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). 3.6.2 For patients randomized to vaccine or placebo 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). 40 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 3.7 BASELINE ASSESSMENTS (P OST-S URGERY AND P RE-TREATMENT RT+TMZ+P EMBRO) 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). 3.8 ASSESSMENTS WHILE ON TRIAL D URING AND P OST RT oPatients 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. oPatients on ancillary arm do not require vaccine associated physical exam. oAdverse events will be assessed during Physical Exams and on treatment days. oFor 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. oFor 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. oPregnancy test will be done at the start of each cycle. oA 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. 41 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 oA 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. oBlood 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), Post- RT 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). oArchival tissue for PDL1 expression and mutation analysis will be collected after surgery irrespective of whether the patient is randomized or not. oIf a patient has tumor progression while on trial and undergoes surgery, patients will have their tumor evaluated for PDL1 expression and repeat mutational analysis. 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 MDASI- BT 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.