19 3 LIST OF TABLES Table 1. Objectives and Endpoints ............................................................................................................. 21 Table 2. Screening and On -Study Tests and Procedures .......................................................................... 31 Table 3. Conditions under Which Accrual will be Suspended .................................................................... 42 Table 4. Probability of Accrual Suspension as a Function of the True Unacceptable Toxicity Rate .......... 42 Table 5: Temozolomide Dosing Interruption or Discontinuation during Concomitant Radiotherapy .......... 80 Table 6. Dose Modifications of Temozolomide during Radiation Therapy ................................................. 80 Table 7. Criteria to Resume Temozolomide after Radiation ....................................................................... 81 Table 8. Temozolomide Dose Delay, Reduction, or Discontinuation During 21- day Treatment ................ 81 Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 6 CONFIDENTIAL 4 LIST OF ABBREVIATIONS 2HG R-2-Hydroxyglutarate Ab Antibody ABC Automated Blood Count ACD Acid Citrate Dextrose ACLS Advanced Cardiac Life Support ACTH Adrenocorticotropic Hormone AE Adverse Event αKG Alpha -ketoglutarate AML Acute M yeloid Leukemia APAAP Alkaline Phosphatase Antialkaline Phosphatase Complex ALT Autologous Lymphocyte Transfer ANC Absolute Neutrophil Count AST Aspartate Aminotransferase AT Ambient Temperature BBB Blood Brain Barrier β-HCG Beta-Human Chorionic Gonadotropin BMT Bone Marrow Transplant BTC Brain Tumor Center BUN Blood Urea Nitrogen Ca++ Calcium caBIG® Cancer Biomedical Informatics Grid CAMRD Center for Advanced MR Development CAR Chimeric Antigen Receptor CBC Complete Blood Count CCL3 C-C motif Chemokine Ligand 3 cDNA Complimentary Deoxyribonucleic Acid CFA Complete Freund’s Adjuvant CFC Cytokine Flow Cytometry CLIA Clinical Laboratory Improvement Act CMP Comprehensive Metabolic Panel CNC Clinical Neurologic Change CNS Central Nervous System CPC Cancer Protocol Committee CIMP CpG-Island Methylator Phenotype CRF Case Report Form CT Computed Tomography CTCAE Common Terminology Criteria for Adverse Events CTL Cytotoxic T -Lymphocyte DAR Drug Accountability Record DBTIP Duke Brain Tumor Immunotherapy Program DC Dendritic Cell DCCC Duke Comprehensive Cancer Center DCI Duke Cancer Institute DOB Date of Birth DSMB Data and Safety Monitoring Board DSMP Data and Safety Monitoring Plan DTH Delayed -type Hypersensitivity DUHS Duke University Health System EAE Experimental Autoimmune Encephalomyelitis EBRT External Beam Radiation Therapy eCRFs Electronic Case Report Forms ELISA Enzyme -Linked ImmunoSorbent Assay Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 7 CONFIDENTIAL ELISPOT Enzyme -linked Immunospot EGFR Epidermal Growth Factor Receptor EGFRvIII EGFR variant III FACS Fluorescence Activated Cell Sorting FDA Federal Drug Administration FEV Forced Expiratory Volume GBM Glioblastoma GCP Good Clinical Practice GLP Good Laboratory Practice GM-CSF Granulocyte Macrophage Colony Stimulating Factor HIV Human Immunodeficiency Virus HLA Human Leukocyte Antigen HMG Hemoglobin HMO Health Maintenance Organization HPLC High Pressure Liquid Chromatography IBA Ion Beam Applications IC Intracerebral(ly) ICF Informed Consent Form ICH International Conference on Harmonization IDH1 Isocitrate D ehydrogenase 1 IEC Independent Ethics Committee IFA Incomplete Freund’s Adjuvant IFN-γ Interferon -gamma IHC Immunohistochemistry IL-4 Interleukin -4 IL-12 Interleukin -12 IL-13 Interleukin -13 IRB Institutional Review Board KLH Keyhole Limpet Hemocyanin KPS Karnofsky Performance Status LAL Limulus Amoebocyte Lysate LGG Low Grade Glioma MAb Monoclonal Antibody MedDRA Medical Dictionary for Regulatory Activities MG Malignant Glioma MHC Major Histocompatibility Complex MRI Magnetic Resonance Imaging MRN Medical Record Number mRNA Messenger Ribonucleic Acid MRS Magnetic Resonance Spectroscopy MTD Maximally Tolerated Dose NA Non-adherent NCI CTC National Cancer Institute Common Toxicity Criteria NIH National Institutes of Health NK Natural Killer OARC Office of Audit, Risk, and Compliance OS Overall Survival PBL Peripheral Blood Lymphocyte PBMC Peripheral Blood Mononuclear Cells PBS Phosphate -Buffered Saline PET Positron Emission Tomography PD Progressive Disease PHA Phytohemagglutinin Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 8 CONFIDENTIAL PFS Progression Free Survival PI Principle Investigator PRTBTC Preston Robert Tisch Brain Tumor Center PT Prothrombin Time PTT Partial Thromboblastin Time PTMs Post-Translational Modifications QC/QA Quality Control/Quality Assurance R-2HG R-2-hydroxyglu tarate RANO Revised Assessment in Neuro -Oncology RDSPs Research Data Security Plans RECIST Response Evaluation Criteria in Solid Tumors reGBM Recurrent Glioblastoma reLGG Recurrent Low Grade Glioma RIO Research Integrity Office RNA Ribonucleic Acid RT Radiation Therapy RT-PCR Reverse Transcriptase Polymerase Chain Reaction SAE Severe Adverse Event scFV Single Chain Variable Antibody Fragment SFC Spot-Forming Cells SOC Standard of care SOP Standard Operating Procedure SPECT Single Photon Emission Computed Tomography TCA Trichlor oacetic Acid TCR T cell Receptor Td Tetanus -diphtheria toxoid TGF-β Transforming Growth Factor -β TMZ Temozolomide TNF-α Tumor Necrosis Factor -α Tregs Regulatory T cells TTP Time to Progression TTRNA Total Tumor mRNA VDLNs Vaccine -site Draining Lymph Nodes WHO World Health Organization WOCBP Women Of Childbearing Potential XRT Radiation Therapy Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 9 CONFIDENTIAL 5 PROTOCOL SYNOPSIS AND SUMMARY Please see separate document (available upon request). Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 10 CONFIDENTIAL 6 STUDY SCHEMA Figure 1. Study Schema Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 11 CONFIDENTIAL 7 BACKGROUND AND SIGNIFICANCE 7.1 Study Diseas e Malignant primary brain tumors are the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma.3 Gliomas are the most common primary brai n tumors with GBMs, astrocytomas and oligodendrogliomas comprising the large majority of subtypes.4 Primary GBM, WHO grade IV, occurs de novo and is the most common and aggressive glioma with a median OS of <15 months .5 Patients with lower grade II or III astrocytomas, oligodendrogliomas or mixed gliomas have a more favorable OS. However, lower grade astrocytomas can progress to secondary GBM and malignant progression is assured in almost all cases with patients succumbing to disease within a decade. Moreover, current therapy for malignant primary brain tumors is incapacitating6 and limited by non-specific toxic ity to systemic tissue or surrounding eloquent brain. In contrast, immunotherapy promises an exquisitely precise approach and substantial evidence suggests that immunotherapeutic intervention can eradicate well -established tumors in mice and humans, even w hen tumors reside within the “immunologically privileged” brain. (See Figure 2, Figure 3, and Figure 4)1,2,7 ,8 There i s a clear and urgent need for the development of targeted therapeutics that can improve outcomes for patients with glioma without adding significant toxicity to current treatment regimens. Immunotherapy targeting tumor -associated antigens holds significant promise to meet this need. Figure 2. Highly reactive transferred cells traffic to brain and destroy tumor deposit in melanoma patients. Arrows represent location of melanoma metastases. (From Johnson et al ., Blood 2009)1 Figure 3. Host -derived antitumor T cells treat lesions in the brain. Arrows represent location of metastases. (A&C) Pretreatment (B) 14 months after cell transfer (D) 6 m onths after cell transfer. (From Hong et al., Clinical Cancer Research)2 Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 12 CONFIDENTIAL Figure 4. Genetically engineered antitumor T cells treat lesions in the brain Arrows represent location of metastases. (A&C) Pretreatment (B) 6 months after cell transfer (D) 30 months after cell transfer. (From Hong et al., Clinical Cancer Research)2 7.2 Study Agents The study agent will consist of a 25 amino acid peptide (PEP IDH1M) and the adjuvant s GM-CSF and Montanide ISA -51. 7.2.1 PEP IDH1M PEP IDH1M is a 25 amino acid peptide that spans the mutated region of IDHR132H. 7.2.2 GM-CSF Recombinant GM- CSF, Leukine® (sargramostim), is a normal human cytokine that increases the number and function of granulocytes (neutrophils, eosinophils, and basophils) and monocytes. It has been FDA-approved for clinical use for more than a decade. It is usually used follo wing induction chemotherapy in older adult patients with AML to shorten time to neutrophil recovery , but has also been used previously as a vaccine adjuvant .9 It is contra- indicated in patients with excessive leukemic myeloid blasts (which we would not expec t in the disease under study in this protocol) , patients with known hypersensitivity to GM - CSF, yeast -derived products, or any component of Leukine. Serious allergic or anaphylactic reactions have been reported with Leukine. 7.2.3 Montanide ISA 51 (Incomplete Fr eund’s Adjuvant) Montanide ISA 51 or Incomplete Freund’s Adjuvant (IFA) is a water -in-oil (w/o) emulsion with immunomoadjuvant activity. Montanide ISA 51 enhances the immune system’s CTL response against antigen(s) in vaccines. Peptide vaccinations employing GM- CSF and Montanide ISA -51 as adjuvants have generally been well tolerated in human patients in numerous phase I -III trials . 7.2.4 Tetanus -diphtheria toxoid (Td) The current use of Td toxoid is for active immunization in children and adults against infection with the bacteria Clostridium tetani and Corynebacterium diphtheria. Tetanus infection is manifested primarily by neuromuscular dysfunction caused by a potent exotoxin released by C. tetani. Diphtheria is an acute toxin- mediated infectious disease caused by toxigenic strains of C. diphtheriae. Protection against disease is due to the development of neutralizing antibodies to the diphtheria toxin. Td toxoids adsorbed are readily available as several approved administrations [i.e. Daptacel (DTaP), Inf anrix (DTap), Tenivac (Td adult), Boostrix (Tdap) 10. Protection against disease is due to the development of neutralizing antibodies to the tetanus toxin. A serum tetanus antitoxin level of at least 0.01 IU/mL, measured by neutralization assays, is considered the minimum protective level. A level ≥0.1 IU/mL by ELISA has been considered as protective 11. A serum diphtheria antitoxin level of 0. 01 IU/mL, measured by neutralization assays, is the lowest level giving some degree of protection; a level of 0.1 IU/mL by ELISA is regarded as protective. Diphtheria antitoxin levels ≥1.0 IU/mL by ELISA have been associated with long- term protection. Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 13 CONFIDENTIAL Following deep s.c./i.m. administration of the tetanus toxoid vaccine, toxoid molecules are taken up at the vaccination site by immature DCs, which are professional antigen- presenting cells. Within these cells, they are processed through the endosomal pathway (involving the phagolysosome) where they are bound to MHC type II molecules on the surface of DCs. The MHC II:toxoid complex then migrates to the cell surface. While this process is happening within the cell, the now activated mature DC at the vaccine site migrates along lymph channels to the draining lymph node where they encounter naive TH2 cells, each with their own unique TCR. Identifying and then binding of the MHC II:toxoid to the specific TH2 receptor then activates the naive T cell, causing it to proliferate. Simultaneously, toxoid molecules not taken up by DCs pa ss along lymph channels to the same draining lymph nodes where they come into contact with B cells, each with their own unique B -cell receptor (BCR). Binding to the B cell through the specific immunoglobulin receptor that recognizes tetanus toxoid results in the internalization of toxoid, processing through the endosomal pathway and presentation on the cell surface as an MHC II:toxoid complex, similarly to DCs undergoing the same process 12. These two processes occur in the same part of the lymph node with the result that the B cell with the MHC II:toxoid complex on its surface now comes into contact with the activated TH2 whose receptors are specific for this complex. The proc ess, termed linked recognition, results in the TH2 activating the B cell to become a plasma cell with the production initially of IgM, with a later switch to IgG antibodies produced. Additionally, a subset of these B cells becomes memory cells12. The novelty of using Td toxoid vaccination lies in the ability of this potent recall antigen to enhance antitumor responses as part of a cancer vaccination protocol. Td toxoid induces an inflammatory milieu within the intradermal vaccine site, thereby promoting the migration of injected tumor -specific DCs. Additionally, in the context of vaccinating the host with tumor -derived peptides, conditioning the vaccine site with Td toxoid has demonstrated enhanced immunogenicity with these peptides. Our data from the ATTAC clinical trial (Duke IRB Protocol # Pro00003877) demonstrating the capacity to enhance DC migration to VDLNs via Td pre- conditioning of the vaccine site offer potential therapeutic interventions whereby we can enhance the immunologic responses to ultimately overcome the inherent challenges in faithfully eradicating established tumors. In a completed randomized clinical trial, we found that migration of injected DCs to VDLNs following vaccine site pre- conditioning with Td toxoid was significantly increased compared to controls and that the efficiency of DC migration was strongly associated with clinical outcomes of patients with newly -diagnosed GBM, the most fatal type of mali gnant brain tumors 13. To address this observation, we took our Td pre- conditioning platform back into the preclinical setting using transgenic mouse models and were able to corroborate the effects of Td pre- conditioning on increasing the lymph node homing of intradermally administered DCs. Moreover, Td administration at a single vaccine site increases the migration of a bilateral DC vaccine to both inguinal lymph nodes. Regardless of the side of the Td intradermal skin prep, DC migration to bilateral inguinal VD LNs was equally increased, supporting a systemic response to recruit peripherally administered DCs. Our Td pre- conditioning platform in the context of DC vaccination also elicited superior anti -tumor responses compared to controls receiving DC vaccines without Td pre- conditioning. In our clinical trial, patients with newly -diagnosed GBM who were administered the Td skin prep before DC vaccination revealed significantly longer progression- free and overall survival rates compared to the control cohort. In evaluating the relationship between DC migration and clinical responses, we observed a modest positive correlation between levels of DC migration and survival. In our preclinical model, Td pre- conditioning prior to vaccination with tumor antigen- specific D Cs dramatically suppressed the growth of established and highly aggressive B16- F10/OVA tumors. The use of Td with a DC vaccine increased antitumor responses in an antigen- specific manner, as non- specific DC vaccines were not potentiated with Td pre- conditi oning. Furthermore, in a challenge setting, where mice are administered the treatment platform prior to challenge with tumor inoculation, Td pre- conditioning at the vaccine site induced a significant survival benefit compared to controls. For this clinical trial evaluating PEPIDH1M, we are also proposing to use Td as an adjuvant based on the pre- clinical data described below. Here, mice were immunized with 25mer mutant and wildtype peptides with two clinically relevant adjuvants: granulocy te-macrophage colony stimulating factor (GM- CSF), a Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 14 CONFIDENTIAL cytokine known to have a principle role in the activity of antigen presenting cells, and imiquimod, a topical TLR7 agonist administered at the vaccine site and tetanus -preconditioning. Immunogenicity of each adjuvant/vaccine combination was measured via IFNγ ELISpot; administration of imiquimod at the site of vaccination results in a more robust mutant -specific immune response compared to GM- CSF, Notably, immunogenicity of the mutant peptide in mice that received imiquimod emulsified in IFA/IFA/IFA is comparable to the immune response seen in mice that received CFA/IFA/IFA, a treatment course with widely known strong adjuvant activity ( Figure 5). Figure 5. Tetanus pre conditioning, imiquimod and GM -CSF increases the number of IDH1 mutant specif ic T cells after vaccination. Mice were immunized with (three weekly 100 ug of peptide emulsified in IFA/IFA/IFA), and immunogenicity was measured via IFNγ ELISPOT against both the wild- type and mutant 25mer peptides. 7.2.5 Pre-clinical experience We have developed a functional murine homologue (Figure 6) of the human IDH1R132H mutant enzyme, cloned syngeneic murine gliomas that express this mutation, mapped peptide sequences specific to this mutation in either species (Figure 7), and demonstrated mutation- specific immunogenicity of these peptides (Figure 8 and Figure 9). Preliminary ELIS pot data from our laboratory demonstrates that splenic lymphocytes isolated from mice immunized with peptides of various lengths containing the IDH1R132H mutation (PEP IDH1M) are activated by mutant but not wild type peptide, demonstrating both specificity and immunogenicity ( Figure 10). Vaccination with PEP IDH1M in 3 different strains of mice has produced no toxicity. Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 15 CONFIDENTIAL Figure 6. The murine homologue of the IDH1R132H mutation is functional. Murine cells were transduced with the IDH1R132H mutation or left untransduced as controls. Whole cell extracts were submitted for quantification of R -2-hydroxyglutarate by LC -negative electrospray ionization- MS/MS. Values were normalized to total protein levels in each sample. Figure 7. Human IDH1R132H protein sequence and human and mouse wild type sequences. The mutation is highlighted in yellow. Purple highlights nearest sequence difference between human (S) and mouse (T) protein which is not included in the 25mer IDH1R132H (PEPIDH1M) peptide. PEPIDH1M (red text) is identical between human and mouse. Figure 8. PEPIDH1M vaccination is immunogenic and specific in several mouse strains. Different mouse strains were immunized weekly for 3 weeks with PEPIDHM1 in CFA. After 7 days splenic lymphocytes were derived and stimulated with mutant or wildtype IDH1 peptide and IFNy secretion was assessed by ELISpot. *p<0.05 by t -test Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 16 CONFIDENTIAL Figure 9. PEPIDH1M vaccination is immunogenic and specific in the context of human HLA. HLA- A2 Transgenic mice were immunized weekly for 3 weeks with 25mer or 9mer length peptides in CFA containing PEPIDHM1. 9mer peptides consisted of a peptide mix of 9 different peptides all spanning the IDH1R132H mutation. After 7 days, splenic lymphocytes were derived and stimulated with mutant or wild type IDH1 25mer peptide and IFNy secretion was assessed by ELISpot. Figure 10. Splenocytes from mice immunized with different lengths of mutant IDH1 peptides. Mice were immunized weekly for 3 weeks with different length peptides in CFA containing the IDH1R132H mutation. After 7 days, splenic lymphocytes were derived and stimulated with the 25mer mutant peptide (PEP IDH1M) or wildtype IDH1 peptide and IFNy secretion was assessed by ELISpot. 7.2.6 Clinical experience This is a Phase I safety study. PEP IDH1M has not been used in humans previously. Recombinant GM- CSF is FDA-approved for human use. Our previous experience with a peptide vaccine corresponding to a similar tumor specific mutation, EGFRvIII , in clinical trials has shown that a dose of 500 µg of peptide with 150 µg of GM- CSF yields effective immunity against tumors in the brain without any toxicity . EGFRvIII is a tumor -specific mutation of the epidermal growth factor receptor that is expressed in primary GBMs and other common neoplasms.14 Because the wild type sequences of human and mouse overlap adjacent to the EGFRvIII mutation, we Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 17 CONFIDENTIAL were previously able to create a murine homologue of this mutation to test its immunogenicity without concerns that the results would be influenced by xenogeneic responses .15 Using this approach, w e demonstrated that a peptide vaccine (PEPvIII) specific for the tumor -specific mutation EGFRvIII is capable of eliciting EGFRvIII -specific immune responses in mice with orthotopic gliomas .16 This was possible despite these mice exhibiting pr ofound, tumor -induced immunosuppressive deficits that mirror those seen in humans with malignant brain tumors.9,16-18 Moreover, these immune responses were capable of eliminating EGFRvIII -expressing tumor cells in these tumors and extending survival of tumor -bearing mice.16 When translated into humans with EGF RvIII-expressing GBM , similar results were seen.9,16-18 Despite no evidence of EGFRvIII -specific immune responses prior to vaccination, EGFRvIII- specific immune responses were gener ated in almost all patients and EGFRvIII -expressing tumor cells were nearly universally eliminated without attendant toxicity.16-18 Based on these data, this EGFRvIII- targeted vaccine is now being tested in an international Phase III trial (NCT01480479) . Unfortunately, EGFRvIII is heterogeneously expressed and tumors recur as a result of outgrowth of the EGFRvIII negative tumor cells.9 Montanide ISA 51 has proved to be a very efficient adjuvant, activating the cellular and the humoral immune response. It has been administered to more than 10,000 patients throughout the world. Our overall goal is to utilize our vast experience in peptide- based immunotherapy to provide a PEP IDH1M vaccine that is both safe and immunogenic in patients wit h IDH1 mutant -expressing WHO Grade II gliomas . 7.3 Study Purpose and Rationale 7.3.1 Temozolomide, Lymphopenia, and Homeostatic Proliferation TMZ is an alkylating chemotherapeutic that has recently been shown to prolong survival in patients with GBM .5 As a result, it has become part of the standard regimen used to treat these patients . TMZ induces a transient lymphopenia, and although counterintuitive, we and others have shown that following periods of lymphopenia, immune responses can be markedly enhanced.19-23 Probably as a result of a surge in cytokines (IL- 7, IL- 15) in response to lymphopenia, lymphocytes undergoing homeostatic proliferation enjoy a reduced activation threshold,24,25 differentiate di rectly into effector memory T -cells capable of rapid and intense response to antigen,26 display increased expression of anti -apoptotic molecules, and are less sensitive to immunosuppressive NK cell -mediated ly sis.26 Still, lymphocytes must encounter their cognate antigen and compete for limiting amounts of these homeostatic cytokines to proliferate under thes e conditions .25 Thus, B - or T-cells specific for antigens that predominate during this recovery period, like those provided in the form of a vaccine, have a competitive advantage and become disproportionately over - represented in the recovering lymphocyte population both in murine models19,27 and in humans .23 These skewed homeostatic responses have been shown to enhance antitumor immunity19,27,28 but can also increase the risk of autoimmunity . Figure 11. Lymphocyte counts of patients with newly -diagnosed GBM receiving standard dose adjuvant TMZ (200 mg/m2/5d) and dose -intensified TMZ (100 mg/m2/21d). Standard dose TMZ induces Grade 2 lymphopenia in 100% of patients (<800 cells/uL; blue line) and transient Grade 3 lymphopenia in 40% of patients. Sustained Grade 3 lymphopenia (<500 cells/uL; red line) was induced by dose- intensified TMZ cycle #3 in all patients. Vaccine 1 Vaccine 2 Vaccine 3Pre-TMZ TMZ Cycle-1 TMZ Cycle-2 TMZ Cycle-3 TMZ Cycle-4 TMZ Cycle-5 TMZ Cycle-6010020030040050060070080090010001100120013001400150016001700 Standard Dose TMZ (200mg/m2/5d) (N=5) Dose-Intesified TMZ (100mg/m2/21d) (N=8) Grade 3 (<500 cells/ µL)Grade 2 (<800 cells/ µL)Absolute Lymphocyte Count (Day 21) (cells/µL blood) Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 18 CONFIDENTIAL Leveraging this principle, Rosenberg and colleagues have produced dramatic clinical responses21,29 -32, along with some autoimmune toxicity, in patients with advanced malignant melanoma.20,22 After lymphodepletion, T -cells can expand dramatically and tumor -specific T -cells can constitute up to 90% of the host’s T -cell repertoire and can be maintained for months ,20,33 and in these studies clinical regressions correlate with the frequency of tumor -spec ific T -cells achieved in the peripheral blood and persistence of these cells in vivo .21,29-32,34-36 TMZ produces a survival benefit in patients with GBM and has become a routine part of the therapy for these patients. Due to the known myelosuppressive and immunosuppressive effects of TMZ37-40 and its potential negative implications for immunotherapy targeting GBMs, we evaluated the impact of TMZ treatment, in the context of our EGFRvIII peptide vaccine trial (now CDX -110, Pfizer, Inc.), on the lymphocyte compartment and immunologic responses of patients with newly -diagnosed GBM un dergoing two different dose regimens of adjuvant TMZ. These regimens were evaluated in a randomized, Phase III trial (RTOG 052541). After concurrent TMZ with EBRT, patients received TMZ for either 5 days (200 mg/m2) or 21 days (100 mg/m2) of each 28 day cycle. 100% of patients receiving the standard 5 day schedule exhibited Grade 2 lymphopenia (<800 cells/uL) ( Figure 11) with nadirs occurring 14- 21 days after the first dose (n=5). Grade 3 lymphopenia was observed in only 1 patient with this regimen. Sustained Grade 3 lymphopenia (<500 cells/uL) was induced in all patients receiving the 21 day regimen, however, by the fifth cycle of TMZ . Despite the profound lymphopenia, we have been able to induce and maintain potent EGFRvIII -specific immune responses (Figure 12 and Figure 13) in patients with GBM receiving serial cycles of TMZ .42 Figure 12. Antibody titers in patients duri ng standard -dose TMZ (200 mg/m2/5d) and dose -intensified TMZ (100 mg/m2/21d) . All patients receiving the 21 day dose regimen developed EGFRvIII -specific antibodies with titers detectable at greater than 1:100,000 dilution. Humoral and cellular immune responses have been dramatically better in patients receiving the higher TMZ dose. Vaccine 3 Vaccine 4 Vaccine 5 Vaccine 6 Vaccine 7 Vaccine 810,000100,0001,000,000 200 mg/m2/5d100 mg/m2/21d1 / TiterP=0.0093, Two-way ANOVA, repeated measures, For InteractionACTIVATE: 7/15 ACT II: 15/15 (P= 0.0022, Fisher’s exact) Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 19 CONFIDENTIAL Vaccine DTH Response Pre Vaccine Dose 1 TMZ Dose 3 TMZ Dose 5 TMZ020,00040,00060,00080,000100,000 Standard 200mg/m2/5dUndectable UndectableDose-Intensified 100mg/m2/21dmm squaredCandida DTH Response Pre Vaccine Dose 1 TMZ Dose 3 TMZ Dose 5 TMZ02,0004,0006,0008,000 Standard 200mg/m2/5d UndetectableDose-Intensified 100mg/m2/21dmm squared Figure 13. DTH responses to EGFRvIII vaccination. DTH responses to EGFRvIII vaccination (Left) in patients receiving lymphodepletive TMZ (100 mg/m2/d x 21 days) cycles (black) were increased when compared with standard dosing (200 mg/m2/d x 5 days) with continued vaccination. DTH responses to the recall antigen Candida (Right) in patients receiving lymphodepletive TMZ (1 00 mg/m2/d x 21 days) cycles (black) were decreased when compared with standard dosing (200 mg/m2/d x 5 days) with continued cycles of TMZ. 7.3.2 Peptide Vaccines for Cancer Peptide vaccines encoding minimal CD8+ CTL epitopes have been demonstrated in many contexts to induce protective immunologic responses in experimental animals and mediate regressions of established tumors .43,44 However, with few several notable exceptions ,36,45-48 vaccine -based cancer immunotherapy in humans is in need of significant improvem ent in immunogenicity and clinical outcomes .21 The addition of CD4+ T cell helper epitopes to peptide vaccine formulations is widely believed to be an advantageous strategy for enhancing anti -tumor immunity .49 This presumption is supported by the observation that CD4+ T cell directed therapy can mediate effective anti -tumor immunity in humans and experimental animal s.50-52 CD4+ T helper cells deliver assistance for CD8+ effector cells by fully activating dendritic cells through the CD40- CD40L signaling pathway .53 However, vaccination with T helper epitope encoding peptides has yielded mixed results in human clinical trials as a method to enhance anti -tumor cytotoxic T -cell responses in vivo50,54-56 and thus warrants systematic evaluation. Recent studies have highlighted the use of longer peptides (15- 30 amino acids in length) instead of minimal CTL epitopes (typically 8- 10 amino acids) for induction of a more robust, long lived CD8+ cytotoxic T-cell responses in experimental animals .36,57 The expanded length of the peptide requires it be processed by antigen -presenting cells. Minimal CTL epitopes do not require processing by antigen- presenting cells and thus are free to bind directly to T -cell receptors. This can induce tolerance due to a lack of co- stimulatory molecules that are presented to T -cells during antigen presentation by dendritic cells, however this area of peptide formulation requires further evaluation.58 Peptide vaccination in humans has exhibited an excellent safety profile with virtually no dose limiting toxicities .36,56 Most toxicities occur when peptide vaccines are used in conjunction with immunostimulatory cytokines such as IL- 2, which are expected results of IL- 2 therapy .59 This feature of peptide vaccinations allows it to be used in combinatorial immunotherapeutic strategies and/or as salvage therapies in resistant disease.60,61 Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 20 CONFIDENTIAL 7.3.3 Summary of Study Purpose/Rationale Primary brain tumors are the most frequent cause of cancer death in children and young adults. GBM, the most malignant primary brain tumor, has a median OS of <15 months,5 and patients with lower grade gliomas progress to this universally lethal tumor type within ten years. Moreover, current therapy for these tumors is incapacitating6 and limited by non- specific toxicity. In contrast, immunotherapy promises an exquisitely precise therapeutic approach, and substantial evidence suggests that activated T-cells can eradicate large tumors, even within the “immunologically privileged” brain.1 EGFRvIII is a tumor -specific mutation that is expressed in primary GBMs.14 We have previously demonstrated that EGFRvIII -specific immu ne responses could be generated in mice, and this predicted the ability to generate similar immune responses in humans.9,16-18,62 These were sufficient to eliminate orthotopic gliomas expressing a murine homologue of EGFRvIII in mice and were suffici ent to consistently eliminate all EGFRvIII -expressing tumors cells in mice and humans without toxicity.9,17,63,64 Based on these observations, our EGFRvIII -targeted vaccine is now being tested in an international Phase III trial. Unfortunately, EGFRvIII is heterogeneously expressed and tumors recur as a result of outgrowth of the EGFRvIII negative tumor cells.9 Recently, unbiased, genome wide sequencing has been shown to be a powerful approach for identifying other recurring mutations like EGFRvIII. Using next -generation sequencing, we discovered tumor -specific mutations exclusively in codon 132 of the active site of IDH165,66 – an evolutionarily - conserved enzyme essential to cell function.67 Further study revealed that IDH1 mutations occur in >70% of almost all glioma subtypes, except primary GBM. Greater than 90% of all IDH1 mutations occur from a substitution of histidine for arginine resulting in the highly conserved and tumor -specific mutation, IDH1R132H.68 Immunohistochemical analysis shows that IDH1R132H is homogeneously expressed in all tumor cells, including single infiltrating tumor cells69 but is absent in normal cells.66,70 The high frequency, specificity, and homogeneous expression of the IDH mutation make it an ideal target for therapeutic intervention. Although IDH mutations can be seen in tumors with longer overall survival,71 IDH status has been occasionally misundersto od to function as an inhibitor of tumor growth. Rather it denotes a genetically distinct subset of tumors. Within those tumors, however, IDH mutations are oncogenic and cause grossly aberrant methylation as well as impaired differentiation that promotes tumorigenesis.72-78 While wildtype IDH1 converts isocitrate to α KG as part of cellular respiration, IDH1R132H mutations impair normal function resulting in the conversion of αKG into the onco- metabolite R -2HG.76,79 R-2HG impairs αKG-dependent histone and DNA demethylases resulting in a global epigenetic CIMP, obstructed differentiation, and tumorigenesis.72-78 Mutant enzyme i nhibition in gliomas using recently discovered small molecule inhibitors, while inhibiting tumor growth slightly, does not induce apoptosis , and tumor cells maintain logarithmic growth.76 Thus enzyme inhibition may only be partially effective as a therapeutic approach.72,76,80 In contrast, T cells targeted to these tumor -specific mutations are agnostic to the biologic function of the mutation and kill cells expressing the mutation indiscriminately . Our ability to eliminate EGFRvIII positive cells in GBM using a vaccine strategy similar to this proposal, also argues strongly for the potential of IDH -targeted immunotherapy. Moreover, the homogeneous expression of IDH1R132H and the apparent dependence on IDH1R132H for tumorigenicity may protect against the antigen escape seen with EGFRvIII -targeted vaccines. Here we propose to leverage our experience targeting tumor -specific antigens to assess the potential of an IDH1 mutation- specific vaccine as a treatment for patients with tumors containing IDH1R132H mutations. Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 21 CONFIDENTIAL 8 OBJECTIVES AND ENDPOINTS Table 1. Objectives and Endpoints Objective Endpoint Analysis Primary To assess the safety of the PEP IDH1M vaccine alone and in combination with adjuvant TMZ and/or XRT/TMZ in adult patients with progressive, resectable WHO grade II gliomas that are confirmed Grade II or transition to a higher grade glioma at the time of surgery. The percentage of patients who experience an unacceptable toxicity defined as any Grade 3 toxicity at least possibly attributed to the vaccine (or vaccine + TMZ and/or RT ) that does not resolve to baseline with in 3 week s, any Grade 3 hypersensitivity reactions requiring steroids, any Grade 4 toxicity, including neurologic events not due to progressive disease, or any life threatening- event not attributable to concomitant medication, co- morbid event, or disease progression . See Section 15.4 Secondary To assess the immunogenicity of the PEP IDH1M vaccine with adjuvant TMZ using ELISpot . Mean difference in the number of SFC per 106 lymphocytes for cells cultured with and without IDH1 peptide See Section 15.5 Exploratory To describe PFS and OS . Proportion of patients alive without disease progression 6 months after initial vaccine treatment; m edian progression- free survival and median overall su rvival. See Section 15.6 Exploratory To determine if ELISpot results after vaccination predict PFS or OS. Number of SFC per 106 lymphocytes above pre- vaccine baseline is a predictor of PFS or OS See Section 15.6 Exploratory To estimate radiographic response rate prior to surgical resection and PFS after resection using RANO criteria. Proportion of patients with radiographic response (complete + partial response) observed prior to surgical resection. Proportion of patients alive without disease progression 6 months after resection. See Section 15.6 Exploratory To determine if tumors are IDH1 negative by immunohistochemical analysis and microarray analysis at the time of post-vaccine surgery. Proportion of tumors that are IDH1 negative at the time of surgery See Section 15.6 Exploratory To characterize immunologic cell infiltrate in tumors at the time of post-vaccine surgery. Median number of immunologic cell infiltrates at the time of surgery See Section 15.6 Exploratory To determine if tumor -specific circulating DNA containing the c.395G>A (R132H) mutation can be detected in plasma before, during, and after study drug treatment. Detection of t umor -specific circulating DNA containing R132H mutation. Mean changes in R132H mutation from baseline. See Section 15.6 Exploratory To determine if 2 -HG, a metabolite specifically produced by tumors with IDH R132H, can be detected in plasma by mass spectrometry before, during, and after study drug treatment. Detection of 2 -HG in plasma. Mean changes in 2- HG in plasma from baseline. See Section 15.6 Exploratory To explore the impact of PEP IDH1M vaccination on genetic alternations, differential protein expressions or post -translational modifications (PTMs) using quantitative proteomic strategies in patient -derived samples. Changes between baseline and surgery in genetic alternations, differential protein expressions and post -translational modifications (PTMs) See Section 15.6 Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 22 CONFIDENTIAL Exploratory To demonstrate quantitative repeatable measures of metabolism change in IDH1+ gliomas via a standardized MRS protocol, patients will be scanned on the research MR scanner to establish same- day and longitudinal metabolite coefficient of variance and possible longitudinal metabolite changes. Difference in levels of metabolism measured at same assessment. Changes from baseline in metabolism levels at each follow -up assessment. See Section 15.6 9 INVESTIGATIONAL PLAN 9.1 Study D esign Subjects with radiographic and/or clinical progressive and resectable Grade II glioma with expression of IDH1R132H from the primary tumor (initial diagnosis ) will be enrolled after signing informed consent. After consent has been signed, all subject s will undergo standard of care vaccination with 0.5 mL of Td (tetanus and diphtheria toxoids adsorbed) intramuscularly (I.M.) into the deltoid muscle to ensure adequate immunity to the tetanus antigen. The tetanus vaccine will be obtained through Duke ICS Pharmacy . Eligible subjects will undergo a 1- 2 hour leukapheresis for immune monitoring. Within 48 hours of leukapheresis, subjects will receive a vaccine site pre- conditioning as a single dose of Td toxoid (1 flocculation unit, Lf, in a total volume of 0.4 mLs saline). This will be administered one day prior to receiving PEP IDH1M vaccine intradermally (i.d.) to the RIGHT gro in area (as described below). The peptide vaccine is administered in the groin area approximately 10 cm below the inguinal ligament as vaccine # 1. Subsequent vaccines will be given on day 15 ±3 days (vaccine #2) and day 29 ±3 days (vaccine #3). These injections will occur without TMZ. Each injection of the peptide vaccine will be given half on the right groin and half on the left groin. Seven to 12 days after the 3rd vaccine, subjects will undergo surgical resection of tumor , which is part of the SOC for these subjects . If possible, the provider will take a photo of the injection site at the pre- op appointment seven to twelve days after the 3rd vaccine to document the subject’s reaction to the vaccine. Tumor samples from the surgical resection will be evaluate d as outlined in the Exploratory Objectives. Based on tissue obtained at surgical resection, s ubjects with stable histologic grade at recurrence will then be treated with vaccine and adjuvant TMZ at a targeted dose of 50- 100mg/m2/d for 21 days every 28 days for up to 12 cycles (please see Section 18.5 in the Appendices for TMZ) . During monthly cycles of TMZ, subjects will receive the vaccine on day 22 (+ 2 days ) for a maximum of 15 total vaccines (which includes the first 3 bi -weekly vacci nes). Patients that have transitioned to a higher Grade brain tumor will receive TMZ and radiation therapy per standard of care and monthly vaccines . All Adverse Events will be collected from time of consent until the end of the treatment phase of the study. The treatment phase of the study will end 1 month after the last vaccine (Section 12.3) . Patients will be followed only for overall survival , progression- free survival, and subsequent therapies thereafter. For higher grade gliomas undergoing RT (please see Section 18.4 in the Appendices for SOC RT ), vaccine #4 will be given during the SOC clinic visit prior to initiating RT, and vaccine # 5 will be given 4 weeks later (± 1 week) during RT. Vaccine #6 will be given at the end of RT during the SOC clinic visit to review MRI and begin post -RT TMZ, if MRI stable. Then, subjects in this treatment group will receive vaccines monthly on day 22 (+2 days ) with post -RT cycles of TMZ to a maximum of 15 vaccines . For immune monitoring and biomarker testing, subjects will have blood collected, as described in Section 12, during the following times: prior to vaccine therapy (at leukapheresis visit), prior to surgical resection (at the pre- op screening visit) , prior to either RT/TMZ or first cycle of adjuvant TMZ (at SOC clinic visit) depending on tumor pathology , at vaccine # 6, and at End of Treatment (follow -up visit 1 month af ter vaccine #15; If a subject decides to stop participating in the study prior to vaccine #15, blood will be obtained at that time, if possible ; If a subject progresses prior to vaccine #15, blood will be obtained at that time, if possible) .For genomic pr oteomic analyses, blood and tumor samples will be obtained as described below in Section 9.4. Subject s will be imaged with contrast -enhanced MRI according to the SOC every 10 weeks (+/ - 4 weeks) while on TMZ and afterward, per the treating neuro- oncologist’s recommendation. RANO criteria will be used for assessment of pseudoprogression and tumor progression (please see Section 12.7.3 for further detail) . Subjects demonstrating definitive progression will be removed from study. Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 23 CONFIDENTIAL In addition, to demonstrate quantitative repeatable measures of 2HG in IDH1+ gli omas, subjects will be imaged using magnetic resonance spectroscopy (MRS) at baseline (prior to vaccine #1 following consent), and up to 4 times prior to surgery (please see Section 9.5 below for further information). As part of standard care for these subject s, upon tumor progression, participants may undergo stereotactic biopsy or resection. As this is not a research procedure, consent will be obtained separately . Subjects that have this procedure done within the Duke University Health System may be approached to participate in the Duke Brain Tumor Center Biorepository study (Pro00007434) . Tissue obtained from subjects who consented to the Duke Brain Tumor Center Biorepository will be used to assess immunologic cell infiltration , antigen expression, and biomarkers for immunologic response. 9.1.1 Definition of Unacceptable Toxicity Toxicities will be graded according to the NCI CTCAE version 4 criteria. An unacceptable toxicity is defined as any Grade 3 toxicity at least possibly attributed to the vaccine (or vaccine + TMZ +/- RT) that does not resolve to baseline with in 3 week s, any Grade 3 hypersensitivity reactions requiring s teroids, any Grade 4 toxicity, including neurologic events not due to progressive disease, or any life– threatening event not attributable to concomitant medication, co- morbid event, or disease progression. Please refer to the Potential Risks Section 12.8.2 of this protocol. 9.1.2 Concomitant Medications Concomitant medications will be managed by the treating neuro- oncologist and recorded at each study visit by the study team . 9.2 Rationale for Selection of Dose, Regimen, and Treatment Duration The PEP IDH1M vaccine dose that will be evaluated in this trial will be 500 µg with 150 µg of GM- CSF mixed 1:1 with Montanide ISA 51 administered intradermally as vaccine # 1. Subsequent vaccines will be given on day 15 ±3 days (vaccine #2) and on day 29 ±3 days (vaccine #3) . The same dose will be administered on day 22 (+ 2 days) of each monthly cycle of adjuvant TMZ following surgical resection for those with confirmed Grade II . For those who transition to a higher grade glioma, they will be undergoing RT (please see Section 18.4 in the Appendices for SOC RT) , thus, vaccine #4 will be given during SOC clinic visit prior to in itiating RT, and vaccine # 5 will be given 4 weeks later (± 1 week) during RT. Vaccine #6 will be given at the end of RT during the SOC clinic visit to review MRI and begin post -RT TMZ, if MRI stable. Then, subjects in this treatment group will receive v accines monthly on day 2 2 (+ 2 days) with post - RT cycles of TMZ to a maximum of 15 vaccines, as the confirmed Grade IIs. The nearly universal expression of IDH1 in secondary GBMs and low -grade gliomas may make excellent anti -tumor immunotherapeutic targets . Vaccination and adoptive T -cell strategies in humans in other contexts have been safe and effective. Therapeutic TMZ induces a profound lymphopenia that may enhance anti -tumor vaccination responses when given during the homeostatic T -cell prolifer ation that occurs in response to lymphodepletion. Other peptide vaccines given to patients with high- grade gliomas during recovery from TMZ -induced lymphopenia have produced potent tumor -specific immune responses and have been well tolerated9,17,81. 9.3 Rationale for Correlative Studies While the primary goal of this study is to assess safety of the PEP IDH1M vaccine, a secondary endpoint will be to determine whether this vaccine is capable of eliciting an immune response in patients with IDH1 mutation- expressing tumor . The primary measure of immunogenicity will be by ELISpot to the immunizing peptide . For the immune system to recognize the tumor specific IDH1 mutant, this mutant peptide must be presented by human leukocyte antigens (HLA) on the surface of a tumor cell for recognition by a T cell. There are a tremendous number of HLA alleles with variable expression from person to person. As certain peptides are able to bind with much greater affinity to certain HLA alleles, the ability of one individual to present the IDH1 mutant peptide will be dependent upon their unique set of HLA alleles and may differ greatly from another individual with differing HLA. Therefore, it is critical to know the HLA type of a patient receiving the peptide vaccine as this may reveal if certain HLA types are associated with enhanced vaccine induced immune responses. Such information may suggest certain HLA types present the mutant peptide in a superior fashion and may be valuable in stratifying patients for clinical trials using this vaccine. We Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 24 CONFIDENTIAL intend to identify HLA types from PBMCs taken at the leukapheresis time point. The purpose of collecting these data will be to determine whether there may be immune correlates that associate with clinical outcomes such as PFS and OS following vaccination with the PEP IDH1M vaccine . Although this Phase I study is not designed to be definitive t owards this end, these analyses will provide the initial data necessary to adequately power future studies . Any evidence of tumor response will be determined according to the Duke PRTBTC SOP ( available upon request ). RANO criteria82 will be used for assessment of pseudoprogression. Tumor progression will need to be documented histologically, unless there are clinical contraindications, to exclude inflammatory responses presenting as radiographic or clinical changes, which could indic ate potentially toxic or therapeutic responses and not tumor progression. Patient s will be followed until death. 9.4 Rationale for Genomic Proteomic A nalyses IDH1 mutation is a critical factor in glioma classification and a key determinant of clinical outcom e. First, though the effect of mutant IDH1 in tumor cells have been well -established at the genetic and epigenetic levels (e.g., closely associated with several other genes, and has a profound impact in DNA methylome), the effect of the IDH1 mutant on the proteomic level is unclear, thus presenting a great opportunity for understanding glioma pathogenesis and developing new therapeutics. Second, in addition to IDH1 mutations, other genetic alterations and their associated mutant peptides may serve as multi variant factors underlying patient treatment outcome. It is critical to have a bird’s eye view of and dissect all the genetic and peptide components that help stratify patients for clinical trials. We intend to identify genetic alterations, differential protein expressions or post-translational modifications (PTMs) via our comprehensive ‘inside- out’ quantitative proteomic strategies in patient -derived tumor samples. Genetic alterations, differential protein expressions or PTM s that have been identified wi ll be further tested in all patients enrolled in this clinical trial. We propose to obtain the following specimens, from patients both before and after treatment, for both genomic and proteomic analyses. (1) Tumor tissues – 500 mg fresh frozen tumor tissue wi ll be requested, as well as tissues from previous surgery. If frozen tissues from previous surgery are not available, fifteen 5 μm sections of FFPE samples are request ed. If available, t hese samples will be used for genomic and proteomic analysis. (2) PBCs – to determine proteomic profiling with a focus on immune system, and also for immune monitoring and tumor circulating DNA detection. Twenty mLs of peripheral blood (3 yellow top tubes) is requested (1) before vaccine with the leukapheresis visit , (2) at the pre-op screening visit before surgery, (3) At the SOC clinic visit prior to starting the 1 st cycle of TMZ for those who are confirmed Grade II, or at the SOC clinic visit prior to starting RT/TMZ for those who have transitioned to a higher grade glioma, ( 4) Vaccine #6, and (5) End of Treatment (follow -up visit 1 month after vaccine #15). If a subject decides to stop participating in the study prior to vaccine #15, blood will be obtained at that time (if possible). If a subject progresses prior to vaccine #15, blood will be obtained at that time (if possible) . PBMCs and plasma will be separated. Plasma will used for the detection of IDH1 mutation before surgery. These experiments will: - Identify genetic alterations and changes in protein before and after treatment with peptide vaccine in tumor cells. - Identify proteins showing differential expressions or PTMs in immune cells, with and without IDH1 mutant, and before and after IDH1 vaccination, with a potential of revealing how immune cells response to IDH1 mutant, before and following vaccination. We will perform proteomics, exome sequencing and RNA -sequencing on the resected tumors (after three IDH1 vaccines). We will use patient samples before IDH1 vaccination as controls. Radiographic and clinical tumor progress, as well as IDH1 mutation status in patient’s plasma will be recorded. 9.5 Single-Voxel MR Spectroscopy To demonstrate quantitative repeatable measures of metabolism change in IDH1+ gliomas, patients will be scanned on the research MR scanner using standardized MRS protocol to assess same- day repeatability and to assess longitudinal metabolite changes. Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 25 CONFIDENTIAL The IDH1 mutation causes extreme overexpression of the onco- metabolite R -2-hydroxyglutarate (2HG) in all tumor cells. This can be non- invasively measured using clinical MRS methods. 2HG is a specific marker for IDH1 mutated gliomas because its concentration in ‘wild’ IDH1 gliomas is below the level of measurement of MRS. Duke Radiology has a standard protocol for 2HG measures using a proven 2HG MEGA- LASER pulse sequence for Siemens MR scanners and Dr. Soher’s open source “Vespa” software for spectral simulation and fitting of the MEGA -LASER data. The protocol is available for immediate use on our research scanner as a part of the Duke Radiology’s Center for Advanced MR Development ( CAMRD ). We will assess subject s at up to 5 time points: baseline (after consent and before PEP IDH1M vaccine #1), and up to 4 times prior to surgery. This will provide between 6- 8 weeks of tumor/vaccine interaction. A typical 2HG protocol consists of 12- 15 minutes of anatomical MRI and ~ 15-30 minutes of MRS data acquisition. An IDH1+ patient can usually be processed in a 1 -2 hour time slot. In order to establish same- day coefficients of variance and/or optimized acquis ition parameters , patients may be scanned twice within a time point . Patients will be positioned and scanned in the MR scanner, then completely removed and put back into the scanner, and then positioned and scanned a second time. If necessary for patient c omfort, this evolution can be done immediately or with a short rest period so long as scanning is finished within the booked period of time. Longitudinal acquisitions will also be analyzed for longitudinal metabolite coefficients of variance and possible l ongitudinal metabolite changes. 9.6 Definition of Evaluable Subjects, On Study, and End of Study For the initial monitoring of adverse events (see Section 15.4.2), evaluable patients will include the first 6 accrued patients who received 3 vaccinations or experienced an unacceptable toxicity due to one of the first 3 vaccinations. For the final analys is of the primary objective (See section 15.4.2), evaluable patients will include all patients who received at least 6 vaccinations or experienced an unacceptable adverse event that was relate d to vaccine (or vaccine + TMZ) . Once the patient signs ICF, that subject will be considered “on study .” Rationale for taking patient off protocol treatment will be documented. End of study is defined as 2 months following last vaccine for purposes of toxicity monitoring. 9.7 Early Study Termination This study can be terminated at any time for any reason by the PI -sponsor . If this occurs, all subjects on study should be notified as soon as possible. Additional procedures and/or follow up should occur in accordance with Section 12.5. Section 12.6 describes procedures and process for prematurely withdrawn patients. Pro00054746: RESIST Study Version: 20171011 PT-PhI-II v05.22.12 Duke Cancer Institute Page 26 CONFIDENTIAL 10 STUDY DRUG 10.1 Names, Classification, and Mechanism of Action The name of the drug will be PEP IDH1M vaccine . The class of action is a biological . 10.2 Packaging and Labeling Name MRN DOB Drug: PEP IDH1M Lot #: Lot 001 Caution New Drug Limited By Federal Law To Investigational Use 10.3 Supply, Receipt, and Storage The PEP IDH1M will be manufactured under GMP conditions by Bachem Americas, Inc., and stored in the investigational pharmacy. Montanide is supplied by Seppic and stored at 4OC in the investigational pharmacy. GM-CSF (LEUKINE®; sargramostim) will be obtained from commercial supply, as a sterile, white, preservative- free powder lyophilized powder in a vial containing 250 mcg to be reconstitut ed in 0.5 mL of sterile water for injection. Store GM- CSF under refrigeration at 2- 8°C (36- 46°F). Vials must not be frozen. Lyophilized GM- CSF vials contain no antibacterial preservative, and therefore solutions prepared with Sterile Water for Injection, USP should be administered as soon as possible, and within 6 hours following reconstitution. The vial should not be re- entered or reused. Do not save any unused portion for administration more than 6 hours following reconstitution. Bacteriostatic Water for Injection, USP (0.9% benzyl alcohol) should NOT be used. Aseptic technique should be employed in the preparation of all GM- CSF solutions. To assure correct concentration following reconstitution, care should be exercised to eliminate any air bubbles from the needle hub of the syringe used to prepare the diluent. During reconstitution of lyophilized GM- CSF, the diluent should be directed at the side of the vial and the contents gently swirled to avoid foaming during dissolution. PEP IDH1M will be stored at -80OC, thawed, and inspected visually for particulate matter and discoloration prior to administration. If particulate matter is present or the solution is discolored, the vial should not be used. Before use, the vial should be gently swirled to ensure uniform mixing of the contents.