IFN -γ enhances cross priming, the primary method by which immunity is generated against cancer, at the site of cancer. Moreover, vaccine induced Th1 immunity has the potential to generate epitope spreading, a broadening of the immune response to multiple potential antigens in the tumor which may resul t in more efficient kill of residual tumor cells by T cells. This is particularly important as impaired IFN signaling with associated T cell activation defects have been identified in breast cancer and may be a contributing factor to decreased Th1 immune responses to breast cancer vaccines. As previously described, our pre- clinical studies have shown that PSK can induce DC maturation and secretion of multiple inflammatory cytokines that result in antigen- specific T cell immunity. Additionally, growing evidence indicates that NK cells have mechanisms to efficiently promote maturation and differentiation of DCs toward a Th1 profile. Specifically, activated DCs in the tumor microenvironment are able to take up tumor antigens and migrate to lymph nodes wher e they present their antigens to CD8+ T cells and CD4+ T cells which then undergo clonal expansion and are able to infiltrate and destroy tumor in an antigen specific manner. Thus, NK cell activation and resultant IFN -γ production indirectly promote adapt ive antitumor T cell responses via activated DCs . [10] Similarly, activated DCs induce potent NK cell activation. Together, NK cell and DC activation are key in the priming process of CD8+ and Th1 immunit y and development of adaptive immunity.[10] Recognizing the importance of NK cells and their role in stimulating adaptive immunity, recent immunotherapeutic strategies such DC vaccination have focused on overcoming impaired Th1 immunity via NK cell activation. Specifically, Baek et al, showed that DC vaccines when given with IL- 12 were able to induce antigen specific immunity; and NK cell activation with reduction of inhibitory immunity.[28, 29] In a follow -up study, Qi et al, demonstrated the ability of DC vaccination to promote secretion of Th1 cytokines and significantly increase the number of peripheral blood NK cells resulting in tumor specific CD8+ T cell expansion and further differentiation into IFN -γ producing effector cells.[29] We have previously demonstrated our ability to induce tumor -specific immunity and also epitope spreading in patients with HER2+ breast cancer.[30, 31] Moreover, epitope spreading was associated with potential survival benefit in vaccinated patients .[32] Key elements to inducing HER2 -specific Th1 immunity and epitope spreading in the majority of HER2+ breast cancer patients were the use of vaccines which elicit T h1 immunity given in combination with GM -CSF as adjuvant and our targeted patient population (optimally treated patients to state of NED). The addition of immunomodulatory agents such as PSK as adjuvants to vaccines may help enhance, sustain, and skew the immunogenicity of antigens toward a Th1 phenotype via robust NK cell activation, allowing for development of robust and durable vaccine- induced Th1 immunity; and potentially prevention of cancer recurrence. Thus, we propose to investigate if PSK when given orally with HER2 peptide based vaccination and trastuzumab can enhance both development and magnitude of HER2 vaccine- induced Th1 T cell immunity specifically, epitope spreading. PSK PBS Figure 4. PSK as vaccine adjuvant stimulates dendritic cell (DC) activation in vivo and enhances antigen -specific T cells. (A) Representative pictures of draining lymph nodes (dLN ) from mice vaccinated with ova peptide with PBS, GM -CSF or PSK as adjuvant. (B) The expression of DC activation markers, CD80, CD86, MHCII, and CD40 on DC in dLN from mice received ova plus PSK (empty histogram) or PBS control (shaded histogram). (C) The number of spots per well in IFN -γ ELISPOT assay evaluating ova -specific T cells using dLN cells from mice received ova plus PBS, GM -CSF or PSK. PBS GM-CSF PSK050100150200*** Adjuvant TreatmentSpots per well 7866/135 08/23/2021 Page 13 of 39 F. PSK may enhance NK cell activation by inhibiting TGF-β production in the tumor microenvironment. TGF- β is a hormonally active polypeptide that inhibits the proliferation of normal epithelial cells, and enhances matrix formation and angiogenesis .[33] Most epithelial cancer cells lose responsiveness to TGF- β induced growth inhibition.[34, 35] TGF-β is secreted by both breast cancer cells and the tumor stroma resulting in increased proliferative capacity, enhanced epithelial -mesenchymal transition (EMT); and subsequent increased metastatic potential of tumors .[36, 37] TGF-β, a potent immunosuppressive cytokine acts in the tumor microenvironment to blunt immune- surveillance via multiple mechanisms, including suppression of NK cells. Additionally, TGF- β is secreted by T regulatory cells which function to inhibit the development of adaptive immunity to “self -antigens” such as tumor antigens .[38] Moreover, elevated levels of serum TGF -β have been found to be related with diseas e progression and worse prognosis in breast cancer patients .[39] Recent studies have shown that inhibitory molecules in the tumor microenvironment such as TGF- β directly contribute to impairment of NK cell phenotype and function. As described above, Mamessier et al showed that expression of activating NK cell receptors were significantly decreased while expression of inhibitory receptors were increased in both peripheral blood and breast tumor infiltrating NK cells; and stroma- derived inhibitory f actors particularly TGF- β were involved in driving the impaired NK cell activity. Additionally, increased in situ levels of TGF- β and regulatory T cell infiltrates correlated with worse NK cell impairment. Thus, inhibition of the TGF- β via immunomodulati on of the tumor microenvironment may restore NK cell activity and result in development of robust tumor antigen specific Th1 immunity. Previous studies have suggested that PSK may modulate the biological activity of TGF-β and thus restore robust NK cell activity. Additionally, enhanced NK cell activity with subsequent increased IFN -γ production may result in decreased TGF-β production in the tumor microenvironment . In a more recent study, PSK was shown to inhibit the Smad pathway, the major regulator of TGF -β signaling.[40] Treatment with PSK inhibited the expression of several TGF- β pathway target genes and prevented TGF-β1-induced EMT. Studies by our group have demonstrated that circulating levels of TGF- β are significantly elevated in sera from breast cancer patients compared to controls; and that vaccine- induced HER2 specific IFN -γ T cell responses specifically epitope spreading , were associated with reductions in serum TGF- β levels. Additionally, the magnitude of the epitope spreading T cell response elicited with active vaccination was inversely correlated with TGF- β levels assessed in serum throughout immunization. As described in Section 13 (Statistical Considerations) our previous study data suggest a modest decrease in ser um TGF - β levels among patients who received HER2 ICD vaccination and trastuzumab alone. Therefore we aim to investigate if administration of PSK with HER2 vaccination and trastuzumab results in enhanced downregulation of TGF- β expression in the tumor and augmented NK cell induced IFN -γ production; and subsequent decrease in detectable circulating serum levels of the cytokine. G. Concomitant administration of pertuzamab and trastuzumab has been shown to be safe and effective in treating HER2+ metastatic breast cancer (MBC) . Pertuzumab is a recombinant humanized monoclonal antibody that binds to the extracellular dimerization domain II of HER2, and inhibits heterodimerization of HER2 with other HER family members, including EGFR, HER3, and HER4. Blockade of HER2- HER3 likely represents the most relevant action of .[41] In pre- clinical models, pertuzumab when given in combination with trastuzumab, is associated with a more complete HER2 blockade. Additionally, the treatment combination is more effective in HER2- positive tumor xenografts than when either antibody is given alone.[41] Moreover, enhanced efficacy of the combined use of trastuzumab and pertuzumab when given with docetaxel has now been demonstrated in several clinical studies in HER2+ MBC. [42] Over the past 2 years, the first -line treatment of HER2+ MBC has been changing and now also includes other HER2 targeted agents specifically pertuzumab (perjeta). In 06/2012, the FDA approved the combination of docetaxel and pertuzumab/trastuzumab for t he treatment of HER2+ MBC. This was based on data from the CLEOPATRA study, a large Phase III trial enrolling 808 patients which showed the combination of docetaxel + pertuzumab/trastuzumab improved progression free survival (PFS) by 6 months compared to docetaxel + placebo/trastuzumab.[43] Given the PFS benefit, oncologists started using the triple regimen more readily. As a result, HER2+ MBC patients started receiving indefinite concomitant pertuzamab and trastuzumab as maintenance therapy once docetaxel was stopped. More recently, final results from CLEOPATRA (reported 02/2014) showed that first -line treatment with docetaxel + pertuzumab/trastuzumab in patients with HER2+ MBC significantly improved overall survival (O S) when 7866/135 08/23/2021 Page 14 of 39 compared to docetaxel + placebo/trastuzumab. Median OS was 56.5 months (pertuzumab arm) vs 40.8 months (placebo arm) with difference of 15.7 months. Extensive cardiac surveillance was conducted in CLEOPATRA and treatment with docetaxel + pertuzumab/trastuzumab did not increase the rate of left -ventricular systolic dysfunction (LVSD) when compared to docetaxel + placebo/trastuzumab. Specifically, the incidence of LVSD (all grades) was 8.6% in placebo arm vs 5.4% in pertuzumab arm. Additionally, the incidence of symptomatic LVSD was 1.8% in placebo arm vs 1.2% in pertuzumab arm. Lastly, incidence of LVEF decline to <50% and by ≥10% points from baseline was 7.4% in placebo arm vs 4.6% in pertuzumab arm; and LVEF recovery to ≥50% was 89.3% in placebo arm vs 88.9% in the pertuzumab arm. Altogether, no increased cardiotoxicity was observed in patients receiving concomitant pertuzumab and trastuzumab with docetaxel when compared to concomitant placebo and trastuzumab with docetaxel. Given the signifi cant OS benefit, and the acceptable toxicity data, it has been recommended that the new standard of care for first -line treatment of HER2+ MBC include docetaxel + pertuzumab/trastuzumab. 3. Study Rationale The proposed oral dose of PSK (3000 mg/day) in this study has been demonstrated to be safe with and without chemotherapy in large randomized breast cancer studies .[44, 45] In a review of PSK us e and adverse events (AEs), 114 ( 1%) AEs were reported from a total of 11,300 individuals who used PSK alone or with other medical treatments (Table 1). Principal AEs were diarrhea (0.25%), nausea (0.24%), vomiting (0.15%), and anorexia (0.15%). These data are taken from the Japanese Mi nistry of Health and Welfare and are in the PSK Package Insert. Severe PSK- related AEs have not been reported since clinical trials began in Japan in the 1970s thus, PSK appears to be extremely well tolerated. Table 1. Adverse Event Summary for PSK Dos e (3g/day) Based on 11,300 Individuals Symptomatic Diarrhea (0.25%) Nausea (0.24%) Vomiting (0.15%) Anorexia (0.15%) Hives (0.1 %) Stomach discomfort (0.1 %) Hematologic None Combination immunotherapy with HER2 -directed immunotherapy has also been shown to be both safe and immunogenic in our previous studies. However, the safety of PSK when added to HER2- directed immunotherapy is less well known and warrants further investigation. This study will allow us to better define the safety and immunomodulatory effects of this regimen on the tumor microenvironment and systemic immunity. 4.