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Version Date: *06/30/2021* Abbreviated Title: GBM Pembro HSPPC
NIH **Protocol \#:** 17C0034 Version Date: 06/30/2021 NCT Number: NCT03018288 Title: A randomized, double blind phase II trial of Surgery, Radiation Therapy plus Temozolomide and Pembrolizumab with and without HSPPC-96 in newly diagnosed Glioblastoma (GBM)
Coordinating Center: BTTC Coordinating Center, Center for Cancer Research, NCI Responsible DSMB: NCI Data Safety Monitoring Board NCI Principal Investigator: Mark Gilbert, M.D.  
Neuro Oncology Branch (NOB), CCR, NCI 9030 Old Georgetown Road, Building 82, Room 235A Bethesda, MD 20892 Phone: 240-760-6023 Email: mark.gilbert@nih.gov Investigational Agents:  
| Drug Name:    | HSPPC-96                                                | Pembrolizumab                                           | Temozolomide                                            |
|---------------|---------------------------------------------------------|---------------------------------------------------------|---------------------------------------------------------|
| IND Number:   | 17203                                                   | 17203                                                   | 17203                                                   |
| Sponsor:      | National Cancer  Institute, Center for  Cancer Research | National Cancer  Institute, Center for  Cancer Research | National Cancer  Institute, Center for  Cancer Research |
| Manufacturer: | Agenus                                                  | Merck                                                   | generic                                                 |
| Supplier:     | Agenus                                                  | Merck                                                   | CC Pharmacy                                             |  
Version Date: *06/30/2021*  
| LEAD PI AND STUDY CHAIR Mark R. Gilbert, MD Neuro-Oncology Branch NCI/NINDS National Institutes of Health Bloch Bldg. 82, Rm. 235A 9030 Old Georgetown Road Bethesda, MD 20892 Email: mark.gilbert@nih.gov   | BTTC COORDINATING CENTER National Cancer Institute, National  Institutes of Health 9030 Old Georgetown Road,  Bloch Bldg. 82, Room 229 Bethesda, MD 20892 Phone: 240-760-6060 Fax : 301-451-5429 Email: nci_bttc@mail.nih.gov   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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bttc participating investigators/sites (enrolling)

The BTTC Participating Investigators/Sites listed in the BTTC Investigator/Site table on the Study Personnel Page will enroll study subjects as described in the protocol. Each site will obtain IRB Approval at their site.
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a disclaimer statement for bttc

This is a research protocol of the Brain Tumor Trials Collaborative describing an experimental treatment procedure. It is a privileged document and is not intended to be circulated or used for other purposes. The Brain Tumor Trials Collaborative assumes no responsibility for its use outside of the constraints of this research protocol or by investigators other than those approved by the Consortium.
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precis background

- Malignant gliomas are unfortunately, in most cases, a uniformly fatal tumor. Despite aggressive surgery, radiation treatment (RT) and chemotherapy at initial diagnosis these tumors almost always recur.  
- Many clinical trials in glioblastoma (GBM) have evaluated the addition of agent(s) to standard therapy which consists of concurrent radiation with temozolomide chemotherapy after maximal surgical resection in patients with newly diagnosed disease and salvage chemotherapy with either rechallenge with temozolomide or an alternative alkylating agent such as CCNU or cisplatin. To date, none of the combination strategies have demonstrated clinical benefit. Furthermore, in subjects with an unmethylated MGMT (O
6-methylguanine-DNA methyltransferase) promoter temozolomide has only modest benefit and salvage therapies have not demonstrated a significant impact in this subject group underscoring the need for more research.  
- Immunotherapy offers the promise of improving outcomes for patients with GBM by evoking specific immune responses that may produce a more sustained and less toxic effect than conventional therapy. Heat-shock proteins (HSPs), which function as intracellular chaperones, can be used to deliver a variety of tumor antigens to antigen presenting cells for immune stimulation.  
- Heat Shock Protein-Peptide Complex-96 (HSPPC-96) consists of the heat shock protein glycoprotein-96 (HSP gp-96) and a wide array of chaperoned proteins, including autologous antigenic peptides (aka "vaccine"). Heat shock proteins (HSP) are molecules that respond to cellular stress and counteract abnormal protein folding. They are known to modulate immune responses, especially the HSP gp-96. In a stressful environment, such as a tumor, HSPs are upregulated and highly expressed on tumor cells. This protects the tumor and leads to resistance to therapy. HSP expression is associated with cellular proliferation, apoptosis evasion, tissue invasion, metastasis, and angiogenesis.  
- Pembrolizumab is a potent and highly selective humanized monoclonal antibody
(mAb) of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Additionally, pembrolizumab is thought to also have activity in the peripherally circulating T-effector cells by reversing lymphocyte exhaustion. It is currently FDA approved for use in patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor and NSCLC with elevated PDL1 in the tumor. recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy. It is also FDA approved for use with advanced (metastatic) non-small cell lung cancer (NSCLC) whose disease has progressed after other treatments and with tumors that express a protein called PD-L1 and for the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy
- This study will be the first to evaluate this combination of vaccine (HSPPC-96) and PD1 inhibition (pembrolizumab) in newly diagnosed GBM patients whose tumors are MGMT promoter unmethylated and are isocytrate dehydrogenase (IDH) wildtype; and will provide important data on immune-modulatory effect of this combination. This may be of particular value in patients with high peripheral PD-L1 expression, but also the value of PD-1 added to standard GBM therapy. As vaccine needs to be generated from the patient's tumor, patients will need to be identified prior to surgery.
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eligibility

- MRI findings consistent with a suspected GBM or histologically confirmed newly diagnosed GBM that has not been treated and would benefit from further surgical resection.  
- Tumor must be supratentorial.  
- GBM diagnosis must be made by surgical excision (stereotactic biopsy will not be allowed unless there is plan for second surgery to remove ≥ 80 % of the tumor) and patients tumors must not have MGMT promoter methylation and must be IDH
wildtype.  
- No prior treatment with radiation or chemotherapy for their GBM.  
- Age ≥ 18 years on day of signing informed consent
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objective

- The primary endpoint is to determine whether the one-year overall survival (OS) rate is improved in newly diagnosed GBM patients whose tumors have an unmethylated MGMT promoter and are IDH wildtype treated with RT + TMZ + Pembrolizumab followed by TMZ + Pembrolizumab + HSPPC-96 vaccine or Placebo vaccine x 6 cycles (1 cycle is 9 weeks).
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design

- This will be a randomized, double blind phase II trial of surgery, RT + TMZ +
Pembrolizumab followed by TMZ + Pembrolizumab +/- HSPPC-96 in newly diagnosed GBM patients whose tumors have an unmethylated MGMT promoter and are IDH wildtype.  
- Subjects will be assigned to intervention based on ability to generate vaccine as follows:
o If ≥ 80 % of tumor removed and ≥7 g of tumor is resected but HSPPC-96 cannot be generated, subjects will be treated on the ancillary arm of RT+TMZ +Pembrolizumab followed by TMZ+ Pembrolizumab.  
o If ≥ 80% of contrasting enhanced tumor removed (based on T1 Post contrast MRI
using cross sectional measurement), ≥7 g of tumor is resected and sufficient HSPPC-96 is generated, subjects will be included in the main cohort and will be randomized on a 1:1 basis to receive:
- RT+TMZ +Pembrolizumab followed by TMZ+Pembrolizumab + Placebo OR
- RT+TMZ +Pembrolizumab followed by TMZ+Pembrolizumab+HSPPC96
- Subjects whose tumor does not meet the criteria (unmethylated MGMT promoter and IDH wildtype by pathology) and for whom < 80 % of tumor is removed or < 7g of tumor is resected are not eligible for further intervention. Approximately 8 potentially eligible  
Version Date: *06/30/2021* patients are screened per month, and it is anticipated that at least 1-2 per month will be accrued per site.  
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abbreviated title gbm pembro hsppc

| TABLE OF CONTENTS                                                              |                                                                                                                                          |
|--------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS .                                                                       |                                                                                                                                          |
| TABLE OF CONTENTS.                                                             |                                                                                                                                          |
| SCHEMA                                                                         |                                                                                                                                          |
| STATEMENT OF COMPLIANCE                                                        |                                                                                                                                          |
| 1.                                                                             | INTRODUCTION .                                                                                                                           |
| 1.1                                                                            | Study Objectives .                                                                                                                       |
| Primary Objective: .                                                           |                                                                                                                                          |
| 1.1.1                                                                          | Secondary Objectives:                                                                                                                    |
| 1.1.2                                                                          |                                                                                                                                          |
| 1.1.3                                                                          | Exploratory Objectives:                                                                                                                  |
| 1.2                                                                            | Background and Rationale .                                                                                                               |
| 1.2.1                                                                          | Disease Background .                                                                                                                     |
| 1.2.2                                                                          | Intervention Background & Overview ...................................................................................... 18             |
| 1.2.3                                                                          | Rationale for the Pembrolizumab dose ................................................................................................ 20 |
| 1.2.4                                                                          | Heat Shock Protein-Peptide Complex-96 (HSPPC-96) ................................................. 20                                    |
| Rationale for the Current Study.                                               |                                                                                                                                          |
| 1.2.5                                                                          |                                                                                                                                          |
| 1.2.6                                                                          | Rationale for the patient-reported outcomes: .                                                                                           |
| 2                                                                              | ELIGIBILITY ASSESSMENT AND ENROLLMENT                                                                                                    |
| 2.1                                                                            | Eligibility Criteria .                                                                                                                   |
| 2.1.1                                                                          | Inclusion Criteria                                                                                                                       |
| 2.1.2                                                                          | Exclusion Criteria                                                                                                                       |
| 2.1.3                                                                          | Recruitment Strategies .                                                                                                                 |
| Screening Evaluation .                                                         |                                                                                                                                          |
| 2.2                                                                            |                                                                                                                                          |
| 2.2.1                                                                          | Screening activities performed prior to obtaining informed consent ..................... 29                                              |
| 2.2.2                                                                          | Screening activities performed after a consent for screening has been signed ..... 30                                                    |
| 2.3                                                                            | Participant Registration Status Update Procedures                                                                                        |
| 2.3.1                                                                          | Informed Consent/Authorization                                                                                                           |
| 2.3.2                                                                          | Registration at the NCI                                                                                                                  |
| 2.3.3                                                                          | Registration at Participating BTTC Sites .                                                                                               |
| Initiation of Therapy (Radiation Therapy, Temozolomide and Pembrolizumab).. 32 |                                                                                                                                          |
| 2.3.4                                                                          |                                                                                                                                          |
| 2.3.5                                                                          | Registration Step 3: Randomization for NCI .                                                                                             |
| 2.3.6                                                                          | Registration Step 3: Randomization for Participating Sites........................................... 32                                 |  
6  
| Version Date: 06/30/2021 2.3.7 Study Status Updates for Participating Sites 33 2.3.8 Screen Failures 33 2.4 Treatment Assignment and Randomization /Stratification Procedures 33 3 STUDY IMPLEMENTATION 34 3.1 Early Termmination 34 3.2 Study Design 35 3.2.1 Blinding 35 3.3 Surgery & Tissue Collection for Vaccine Administration 36 3.3.1 Surgery 36 3.3.2 Tissue Collection for Vaccine Production at NCI 37 3.3.3 Tissue Collection for Vaccine Production at Participating BTTC Sites 37 3.4 Radiation Therapy (RT) 37 3.5 Drug Administration 37 3.5.1 (RT+TMZ+Pembro) 37 3.5.2 HSPPC-96 Vaccine or Placebo 38 3.6 Post RT Treatment Regimen 38 3.6.1 For patients not randomized (ancillary arm 2) 38 3.6.2 For patients randomized to vaccine or placebo 38 3.7 Baseline Assessments (Post-Surgery And Pre-Treatment RT+TMZ+Pembro) 39 3.8 Assessments While On Trial During And Post RT 39 3.8.1 Questionnaires 40 3.9 Telehealth/Remote Visits 41 3.10 Dose Modifications 42 3.10.1 Pembrolizumab Dose Modifications 42 3.10.2 Guidelines for Drug-Related Adverse Events due to Pembrolizumab 49 3.10.3 Pembrolizumab Supportive Care 49 3.10.4 Temozolomide Dose Modifications 51 3.10.5 HSPPC-96/Placebo Dose Modifications 52 3.10.6 Vaccine Injection Site Reactions 52 3.11 Study Calendar 53 3.12 Surgical Guidelines 56 3.13 Cost and Compensation 56   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
7 Abbreviated Title: GBM Pembro HSPPC  
| Version Date:   06/30/2021                                                                                 |                                                                                                  |                                                |
|------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------|------------------------------------------------|
| COSTS                                                                                                      |                                                                                                  |                                                |
| 3.13.1                                                                                                     |                                                                                                  |                                                |
| 3.13.2                                                                                                     | COMPENSATION                                                                                     |                                                |
| 3.13.3                                                                                                     | Reimbursement                                                                                    |                                                |
| 3.14                                                                                                       | Criteria for Removal from Protocol Therapy and Off Study Criteria............................ 56 |                                                |
| 3.14.1                                                                                                     | Criteria for Removal from Protocol Therapy .                                                     |                                                |
| 3.14.2                                                                                                     | OFF Study Criteria                                                                               |                                                |
| 3.14.3                                                                                                     | Lost to Follow-up                                                                                |                                                |
| 4                                                                                                          | CONCOMITANT MEDICATIONS/VACCINATIONS (ALLOWED & PROHIBITED).. 57                                 |                                                |
| 4.1                                                                                                        | Acceptable Concomitant Medications .                                                             |                                                |
| 4.2                                                                                                        | Prohibited Concomitant Medications .                                                             |                                                |
| 4.3                                                                                                        | Diet/Activity/Other Considerations .                                                             |                                                |
| 4.3.1                                                                                                      | D DIET .                                                                                         |                                                |
| 4.3.2                                                                                                      | Contraception                                                                                    |                                                |
| S                                                                                                          | BIOSPECIMEN COLLECTION .                                                                         |                                                |
| Correlative Studies for Research/Pharmacokinetic Studies............................................... 60 |                                                                                                  |                                                |
| 5.1                                                                                                        | 5.1.1                                                                                            | Collection of correlative blood time points: . |
| 5.1.2                                                                                                      | PD-L1 expression on tumor cells                                                                  |                                                |
| 5.1.3                                                                                                      | Tissue will be interrogated by Next Generation Sequencing for tumor antigens .. 61               |                                                |
| Tissue Collection at NCI .                                                                                 |                                                                                                  |                                                |
| 5.1.4                                                                                                      |                                                                                                  |                                                |
| 5.1.5                                                                                                      | Sample Storage, Tracking and Disposition .                                                       |                                                |
| 5.2                                                                                                        | Samples for Genetic/Genomic Analysis .                                                           |                                                |
| 5.2.1                                                                                                      | Scope of the analysis                                                                            |                                                |
| 5.2.2                                                                                                      | Certificate of Confidentiality .                                                                 |                                                |
| 5.2.3                                                                                                      | Incidental Findings                                                                              |                                                |
| 6                                                                                                          | DATA COLLECTION AND EVALUATION .                                                                 |                                                |
| DATA COLLECTION                                                                                            |                                                                                                  |                                                |
| 6.1                                                                                                        | Exceptions to Data Collection                                                                    |                                                |
| 6.1.1                                                                                                      |                                                                                                  |                                                |
| 6.1.2                                                                                                      | Source Documentation Timeframes                                                                  |                                                |
| 6.1.3                                                                                                      | Database Entry Timeframes                                                                        |                                                |
| Confidentiality                                                                                            |                                                                                                  |                                                |
| 6.1.4                                                                                                      |                                                                                                  |                                                |
| 6.1.5                                                                                                      | Safety Data                                                                                      |                                                |
| 6.1.6                                                                                                      | DATA Monitoring                                                                                  |                                                |  
8 Abbreviated Title: GBM Pembro HSPPC  
| Version Date:   06/30/2021           |                                                                                                                                    |
|--------------------------------------|------------------------------------------------------------------------------------------------------------------------------------|
| DATA SHARING Plans                   |                                                                                                                                    |
| 6.2                                  |                                                                                                                                    |
| 6.2.1                                | Human Data Sharing Plan                                                                                                            |
| 6.2.2                                | Genomic Data Sharing Plan                                                                                                          |
| 6.3                                  | Response Criteria                                                                                                                  |
| Definitions                          |                                                                                                                                    |
| 6.3.1                                |                                                                                                                                    |
| 6.3.2                                | Disease Parameters                                                                                                                 |
| 6.3.3                                | RANO Criteria for Response .                                                                                                       |
| iRANO (Exploratory)[45]              |                                                                                                                                    |
| 6.3.4                                |                                                                                                                                    |
| 6.3.5                                | Evaluation of Best Overall Response ........................................................................................... 70 |
| 6.3.6                                | Dose Limiting Toxicity .                                                                                                           |
| 6.3.7                                | Progression-Free Survival                                                                                                          |
| 6.3.8                                | OVERALL SURVIVAL .                                                                                                                 |
| 6.3.9                                | Response Review                                                                                                                    |
| 6.4                                  | Toxicity Criteria                                                                                                                  |
| 7                                    | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN .... 71                                                                 |
| Definitions                          |                                                                                                                                    |
| 7.1                                  |                                                                                                                                    |
| 7.2                                  | OHSRP Office of Compliance and Training / IRB Reporting ....................................... 71                                 |
| 7.2.1                                | Expedited Reporting                                                                                                                |
| 7.2.2                                | IRB Requirements for NCI PI Reporting at Continuing Review .............................. 71                                       |
| NCI Clinical Director Reporting.     |                                                                                                                                    |
| 7.3                                  |                                                                                                                                    |
| 7.4                                  | NCI Guidance For Reporting Expedited Adverse Events For Multi-Center Trials.... 72                                                 |
| 7.4.1                                | Reporting Deviations and Unanticipated Problems for Multi-Center Trials ........ 72                                                |
| 7.5                                  | NIH Required Data and Safety Monitoring Plan .......................................................................... 73         |
| 7.5.1                                | Data Safety Monitoring Board (DSMB)                                                                                                |
| 7.5.2                                | Patient-Reported Outcomes                                                                                                          |
| Exploratory Objectives & Endpoints . |                                                                                                                                    |
| 7.6                                  |                                                                                                                                    |
| 8                                    | SPONSOR PROTOCOL/SAFETY REPORTING                                                                                                  |
| 8.1                                  | Definitions                                                                                                                        |
| A Adverse Event                      |                                                                                                                                    |
| 8.1.1                                | Serious Adverse Event (SAE)                                                                                                        |
| 8.1.2                                |                                                                                                                                    |
| 8.1.3                                | Life-threatening .                                                                                                                 |
| 8.1.4                                | Severity                                                                                                                           |  
9  
| Version Date: 06/30/2021 8.1.6 Events Of Clinical Interest (ECI) For This Trial Include: 77 8.1.7 Definition of an Overdose for This Protocol and Reporting of Overdose to the  Sponsor 77 8.2 Assessment of Safety Events 77 8.3 Reporting of Serious Adverse Events 78 8.4 Safety Reporting Criteria to The Pharmaceutical Collaborators 78 8.4.1 To be Sent by CCR Safety 78 8.4.2 To be Sent by BTTC study team 78 8.4.3 Events of Clinical Interest 80 8.4.4 Reporting Serious Adverse Events to Merck and Agenus 80 8.5 Reporting Pregnancy 81 8.5.1 Maternal exposure 81 8.5.2 Paternal exposure 81 8.6 Regulatory Reporting for Studies Conducted Under CCR Sponsored IND 82 9 CLINICAL MONITORING 82 10 STATISTICAL CONSIDERATIONS 82 10.1 Sample Size and Accrual 83 10.2 Data Analyses Plans 84 10.3 Secondary Objectives & Endpoints 84 11 COLLABORATIVE AGREEMENTS 84 11.1 Agreement Type 84 11.2 Multi-Institutional Guidelines 85 11.2.1 Participating Institution Site Initiation 85 11.2.2 IRB Approvals 85 12 HUMAN SUBJECTS PROTECTIONS 85 12.1 Rationale For Subject Selection 85 12.2 Participation of Children 85 12.3 Participation of Subjects Unable to Give Consent (NCI) 86 12.4 Participation of Subjects Unable to Give Consent (BTTC) 86 12.5 Evaluation of Benefits and Risks/Discomforts 86 12.5.1 Risks 86 12.5.2 Benefits 87   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
10 Abbreviated Title: GBM Pembro HSPPC  
| Version Date: 06/30/2021                                                                                                                                            |                                                                                                                                                       |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------|
| Consent Process and Documentation.                                                                                                                                  |                                                                                                                                                       |
| 12.6                                                                                                                                                                | Consent Process for Adults Who Lack Capacity to Consent to Research                                                                                   |
| 12.6.1                                                                                                                                                              | Participation                                                                                                                                         |
| 12.6.2                                                                                                                                                              | Request for Waiver of Consent for Screening Activities ............................................ 88                                                |
| 12.6.3                                                                                                                                                              | Reconsent via phone at BTTC Sites (outside of NCI) ................................................... 89                                             |
| 13                                                                                                                                                                  | REGULATORY AND OPERATIONAL CONSIDERATIONS.                                                                                                            |
| 13.1                                                                                                                                                                | Study Discontinuation and Closure ................................................................................................................ 89 |
| 13.2                                                                                                                                                                | Quality Assurance and Quality Control ................................................................................................. 89            |
| 13.3                                                                                                                                                                | Conflict of Interest Policy                                                                                                                           |
| 13.4                                                                                                                                                                | Confidentiality and Privacy                                                                                                                           |
| 14   PHARMACEUTICAL INFORMATION ................................................................................................................................ 91 |                                                                                                                                                       |
| 14.1                                                                                                                                                                | Pembrolizumab/Keytruda ® ................................................................................................................ 91          |
| 14.1.1                                                                                                                                                              | C Construction Construction Construction Processes                                                                                                    |
| 14.1.2                                                                                                                                                              | CAS Name and Number .......................................................................................................................... 91     |
| 14.1.3                                                                                                                                                              | Molecular Formula: C 6534H 10004N 1716 O 2036S 46 (peptide) ....................................................... 91                                |
| 14.1.4                                                                                                                                                              | Molecular Weight: 149 kDa. .............................................................................................................. 91          |
| 14.1.5                                                                                                                                                              | A Appearance                                                                                                                                          |
| Storage and Stability .................................................................................................................................. 91         |                                                                                                                                                       |
| 14.1.6                                                                                                                                                              |                                                                                                                                                       |
| 14.1.7                                                                                                                                                              | SUPPLIER .                                                                                                                                            |
| 14.1.8                                                                                                                                                              | Mechanism of Action                                                                                                                                   |
| 14.1.9                                                                                                                                                              | Administration for this study                                                                                                                         |
| 14.1.10 Preparation                                                                                                                                                 |                                                                                                                                                       |
| 14.1.11 Preparation for Intravenous Infusion                                                                                                                        |                                                                                                                                                       |
| 14.1.12 Incompatibilities                                                                                                                                           |                                                                                                                                                       |
| 14.1.13 Side Effects .                                                                                                                                              |                                                                                                                                                       |
| 14.2                                                                                                                                                                | TEMOZOLOMIDE .                                                                                                                                        |
| 14.2.1                                                                                                                                                              | Chemical Name                                                                                                                                         |
| 14.2.2                                                                                                                                                              | Molecular Formula: C6H6N6O2 .......................................................................................................... 95             |
| Molecular Weight: 194.15 .                                                                                                                                          |                                                                                                                                                       |
| 14.2.3                                                                                                                                                              |                                                                                                                                                       |
| 14.2.4                                                                                                                                                              | A Appearance .                                                                                                                                        |
| 14.2.5                                                                                                                                                              | Supply: Commercially Available. ...................................................................................................... 96             |
| 14.2.6                                                                                                                                                              | Pill Diary .                                                                                                                                          |  
11  
| 12                                  |                                                                                                                                              |
|-------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------|
| Abbreviated Title: GBM Pembro HSPPC |                                                                                                                                              |
| Version Date: 06/30/2021            |                                                                                                                                              |
| 14.2.7                              | Storage and Stability  .                                                                                                                     |
| Metabolism and Elimination .        |                                                                                                                                              |
| 14.2.8                              |                                                                                                                                              |
| 14.2.9                              | Mechanism of Action                                                                                                                          |
| 14.2.10 Special Populations         |                                                                                                                                              |
| Drug-Drug Interactions .            |                                                                                                                                              |
| 14.2.11                             |                                                                                                                                              |
| 14.2.12                             | A Adverse Events:                                                                                                                            |
| 14.2.13                             | Special Handling and Precautions ...................................................................................................... 97   |
| 14.2.14 Contraindications           |                                                                                                                                              |
| 14.2.15 Pregnancy Category D.       |                                                                                                                                              |
| 14.2.16 Fertility                   |                                                                                                                                              |
| 14.3                                | Heat Shock Protein-Peptide Complex-96 Vaccine (HSPPC-96)                                                                                     |
| 14.3.1                              | Molecular Formula                                                                                                                            |
| 14.3.2                              | Peptide Component                                                                                                                            |
| 14.3.3                              | Appearance .                                                                                                                                 |
| 14.3.4                              | Availability .                                                                                                                               |
| 14.3.5                              | HSPPC-96 Vaccine Label .................................................................................................................. 99 |
| 14.3.6                              | Storage and Stability  .                                                                                                                     |
| 14.3.7                              | Preparation                                                                                                                                  |
| 14.3.8                              | Administration                                                                                                                               |
| 14.3.9                              | TOXICITIES .                                                                                                                                 |
| 14.4                                | Placebo                                                                                                                                      |
| 14.4.1                              | Supply                                                                                                                                       |
| Product Description                 |                                                                                                                                              |
| 14.4.2                              |                                                                                                                                              |
| 14.4.3                              | Placebo Label                                                                                                                                |
| 14.4.4                              | S Storage                                                                                                                                    |
| 14.4.5                              | Administration .                                                                                                                             |
| DOSE                                |                                                                                                                                              |
| 14.4.6                              |                                                                                                                                              |
| 14.5                                | Ordering Study Agent                                                                                                                         |
| 14.5.1                              | Heat Shock Protein-Peptide Complex-96                                                                                                        |
| 14.5.2                              | Pembrolizumab                                                                                                                                |
| 14.5.3                              | Temozolomide                                                                                                                                 |
| 14.5.4                              | Agent Storage and Accountability: ..................................................................................... 102                  |  
| 13                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Abbreviated Title: GBM Pembro HSPPC Version Date: 06/30/2021 14.5.5 BTTC Procedures for Agent Accountability and Storage 102 14.5.6 Verification of Compliance 103 14.6 Returning or destroying unused and/or defective agent: 103 14.6.1 Pembrolizumab Return Procedure 103 14.6.2 HSPPC-96 or Placebo Return Procedure 104 15 REFERENCES 105 16 APPENDICES 108 16.1 Performance Status Criteria 108 16.2 Tumor Tissue Submission for Vaccine Manufacture 109 16.3 Vaccine Tissue Procurement And Shipping Instructions 110 16.4 Tumor Procurement Form 115 16.5 LabConnect Manual for Whole Blood PBMC Processing and Shipping 116 16.6 HSPPC-96 Vaccine or Placebo Return Instructions 117 16.7 HSPPC-96 Intradermal Injection Instructions 118 16.8 Infusion Reaction Treatment Guidelines 121 16.9 MD Anderson Symptom Inventory (MDASI-BT) 123 16.10 CCR Reportable Event Form (REF) 124 16.11 Unblinding Request Memo 127 16.12 Unblinding Notification Memo 129 16.13 Study Phases and Correlative Blood Draws 130 16.14 Pill Diary 131 16.15 Agreements 133 16.16 Investigator Agreement 134 |
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schema

MRI findings consistent with a suspected GBM or a histologically confirmed newly diagnosed GBM that has not been treated and would benefit from further surgical resection  
  
Patient comes off study if tumor type and both resection criteria  
  
  
Once off treatment, continue follow-up every 8-16 weeks.  
CR/PR/SD: continue treatment until PD or 2 years.  
CR: complete response; PD: Progressive Disease; GBM: glioblastoma multiforme; PR: partial response; RT: radiation therapy; SD: stable disease; TMZ: temozolomide.  
15
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statement of compliance

The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical Practice (ICH GCP) and the following:
- United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible for the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects Protection and ICH GCP Training. The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both the protocol and the consent form must be obtained before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before the changes are implemented to the study. In addition, all changes to the consent form will be IRB-approved; an IRB determination will be made regarding whether a new consent needs to be obtained from participants who provided consent, using a previously approved consent form.
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introduction 1.1 study objectives

1.1.1 **Primary Objective:**
o To determine whether the one-year overall survival rate (OS) is improved in newly diagnosed GBM patients whose tumors have an unmethylated MGMT promoter and are IDH wildtype treated with RT + Temozolomide (TMZ) + Pembrolizumab followed by Pembrolizumab + TMZ and either HSPPC-96 x 6 cycles (1 cycle is 9 weeks) or placebo vaccine.  
Nota *Bene*: Those patients whose tumor does not meet the criteria (unmethylated MGMT promoter and IDH wildtype by pathology) and with < 80% of contrast enhanced tumor removed or < 7g of tumor resected are ineligible.
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1.1.2 secondary objectives

o To determine the following o Progression Free Survival-6 **(PFS-6)** (percentage of patients alive and progressionfree at 6 months post registration).  
o Overall Survival **(OS).** o Overall Survival (**OS) OS-6** and 24 (percentage of patients alive at 6 and 24 months respectively).  
o Response will be determined by **Table 2** below using RANO as main criteria. If a lesion is 25% larger but there remains a question as to disease progression or pseudoprogression due to treatment, the patient can remain on treatment until this can be defined, if true progression occurred earlier, the earlier date will be used.  
o Safety of Pembrolizumab +/- HSPPC-96 in GBM.  
o To evaluate the occurrence of symptoms and correlate to disease progression and tolerance to treatment using the MD Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT). This will include:
- To longitudinally evaluate patient reported outcome measures using self-reported symptom severity and interference with daily activities using the MDASI-BT questionnaire.  
- To measure symptom burden over the course of therapy to evaluate differences between patients individual symptom severity, overall mean symptom severity, and difference in scores on the interference items between responders and nonresponders.  
- To describe the variability of symptom severity longitudinally over the treatment course and follow-up period.
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1.1.3 exploratory objectives

o To determine if there is a correlation with clinical outcome of PDL1 expression and TCell infiltrate in pathology from first, and if applicable, second surgical specimens with outcome.  
o To assess tumor tissue for neo-antigens. o To collect peripheral monocytes for PD-L1 levels to confirm that low level expression leads to better outcome and if higher levels that are treated with Pembrolizumab might improve outcome.  
o Assess changes in peripheral T-Cell activation and tryptophan metabolites. o Evaluate response using iRANO criteria.  
o Agenus will isolate additional heat shock protein (HSP)-peptide complexes from
"waste streams" generated during the manufacture of HSPPC-96 (Prophage) from GBM tumor tissue. From these HSP-peptide complexes, Agenus may generate a novel "multi-chaperone" vaccine that Agenus will evaluate in preclinical studies.
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1.2 background and rationale 1.2.1

Malignant gliomas, particularly GBM are nearly always fatal. Despite aggressive surgery, radiation treatment (RT) and chemotherapy at initial diagnosis these tumors almost always recur. There have been many studies in patients with newly diagnosed GBM, either altering the dosing schedule of the temozolomide (RTOG 0525) [1] or adding anti-angiogenic agents to the standard treatment regimen. None have proven to prolong survival and the latter treatments targeting neovascularization were particularly disappointing given that angiogenesis is a cardinal feature of GBM. [2] [3]
Many clinical trials in GBM have evaluated the addition of agent(s) to standard therapy which consists of concurrent radiation with temozolomide chemotherapy after maximal surgical resection in patients with newly diagnosed disease and salvage chemotherapy with either rechallenge with temozolomide or an alternative alkylating agent such as CCNU or cisplatin. To date, none of the combination strategies have demonstrated clinical benefit. Furthermore, in subjects with an unmethylated MGMT (O
6-methylguanine-DNA methyltransferase)
promoter temozolomide has only modest benefit and salvage therapies have not demonstrated a significant impact in this subject group underscoring the need for more research. Novel approaches being evaluated in GBM treatment include immunologic therapies, such as therapeutic vaccines that are either derived from the subject's own tumor (HSPPC-96) or have been designed to target specific epitopes on tumor cells (CDX-110, ICT-107). Preliminary results were promising from Phase 2 trials of CDX-110 (rindopeptimet), ICT 107, HSPPC-96, and others. [4-6]. However, a randomized Phase 3 trial evaluating the CDX-110 did not demonstrate efficacy [7]. There are several agents currently under investigation that target the local immune response in solid tumors, particularly in melanoma, such as ipilimumab (blockade of cytotoxic T
lymphocyte–associated protein 4 [CTLA-4]) and programmed cell death protein 1 (PD-1; PD-L1 blockade). Such agents are either FDA approved or being evaluated in various solid tumors with the most activity seen in [8]. There are many immunologic approaches to treatment of GBM. Gliomas can upregulate B7-H1 expression in circulating monocytes and tumor-infiltrative macrophages through modulation of autocrine/paracrine interleukin 10 (IL-10) signaling, resulting in an immunosuppressive phenotype.[9] Glioma-associated cancer-initiating cells have been shown to express the costimulatory inhibitory molecule B7-H1, [10] which is a key factor in mediating immune resistance in glioma[11] and can induce T-cell apoptosis. [12]. Direct cell-to-cell contact experiments have shown that the glioma-associated cancer-initiating cells induced T-cell apoptosis in the setting of B7-H1 expression. [10] found that cancerinitiating cells markedly inhibited T-cell proliferation and activation, induced regulatory T
cells, and triggered T-cell apoptosis that was mediated by B7-H1 and soluble galectin-3. Loss of tumor suppressor phosphotase and tensin homolog (PTEN) function increases B7-H1 expression and immunoresistance in glioma. [11] It also been shown that glioma-associated cancer-initiating cells induce immunosuppression, thereby allowing tumor cells to evade immunosurveillance. Yao et al. [13] found that B7-H1 was correlated with the malignancy grade of human astrocytic tumors, but preferentially in the non-dividing tumor cells. It was also upregulated at the growing edge of the tumors and had a negative correlation with tumor-infiltrating CD8 T cells. Tumor-infiltrating lymphocytes and PD-L1 expression are seen in the majority of GBM samples. [14, 15] Data show that lymphopenia induced by temozolomide (TMZ) can actually potentiate cancer immunotherapy, which may be due in part to cell death, which releases cellular contents that increase the immunoreactivity of the tumor environment. [16]
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1.2.2 intervention background & overview

The importance of intact immune surveillance in controlling outgrowth of neoplastic transformation has been known for decades.[17] Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in various malignancies. [12] [18] [19, 20] [21]. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to correlate with improved prognosis and long-term survival in many solid tumors.  
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T-cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member related to CD28 and CTLA-4 which has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2). [22, 23] The structure of murine PD-1 has been resolved. [24] PD-1 and family members are type I transmembrane glycoproteins containing an Ig Variable-type (V-type) domain responsible for ligand binding and a cytoplasmic tail which is responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3ζ, PKCθ and ZAP70 which are involved in the CD3 T-cell signaling cascade [23, 25-27]. The mechanism by which PD-1 down modulates T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an overlapping set of signaling proteins[25] [26]. PD-1 was shown to be expressed on activated lymphocytes including peripheral CD4+ and CD8+ Tcells, B-cells, T regs and Natural Killer cells. [28] [29] Expression has also been shown during thymic development on CD4-CD8- (double negative) T-cells as well as subsets of macrophages and dendritic cells. [30] The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic tissues as well as in various tumors. [30] [31] [32, 33] Both ligands are type I transmembrane receptors containing both IgV- and IgC-like domains in the extracellular region and contain short cytoplasmic regions with no known signaling motifs. Binding of either PD-1 ligand to PD-1 inhibits T-cell activation triggered through the T-cell receptor. PD-L1 is expressed at low levels on various non- hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed on antigen-presenting cells found in lymphoid tissue or chronic inflammatory environments. PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues. [34] Although healthy organs express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant levels of this T-cell inhibitor. PD-1 has been suggested to regulate tumorspecific T-cell expansion in subjects with melanoma. [35] This suggests that the PD- 1/PD-L1 pathway plays a critical role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention. Pembrolizumab (previously known as SCH 900475) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. It is currently FDA approved for use in patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. It is also FDA approved for advanced (metastatic) non-small cell lung cancer (NSCLC) whose disease has progressed after other treatments and with tumors that express a protein called PD-L1 and for the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy.
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1.2.3 rationale for the pembrolizumab

Selection of 200 mg as the appropriate dose for a switch to fixed dosing is based on simulation results indicating that 200 mg will provide exposures that are reasonably consistent with those obtained with 2 mg/kg dose and importantly will maintain individual patient exposures within the exposure range established in melanoma as associated with maximal clinical response. A population PK model, which characterized the influence of bodyweight and other patient covariates on exposure, has been developed using available data from 476 subjects from PN001. The distribution of exposures from the 200 mg fixed dose are predicted to considerably overlap those obtained with the 2 mg/kg dose, with some tendency for individual values to range slightly higher with the 200 mg fixed dose. The slight increase in PK variability predicted for the fixed dose relative to weight-based dosing is not expected to be clinically important given that the range of individual exposures is well contained within the range of exposures shown in the melanoma studies of 2 and 10 mg/kg to provide similar efficacy and safety. The population PK evaluation revealed that there was no significant impact of tumor burden on exposure. In addition, exposure was similar between the NSCLC
and melanoma indications. Therefore, there are no anticipated changes in exposure between different tumor types and indication settings. Available pharmacokinetic results in subjects with melanoma, NSCLC, and other solid tumor types support a lack of meaningful difference in pharmacokinetic exposures obtained at a given dose among tumor types. Population PK analysis has been performed and has confirmed the expectation that intrinsic factors do not affect exposure to Pembrolizumab to a clinically meaningful extent. Taken together, these data support the use of lower doses (with similar exposure to 2 mg/kg Q3W) in all solid tumor indications. 2 mg/kg Q3W is being evaluated in NSCLC in PN001, Cohort F30 and PN010, and 200 mg Q3W is being evaluated in head and neck cancer in PN012, which are expected to provide additional data supporting the dose selection. This trial will use Pembrolizumab 200 mg every three weeks to fit with treatment schedules currently used.
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1.2.4 heat shock protein-peptide complex-96

Immunotherapy offers the promise of improving outcomes for patients with GBM by evoking specific immune responses that may produce a more sustained and less toxic effect than conventional therapy. Heat-shock proteins (HSPs), which function as intracellular chaperones, can be used to deliver a variety of tumor antigens to antigen presenting cells for immune stimulation. Heat Shock Protein-Peptide Complex-96 (HSPPC-96) consists of the heat shock protein glycoprotein-96 (HSP gp-96) and a wide array of chaperoned proteins, including autologous antigenic peptides. Heat shock proteins (HSP) are molecules that respond to cellular stress and counteract abnormal protein folding. They are known to modulate immune responses, especially the HSP gp-96. In a stressful environment, such as a tumor, HSPs are upregulated and highly expressed on tumor cells. This protects the tumor and leads to resistance to therapy. HSP expression is associated with cellular proliferation, apoptosis evasion, tissue invasion, metastasis, and angiogenesis. HSPPC-96 immunization works mechanistically by interacting with antigen presenting cells (APCs) via specific receptors, including CD91. [36, 37] highly specific nature of the interaction between HSPPC-96 and APCs is a significant advantage over other cancer vaccine approaches; and has been shown to facilitate robust CD4+ and CD8+ T-cell immune responses. There are multiple mechanisms by which the vaccine works. The first is the following: The autologous antigens in the vaccine are bound to the chaperone proteins (HSP) with noncovalent bonds. Macrophages and other transfer the antigens from HSP to antigen presenting cells (e.g., DCs) bind and take up the HSP-antigen complexes, via receptor- mediated endocytosis (with receptors such as CD91). The antigens are then expressed via MHC class I and II on antigen presenting cells (APCs), interacting with naïve T cells, which are activated into specific CD8 T and CD4 T
cells. Another mechanism of the vaccine is an innate immunity pathway cells. HSPPC-96 Ag is internalized into the APC, which activates the APC. This leads to the following:
o Activated APCs express co-stimulatory molecules (CD80, CD86, and CD40) and express MHC class II, which interacts with T cells and produces tumor specific CD4 cells.  
o Activated APCs cause cytokine release (IL-1b, IL-12, IL-6, TNF-alph, GMCSF, chemokines, and nitric oxide). IL-12 stimulates NK cells, which enhance the antitumor activity of T cells.  
o The interaction between HSP-antigen and APCs causes dendritic cell maturation, which stimulates NK cells.  
A final mechanism of the vaccine is that exposure to Gp96 leads to translocation of NFkB into the APC nucleus, which then regulates cancer cell growth, progression, and apoptosis. The advantage of the HSPPC-96 vaccine over others is the following:
o Multifactorial mechanism: as described above, the vaccine works by multiple pathways to exert its effect, rather than 1 pathway.  
o The vaccine is individualized and does not require identification of immunogenic antigens required by other vaccines.  
o Multiple antigens are used, which decreases the chance of immune evasion that occurs with a single antigen vaccine.  
Clinical grade HSPPC-96 can be easily purified from solid tumor and has been safely tested in hundreds of patients with newly diagnosed and recurrent melanoma and renal cell cancer. The toxicity encountered in renal cell carcinoma in prior trials is the following: injection site erythema (50%), injection site induration (48%), back pain (12%), headache (12%), fatigue (10%), nasopharyngitis (9%), incision site complication (9%), arthralgia (9%), hypertension (9%), urinary tract infection (8%), nausea (7%), diarrhea (6%), asthenia (6%), influenza (6%), constipation (5%), pyrexia (5%), and dizziness (5%). In melanoma, toxicity seen was pyrexia (8%), fatigue (6%), and nausea (5%). Between the trials there were 3 serious adverse events, 2 were thyroid dysfunction, and 1 was cellulitis. [38] [39] [11] The rate of successful HSPPC-96 vaccine manufacture is anticipated to be 80% or greater based on prior clinical trials. A prior phase 2 study of new diagnosed glioblastoma, vaccine was successfully manufactured in 84% (63/77) of attempted cases. The majority of manufacture failures were caused by insufficient number of vials available, below the required 4, for clinical use (9 cases). A single center single arm Phase I clinical trial and a multicenter single arm Phase II clinical trial for recurrent GBM patients treated with HSPPC-96 have been completed. Collectively these studies have shown that: 1) HSPPC-96 vaccine made from recurrent GBM tumor is safe and well tolerated, 2) HSPPC-96 vaccine extends survival in recurrent GBM patients over historical controls and 3) HSPPC-96 is immunogenic in recurrent GBM patients, inducing both an immunologic response locally and peripherally and via innate and adaptive immune mechanisms
[39] and confers clinical efficacy in patients with newly diagnosed and recurrent GBM.  
Importantly, in patients with newly diagnosed GBM, HSPPC-96 vaccine in combination with standard of care temozolomide extended response. Specifically, in the phase I trial, testing was performed pre- and post-vaccine on all patients. Eleven of 12 patients were shown to have a significant immune response, which was measured by testing peripheral blood lymphocytes (PBLs). PBLs were tested for phenotype, reactivity to vaccine, IFN gamma production, and proliferation. Vaccine was well tolerated with no serious adverse events and the only toxicity noted was injection site erythema or induration. The median progression survival in responders was 47 weeks, compared to 16 weeks in the non-responder. In the phase II trial, 33 patients were treated. Again, there were no serious adverse events attributed to vaccine. Toxicities reported were injection site reaction in 12 patients, and fatigue in 9 patients. Eleven patients were still alive at time of analysis, with median survival of 333 days for the evaluable population. Also, all patients studied showed immune response, based on CD8 IFN gamma production [9]. In a separate single arm phase II trial of newly diagnosed GBM patients who were treated with RT + TMZ + HSPPC-96, median PFS and overall survival were two to three times longer than expected based on historical controls (median PFS was 18 v 6-9 mos and median OS was 24 v 14-16 mos) of 24 months. Importantly, patients with low PDL1 expression on peripheral monocytes had longer survival than those with high PD-L1 expression, suggesting that an optimal group for HSPPC-96 use with a checkpoint inhibitor would be in the high PD-L1 expressing patients. Importantly, in data presented at ASCO 2015, in patients with less elevated PD-L1 expression on peripheral monocytes, treatment with HSPPC-96 was associated with an unexpected and impressively longer survival than those with high more elevated PD-L1 on circulating monocytes (mPFS of 27 v 6-9 mos and mOS 47 v 14-16; [38] ).
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1.2.5 rationale for the current

The preclinical data about the immunologic aspects of gliomas and the data using vaccines, suggest that there is a strong rationale to evaluate these agents in GBM. While the blood brain barrier (BBB) is often an issue for drug penetration, this would be less of an issue for checkpoint inhibitors as the effect is on trafficking T-Cells which can migrate peripherally to the brain. [15] Nonetheless, in the setting of contrast enhancement, the BBB is leaky, allowing drug penetration. Taken together, these data support the rationale that the combination of an autologous cancer vaccine and an anti-PD-1 inhibitor may synergistically result in an increase in tumor specific immune response with downregulation of tumor immunosuppression resulting in important clinical responses. There is pre-clinical and some clinical precedence for using an immune educating strategy with a release of tumoral immunosuppression. Several studies have shown a benefit of the combination over check point modulators alone or vaccine alone in several types of solid tumors. [19, 40] This study will be the first to evaluate this combination of vaccine (HSPPC-96) and PD-1 inhibition (Pembrolizumab) in newly diagnosed GBM with unmethylated MGMT promoter and will provide important data on immune-modulatory effect of this combination. This may be of particular value in patients with high peripheral PD-L1 expression, but also there also be value adding PD-1 to standard GBM therapy. We will be assessing OS-12 and other PFS and OS rates. A phase I study adding Pembrolizumab (200 mg) to RT+TMZ using standard starting doses was found to be safe and without a DLT.[41] The DLT window was from the start of RT until 4 weeks post Radiation. There were no grade 3 or 4 toxicities seen. Related grade 1 and 2 toxicities were anemia, lymphopenia, neutropenia, thrombocytopenia, fatigue, rash, and GI upset/poor PO intake. Pseudo-progression and inflammatory responses were not seen in in a phase I of RT + TMZ + Pembrolizumab. We do not believe there will be any added toxicity of adding HSPPC-96 to RT + TMZ + Pembrolizumab; while an increased inflammatory reaction might occur, these patients will all have achieved ≥ 80% resection. Tumor tissue will be analyzed to assess for MGMT, PDL-1 levels and T-cell infiltration as well as collection of peripheral markers of T-Cell activation and tryptophan metabolites. Patients who progress and will benefit from further surgery will have their tumor tissue collected and analyzed.
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1.2.6 rationale for the patient-reported

This study seeks to establish effective therapies. We hypothesize that using a combination of pembrolizumab, temozolomide and HSPPC-96 will result in improved survival. However, given the intensive nature of this regimen, it will be important to determine whether any determined survival benefit is associated with improvements in symptoms or whether a worsening of symptoms offsets the increase in survival. Precedence for measuring "non-therapeutic" endpoints exists in oncology research. For example, Gemcitabine was approved by the FDA partially as a consequence of the decrease in pain reported in pancreatic patients who were treated, not on the basis of survival improvement which was modest at best [42]. There have been efforts in neuro-oncology to evaluate secondary endpoints using validated instruments as an additional indicator of benefit.  
The M.D. Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) allows the selfreporting of symptom severity and interference with daily activities. The MDASI-BT has demonstrated reliability and validity in the adult primary brain tumor patient population. [43] This tool represents a modification of the widely used and validated M.D. Anderson Symptom Inventory, with particular attention to symptoms common in patients with brain tumors. The availability of validated instruments provides an opportunity to prospectively assess the impact of treatment, both positive and negative, on patients. The evaluation of symptom burden in this study will assist in finding the best possible treatment with the least toxicity.
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2 eligibility assessment and enrollment

The target population for this study is patients with newly diagnosed GBM with an unmethylated MGMT promoter and IDH wild type. **NOTE**: Enrollment to this protocol is closed.  
See Study Implementation (Section 3.1) regarding study termination and procedures for unblinding participants . Eligibility will be evaluated by the study team according to the following criteria: Eligibility waivers are not permitted.  
Version Date: *06/30/2021* Subjects must meet all of the inclusion and none of the exclusion criteria to be registered to the study. Study treatment may not begin until a subject is registered. Please refer to Section 2.2 for complete instructions regarding registration procedures.
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2.1.1 inclusion criteria pre-surgery (step

2.1.1.1 MRI findings consistent with a suspected GBM or a histologically confirmed newly diagnosed GBM that has not been treated and would benefit from further surgical resection. As vaccine needs to be generated from the patient's tumor, patients will need to be identified prior to definitive surgery.  
2.1.1.2 Preliminary assessment by the neurosurgeons that >80% of the tumor can be resected with an expectation that >7gm of tissue would be resected 2.1.1.3 Age ≥ 18 years on day of signing informed consent. 2.1.1.4 Karnofsky performance status ≥ 70%. 2.1.1.5 Tumor must be supratentorial only. 2.1.1.6 Stereotactic biopsy will not be allowed unless there is plans for second surgery to remove ≥ 80 % of the tumor.  
2.1.1.7 No prior treatment with radiation or chemotherapy for their GBM. 2.1.1.8 No prior treatment with carmustine wafers.
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post-surgery (step 2) inclusion

2.1.1.9 Pathology must be a GBM, MGMT promoter region determined to be unmethylated and IDH wild type, >80 % resection of contrast enhanced tumor on post operative MRI and > 7 grams of tumor resected are required otherwise patient is ineligible .  
2.1.1.10 Treatment must be initiated > 14 days and < 6 weeks from surgery. 2.1.1.11 Craniotomy site must be adequately healed and free of drainage or cellulitis, and the underlying cranioplasty must appear intact at the time of radiation. Radiation must start within 6 weeks of surgery.  
2.1.1.12 Dexamethasone dose should be < 4 mg/day or steroid equivalent prior to starting treatment. If higher doses are needed, consult with Study Chair.  
2.1.1.13 Female subjects of childbearing potential should have a negative urine or serum pregnancy within 7 days prior to receiving the first dose of study medication. If the
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version date 06/30/2021

urine test is positive or cannot be confirmed as negative, a negative serum pregnancy test will be required.  
2.1.1.14 Patients must have adequate organ and bone marrow function within 14 days prior to step 2 registration, as defined below:
2.1.1.15 Absolute neutrophil count (ANC) > 1.5 × 109/L; platelet count > 100 × 109/L; and hemoglobin (Hb) >9.0 g/dL within 7 days prior to step 2 registration. Note: The use of transfusion or other intervention to achieve Hb ≥ 9.0 g/dL is acceptable.  
2.1.1.16 Total bilirubin < 1.5 × ULN (except in patients diagnosed with Gilbert's disease) 2.1.1.17 AST (SGOT), ALT (SGPT), and alkaline phosphatase (ALP) < 2.5 × ULN 2.1.1.18 Serum creatinine < 1.5 × ULN 2.1.1.19 International normalized ratio (INR), prothrombin time (PT), or activated partial thromboplastin time (APTT) as follows: In the absence of therapeutic intent to anticoagulate the patient: INR < 1.5 or PT < 1.5×ULN or aPTT < 1.5×ULN. In the presence of therapeutic intent to anticoagulate the patient: INR or PT and aPTT within therapeutic limits (according to the medical standard in the institution) and the patient has been on a stable dose of anticoagulants for at least 2 weeks before registration.  
2.1.1.20 Females of child-bearing potential (FOCBP) and males must agree to use two adequate contraception methods (give examples, e.g. hormonal or barrier method of birth control; abstinence) prior to study entry, for the duration of study participation, and for 120 days following completion of therapy. Should a female patient become pregnant or suspect she is pregnant while participating in this study, she should inform her treating physician immediately. Male patients who father a child should notify the treating physician.  
NOTE: A FOCBP is *any woman* (regardless of sexual orientation, having undergone a tubal ligation, or remaining celibate by choice) who meets the following criteria:
o *Has not* undergone a hysterectomy or bilateral oophorectomy o *Has had* menses at any time in the preceding 12 consecutive months (and therefore has not been naturally postmenopausal for > 12 months)
2.1.1.21 Patients must have the ability to understand and the willingness to sign a written informed consent prior to registration on study.  
2.1.1.22 Diagnosis must be made by surgical excision. 2.1.1.23 Patients should not be on antibiotics for any infection but post operative antibiotics are allowed if used prophylactically but should be completed prior to starting RT.
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2.1.2 exclusion criteria pre-surgery (step

2.1.2.1 Known history of immunodeficiency (HIV). This medical entity can be exacerbated by PD-1 blockade.  
2.1.2.2 History of another malignancy in the previous 3 years, with a disease-free interval of <
3 years. Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the skin, or in situ cervical cancer that has undergone potentially curative therapy. Patients who have undergone a bone marrow or stem-cell transplant for any malignancy are excluded.  
2.1.2.3 Has an active autoimmune disease requiring systemic treatment within the past 3 months or a documented history of clinically severe autoimmune disease, or a syndrome that requires chronic systemic steroids or immunosuppressive agents except as noted above in **2.1.2.13** and **2.1.2.14** Subjects with vitiligo or resolved childhood asthma/atopy would be an exception to this rule. Subjects that require intermittent use of bronchodilators or local steroid injections will not be excluded from the study. Subjects with hypothyroidism stable on hormone replacement or Sjorgen's syndrome will not be excluded from the study.  
2.1.2.4 Has a history of interstitial lung disease, non-infectious pneumonitis or pneumonitis. 2.1.2.5 Has a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the trial, interfere with the subject's participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator. Examples include:
- Hypertension (defined as 160/95) that is not controlled on medication
- Ongoing or active infection requiring systemic treatment
- Symptomatic congestive heart failure - Unstable angina pectoris
- Cardiac arrhythmia
- Psychiatric illness/social situations or substance abuse disorders that would limit compliance with study requirements
- Any other illness or condition that the treating investigator feels would interfere with study compliance or would compromise the patient's safety or study endpoints.  
2.1.2.6 Is pregnant or breastfeeding or expecting to conceive or father children within the projected duration of the trial, starting with the pre-screening or screening visit through 120 days after the last dose of trial treatment.  
2.1.2.7 The effects of pembrolizumab and HSPPC-96 on the developing human fetus are unknown. For this reason and because checkpoint inhibitors and immunotherapeutic vaccines as well as other therapeutic agents used in this trial are known to be teratogenic, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry, and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.2.8 Has received prior therapy with an anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, or anti- Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody (including ipilimumab or any other antibody or drug specifically targeting T-cell co-stimulation or checkpoint pathways).  
2.1.2.9 On treatment for Hepatitis B or Hepatitis C or history of TB. 2.1.2.10 Patients who have a history of allergic reactions attributed to compounds of similar chemical or biologic composition to Pembrolizumab are not eligible. Known hypersensitivity to any excipients of Pembrolizumab.
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post-surgery (step 2) exclusion

2.1.2.11 Patients are ineligible if the tumor is not a GBM, MGMT promoter region determined to be unmethylated and IDH wild type, or if < 80 % resection of contrast enhanced tumor on post-operative MRI or < 7 grams of tumor is resected.  
2.1.2.12 Patients who are receiving any other investigational agents. 2.1.2.13 Known history of immunodeficiency (HIV). This medical entity can be exacerbated by PD-1 blockade.  
2.1.2.14 Any form of immunosuppressive therapy within 7 days prior to the first dose of trial treatment excluding steroids. Attempts should be made to have patient on lowest possible dose of steroids. These medical entities can be exacerbated by PD-1 blockade.  
2.1.2.15 History of another malignancy in the previous 3 years, with a disease-free interval of <
3 years. Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the skin, or in situ cervical cancer that has undergone potentially curative therapy. Patients who have undergone a bone marrow or stem-cell transplant for any malignancy are excluded.  
2.1.2.16 Has an active autoimmune disease requiring systemic treatment within the past 3 months or a documented history of clinically severe autoimmune disease, or a syndrome that requires chronic systemic steroids or immunosuppressive agents except as noted above in **2.1.2.13** and **2.1.2.14** Subjects with vitiligo or resolved childhood asthma/atopy would be an exception to this rule. Subjects that require intermittent use of bronchodilators or local steroid injections will not be excluded from the study. Subjects with hypothyroidism stable on hormone replacement or Sjorgen's syndrome will not be excluded from the study.  
2.1.2.17 Has a history of interstitial lung disease, non-infectious pneumonitis or pneumonitis. 2.1.2.18 Has an active infection requiring systemic antibiotics within 10 days of surgery. 2.1.2.19 Has a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the trial, interfere with the subject's participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator. Examples include:
- Hypertension (defined as 160/95) that is not controlled on medication
- Ongoing or active infection requiring systemic treatment - Symptomatic congestive heart failure
- Unstable angina pectoris
- Cardiac arrhythmia
- Psychiatric illness/social situations or substance abuse disorders that would limit compliance with study requirements
- Any other illness or condition that the treating investigator feels would interfere with study compliance or would compromise the patient's safety or study endpoints.  
2.1.2.20 Is pregnant or breastfeeding or expecting to conceive or father children within the projected duration of the trial, starting with the pre-screening or screening visit through 120 days after the last dose of trial treatment.  
2.1.2.21 The effects of pembrolizumab and HSPPC-96 on the developing human fetus are unknown. For this reason and because checkpoint inhibitors and immunotherapeutic vaccines as well as other therapeutic agents used in this trial are known to be teratogenic, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry, and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.2.22 Has received prior therapy with an anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, or anti- Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody (including ipilimumab or any other antibody or drug specifically targeting T-cell co-stimulation or checkpoint pathways).  
2.1.2.23 On treatment for Hepatitis B or Hepatitis C or history of TB. 2.1.2.24 Has received a live vaccine within 30 days prior to the first dose of trial treatment (see Section 4.2)
2.1.2.25 Patients who have a history of allergic reactions attributed to compounds of similar chemical or biologic composition to Pembrolizumab are not eligible. Known hypersensitivity to any excipients of Pembrolizumab.
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2.1.3 recruitment strategies

This protocol may be abstracted into a plain language announcement posted on NIH websites and on NIH social media platforms. Patients will be referred by BTTC site PIs. An NIH/IRB approved patient flyer is used to recruit patients.
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2.2 screening evaluation 2.2.1 screening

Minimal risk activities that may be performed before the subject has signed a consent include the following:
- Email, written, in person or telephone communications with prospective subjects - Review of existing medical records to include H&P, laboratory studies, etc. - Review of existing MRI, x-ray, or CT images - Review of existing photographs or videos
- Review of existing pathology specimens/reports from a specimen obtained for diagnostic purposes A waiver of consent for these activities has been requested in section 12.6.2.
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2.2.2 screening activities performed after

The following activities will be performed only after the subject has signed the consent for this study for screening. Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a patient has signed the consent. The screening evaluation elements must be completed within (+/-) 14 days of signing consent unless otherwise noted: Outside lab/procedure results for standard of care studies or another protocol that are performed within this timeframe may be used to determine eligibility.  
- Medical History
- Prior and Concomitant medication review
- Karnofsky performance status
- Physical and Neurological Exam - Vital signs and Weight
- Tumor Imaging (by MRI unless contraindicated) showing tumor consistent with a GBM.  
- Tumor Pathology (before Step 3)
- Pregnancy Test
- PT/INR or aPTT
- CBC with differential
- Na, K, CL, Bicarbonate, BUN/Cr, AST, ALT, ALP, T. Bili, Calcium, Albumin
- Urinalysis
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2.3 participant registration status update

Patients who are candidates for the study will first be evaluated for eligibility by the local investigator. All patients must be registered both locally and centrally with BTTC prior to surgery (see Section **3.3.1**, Surgery).  
BTTC patients will be registered with the BTTC Coordinating Center. All subjects must be registered through the BTTC NCI Central Registration Office (CRO). The CRO is open from 8:30 am to 5:00 pm EST Monday through Friday, excluding federal holidays.  
If any emails described in sections **2.3.2** through **2.3.6** contain protected health information
(PHI), they must be encrypted.
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2.3.1 informed consent/authorization

Prior to protocol enrollment and initiation of treatment, subjects must sign and date an Institutional Review Board (IRB) approved consent form. Registrations must be completed after the patient has signed the informed consent and has been determined to be eligible by the local investigator. At the time of registration, the following information will be requested by the BTTC Coordinating Center:  
o A copy of a completed and signed, protocol specific, Registration Form (eligibility checklist) o One copy of the signed and dated Informed Consent/Authorization. o Copies of all source documents to support protocol specific eligibility. Please refer to the BTTC Operations Manual for specific source documents required.
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2.3.2 registration at the nci

Registration and status updates (e.g., when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found here. Registration will be a three part process as patients are screened on this protocol. Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) within 24 hours of signing consent. See Section 2.3.5 **for Step 3 of Registration Form (Randomization**
at NCI).
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2.3.3 registration at participating bttc

Registration will be a three part process as patients are screened on this protocol. A protocol registration form and cover memo will be supplied by the Coordinating Center, NCI CCR and updates will be provided as needed. Registration will be completed within 2 business days of receipt of documents required for registration.  
Step 1: All patients must be registered both locally and centrally with BTTC prior to surgery. To initially register a subject, after the participant has signed the consent, site will complete and send Step 1 of the Registration Form, source documents, and consent form, indicating that the patient is being registered for screening and surgery, to the BTTC Coordinating Center via secure fax (240-541-4432) or send to the BTTC CC research nurse via Secure Email and File Transfer [SEFT] (preferred). Upon confirmation of consent and available slot, the BTTC CC research nurse will send the Registration Form to NCI Central Registration Office ncicentralregistration-l@mail.nih.gov via encrypted email. Within 1 business day, NCI Central Registration Office will send the Verification of Registration with the assigned subject ID number to the BTTC CC research nurse via encrypted email. The BTTC CC research nurse will email the Verification of Registration to the site and Agenus (redacted with subject ID number and patient initials). The subject ID number is unique to the patient and must be written on all data and correspondence for the patient and used to enter data into the NCI-designated eCRF database.  
Step 2: Study treatment may not begin until a subject is registered. After completion of screening and surgery, site will complete Step 2 of the Registration Form indicating that the patient has met all inclusion and no exclusion criteria. The Registration Form and source documents will be sent to the BTTC CC via secure fax (240-541-4432) or send to the BTTC CC research nurse via Secure Email and File Transfer [SEFT] (preferred). Upon confirmation of eligibility, the BTTC CC research nurse will sign the Registration Form (eligibility checklist) and send it via encrypted email to the CRO at NCI Central Registration Office ncicentralregistration-l@mail.nih.gov. The CRO will send the Verification of Registration to the BTTC CC research nurse via encrypted email. The BTTC CC research nurse will email the  
Version Date: *06/30/2021* Verification of Registration to the site and Agenus (redacted with study ID number and patient initials). When a participant has a status change (e.g., subject screened on the study, does not meet eligibility criteria and is removed from the study, participant is taken off protocol therapy or off study, etc.), the Participant Status Update Form will be supplied by the BTTC Coordinating Center research nurse coordinator. Send the completed form to the BTTC Research Nursevia secure fax: 240-541-4432, or via Secure Email and File Transfer (SEFT)..
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see section 2.3.6 for step

Treatment may not be initiated until the participating institution receives a faxed or emailed copy of the patient's Registration Verification Letter from the Central Registration Office. Treatment must be initiated > 14 days and < 6 weeks from surgery. The BTTC Coordinating Center must be notified in writing of any exceptions to this policy.
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2.3.5 registration step 3 randomization

Following vaccine manufacture and completion of quality testing, Agenus will send the Vaccine Production Notification form to NCI. This Form indicates whether manufacture of vaccine was successful. The site will send Step 3 of the Registration form and Vaccine Production Notification Form to BTTC CC research nurse via secure fax: 240-541-4432, hand-delivery, or encrypted email. BTTC CC will confirm verification and send via encrypted email to CRO and via Secure Email and File Transfer [SEFT] to Agenus. CRO will send the Verification of Registration with the assigned sequence number to the site and the BTTC CC research nurse via encrypted email. The BTTC CC research nurse will send the Verification of Registration with the assigned sequence number (vaccine assignment) to Agenus (redacted with subject ID number and patient initials). Agenus will send the blinded vaccine or placebo directly to the site. In parallel, CRO will also notify the NCI Pharmacy to release blinded vaccine or placebo.
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2.3.6 registration step 3 randomization

Following vaccine manufacture and completion of quality testing, Agenus will send the Vaccine Production Notification form to the site. This Form indicates whether manufacture of vaccine was successful. The site will send Step 3 of the Registration form and Vaccine Production Notification Form to BTTC CC research nurse via secure fax: 240-541-4432 or via Secure Email and File Transfer [SEFT] (preferred). BTTC CC will confirm verification and send via encrypted email to CRO and via Secure Email and File Transfer [SEFT] to Agenus. CRO will send the Verification of Registration with the assigned sequence number to the BTTC CC research nurse. The BTTC CC research nurse will send the Verification of Registration with the assigned sequence number (vaccine assignment) to the site and Agenus (redacted with subject ID number and patient initials). Agenus will send the blinded vaccine or placebo directly to the site.
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2.3.7 study status updates for

For participating BTTC Sites: The Participant Status Updates Form will be supplied by the BTTC Coordinating Center, NCI, CCR. Send the completed Participant Status Updates Form via secure fax: 240-541-4432, or via Secure Email and File Transfer (SEFT).
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2.3.8 screen failures

Screen failures are defined as participants who consent to participate in the clinical trial but are not subsequently assigned to the study intervention or entered in the study. A minimal set of screen failure information is required to ensure transparent reporting of screen failure participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond to queries from regulatory authorities. Minimal information includes demography, screen failure details, eligibility criteria, and any serious adverse event (SAE).
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2.4 treatment assignment and randomization

| Number      | Name                                                               | Description                                                                                                                                                                                                                                                                            |
|-------------|--------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1           | Patients with Glioblastoma  eligible to receive radiation  therapy | GBM patients with MRI findings consistent with a suspected  GBM or a histologically confirmed newly diagnosed GBM that  has not been treated, have met all inclusion and no exclusion  criteria, and have completed surgery (i.e., Step 2 of registration)                             |
| 2           | Patients with Glioblastoma with sufficient vaccine  created        | GBM patients with MRI findings consistent with a suspected  GBM or a histologically confirmed newly diagnosed GBM that  has not been treated, whose tumors have an unmethylated MGMT  promoter and are IDH wildtype with sufficient vaccine created (i.e., Step 3 of registration)     |
| 3           | Patients with Glioblastoma  without sufficient vaccine  created    | GBM patients with MRI findings consistent with a suspected  GBM or a histologically confirmed newly diagnosed GBM that  has not been treated, whose tumors have an unmethylated  MGMT promoter and are IDH wildtype without sufficient  vaccine created (i.e., Step 3 of registration) |
| Arms Number | Name                                                               | Description                                                                                                                                                                                                                                                                            |
| 1           | Radiation Therapy (RT)                                             | Standard radiation therapy                                                                                                                                                                                                                                                             |
| 2           | RT+TMZ + Pembrolizumab                                             | Standard treatment with experimental  treatment (pembro) added                                                                                                                                                                                                                         |
| 3           | RT+TMZ+Pembrolizumab+HSPPC-96                                      | Standard treatment with experimental                                                                                                                                                                                                                                                   |
| Vaccine     | treatment (pembro+ vaccine) added                                  |                                                                                                                                                                                                                                                                                        |  
| Number   | Name                         | Description                          |
|----------|------------------------------|--------------------------------------|
| 4        | RT+TMZ+Pembrolizumab+Placebo | Standard treatment with experimental |
| Vaccine  | treatment and placebo added  |                                      |
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randomization and arm assignment

All study patients satisfying the eligibility criteria and who have completed surgery will be assigned to Cohort 1 and Arm 1, Radiation Therapy. All study patients that satisfy the eligibility criteria post-radiation therapy, will be assigned to Cohort 2 or 3, based on ability or inability to make sufficient vaccine. Study patients in Cohort 2 will then be randomized 1:1 in a blinded fashion between arms 3 and 4. Patients in cohort 3 will be assigned directly to the ancillary arm, Arm 2.  
Stratifications  
| Name   | Distinct Options   | Notes   |
|--------|--------------------|---------|
| None   |                    |         |
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3 study implementation 3.1 early

On May 20, 2021 the NCI/CCR Data Safety and Monitoring Board met to review the progress of this protocol, including the results of an unscheduled interim analysis. Prior to the meeting, the board agreed to a request by the Lead NCI PI to look at outcome data by arm and agreed on principles for interpretation of outcome and accrual data. Based on the presented data, the board recommended the study be stopped because of poor accrual. The board deferred to the principal investigator on how to enact an orderly termination of the study including continued treatment and unblinding of patients.  
Because this is a multi-institutional trial, the IRBs of all institutions participating in this study must be informed by the Lead NCI PI about this DSMB's decision. Sections throughout the protocol have been revised to reflect that the study is closed to new enrollment and to describe the early termination procedures. The revisions include decisions for how long participants currently enrolled will remain on study after the unblinding. Below are guidelines for unblinding and follow up per Study Arm:  
- **Ancillary Arm 2**: For participants assigned to RT+TMZ + Pembrolizumab, they will remain on treatment and will be followed for overall survival per Study Calendar.  
- **Arm 3**: For participants randomized to the RT+TMZ+Pembrolizumab+HSPPC-96 Vaccine, they will be unblinded but may remain on treatment to receive TMZ, Pembro and/or HSPPC-96 vaccine and will be followed for overall survival per Study Calendar.
- **Arm 4**: For participants randomized to RT+TMZ+Pembrolizumab+Placebo Vaccine, they will be unblinded but may remain on treatment to receive TMZ and/or Pembro and will be followed for overall survival per Study Calendar. Placebo vaccine will be discontinued as permitted per Section **3.10**.  
Participants who decide to discontinue treatment will continue to be followed for overall survival.
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3.2 study design

This will be a randomized, double blind phase II trial of surgery, RT + TMZ + Pembrolizumab followed by TMZ + Pembrolizumab with HSPPC-96 vaccine or placebo vaccine in newly diagnosed GBM patients whose tumor has an unmethylated MGMT promoter and is IDH
wildtype.  
Subjects will be randomized without stratification on a 1:1 basis, using variable block sizes. The first block will be of size n=6 in order to facilitate the evaluation of toxicity and safety in the first 3 patients on each arm. The study will utilize the Recommended Phase 2 Dose (RP2D) for RT + TMZ + Pembrolizumab which was TMZ 75 mg/m2, RT approximately 60Gy and Pembrolizumab 200 mg every 3 weeks.  
HSPPC-96 is not expected to result in additional toxicity although the first 3 subjects treated in each arm will be observed for toxicity before further enrollment. The primary endpoint is:
o To determine whether the one-year overall survival rate (OS) is improved in newly diagnosed GBM patients whose tumor is MGMT promoter unmethylated and is IDH wildtype treated with RT + TMZ + Pembrolizumab followed by Pembrolizumab + TMZ + HSPPC-96 vaccine (or placebo vaccine) x 6 cycles (1 cycle is 9 weeks).  
Nota Bene: Those patients whose tumor does not meet the criteria (unmethylated MGMT promoter and IDH wildtype by pathology) and with < 80% of contrast enhanced tumor or < 7grams of tumor resected are ineligible.  
All eligible patients will be treated with standard RT to approximately 60 Gy (180 cGy (1.8 Gy)
x 33 or 200 cGy (2.0 Gy) x 30). Initial dose of TMZ during radiation will be 75 mg/m2/day 7 days per week and Pembrolizumab 200 mg every 3 weeks.
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3.2.1 blinding

This study will utilize a placebo control for the vaccine in order to fulfill the double blind design of the study. This protocol design minimizes the risk of bias and enhances the impartial bias analysis of the study vaccine versus placebo effect. However, it is important to note that in case this information is required for safety purposes (affects care or medically needed), it will be provided in an expedited manner. In this double blind study, neither the study personnel (including pharmacists) nor the subject will have knowledge of the vaccine assignment being administered. Data analysis personnel and the Central Registration Office will have access to vaccine assignment. Blinded vaccine (HSPPC-96 or placebo) will be prepared by Agenus and shipped directly to sites. HSPPC-96 or placebo will be dispensed in identical appearing single-use vial as a clear, colorless solution. Each vial of vaccine or placebo is labeled with the subject/patient number, subject/patient initials, and subject/patient date of birth (DOB). Unblinding may occur at the discretion of the principal investigator and only where knowledge of treatment affects patient care. Site would need to submit the document found in Appendix 16.11 to request unblinding. Unblinding will only be done upon approval by the lead PI as documented in Appendix **16.12**. NOTE: Based on the DSMB's May 2021 decision for early termination, unblinding treatment assignments for participants receiving HSPPC-96 vaccine or placebo will be provided to the Lead NCI PI by the NCI Central Registration Office (CRO) in consultation with and confirmed by Agenus, the vaccine manufacturer. The Lead NCI PI or designee at the BTTC Coordinating Center will be responsible for notifying site PIs (at the participating BTTC site) of the unblinded treatment assignments for participants receiving HSPPC-96 vaccine or placebo. Participants receiving pembrolizumab, temozolomide and/or HSPPC-96 vaccine will continue to receive those study treatments per Study Calendar (See Section **3.11**). Participants who are on placebo will stop receiving placebo but may continue to receive pembrolizumab and/or temozolomide per Section **3.10**. Participants who decide to discontinue treatment will continue to be followed for overall survival, and the procedures for removal from protocol therapy will be followed (see Section **3.14**). Off-Study Procedures will be followed per protocol for patients who decide to withdraw from the study (see Section **3.14**).
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3.2.1.1 rationale for double blinding

This study has been designed to be double blinded despite having an objectively ascertained primary endpoint for the following reasons: (1) This limits study defection due to randomization disappointment (so subjects randomized to placebo will not quit the study), (2) there will be no difference in the interpretation of biomarkers or lab tests due to knowing the intervention being delivered. (3) Adverse event reporting will not be impacted with the knowledge of the treatment arm. (4) Subject/patient responses in the MDASI will not be impacted with the knowledge of the treatment arm.
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3.3 surgery & tissue collection

Patients with presumed GBM based on MRI or if they require a more definitive surgery may be consented for the trial. At the time of surgery, tissue will be collected for vaccine production (≥
7 grams is required) and processed per Appendix **16.2** Tumor must be confirmed to be MGMT
promoter unmethylated and IDH wildtype prior to starting treatment. GBM patients whose tumor is MGMT promoter methylated and is not IDH wildtype are not eligible for treatment on this protocol and will be removed from the protocol. Those patients with < 80% of contrast enhanced tumor resected or < 7 grams of tissue will be no longer eligible for treatment with SOC (RT + TMZ) + Pembrolizumab. Tumor will be measured  
Version Date: *06/30/2021* per RANO criteria. The determination of ≥ 80% resection will be based on the pre-operative and post operative cross sectional measurements, the residual tumor, if any, should be < 20% or original cross sectional measurements. Post tumor resection, all patients must have a post-operative MRI done no more than 72 hours after surgery. This MRI can be used as baseline prior to treatment.
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3.3.2 tissue collection for vaccine

Tumor sample will be collected at time of surgery by the Laboratory of Pathology NCI. Tissue sections will be obtained and will be labeled with the Study Subject ID number. The relationship between the Study Subject ID number and the patient clinical information will be stored in a secure database that is maintained and regularly backed up by the Laboratory of Pathology NCI. The Laboratory of Pathology NCI will submit tumor tissue for vaccine manufacture according to Appendix **16.2**.  
Tumor tissue that does not qualify either because of incorrect diagnosis, MGMT status, or presence of IDH mutation will remain at or be returned to the site of origin.  
Tissue will also be collected at NCI for Correlatives. Refer to 5.1.2, **5.1.3**, and **5.1.4** for tissue collection for Correlatives. Tumor tissue that does not qualify either because of incorrect diagnosis, MGMT status, or presence of IDH mutation will remain at or be returned to the site of origin.
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3.3.3 tissue collection for vaccine

Tumor sample will be collected at time of surgery at the participating sites. Tissue sections will be obtained and will be labeled with the Study Subject ID number. The participating BTTC site will submit tumor tissue for vaccine manufacture according to Appendix **16.2**.  
Tumor tissue that does not qualify either because of incorrect diagnosis, MGMT status, or presence of IDH mutation will remain at or be returned to the site of origin.  
Tissue will also be collected at the participating BTTC sites for Correlatives. Refer to **5.1.2** and 5.1.3 for tissue collection for Correlatives.
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3.4 radiation therapy (rt)

All eligible patients will be treated with standard RT to approximately 60 Gy (180 cGy (1.8 Gy) x 33 fractions or 200 cGy (2.0 Gy) x 30 fractions).
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3.5 drug administration 3.5.1 (rt+tmz+pembro)

TMZ will be administered orally on day 1 of radiation therapy (RT) 1-2 hours before each session of radiotherapy or 1 night before RT (within 24 hours before Day 1); and it should be administered the same time throughout the course of RT. TMZ should continue throughout RT at the dose of 75 mg/m2/day. During weekends without radiotherapy (Saturday and Sunday), the drug will be taken at the same time as on radiotherapy days. Pembrolizumab at 200 mg will be administered on day 1 as a 30 minute IV infusion (+/- 24 hours day of RT) every 3 weeks during RT Phase on days 1, 22 and 43 (+/- 24 hours for each timepoint). Sites should make every effort to target infusion timing to be as close to 30 minutes as possible. However, given the variability of infusion pumps from site to site, a window of -10 minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes: -10 min/+10 min). Observation for one hour after the infusion of the first dose of Pembrolizumab is recommended.
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3.5.2 hsppc-96 vaccine or placebo

HSPPC-96 vaccine will be produced prior to the end of RT. If > 4 doses can be made, the patient will then be randomized 1:1 to the treatment with TMZ + Pembrolizumab with HSPPC96 vaccine or placebo.
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3.6 post rt treatment regimen

Four weeks post end of RT, an MRI will be performed and then patients will start TMZ (days 15 and 29-33) + Pembrolizumab (days 1, 22 and 43 of each cycle +/- 24 hours for each timepoint). 1 cycle = 9 weeks. Please note that if days 1-5 of TMZ is well tolerated, the dose of TMZ will be increased for days 29-33 as described below. The starting TMZ dose will be 150 mg/m2/day for cycle 1, days 1-5, with a single dose escalation to 200 mg/m2/day for all subsequent treatment if no treatment-related adverse events greater than Grade 2 are noted. After completion of 6 cycles, patients may continue on Pembrolizumab for an additional 12 months if felt to be of benefit by the treating physician.  
NOTE: For early terminationArm 2 ancillary participants that were not randomized and were assigned to Arm 2 will continue on treatment andfollowed for overall survival per Study Calendar (See Section **3.11**).
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3.6.2 for patients randomized to

One week post end of RT, patients will receive a dose of HSPPC-96 vaccine or placebo vaccine given as approximately 0.4mL intradermal weekly x 4 (see Section **14.3.8** details on intradermal administration). Within 7 days of the last dose of HSPPC-96 vaccine or placebo, an MRI will be performed and then patients will start TMZ (days 1-5 and 29-33) + Pembrolizumab (days 1, 22 and 43 of each cycle +/- 24 hours for each timepoint). 1 cycle = 9 weeks.  
The starting TMZ dose will be 150 mg/m2/day for cycle 1, days 1-5, with a single dose escalation to 200 mg/m2/day for all subsequent treatment if no treatment-related adverse events greater than Grade 2 are noted.  
HSPPC-96 vaccine or placebo will then be given 21 days (+/- 24 hours) after the day 5 and day 33 doses of TMZ. TMZ will be given for 6 cycles. HSPPC-96 vaccine or placebo vaccine will be given for 6 cycles or until supply runs out. Patients who receive placebo will be matched for number of vaccine injections that were generated by their tumor tissue. After the completion of 6 cycles (54 weeks), they may continue pembrolizumab for 12 more months alone or in conjunction with HSPPC-96 or placebo vaccine if any is available, if felt to be of benefit by the treating physician.  
NOTE: Early Termination procedures require that participants randomized to HSPPC-96 or placebo be unblinded. Participants randomized to HSPPC-96 vaccine may continue on treatment to receive TMZ, Pembro and/or HSPPC-96 vaccine. Patients randomized to placebo vaccine may continue on treatment to receive TMZ and Pembro. Placebo vaccine will be discontinued. Participants randomized to HSPPC-96 vaccine or placebo will be followed for overall survival per Study Calendar (see Section **3.11**).
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3.7 baseline assessments (post-surgery and

Prior to RT, once patient has recovered from surgery, all patients will undergo the following baseline evaluations or procedures:  
- Karnofsky performance status - Physical and Neurological Exam
- Vital signs and Weight
- Pregnancy Test - PT/INR or aPTT
- CBC with differential - Na, K, CL, Bicarbonate, BUN/Cr, AST, ALT, ALP, T. Bili, Calcium, Albumin - Urinalysis - T3, T4, and TSH - MDASI-BT*
- Correlative Studies Blood Collection (LabConnect Kit B refer to Appendix **16.3**)
- Baseline tumor imaging (by MRI unless contraindicated) to be performed within 72 hours after surgery.
*The MDASI-BT (Appendix **16.9**) will be administered at time of baseline MRI or up to 14 days prior to treatment initiation.  
Baseline assessments can be performed up to 7 days before RT+TMZ+Pembro treatment starts. If treatment is delayed (e.g. complications of wound healing, infection), repeat all baseline assessments (except for the MDASI-BT and Correlative research blood samples which do not need to be repeated).
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3.8 assessments while on trial

o Patients will have a physical (including vital signs and weight), KPS, and neurological exam every 3 weeks (- 3 days to + 7 days) during RT and then every 4 weeks (+/- 7 days) after RT.  
o Patients on ancillary arm do not require vaccine associated physical exam. o Adverse events will be assessed during Physical Exams and on treatment days.
o For subjects not randomized: An MRI will be done pre surgery, post surgery (within 72 hours), 4 weeks post RT (+/- 1 week), and then every 9 weeks +/- 1 week.  
o For subjects who are randomized: An MRI will be done pre surgery, post surgery (within 72 hours), within 7 days of last dose of vaccine or placebo induction, and then every 9 weeks +/- 1 week.
o Pregnancy test will be done at the start of each cycle.  
o A CBC with differential will be done weekly +/- 3 days during RT and then weekly +/- 3 days after RT for the first year and then every 2 weeks for the year after if the patient remains on Pembrolizumab.  
o A Comprehensive Chemistry Panel with LFTs and TFTs, T3, T4, TSH will be done on day 21 of RT +\- 3 days, at the completion of RT and then day 1 (+/- 1 day) and day 29 (- 7 days) of each cycle.
o Blood for correlative labs will be collected for different immune monitoring assessments that may include, but are not limited to: whole exome sequencing (WES), immune phenotyping of immune cell subsets, functional characterization of immune cell subsets, measurement of immune mediators, peripheral lymphocyte, monocyte and tryptophan and the assessment of PD-L1 expression and circulating tumor at the following time points: For subjects who are randomized: Pre-RT treatment (RT+TMZ+Pembro), PostRT treatment (RT+TMZ+Pembro)/Pre-vaccine (or placebo) induction, Post Vaccine (or placebo) induction/Pre-cycle 1 maintenance, Every Day 1 on Cycle 2, C4, C6, C8 (if continuing on Pembrolizumab), then at discretion of Study Chair and BTTC Coordinating Center, every other cycle, at time of progression or at end of treatment and for subjects not randomized: Pre-RT treatment (RT+TMZ+Pembro), Post-RT treatment (RT+TMZ+Pembro)/Pre-cycle 1 maintenance, Every Day 1 on Cycle 2, C4, C6, C8 (if continuing on Pembrolizumab); then at discretion of Study Chair and BTTC Coordinating Center, every other cycle, at time of progression, or at end of treatment
(See Study Calendar **3.11**).  
o Archival tissue for PDL1 expression and mutation analysis will be collected after surgery irrespective of whether the patient is randomized or not.  
o If a patient has tumor progression while on trial and undergoes surgery, patients will have their tumor evaluated for PDL1 expression and repeat mutational analysis.
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3.8.1 questionnaires

The MDASI-BT will be administered prior to the initiation of radiation, temozolomide and pembrolizumab (baseline) prior to initiation of adjuvant therapy and then at the time of each evaluation that also includes imaging. Of note, every attempt should be made to administer the MDASI-BT prior to informing the patients about the results of the imaging study. The MDASIBT will be completed only by the patient, unless changes in vision or weakness make this difficult. If this occurs, then the caregiver or research assistant may read the questions to the patient or assist with marking the severity number or score as described by the patient. A caregiver may complete the questionnaires as a patient proxy if the patient's deficits preclude self-report; however, this must be done at every assessment from baseline to end of treatment.  
Full instruments are provided in the Appendix **16.9**. In addition, information regarding demographics and treatment history will be collected as part of the larger study and used in this analysis. The MDASI-BT consists of symptoms rated on an 11-point scale (0 to 10) to indicate the presence and severity of the symptom, with 0 being "not present" and 10 being "as bad as you can imagine." Each symptom is rated at its worst in the last 24 hours. Symptoms included on the instrument include those commonly associated with cancer therapies, those associated with increased intracranial pressure, and those related to focal deficits. The questionnaire also includes ratings of how much symptoms interfered with different aspects of a patient's life in the last 24 hours. These interference items include: general activity, mood, work (includes both work outside the home and housework), relations with other people, walking, and enjoyment of life. The interference items are also measured on 0 - 10 scales. The average time to complete these instruments is 5 minutes. The MDASI-BT has been translated into multiple languages, but the English language version will be used for this study. [43]
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3.9 telehealth/remote visits

In the extenuating event that a subject cannot travel to be seen in person at the study site, or any other crisis causing travel and staffing restriction, the provider assessment may occur via telehealth visits. Telehealth visits (synonyms: telemedicine, videoconferencing, virtual visits, video phone call or remote visits) will be allowed for patients on-study unless these visits are essential to maintaining patient safety while on-study, refer to study calendar section **3.11** for more details. Decisions regarding the use of telehealth will be based on the patient's physician's decision regarding the risk of an in-person clinic visit compared with the potential benefit of a comprehensive in-person evaluation. The medical record or progress note for the telehealth visit should state that the risks were greater than the benefit. Telehealth visits for clinical assessments will be conducted in compliance with local guidelines and FDA regulations. For example, to ensure privacy, the patients will confirm their identities before the interaction or exchange of private information begins; guidance on this issue can be found in the National Institute of Standards and Technology (NIST) Digital Identity Guidelines, Special Publication 800-63A —Enrollment and Identity Proofing Requirements When Developing an Identity Verification Plan. In addition, documentation of telehealth visits must comply with local requirements (e.g., date and time of the real-time video interaction, location of the subject, individuals present, assessments performed, etc.). General interviews may be conducted adequately via telehealth visits. For example, to obtain a patient's medical history, check-in with a participant regarding their current health status, or to administer a questionnaire to a participant related to their level of functioning or activities of daily living. Clinical assessments via telehealth visits for patients on study will not compromise either the primary or secondary objectives and will likely have minimal impact on the ability to complete this study as long as administration of treatment (e.g., radiation, temozolomide, pembrolizumab and vaccine/placebo) is not compromised. Given that the study requires only standard, clinical laboratory assessments and imaging per routine radiologic parameters/procedures, any possible interlaboratory or facility variability is not expected to be a concern. If any question about possible progression, a patient may be asked to have repeat imaging and/or additional assessments to confirm response. Further, a patient may be referred to another local provider or asked to come to the NIH for an in-person assessment, if clinically indicated, and at the discretion of the investigator. In the case of any visits with participants' local providers, records will be obtained for the research records. The following clinical assessments generally obtained during a physical examination (i.e., H&P) can be conducted via telehealth visits through discussion and remote/video observation:
- Karnofsky Performance Status.  
- Neurological exam to assess cognition remotely will use the American Academy of Neurology Telemedicine guidance document for evaluation of memory, attention, concentration, language, eye movements, facial weakness, strength, walking and coordination.  
- Physical exam to observe rashes, thrust, surgical scar, and swelling will use the American Academy of Dermatology Teledermatology guidance document for physical evaluation during telehealth vistis.  
The following clinical assessments are not feasible via telehealth visits:
- Hearing and vision
- Listening to your heart and lungs
- Assessing weakness - Checking reflexes
- Checking for abnormalities in sensation
- Feeling lymph nodes, areas of swelling
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3.10 dose modifications

If Pembrolizumab and/or HSPPC-96 are stopped or discontinued, the patient can continue treatment on TMZ alone. If TMZ is stopped or discontinued, Pembrolizumab or HSPPC96/placebo can continue. If HSPPC-96/Placebo is stopped or discontinued, TMZ and Pembrolizumab can continue.
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3.10.1 pembrolizumab dose modifications

Adverse events (AE) (both non-serious and serious) associated with pembrolizumab exposure, including coadministration with additional compounds, may represent an immunologic etiology. These immune-related adverse events (irAEs) may occur shortly after the first dose or several months after the last dose of treatment and may affect more than one body system simultaneously. Pembrolizumab must be withheld for drug-related toxicities and severe or lifethreatening AEs as per Table 1: Dose Modification and Toxicity Management Guidelines for Immune-related AEs Associated with Pembrolizumab. See Section **3.10.3** for supportive care guidelines, including use of corticosteroids.  
3.10.1.1 Attribution of Toxicity: When study interventions are administered in combination, attribution of an adverse event to a single component is likely to be difficult. Therefore, while the investigator may attribute a toxicity event [to the combination, to Temozolomide alone or to pembrolizumab alone, for adverse events listed in [**Table 1**], both interventions must be held according to the criteria in
[Table 1 Dose Modification and Toxicity Management Guidelines for Immune-Related Adverse Events Associated with Pembrolizumab]. 3.10.1.2 Holding Study Interventions:
When study interventions are administered in combination, if the AE is considered immunerelated, both interventions should be held according to recommended dose modifications.  
3.10.1.3 Restarting Study Interventions: Participants may not have any dose modifications (no change in dose or schedule) of pembrolizumab in this study, as described in [**Table 1**].  
- If the toxicity does not resolve or the criteria for resuming treatment are not met, the participant must be discontinued from all study interventions.  
If the toxicities do resolve and conditions are aligned with what is defined in [**Table 1**], the combination of temozolomine and pembrolizumab may be restarted at the discretion of the investigator.[In these cases where the toxicity is attributed to [the combination or to temozolomine alone, re-initiation of pembrolizumab as a monotherapy may be considered at the principal investigator's discretion .
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44 version date 06/30/2021

Table 1: **Dose Modification and Toxicity Management Guidelines for Immune-related AEs Associated with Pembrolizumab**  
| General instructions:   |
|-------------------------|  
1. Severe and life-threatening irAEs should be treated with IV corticosteroids followed by oral steroids. Other immunosuppressive treatment should begin if the irAEs are not controlled by corticosteroids.  
2. Study intervention must be permanently discontinued if the irAE does not resolve or the corticosteroid dose is not ≤10 mg/day within 12 weeks of the last study intervention treatment.  
3. The corticosteroid taper should begin when the irAE is ≤ Grade 1 and continue at least 4 weeks. 4. If study intervention has been withheld, study intervention may resume after the irAE decreased to ≤ Grade 1 after corticosteroid taper.  
| Toxicity  Grade                        | Action With                                  | Corticosteroid and/or Other                    |                                                  |                                                  |
|----------------------------------------|----------------------------------------------|------------------------------------------------|--------------------------------------------------|--------------------------------------------------|
| (CTCAE v5.0)                           | Pembrolizumab                                | Therapies                                      | Monitoring and Follow-up                         |                                                  |
| irAEs                                  | Grade 2                                      | Withhold                                       | - Administer corticosteroids (initial dose of    | - Monitor participants for signs and symptoms of |
| 1 to 2 mg/kg prednisone or equivalent) | pneumonitis                                  |                                                |                                                  |                                                  |
| followed by taper                      |                                              |                                                |                                                  |                                                  |
| Recurrent  Grade 2,  Grade 3 or 4      | Permanently  discontinue                     | - Evaluate participants with suspected         |                                                  |                                                  |
| Pneumonitis                            | - Add prophylactic antibiotics for           | pneumonitis with radiographic imaging and      |                                                  |                                                  |
| opportunistic infections               | initiate corticosteroid treatment            |                                                |                                                  |                                                  |
| Grade 2 or 3                           | Withhold                                     | - Administer corticosteroids (initial dose of  | - Monitor participants for signs and symptoms of |                                                  |
| 1 to 2 mg/kg prednisone or equivalent) | enterocolitis (ie, diarrhea, abdominal pain, |                                                |                                                  |                                                  |
| Diarrhea/Colitis                       | followed by taper                            | blood or mucus in stool with or without fever) |                                                  |                                                  |  
45  
Version Date: *06/30/2021*  
| Version Date: 06/30/2021 Toxicity  Grade                                                                                                                                                                                                                                                                                                                      | Action With                                               | Corticosteroid and/or Other                   |                                                   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------|-----------------------------------------------|---------------------------------------------------|
| (CTCAE v5.0)                                                                                                                                                                                                                                                                                                                                                  | Pembrolizumab                                             | Therapies                                     | Monitoring and Follow-up                          |
| irAEs                                                                                                                                                                                                                                                                                                                                                         | Recurrent  Grade 3 or  Grade 4                            | Permanently                                   | and of bowel perforation (ie, peritoneal signs    |
| discontinue                                                                                                                                                                                                                                                                                                                                                   | and ileus)                                                |                                               |                                                   |
| - Participants with ≥Grade 2 diarrhea suspecting  colitis should consider GI consultation and  performing endoscopy to rule out colitis - Participants with diarrhea/colitis should be  advised to drink liberal quantities of clear  fluids. If sufficient oral fluid intake is not  feasible, fluid and electrolytes should be  substituted via IV infusion |                                                           |                                               |                                                   |
| Grade 2 a                                                                                                                                                                                                                                                                                                                                                     | Withhold                                                  | - Administer corticosteroids (initial dose of | - Monitor with liver function tests (consider     |
| 0.5 to 1 mg/kg prednisone or equivalent)                                                                                                                                                                                                                                                                                                                      | weekly or more frequently until liver enzyme              |                                               |                                                   |
| AST or ALT  Elevation or  Increased  Bilirubin                                                                                                                                                                                                                                                                                                                | followed by taper                                         | value returned to baseline or is stable)      |                                                   |
| Grade 3 b or 4 c                                                                                                                                                                                                                                                                                                                                              | Permanently                                               | - Administer corticosteroids (initial dose of |                                                   |
| discontinue                                                                                                                                                                                                                                                                                                                                                   | 1 to 2 mg/kg prednisone or equivalent)  followed by taper |                                               |                                                   |
| New onset  T1DM or  Grade 3 or 4  hyperglycemia  associated with                                                                                                                                                                                                                                                                                              | Withhold d                                                | - Initiate insulin replacement therapy for    | - Monitor participants for hyperglycemia or other |
| T1DM or  Hyperglycemia                                                                                                                                                                                                                                                                                                                                        | participants with T1DM                                    | signs and symptoms of diabetes                |                                                   |  
46  
Version Date: *06/30/2021*  
| Version Date: 06/30/2021 Toxicity  Grade                                         | Action With                                            | Corticosteroid and/or Other                      |                                                                                         |                                     |
|----------------------------------------------------------------------------------|--------------------------------------------------------|--------------------------------------------------|-----------------------------------------------------------------------------------------|-------------------------------------|
| (CTCAE v5.0)                                                                     | Pembrolizumab                                          | Therapies                                        | Monitoring and Follow-up                                                                |                                     |
| irAEs                                                                            | evidence of                                            | - Administer antihyperglycemic in                |                                                                                         |                                     |
| -cell failure                                                                   | participants with hyperglycemia                        |                                                  |                                                                                         |                                     |
| Grade 2                                                                          | Withhold                                               | - Administer corticosteroids and initiate        | - Monitor for signs and symptoms of hypophysitis (including hypopituitarism and adrenal |                                     |
| Grade 3 or 4                                                                     | Withhold or                                            | insufficiency)                                   |                                                                                         |                                     |
| permanently  discontinue d                                                       | hormonal replacements as clinically                    |                                                  |                                                                                         |                                     |
| Hypophysitis                                                                     | indicated                                              |                                                  |                                                                                         |                                     |
| Grade 2                                                                          | Continue                                               | - Treat with nonselective beta-blockers          |                                                                                         |                                     |
| Hyperthyroidism                                                                  | - Monitor for signs and symptoms of thyroid  disorders |                                                  |                                                                                         |                                     |
| Grade 3 or 4                                                                     | Withhold or  permanently  discontinue d                | (eg, propranolol) or thionamides as  appropriate |                                                                                         |                                     |
| Grade 2, 3 or 4                                                                  | Continue                                               | - Initiate thyroid replacement hormones          | - Monitor for signs and symptoms of thyroid                                             |                                     |
| Hypothyroidism                                                                   | (eg, levothyroxine or liothyronine) per                | disorders                                        |                                                                                         |                                     |
| standard of care                                                                 |                                                        |                                                  |                                                                                         |                                     |
| Nephritis: grading  according to  increased  creatinine or  acute kidney  injury | Grade 2                                                | Withhold                                         | - Administer corticosteroids (prednisone 1                                              | - Monitor changes of renal function |
| to 2 mg/kg or equivalent) followed by                                            |                                                        |                                                  |                                                                                         |                                     |
| Grade 3 or 4                                                                     | Permanently  discontinue                               | taper                                            |                                                                                         |                                     |
| Grade 2                                                                          | Withhold                                               |                                                  |                                                                                         |                                     |  
47  
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| Version Date: 06/30/2021 Toxicity  Grade                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Action With                                      | Corticosteroid and/or Other                                               |                                                  |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------|---------------------------------------------------------------------------|--------------------------------------------------|
| (CTCAE v5.0)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Pembrolizumab                                    | Therapies                                                                 | Monitoring and Follow-up                         |
| irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Grade 3 or 4                                     | Permanently                                                               | - Based on severity of AE administer             |
| discontinue                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | - Ensure adequate evaluation to confirm etiology |                                                                           |                                                  |
| Neurological  Toxicities                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | corticosteroids                                  | and/or exclude other causes                                               |                                                  |
| Grade 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Withhold                                         | - Based on severity of AE administer                                      | - Ensure adequate evaluation to confirm etiology |
| Myocarditis                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | and/or exclude other causes                      |                                                                           |                                                  |
| Grade 2, 3 or 4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Permanently  discontinue                         | corticosteroids                                                           |                                                  |
| Suspected SJS,  TEN, or  DRESS                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Withhold                                         | - Based on severity of AE administer                                      | - Ensure adequate evaluation to confirm etiology |
| Exfoliative  Dermatologic  Conditions                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | corticosteroids                                  | or exclude other causes                                                   |                                                  |
| Confirmed SJS,  TEN, or  DRESS                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Permanently  discontinue                         |                                                                           |                                                  |
| PersistentGrade 2 Withhold                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | - Based on severity of AE administer             | - Ensure adequate evaluation to confirm etiology  or exclude other causes |                                                  |
| Grade 3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Withhold or  discontinue based  on the event e   | corticosteroids                                                           |                                                  |
| All Other irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Recurrent  Grade 3 or  Grade 4                   | Permanently  discontinue                                                  |                                                  |
| AE(s)=adverse event(s); ALT= alanine aminotransferase; AST=aspartate aminotransferase; CTCAE=Common Terminology Criteria for Adverse Events; DRESS=Drug  Rash with Eosinophilia and Systemic Symptom; GI=gastrointestinal; IO=immuno-oncology; ir=immune related; IV=intravenous; SJS=Stevens-Johnson Syndrome;  T1DM=type 1 diabetes mellitus; TEN=Toxic Epidermal Necrolysis; ULN=upper limit of normal. Note: Non-irAE will be managed as appropriate, following clinical practice recommendations. |                                                  |                                                                           |                                                  |
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48 version date 06/30/2021

Toxicity  
Grade  
(CTCAE v5.0)  
Action With  
Pembrolizumab  
Corticosteroid and/or Other  
Therapies **Monitoring and Follow-up**  
a AST/ALT: >3.0 to5.0 x ULN if baseline normal; >3.0 to 5.0 x baseline, if baseline abnormal; bilirubin:>1.5 to 3.0 x ULN if baseline normal; >1.5 to 3.0 x baseline if baseline abnormal b AST/ALT: >5.0 to 20.0 x ULN, if baseline normal; >5.0 to 20.0 x baseline, if baseline abnormal; bilirubin:>3.0 to 10.0 x ULN if baseline normal; >3.0 to 10.0 x baseline if baseline abnormal c AST/ALT: >20.0 x ULN, if baseline normal; >20.0 x baseline, if baseline abnormal; bilirubin: >10.0 x ULN if baseline normal; >10.0 x baseline if baseline abnormal d The decision to withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating physician. If control achieved or ≤ Grade 2, pembrolizumab may be resumed.  
e Events that require discontinuation include, but are not limited to: encephalitis and other clinically important irAEs (eg, vasculitis and sclerosing cholangitis).  
| irAEs   |
|---------|
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3.10.2 guidelines for drug-related adverse

Dosing interruptions are permitted in the case of medical / surgical events or logistical reasons not related to study therapy (e.g., elective surgery, unrelated medical events, patient vacation, and/or holidays). Subjects should be placed back on study therapy within 3 weeks of the scheduled interruption, unless otherwise discussed with the Study Chair. The reason for interruption should be documented in the patient's study record.
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3.10.3 pembrolizumab supportive care

Participants should receive appropriate supportive care measures as deemed necessary by the treating investigator. Suggested supportive care measures for the management of adverse events with potential immunologic etiology are outlined below. Where appropriate, these guidelines include the use of oral or intravenous treatment with corticosteroids as well as additional antiinflammatory agents if symptoms do not improve with administration of corticosteroids. Note that several courses of steroid tapering may be necessary as symptoms may worsen when the steroid dose is decreased. For each disorder, attempts should be made to rule out other causes such as metastatic disease or bacterial or viral infection, which might require additional supportive care. The treatment guidelines are intended to be applied when the investigator determines the events to be related to pembrolizumab. Note: If after the evaluation the event is determined not to be related, the investigator does not need to follow the treatment guidance (as outlined below). Refer to Section **3.10** for dose modification. It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or skin photography as part of evaluation of the event.
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- pneumonitis

o For **Grade 2 events**, treat with systemic corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks.  
o For **Grade 3-4 events**, immediately treat with intravenous steroids. Administer additional anti-inflammatory measures, as needed.  
o Add prophylactic antibiotics for opportunistic infections in the case of prolonged steroid administration.
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- diarrhea/colitis

Subjects should be carefully monitored for signs and symptoms of enterocolitis (such as diarrhea, abdominal pain, blood or mucus in stool, with or without fever) and of bowel perforation (such as peritoneal signs and ileus).  
o All subjects who experience diarrhea/colitis should be advised to drink liberal quantities of clear fluids. If sufficient oral fluid intake is not feasible, fluid and electrolytes should be substituted via IV infusion. For Grade 2 or higher diarrhea, consider GI consultation and endoscopy to confirm or rule out colitis.  
o For **Grade 2 diarrhea/colitis,** administer oral corticosteroids.  
o For **Grade 3 or 4 diarrhea/colitis,** treat with intravenous steroids followed by high dose oral steroids.  
o When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks.
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- type 1 diabetes mellitus

- Insulin replacement therapy is recommended for Type I diabetes mellitus and for Grade 3-4 hyperglycemia associated with metabolic acidosis or ketonuria.  
- Evaluate patients with serum glucose and a metabolic panel, urine ketones, glycosylated hemoglobin, and C-peptide.
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- hypophysitis

o For **Grade 2** events, treat with corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks. Replacement of appropriate hormones may be required as the steroid dose is tapered.  
o For **Grade 3-4** events, treat with an initial dose of IV corticosteroids followed by oral corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks. Replacement of appropriate hormones may be required as the steroid dose is tapered.
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- hyperthyroidism or hypothyroidism

Thyroid disorders can occur at any time during treatment. Monitor patients for changes in thyroid function (at the start of treatment, periodically during treatment, and as indicated based on clinical evaluation) and for clinical signs and symptoms of thyroid disorders.  
o **Grade 2** hyperthyroidism events (and **Grade 2-4** hypothyroidism):
- In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are suggested as initial therapy.  
- In hypothyroidism, thyroid hormone replacement therapy, with levothyroxine or liothyronine, is indicated per standard of care.  
o **Grade 3-4** hyperthyroidism
- Treat with an initial dose of IV corticosteroid followed by oral corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks. Replacement of appropriate hormones may be required as the steroid dose is tapered.
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- hepatic

o For **Grade 2** events, monitor liver function tests more frequently until returned to baseline values (consider weekly).  
- Treat with IV or oral corticosteroids o For **Grade 3-4** events, treat with intravenous corticosteroids for 24 to 48 hours.  
o When symptoms improve to Grade 1 or less, a steroid taper should be started and continued over no less than 4 weeks.
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- renal failure or nephritis

o For **Grade 2** events, treat with corticosteroids.  
o For **Grade 3-4** events, treat with systemic corticosteroids.  
o When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks.  
- **Management of Infusion Reactions**: Signs and symptoms usually develop during or shortly after drug infusion and generally resolve completely within 24 hours of completion of infusion.  
Refer to **Infusion Reaction Treatment Guidelines** Appendix **16.8** for subjects who experience an infusion reaction associated with administration of pembrolizumab.
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3.10.4 temozolomide dose modifications

NOTE: TMZ dose is modified ONLY if toxicity is specifically attributed to TMZ.  
TMZ can be delayed at the beginning of a cycle (e.g., C2D1) and then restarted when the reason for a delay has resolved. If TMZ is held (e.g., for toxicity) at mid-cycle (e.g., C2D29) it can be delayed up to 2 weeks. When TMZ is restarted, for example, 1 week later (e.g., D36) the cycle date entered in the CRF will automatically be labelled as D36, not D29; please enter a comment in the CRF that TMZ was held for X amount of days.  
If during a TMZ dosing period, there is a drop in ANC to < 1.5 or platelets < 75 000 a dose reduction should occur in the subsequent cycle. See tables.  
| RT Phase          |                   |                |
|-------------------|-------------------|----------------|
| Dose level        | Temozolomide dose | Dose reduce to |
| 0 (starting dose) | 75 mg/m2          | 60 mg/m2       |
| -1b               | 60 mg/m2          | 50 mg/m2       |
| -2b               | 50 mg/m2          | 40 mg/m2       |  
| Post RT phase                  |                   |                |
|--------------------------------|-------------------|----------------|
| Dose level                     | Temozolomide dose | Dose reduce to |
| 0                              |                   |                |
| (starting dose for cycle 2 and | 200 mg/m2         | 150 mg/m2      |
| beyond) -1                     |                   |                |
| (starting dose for cycle 1)    | 150 mg/m2         | 125 mg/m2      |
| -2                             | 125 mg/m2         | 100 mg/m2      |
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3.10.5 hsppc-96/placebo dose modifications

For grade 1 or 2 reactions considered related to HSPPC-96/Placebo, manage symptoms according to institutional procedures. Continue on study.  
For grade 3 or 4 allergic reactions considered related to HSPPC-96/Placebo, discontinue HSPPC96/Placebo.
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3.10.6 vaccine injection site reactions

For grade 1 or 2 injection site reactions, continue on study; make sure to rotate injection sites as instructed in Administration Section **14.3.8.** Manage symptoms according to institutional procedures. For grade 3 or 4 injection site reactions, discontinue HSPPC-96/Placebo.  
For other grade 1 or 2 non-hematologic toxicity, continue on protocol once toxicity is resolved to < grade 1. For other grade 3 or 4 non-hematologic toxicity considered at least possibly related to HSPPC-96, discontinue HSPPC-96/Placebo.  
53 Version Date: *06/30/2021*
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3.11 study calendar

| 3.11 STUDY CALENDAR Screening7            | Treatment                                          | Follow Up                            |                           |                       |    |    |
|-------------------------------------------|----------------------------------------------------|--------------------------------------|---------------------------|-----------------------|----|----|
| Pre                                           |                                                    |                                      |                           |                       |    |    |
| Surgery  Screening Surgery                | TMZ+Pembrolizumab +/- HSPPC-96/Vaccine  (6 cycles) |                                      |                           |                       |    |    |
| Time Period                               | RT + TMZ +                                         |                                      |                           |                       |    |    |
| Pembrolizumab16                           | HSPPC-96/Vaccine Induction or Break14 (4 weeks)    | End of Treatment  or Discontinuation | 30 days  after last  dose | Long-term 8 Follow-up |    |    |
| +/-14 of  signing consent                 |                                                    |                                      |                           |                       |    |    |
| SchedulingWindow (Days)                   | - 7                                                | - 7                                  | +/- 3                     | Every 8-16            |    |    |
| (before treatment)                        | (before treatment)                                 | weeks                                |                           |                       |    |    |
| Informed Consent                          | X                                                  |                                      |                           |                       |    |    |
| Medical history                           | X                                                  |                                      |                           |                       |    |    |
| Prior & concomitant medicationsreview     | X                                                  |                                      |                           |                       |    |    |
| Assess and  document first postprotocol treatment                                           | X                                                  |                                      |                           |                       |    |    |
| Assess and document Adverse               | 12                                                 | X 12                                 | X 12                      | X                     | X  |    |
| X                                         |                                                    |                                      |                           |                       |    |    |
| Events12 Physical, KPS &                  | 13                                                 | X 13                                 | X 13                      | X                     | X  |    |
| 13                                        | X                                                  | X                                    |                           |                       |    |    |
| neurological exam Vital Signs and Weight1 | X                                                  | X                                    | X                         | X                     | X  | X  |
| Pregnancy test 2                          | X                                                  | X 2                                  | X 2                       | X 2                   |    |    |
| PT/INR or aPTT                            | X                                                  | X                                    |                           |                       |    |    |
| CBC with diff                             | X                                                  | X 4                                  | X 4                       | X 4                   | X  | X  |
| Comprehensive                             | 5                                                  |                                      |                           |                       |    |    |
| Chemistry panel9                          | X                                                  | X 5                                  | X                         | 5                     | X  |    |
| X                                         |                                                    |                                      |                           |                       |    |    |
| Urinalysis                                | X                                                  | X                                    |                           |                       |    |    |
| T3, T4, and TSH                           | X 5                                                | X 5                                  | X 5                       | X                     | X  |    |
| Tumor Imaging3                            | X                                                  | X3                                   | X3                        | X3                    | X  | X  |
| Archival or Collection Tumor              | X                                                  |                                      |                           |                       |    |    |
| MDASI-BT11                                | X                                                  | X                                    | X                         | X                     |    |    |
| Correlative Studies Blood Collection      | X6                                                 | X6                                   | X6                        | X6                    |    |    |  
| Screening7                    | Treatment                                          | Follow Up                            |                           |                       |
|-------------------------------|----------------------------------------------------|--------------------------------------|---------------------------|-----------------------|
| Pre                               |                                                    |                                      |                           |                       |
| Surgery  Screening Surgery    | TMZ+Pembrolizumab +/- HSPPC-96/Vaccine  (6 cycles) |                                      |                           |                       |
| Time Period                   | RT + TMZ +                                         |                                      |                           |                       |
| Pembrolizumab16               | HSPPC-96/Vaccine Induction or Break14 (4 weeks)    | End of Treatment  or Discontinuation | 30 days  after last  dose | Long-term 8 Follow-up |
| +/-14 of  signing consent     |                                                    |                                      |                           |                       |
| SchedulingWindow (Days)       | - 7                                                | - 7                                  | +/- 3                     | Every 8-16            |
| (before treatment)            | (before treatment)                                 | weeks                                |                           |                       |
| Drug Administration           | X15                                                | X10                                  | X10                       |                       |
| Assess and  document Survival | X                                                  |                                      |                           |                       |
| Status                        |                                                    |                                      |                           |                       |  
1Includes vital signs (pulse, respirations, blood pressure) and height (baseline only) and weight.  
2 Serum or urine test for females of child-bearing potential prior to the start of RT+TMZ+Pembro and at the start of every cycle.  
3 By MRI (unless contraindicated): For subjects not randomized: An MRI will be done pre surgery, post surgery (within 72 hours), 4 weeks post RT, and then every 9 weeks +/- 1 week. For subjects who are randomized: An MRI will be done pre surgery, post surgery (within 72 hours), within +/- 10 days of last dose of vaccine or placebo induction, and then every 9 weeks +/- 1 week. This schedule of MRIs is consistent with standard of care.  
4 A CBC with differential will be done weekly during RT and then weekly after RT for the first year and then every 2 weeks for the year after if the patient remains on Pembrolizumab.  
5 Chemistry should be done at beginning of RT, Day 21 of RT +/- 3 days, at completion of RT, and on Days 1(+/- 1 day) and Day 29
(- 7 days) of each cycle.  
6 Randomized - 8x8mL CPT blue and black top tubes (LabConnect Kit B) will be used to collect correlative blood samples at the following time points for patients randomized to study vaccine or placebo: 1) Pre-RT treatment (RT+TMZ+Pembro) 2) Post-RT treatment (RT+TMZ+Pembro)/Pre-vaccine (or placebo) induction 3) Post Vaccine (or placebo) induction/Pre-cycle 1 maintenance 4) Every Day 1 on Cycle 2, C4, C6, C8 (if continuing on Pembrolizumab); then, at discretion of Study Chair and BTTC Coordinating Center, every other cycle; 5) At time of progression or at End of Treatment. Ancillary - 8x8mL CPT blue and black top tubes (LabConnect Kit B) will be used to collect correlative blood samples at the following time points for patients not randomized (ancillary cohort): 1) Pre-RT treatment (RT+TMZ+Pembro) 2) /Pre-cycle 1 maintenance 3) Every Day 1 on Cycle 2, C4, C6, C8 (if continuing on Pembrolizumab); then, at discretion of Study Chair and BTTC Coordinating Center, every other cycle 4) At time of progression, or at End of Treatment.  
7 Screening labs will be completed within (+/-) 14 days of signing consent.  
8 Patients will be followed for overall survival. Patients withdrawn from treatment will follow the same schedule.  
9 Comprehensive Serum Chemistry panel=Na, K, CL, Bicarbonate, BUN/Cr, AST, ALT, ALP T. Bili, Calcium, Albumin.  
55 Version Date: *06/30/2021* 10 HSPPC-96 Vaccine/Placebo given weekly post RT x 4 weeks, then 21 days (+/- 24 hours) after the day 5 and day 33 doses of TMZ
of each cycle. TMZ doses Cycle 1, days 1-5 at 150 mg/m2. If tolerated, the dose will be escalated to 200 mg/m2 for all remaining doses (This would begin with Cycle 1 days 29-33.). Pembrolizumab dosed on day 1, 22, and 43 (+/- 24 hours for each timepoint) of RT then day 1, 22, 43 (+/- 24 hours for each timepoint) of each 9 week cycle.  
11 MDASI-BT assessments will take place along with imaging studies. MDASI-BT will need to be given prior to informing the patients about the results of the imaging study, within +/- 1 week of the MRI assessment date.  
12 Assess patients for adverse events during Physical Exam and treatment days. 13 Physical, KPS and neurological exam every 3 weeks (- 3 days to + 7 days) during RT and then every 4 weeks (+/- 7 days) after RT.  
If needed, telehealth assessments will be allowed for the physical, KSP & neurological exams. For additional details on allowed telehealth assessments, please see Section 3.9.  
14 For those not randomized (ancillary arm), no drug administration during the 4 weeks prior to Pembro and TMZ, therefore no laboratory tests or scheduled assessments are required.  
15 TMZ may be given night before RT (within 24 hours before day 1).  
16 Baseline assessments will be done prior to the start of RT+TMZ+Pembro (see 3.7).
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3.12 surgical guidelines

Surgical guidelines will be according to Standard of Care at each institution.
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3.13 cost and compensation 3.13.1

Subjects costs will be based on local guidelines as described in the site specific consent.
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3.13.3 reimbursement

Reimbursement is based on site policy. At the NCI site, the NCI will cover the costs of some expenses associated with protocol participation. Some of these costs may be paid directly by the NIH and some may be reimbursed to the participant/guardian as appropriate. The amount and form of these payments are determined by the NCI Travel and Lodging Reimbursement Policy.
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3.14 criteria for removal from

Patients can be taken off the protocol therapy and/or study as a whole at any time at their own request, or they may be withdrawn at the discretion of the investigator for safety, behavioral or administrative reasons. Prior to documenting removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy. The following evaluation will occur:  
- KPS
- Toxicity Assessment - Physical & Neurological Exam
- Vital Signs and Weight - CBC with Diff, Comprehensive Chemistry Panel, T3, T4, and TSH - Tumor Imaging - MDASI-BT - Correlative Blood Collection
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3.14.1 criteria for removal from

The reason(s) for discontinuation must be clearly documented on the appropriate eCRF and may include:  
- Patient voluntarily withdraws from treatment (follow-up permitted)
- Patient demonstrates disease progression after beginning treatment
- Patient experiences unacceptable toxicity
- Treating physician determines that continuation with treatment on the study would not be in the patient's best interest
- Patient develops a second malignancy that requires treatment which would interfere with this study
- Patient becomes pregnant
- Patient completes treatment as outlined in section 3.3
- Screen failure
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3.14.2 off study criteria

The reason(s) for discontinuation must be clearly documented on the appropriate eCRF and may include:
- Screen failure - Tumor is identified as a methylated MGMT promoter
- Tumor is not IDH wildtype by pathology
- Investigator decision to end study
- Patient withdraws consent (no follow-up permitted)
- Patient is unable to comply with protocol requirements - Patient becomes lost to follow-up (LTF)
- Death
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3.14.3 lost to follow-up

A participant will be considered lost to follow-up if he or she fails to return for 3 scheduled visits and is unable to be contacted by the study site staff. The following actions must be taken if a participant fails to return to the clinic for a required study visit:
- The site will attempt to contact the participant and reschedule the missed visit within 24 hours, and reschedule within 7 days and counsel the participant on the importance of maintaining the assigned visit schedule and ascertain if the participant wishes to and/or should continue in the study.  
- Before a participant is deemed lost to follow-up, the investigator or designee will make every effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, a certified letter to the participant's last known mailing address or local equivalent methods). These contact attempts should be documented in the participant's medical record or study file.  
- Should the participant continue to be unreachable, he or she will be considered to have withdrawn from the study with a primary reason of lost to follow-up.
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4 concomitant medications/vaccinations (allowed &

Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed during the ongoing trial. If there is a clinical indication for one of these or other medications or vaccinations specifically prohibited during the trial, discontinuation from trial therapy or vaccination may be required. The investigator should discuss any questions regarding this with the coordinating center. The final decision on any supportive therapy or vaccination rests with the investigator and/or the subject's primary physician.
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4.1 acceptable concomitant medications

All treatments that the investigator considers necessary for a subject's welfare may be administered at the discretion of the investigator in keeping with the community standards of medical care. All concomitant medication will be recorded on the case report form (CRF) including all prescription, over-the-counter (OTC), herbal supplements, and IV medications and fluids. If changes occur during the trial period, documentation of drug dosage, frequency, route, and date may also be included on the CRF. All concomitant medications received within 28 days before the first dose of trial treatment and 30 days after the last dose of trial treatment should be recorded. Concomitant medications administered after 30 days after the last dose of trial treatment should be recorded for SAEs and ECIs as defined in Section 8.1.
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4.2 prohibited concomitant medications

Subjects are prohibited from receiving the following therapies during the Screening and Treatment Phase (including retreatment for post-complete response relapse) of this trial:
- Antineoplastic systemic chemotherapy or biological therapy
- Immunotherapy not specified in this protocol
- Chemotherapy not specified in this protocol - Investigational agents other than pembrolizumab and HSPPC-96
- Live vaccines within 30 days prior to the first dose of trial treatment and while participating in the trial. Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella/zoster, yellow fever, rabies, BCG, and typhoid vaccine.  
- Systemic glucocorticoids for any purpose other than to modulate symptoms from an event of clinical interest of suspected immunologic etiology. The use of physiologic doses of corticosteroids may be approved after consultation with the Sponsor. The exception is for cerebral edema, but dose should be < 4 mg once treatment begins.  
- Bevacizumab can be used to control edema, but not as a biologic treatment intended for the patient's disease under study.  
Subjects who, in the assessment by the investigator, require the use of any of the aforementioned treatments for clinical management should be removed from the trial. Subjects may receive other medications that the investigator deems to be medically necessary. The Exclusion Criteria describes other medications which are prohibited in this trial. There are no prohibited therapies during the Post-Treatment Follow-up Phase.
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4.3 diet/activity/other considerations 4.3.1 diet

Subjects should maintain a normal diet unless modifications are required to manage an AE such as diarrhea, nausea or vomiting.
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4.3.2 contraception

Pembrolizumab may have adverse effects on a fetus in utero. Furthermore, it is not known if pembrolizumab has transient adverse effects on the composition of sperm. For this trial, male subjects will be considered to be of non-reproductive potential if they have azoospermia (whether due to having had a vasectomy or due to an underlying medical condition). Female subjects will be considered of non-reproductive potential if they are either: (1) postmenopausal (defined as at least 12 months with no menses without an alternative medical cause; in women < 45 years of age a high follicle stimulating hormone (FSH) level in the postmenopausal range may be used to confirm a post-menopausal state in women not using hormonal contraception or hormonal replacement therapy. In the absence of 12 months of amenorrhea, a single FSH measurement is insufficient.);