7866/135 08/23/2021 Page 1 of 39 UNIVERSITY OF WASHINGTON DEPARTMENT OF MEDICINE DIVISION OF ONCOLOGY Protocol 135 Phase I/II Ra ndomized Study of Combination Immunotherapy With or Without Polysaccharide Krestin (PSK ®) Concurrently with a HER2 ICD Peptide- Based Vaccine in Patients with Stage IV Breast Cancer Receiving HER2- Targeted Monoclonal Antibody Therapy Investigators William R. Gwin, MD Assistant Professor Division of Oncology, UW (206) 221- 5956 Mary L. Disis, M.D. Professor of Medicine Division of Oncology, UW (206) 616- 1823 John Liao, M.D. Assistant Professor GYN/ONC, UW (206) 543- 3915 Andrew Coveler, M.D. Assistant Professor Division of Oncology, UW (206) 221- 7509 Sasha Stanton, M.D. Acting Instructor Division of Oncology, UW (206) 685- 8849 Doreen Higgins, B.S.N. Research Nurse Division of Oncology, UW (206) 616- 9538 Jennifer Childs, M.P.H. Research Coordinator Division of Oncology, UW (206) 616- 2305 Ted Gooley , Ph.D Biostatistician Fred Hutchinson Cancer Research Center (206) 667- 6358 Emergency (24 hour) pager: William R. Gwin , M.D. (206) 314- 1279 Mary L. (Nora) Disis, M.D. (206) 427- 8700 IRO received 08/23/21 FHCRC IRB Approval SEP 03 2021 Document Released Date 7866/135 08/23/2021 Page 2 of 39 Table of Contents Page S ynopsis 3 Li st of Appendices 6 Li st of Abbreviations 7 I ntroduction 8 B ackground 8 S tudy Rationale 13 O bjectives 13 E xperimental Agent Preparation HER2 ICD Peptide -Based Vaccine 14 GM-CSF 14 Vaccine Dose Compounding and Dispensing 15 PSK Product and Placebo 15 Experimental Agent Accountability 16 I nclusion and Exclusion criteria 16 E xperimental Design Study Design 17 Sample Size 18 Outcome Measures 18 P lan of Treatment – Arms 1 and 2 Pre Screen 19 Initial Evaluation 19 Monthly Immunizations 20 Evaluation after Final Vaccine 20 Management of Potential Study Risks 20 P atient Withdrawal from Study 21 E valuation and Management of Toxicity 21 D ose Modifications 22 A ccrual and Criteria for Premature Study Termination 22 S tatistical Consideration 22 A dministrative Considerations 25 R eferences 26 A ppendices 29 7866/135 08/23/2021 Page 3 of 39 SYNOPSIS Title Phase I/II Ra ndomized Study of Combination Immunotherapy With or Without Polysaccharide Krestin (PSK®) Concurrently with a HER2 ICD Peptide -Based Vaccine in Patients with Stage IV Breast Cancer Receiving HER2 -Targeted Monoclonal Antibody Therapy Study Population Patients with S tage IV HER2+ b reast cancer who have been treated with definitive therapy and are receiving maintenance HER2 -targeted monoclonal antibody therapy (defined as trastuzumab monotherapy or combination trastuzumab and pertuzumab therapy) ; and are currently without evidence of disease (NED) or have stable- bone only disease. Rationale Hot-water extracts of mushrooms have been used in traditional medicine in China, Japan, and other Asian countries for centuries. PSK was isolated in Japan from the Trametes versicolor mushroom in the 1960s and has been studied in more than 48 cl inical trials for various types of cancers. R andomized clinical trials have show n low toxicity profile and improved survival in breast cancer patients treated with PSK . Pre-clinically, in vitro studies have shown PSK to be a potent TLR -2 agonist that activates dendritic cell maturation , activates human NK cells , induces pro-inflammatory cytokine /chemokine secretion and potentiates trastuzumab- mediated ADCC against breast cancer cells. PSK has al so been shown to potentiate antitumor effect s of anti -HER2 mAb therapy in neu- transgenic mice . We hypothesize that the use of PSK will augment the innate anti -tumor immune responses specifically NK -cell activity. Additionally , PSK could potentially augment HER vaccine induced Th1 immunity and collectively the combination therapy could result in enhanced eradication of micrometastatic disease which could prevent relapse in patients with optimally treated HER2+ Stage IV Breast Cancer. Oral PSK at 3000 mg/day has been shown to be safe with and without chemotherapy in breast cancer . Moreover, combination immunotherapy with HER2 -directed immunotherapy ( HER2 ICD vaccination and HER2 -targeted monoclonal antibody therapy ) has been shown to be safe and immunogenic. 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 combined immunotherapy regimen . Objectives Primary 1. To evaluate the safety of PSK when given with HER2- directed immunotherapy . Secondary 1. To evaluate the effect of PSK on NK cell functional activity when given with HER2 -directed immunotherapy . Tertiary 1. To investigate the effect of PSK when given with HER2 -directed immunotherapy on: a. Serum levels of pro-inflammatory cytokine and/or chemokines. b. Intermolecular epitope spreading. c. Serum TGF -β levels. d. Progression free survival (PFS) and overall survival (OS). Study Design A phase I/ II double- blind randomized two -arm clinical trial designed to examine the safety and immune effects of PSK when given as combination HER2 -directed immunotherapy (a HER2 ICD peptide vaccine and HER2 -targeted monoclonal antibody therapy ) in patients with Stage IV HER2+ breast cancer. Eligible patients will be enrolled and randomized in equal numbers to one of 2 arms (15 patients per arm). 7866/135 08/23/2021 Page 4 of 39 Arm 1 : HER2 ICD vaccine + HER2 -targeted monoclonal antibody therapy* + Placebo: Patients will receive HER2 ICD peptide vaccine administered concomitantly with HER2 -targeted monoclonal antibody therapy and placebo product. Arm 2 : HER2 ICD vaccine + HER2 -targeted monoclonal antibody therapy* + PSK : Patients will receive HER2 ICD peptide vaccine administered concomitantly with HER2 -targeted monoclonal antibody therapy and PSK. *Trastuzumab (or trastuzumab and pertuzumab) will be administered per standard of care by patient’s primary oncologist. Number of Patients A total of 30 patients will be enrolled and randomized in equal numbers to one of 2 arms with PSK or placebo. Outcome Measures Primary Endpoint: 1. Evaluation of safety of PSK when administered with HER2 -directed immunotherapy . Evaluation of safety and systemic toxicity will include physical examination and clinical labs performed at various timepoints. Toxicity grading will be evaluated per CTEP CTCAE 4.0 and monitoring of adverse events will be done per FDA and NCI guidelines. Additionally, stopping rules for accrual to study will become operational if there is sufficient evidence of excessive toxicity. Secondary Endpoint: 1. Evaluation of the effect of PSK on NK cell functional activit y. PBMC will be obtained and cryopreserved at baseline (prior to start of PSK) and then at 4, 8, and 16 weeks . PBMC will subsequently be thawed and used to determine the effect of PSK on NK cell functional activity specifically, induction of IFN - gamma production and CD107a expression in NK cells, via flow cytometry . Augmentation of NK cell activity is defined by a 2 -fold increase in NK cell IFN - gamma production and CD107a expression from baseline after 4 weeks of oral administration of PSK. Tertiary Endpoints: 1. Assessment of the effect of PSK on pro- inflammatory serum cytokine and/or chemokines when administered with a HER2 -directed immunotherapy . Serial blood draws will be done before initial dose of PSK (time 0) and 30 min, 4 hours, and 24 hours post -initial PSK dose to assess serum cytokines/chemokines (i.e., IL -6, IFN -γ, and TNF- α) via Luminex analysis. Serum cytokine/chemokine levels at different timepoints post -PSK will be compared to baseline levels. Serum will be cryopreserved and subsequently be thawed at time o f analysis. 2. Assessment of the effect of PSK on intermolecular epitope spreading when administered with HER2 -directed immunotherapy . IFN -γ ELISPOT assay will be used to evaluate T cell precursor frequency to specific breast tumor antigens (HER2, IGFBP -2, TOPO -IIα, p53, and PRAME) at various timepoints. A positive immune response will be defined as a post -vaccination T cell precursor frequency >1:20,000 antigen -specific PBMCs. In patients with a baseline precursor frequency >1:20,000, a positive post -vaccination immune response will be defined as a 2 -fold increase in antigen- specific PBMC. PBMC will be cryopreserved and subsequently be thawed at time of analysis. 3. Assessment of the effect of PSK on serum TGF -β levels when administered with HER2 -directed immunotherapy . Serum levels of TGF -β will be assessed with ELISA at various timepoints to determine incidence and magnitude of 7866/135 08/23/2021 Page 5 of 39 serum TGF- β reduction pre- and post -combination immunotherapy. Serum will be cryopreserved and subsequently be thawed at time of analysis. 4. Evaluation of PFS and OS . Evaluation of PFS and OS will be followed and compared between the treatment arms. Thoug h not statistically powered to this endpoint, large differences if observed between the treatment groups will be noted and described. 7866/135 08/23/2021 Page 6 of 39 Appendices Appendix A Zubrod Performance Status Scale Appendix B Schedule of Events Appendix C Data Safety Monitoring Plan 7866/135 08/23/2021 Page 7 of 39 Abbreviations: ADCC antibody -dependent cell -mediated cytotoxicity ALND axillary node dissection APC antigen presenting cells BUN blood urea nitrogen CBC complete blood count CFC cytokine flow cytometry CMP Complete metabolic panel CTCAE Common Terminology Criteria for Adverse Events CTEP Cancer Therapy Evaluation Program CTL cytotoxic T lymphocytes DC dendritic cell DTH delayed type hypersensitivity DSMP Data Safety Monitoring Plan ECD extracellular domain of HER2 FISH fluorescen ce in situ hybridization rhuGM -CSF rhu granulocyte macrophage colony stimulating factor GMP good manufacturing practice HER2 HER -2/neu HPLC high pressure liquid chromatography ICD intracellular domain of HER2 id. intradermal IFNγ interferon -gamma IHC immunohistochemistry LVEF left ventricular ejection fraction MBC Metastatic breast cancer MHC major histocompatability complex MUGA multigated acquisition NED no evidence of disease NK Natural Killer Cell OS overall survival PBMC peripheral blood mononuclear cells PSK Polysaccharide Krestin PFS progression free survival Th T helper cells TLR Toll-like receptor agonist TNF Tumor necrosis factor 7866/135 08/23/2021 Page 8 of 39 UPN unique patient number 7866/135 08/23/2021 Page 9 of 39 1. Introduction Patients with advanced breast cancer have been shown to have defects in anti -tumor immunity including impairment of n atural killer (NK) cells.[1-3] NK cells are key components of innate immunity which play a fundamental role in tumor immune surveillance. Recent findings in breast cancer patients show peripheral blood and tumor -infiltrating NK cells to have tumor -promoting alter ations in their activating and inhibitory receptors which correlate with decreased cytotoxic NK cell function .[3] Additionally, inhibitory factors in the tumor microenvironment including TGF -beta (TGF- β) have been shown to be directly involved in the impairment of NK cells’ phenotype and functions. It is postulated that invasive breast tumors escape NK cell antitumor immunity via a strong inhibitory microenvironment resulting in tumor progress ion and recurrence. Thus, novel approaches to improve NK cell function and overcome tumor cell -mediated NK suppression are needed. Toll-like receptor agonists (TLRs) are a group of pathogen recognition receptors that can recognize the pathogen associated molecular patterns (PAMPs) from virus, bacteria, and fungus resulting in initiation of innate immunity. Several TLR agonists have been shown to activate NK cells, either directly or indirectly via dendritic cells (DC).[4-6] Activation of NK cells can result in direct NK cell mediated cytotoxicity of tumor. Activated NK cells also secrete proinfl ammatory cytokines and chemokines which regulate and direct immune responses.[7] Specifically, activated NK cells secrete IFN -γ which can promote maturation and differentiation of DCs toward a Th1 profile and drive the activation of adaptive immunity. Our group has been studying the immune modulatory and anti -tumor potential of Polysaccharide Krestin (PSK), a hot water extract from Tremetes Versicolor mushroom as a means to enhance NK cell function via stimulation of TLR2. Our pre -clinical studies have shown that PSK: (1) is a selective TLR2 agonist with potent antitumor effects whi ch are dependent on enhanced PSK -induced NK and CD8 T cells activity[8], (2) can activate human NK cell function and enhance tr astuzumab- mediated antibody -dependent cell -mediated cytotoxicity (ADCC)[9], (3) can induce NK cell and gamma -delta T cell secretion of multiple inflammatory cytokines including IFN -γ resulting in DC maturation and activation[8, 9] and (5) may also work as a vaccine adjuvant to significantly augment antigen- specific T cell immunity. Collectively, these findings suggest the potential of PSK to augment antitumor immunity via stimulation of DCs and NK cel ls and also to enhance the therapeutic effect of trastuzumab and HER2 vaccination in breast cancer patients. We hypothesize that the use of PSK will enhance the innate anti -tumor immune responses specifically NK-cell activity and result in prolonged upre gulation of IFN -γ production; this could also result in stimulation of adaptive Th1 immunity. Thus, we have designed a phase I/II randomized study of combination immunotherapy with oral PSK administered concurrently with trastuzumab and HER2- targeted pept ide vaccination. Our primary and secondary objectives are to assess the safety of PSK as a component of combination immunotherapy and to evaluate effect of PSK on NK cell functional activity. Additionally, we will investigate if PSK stimulates circulatin g proinflammatory cytokine/chemokines, enhances HER2 vaccine- induced T cell immunity (epitope spreading) and decreases serum TGF -β. 2.