22 3 LIST OF TABLES Table 1. Objectives and Endpoints ............................................................................................... 24 Table 2. Storage of Study Agents ................................................................................................. 31 Table 3. Dispensing of Study Agents ........................................................................................... 31 Table 4. Schedule of Events .......................................................................................................... 36 I-ATTAC Version: 20220204 8 4 LIST OF ABBREVIATION S Ab Antibody ABC Automated Blood Count ACD Acid Citrate Dextrose ACLS Advanced Cardiac Life Support ACT H Adrenocorticotropic Hormone AE Adverse Event AIDS Acquired Immune Deficiency Syndrome ALT Adoptive Lymphocyte Transfer APAAP Alkaline Phosphatase Antialkaline Phosphatase Complex ANC Absolute Neutrophil Count AST Aspartate Aminotransferase AT Ambient Temperature AUC Area Under the Curve BCR B Cell Receptor β-HCG Beta-Human Chorionic Gonadotropin BMT Bone Marrow Transplant BTIP Brain Tumor Immunotherapy P rogram BTSC Brain Tumor Stem Cells Ca++ Calcium caBIG Cancer Biomedical Informatics Grid CAP College of American Pathologists CBC Complete Blood Cou nt CCL3 Chemokine (C -C motif) ligand 3 cDNA Complimentary Deoxyribonucleic Acid CFA Complete Freund’s Adjuvant CFC Cytokine Flow Cytometry cGMP Current Good Manufacturing Practice CLIA Clinical Laboratory Improvement Act CLN Cervical lymph Nodes Cmax Maximum Concentration of Drug in Plasma or Serum CMP Comprehensive Metabolic Panel CMV Cytomegalovirus CNC Clinical Neurologic Change CNS Central Nervous System COI Conflict of Interest Con-A Concanavalin A CPC Cancer Protocol Committee CT Comp uted Tomography CTL Cytotoxic T -Lymphocyte DAR Drug Accountability Record DC Dendritic Cell DCI Duke Cancer Institute DI-TMZ Dose -Intensified Temozolomide DLT Dose Limiting Toxicity DNA Deoxyribonucleic Acid DOB Date of Birth DSMB Data Safety Moni toring Board DSMP Data Safety and Monitoring Plan DTH Delayed -type Hypersensitivity I-ATTAC Version: 20220204 9 DTPA Diethylene Triamine Penta-acetic Acid DUHS Duke University Health System eCRF Electronic Case Report Form IEC Institutional Ethics Committee EAE Experimental A utoimmune Encephalomyelitis EBRT External Beam Radiation Therapy ELISA Enzyme -Linked ImmunoSorbent Assay ELISPOT Enzyme -linked Immunospot EGFR Epidermal Growth Factor Receptor EGFRvIII Epidermal Growth Factor Receptor variant type III EGFRvIII -KLH EGFRvIII conjugated to Keyhole Limpet Hemocyanin FACS Fluorescence Activated Cell Sorting FDA Federal Drug Administration FEV Forced Expiratory Volume FFPE Formalin -Fixed, Paraffin -Embedded GMP Good Clinical Practice GBM Glioblastoma GM-CSF Granuloc yte Macrophage Colony Stimulating Factor HAMA Human Anti -Murine Antibody H&E Hematoxylin and Eosin HIV Human Immunodeficiency Virus HLA Human Leukocyte Antigen HMO Health Maintenance Organization HRPP Human Research Protection Program I.C. Intrace rebral ICF Informed Consent Form ICH International Conference on Harmonisation ICS Investigational Chemotherapy Services ID Identification I.D. Intradermal IDH Isocitrate Dehydrogenase IE1 Immediate Early Gene 1 P rotein IFN-γ Interferon -gamma IgG Immunoglobulin G IgM Immunoglobulin M IHC Immunohistochemistry IL-2 Interleukin -2 IL-4 Interleukin -4 IL-12 Interleukin -12 IL-13 Interleukin -13 I.M. Intramuscular In Indium IND Investigational New Drug iRANO Immun otherapy Response Assessment in Neuro -Oncology IRB Institutional Review Board ISH In Situ Hybridization IT Information Technology IUD Intra -Uterine Device I.V. Intravenous KLH Keyhole Limpet Hemocyanin KPS Karnofsky Performance Status LAMP Lysosomal -associated Membrane Protein Lf Flocculation unit I-ATTAC Version: 20220204 10 LIMS Laboratory Information Management System MAb Monoclonal Antibody MFI Median Fluorescence Index MG Malignant Glioma MGMT Methylguanine Methyltransferase MHC Major Histocompatibility Complex mL MilliLiter MMSE Mini -Menta l Status Examination MPACT Molecular Products and Cellular Therapies MRI Magnetic Resonance Imaging mRNA Messenger Ribonucleic Acid MTD Maximally Tolerated Dose NA Non-adherent NCI CTC National Cancer Institute Common Toxicity Criteria NCI CTCAE National Cancer Institute Common Toxicity Criteria of Adverse Events ng NanoGram NIH National Institutes of Health NK Natural Killer OARC Office of Audit, Risk and Compliance OS Overall Survival OT-I Ovalbumin -specific T cells OVA Ovalbumin PBLs Perip heral Blood Lymphocytes PBMC Peripheral Blood Mononuclear Cells PBS Phosphate Buffered Saline PCR Polymerase Chain Reactions PD Progressive Disease PHA Phytohemagglutinin PFS Progression Free Survival PI Principle Investigator PO By Mouth PRTBTC Preston Robert Tisch Brain Tumor Center PT Prothrombin Time PTT Partial Thromboblastin Time QA/QC Quality Assuran ce/Quality Control RDSP Research Data Security Plan RIO Research Integrity Office RNA Ribonucleic Acid RT Radiation Therapy RT-PCR Rever se Transcriptase Polymerase Chain Reaction SAE Severe Adverse Event S.C. Subcutaneous SFC Spot-Forming Cells SOC Standard of Care SOCOMM Safety Oversight Committee SOP Standard Operating Procedure SPECT Single -Photo Emission Computerized Tomograph y TCR T cell Receptor TD Tetanus -Diphtheria TGF -β Transforming Growth Factor -β TH2 T helper type 2 TILs Tumor -Infiltrating Lymphocytes TMZ Temozolomide I-ATTAC Version: 20220204 11 TNF -α Tumor Necrosis Factor -α TRegs Regulatory T cells TTP Time to Progression TTRNA Total T umor mRNA VDLNs Vaccine -site Draining Lymph Nodes WBI Whole Body Irradiation WBRT Whole Brain Radiation Therapy WHO World Health Organization 5 PROTOCOL SYNOPSIS AND RESEARCH SUMMAR Y Research Summary is available upon request. I-ATTAC Version: 20220204 12 6 STUDY SCHEMA Figure 1. Study Schema Newly diagnosed GBM CMV+, unmethylated Resection Enrollment † Leukapheresis* † DC generation Failed QC – Patient replaced Standa rd of care external beam radiation (XRT) with concurrent TMZ (as per SOC) Standard of care single cycle DI TMZ DC vaccine #1* – 3 CMV pp65 RNA -pulsed DCs on Days 23, 37, and 51 of this cycle Leukapheresis* Skin preparation* with Td (1 Lf) on Day before Vaccine #4 DC vaccine #4* Continue with vaccines #5 – #10 CMV DCs every month Radiographic or clinical tumor progression* Stereotactic biopsy or resection Immunohistochemical studies *Immunologic monitoring from each leukapheresis, prior to vaccines #1 and #4 -10; and prior to Td pre -conditioning and at progression Safety monitoring throughout DC vaccination †Patient may enroll and undergo leukapheresi s under an umbrella study (Pro00102797), instead of i -ATTAC. If so, enrollment on i - ATTAC will occur after DC generation, RT/TMZ, and single cycle TMZ before 1st DC vaccine. I-ATTAC Version: 20220204 13 7 BACKGROUND AND SIGNI FICANCE 7.1 Study Disease Malignant primary brain tumors are more common than Hodgkin’s disease and account for more human deaths than melanoma or than cancer of the bladder or kidney. Despite aggressive, computer- guided tumor resection [ 1], high- dose external beam radiation therapy (XRT) , and multi - mechanistic chemotherapy delivered at toxic doses, most patients with malignant primary brain tumors live < 15 months from the time of diagnosis, and patients with recurrent tumors usually survive < 12 weeks [ 2-7]. The estimated cost of treatment for each patient with a malignant brain tumor is between $30,000 and several hundred thousand dollars annually. Thus, the annual treatment cost alone for these patients, not mention the lost earning potential of afflicted individuals, is greater than the entire annual budget of the National Institute of Neurological Diseases and Stroke. In fact, conventional therapy for patients with malignant brain tumor s is the most expensive medical therapy per quality -adjusted life -years (QALYs) saved currently provided in the United States [ 8, 9]. Moreover, the non- specific nature of standard of care therapy for brain tumors often results in incapacitating damage to surrounding normal brain and systemic tissues [10, 11]. Thus, in order to be more effective, therapeutic strategies need to precisely target tumor cells while minimizing collateral damage to neigh boring eloquent cerebral cortex. The rationale for employing the immune system to target brain tumors is based on the premise that the inherent biologic specificity of immunologic reactivity could meet the urgent need for more specific and less toxic therapy . 7.1 Study Agent Human Cytomegalovirus pp65 - Lysosomal-A ssociated M embrane Protein mRNA -Pulsed Autologous D endritic C ell Vaccines C ontaining Granulocyte Macro phage-Colony Stimulating F actor Dendritic cells ( DCs) are potent immunostimulatory cells that continuously sample the antigenic environment of the host and specifically activate CD4+ and CD8+ T cells and B cells [12, 13]. They are at the crossroads of many of the elegant networks of the immune system, and DCs represent the most promising contemporary biologic entity for realizing the promise of immunotherapy. Potent immune responses and encouraging cli nical results have been seen in Phase I and II human clinical trials in systemic cancers [14-30] . Adjuvants frequently used with vaccination include Freund’s incomplete adjuvant, bacilli Calmette -Guerin, QS -21, and diphtheria toxoid. Supplemental cytokines have been used as well for the adjuvant immunological effects [ 31]. Granulocyte macrophage -colony stimulating factor (GM -CSF) has been commonly used, as it is commercially available and well tolerated. GM -CSF is capable of stimulating macrophage function, inducing proliferation and maturation of DCs , and is able to enhance T cell stimulatory function. Intradermal administration of GM -CSF enhances the immunization efficacy at the site of administration in a dose -dependent fashion at an optimal dose of 125 µg [32]. Significant anti- tumor immunity has been demonstrated in preclinical murine studies in which irradiated, stably transfected tumor cell lines secreting GM -CSF have protected against subsequent tumor challenge, especially against intracerebral tumors [ 33, 34]. Furthermore, the potency of GM -CSF has been demonstrated i n a Phase I clinical trial in melanoma patients vaccinated with irradiated autologous melanoma cells engineered to secrete GM -CSF [35]. The I-ATTAC Version: 20220204 14 immunization sites were intensely infiltrated with T cells , DCs, macrophages, and eosinophils in 100% of evaluable patients. Extensive tumor destruction was seen in 11 of 16 patients. Both cytotoxic T -cell and antibody responses were associated with this tumor destruction. Hence, GM - CSF has an extensive track record both as a growth factor and an adjuvant, is commercially available and has an acceptable toxicity profile. In a recently published study from our laboratory, we reported that immediately following a course of do se-intensified temozolomide ( DI-TMZ), given over 21 consecutive days, the administration of three sequential Cytomegalovirus (CMV )- specific DC vaccines resulted in significantly increased CMV -specific cellular responses and superior overall survival (OS) . Median progression free survival (PFS) and OS for this cohort were 25.3 months [95% confidence interval (CI), 11.0–∞ ] and 41.1 months (95% CI, 21.6– ∞), exceeding projected survival using recursive partitioning analysis (RPA) and matched historical controls . Moreover, f our patients remained progression- free at 59 to 64 months from diagnosis. No known prognostic factors (i.e. age, KPS, IDH -1/2 mutation, and MGMT promoter methylation ) were biased to predict more favorable outcomes for the patients in this cohort [36]. Human CMV is an endemic β-Herpesvirus that does not usually cause significant clinical disease [37]. During primary maternal infection, however, human CMV can cause severe enc ephalitis in fetuses and lead to congenital brain defects . Human CMV disease is also a significant problem in immunocompromised adults such as organ transplant recipients or patients with Acquired Immune Deficiency Syndrome ( AIDS ) [37]. Herpesviruses have also been implicated in a number of human malignancies including lymphoma, nasopharyngeal cancer, cervical cancer, and Kaposi’s sarcoma [38, 39]. Recently, expression of proteins unique to human CMV has been reported within a large proportion of malignant tumors including colorectal carcinoma, prostate cance r, and malignant astrocytomas [40-42]. Universal detection of the human CMV immunodominant protein pp65, immediate early gene 1 protein ( IE1), and several other early antigens was demonstrated using immunohistochemistry ( IHC) in Grade II -IV astrocytomas [42]. Presence of the virus in these samples was confirmed with in situ hybridization (ISH), polymerase chain reactions (PCR) for human CMV -specific glycoprotein B ( UL55), electron microscopic detection of intact virions [42], and di rect detection of the virus from fresh operative samples in the shell vial assay (unpublished data) . Notably human CMV antigens were not detected in surrounding normal brain samples, meningiomas, or brains affected by ischemia, Alzheimer’s disease, paraneoplastic encephalitis, or Cryptococcal cerebritis . The presence of highly -immunogenic human CMV antigens within malignant gliomas ( MGs ) affords a unique opportunity to target these tumors immunologically . There is a vast amount of experience with both the safety and efficacy of immunotherapy targeting human CMV [37], and the presence of this virus within brain tumors may allow this experience to be leveraged toward the effective eradication of MG expr essing human CMV antigens . Adoptive T cell therapy has been used to safely and successfully protect against CMV reactivation in myelodepleted bone marrow transplant ( BMT ) patients [43-46]. In addition, T cell mediated immunotherapy has proven highly effective in the treatment of CMV -associated disease within the central nervous system ( CNS ) [47] and in the treatment of acute CMV infections [44, 48]. Tumors associated with other human Herpesviruses , such as Epstein- Barr virus -associated lymphoma, including tumors within the CNS, have also been effectively treated and even large tumors have been cured by immunotherapy [49-54 ]. More recently, a vaccine directed against the potent viral antigens of I-ATTAC Version: 20220204 15 human papilloma virus has also been shown to reduce the incidence of human papilloma virus - related cervical intraepithelial neoplasia in a prospective, randomized, double- blind trial [ 55]. The potential for non- specific targeting of normal tissues is thought to be minimal in seropositive patients . After initial infection, CMV establishes lifelong latency in the infected individual, with cells of the myeloid lineage constituting a major reservoir for persistence of the virus . Virus can be detected within myeloid progenitors in the bone marrow, with a small portion of these cells demonstrating viral deoxyribonucleic acid (DNA) replication without any detectable gene expression [56, 57]. Also a small proportion ( typically 1 in 1,000 to 1 in 10,000) of peripheral blood monocytes can be found to contain CMV DNA, while detection of viral ribonuclei c acid (RNA) ( gene expression) is not detected [58-60] . Vaccination specifically against CMV [61-65] has effectively reduced the risk of viral infection and transmission to fetuses in animal models [66-68] and in clinical trials [61, 65, 69-72] . Human clinical trials have also demonstrated some benefit of administering neutralizing antibodies in the treatment of human CMV infection [73-76] , highlighting the importance of the development of vaccination strategies that elicit both cellular and humoral immune responses . DCs strongly activate both T cell and B cell responses in vivo [13], and DCs pulsed in vitro with CMV antigens have been shown to be potent inducers of CMV -specific cytotoxic T -lymphocyte (CTL ) responses in several studies [77-81] , in addition to our own work which is outlined below. The use of RNA to encode tumor antigens for DCs was pioneered at Duke University in Dr. Gilboa’s laboratory, but the ability of RNA -loaded DCs to stimulate potent antitumor immunity has been independently confirmed in murine and human systems [82-87] . In fact, there is accumulating evidence that RNA transf ection represents a superior method for loading antigens onto DCs [84, 88]. This novel and innovative approach to DC antigen loading has multiple conceptual advantages over other forms of antigen delivery as well. RNA -based antigen loading does not require knowledge of major histocompatibility complex ( MHC ) restriction, and responses are not restricted to s ingle MHC haplotypes or to a narrow B or T cell repertoire. This diversity increases the likelihood of inducing effective and sustained antitumor immune responses by simultaneous activation of both CTLs and helper T -cells [89-91] . Furthermore, in direct comparisons, RNA -loaded DCs have been found to be better stimulators of antigen- specific T - cells than other approaches [88]. Finally, RNA also carries a significant safety advantage, not possessed by other nucleic acid or viral vectors, in that it cannot be integrated permanently into the host genome . In addition to the preliminary data we present below, Kobayashi et al. [92] have demonstrated that tumor mRNA -loaded DCs can elicit a specific CD8+ CTL response against autologous tumor cells in patients with MG. Temozolomide (TMZ) TMZ, a methylating agent with superior blood- brain barrier penetration, has recently been shown to increase survival by a small, but statistically significant, 2.5 months in a subset of patients with newly -diagnosed GBM if given in conjunction with X RT following initial tumor resection of the tumor [93-95 ]. Leukopenia , diminished white blood cell counts in the host, is essentially the only known human toxicity of TMZ treatment . Although initially counter -intuitive, this TMZ -induced leukopenia may actually be advantageous in treating patients with immunotherapy due to the I-ATTAC Version: 20220204 16 subsequent homeostatic proliferation of newly generated lymphocytes that it induces . In this protocol , patients will receive standard of care six week course of TMZ concurrent with X RT following initial resection of the tumor , followed by a dose -intensified 21- day cycle of TMZ prior to DC vaccination . We believe that the lymphopenia induced by therapeutic TMZ treatment, if carefully timed before vaccination , will actually enhance the proliferation and maintenance of these tumor -specific T -cells through the natural forces that drive T cell homeostatic proliferative recovery . Thus, thi s combination strategy will uniquely exploit the toxicity of one effective therapy for MG to enhance another already promising therapy, immunotherapy . In preparation for this protocol, we have evaluated, in animal models, TMZ and s ublethal whole body irradiation (WBI ), as a positive control, as methods for induction of treatment -induced lymphopenia in order to determine the ability of TMZ -induced lymphodepletion to enhance active vaccination and adoptive immunotherapies . These studies were initially performed in a murine T cell receptor (TCR) transgenic model in which the antigen- specific T -cells can be followed in vivo in mice receiving adoptive transfer of lymphocytes. In this model system, we found that adoptive lymphocyte transfer (ALT) coupled with DC vaccination ( DC + ALT) is a potent mechanism for inducing antigen -specific T -cell expansion after TMZ treatment . TMZ was found to be an effective agent for inducing homeostatic proliferation of transferred CD4+ and CD8+ T cells and for enhancing DC or pepti de vaccinations with or without ALT ( Figure 2). Markedly elevated levels of antigen -specific T -cells were achieved and maintained in mice receiving ALT after therapeutic TMZ -induced lymphodepletion compared to normal hosts receiv ing ALT . The precursor frequency of ovalbumin (OVA) -specific CD8+ T cells in the peripheral blood of untreated mice receiving OVA peptide vaccine after transfer of OVA antigen - specific (OT-I) transgenic T cells was 1.5% . Mice pretreated with TMZ and subsequent OVA peptide vaccin ation had achieved a mean precursor frequency of 54%, exhibiting markedly increased levels of OVA -specific T -cells in the blood ( P < 0.0001) . Similar results were found in mice receiving DC vaccin es, with untreated animals having an average precursor frequency of 3.6% and TMZ pretreated animals achieving an average of 11.9% OVA -specific CD8+ T -cells after a single vaccination . These results demonstrate that active and adoptive immunotherapy administered during hematopoeitic recovery f rom TMZ treatment may be a very effective way to enhance the efficacy of immunotherapy . I-ATTAC Version: 20220204 17 Figure 2: Homeostatic L ymphodepletion Induced by T emozolomide Enhances Antigen-S pecific Immune Responses. Untreated mice or mice receivin g non- myeloablative high -dose temozolomide treatment (5 days x 60 mg/kg) were administered a mixture of OVA -specific and normal lymphocytes intravenously and received either no vaccine, vaccination with DCs transfected with OVA RNA, or OVA peptide vaccinat ion in complete Freund’s adjuvant. The peripheral blood of treated animals was collected and analyzed 6 days after vaccination for the expansion of OVA -specific T cells in the circulation using αCD8 monoclonal antibodies and a specific tetramer. As shown above, mice treated with temozolomide prior to ALT demonstrated markedly increased T cell expansion after vaccination compared to untreated hosts receiving ALT. 111Indium -labeling of Cells for in vivo Trafficking Studies The in vivo distribution of DCs will be evaluated using 111Indium (111In)-labeled pp65-lysosomal- associated membrane protein (pp65 -LAMP) messenger RNA (mRNA)- loaded mature DCs . 111In- labeling has been used extensively for evaluation of adoptively transferred tumor infiltrating lymphocyte s, natural killer cells, granulocytes, DCs, and whole blood leukocytes, for in vivo localization studies in humans [96-99] . DCs will be labeled at the Duke Radio pharmacy Lab according to standardized protocols. Briefly, 2 x 107 pp65- LAMP mRNA- loaded mature DCs will be labeled with 111In (50 μCi / 5 x 107 DCs) (Nycomed -Amersham, Chicago, IL) prior to injection. Intradermal DC injections sites will be carefully mark ed at 10 cm from the groin crease. Gamma camera (dual -headed) images will then be taken immediately after injection s (0 hour), and at 24 and 48 hours after the DC vaccination to compare DC migration from the inguinal intradermal injection sites to the ing uinal vaccine site-draining lymph nodes (VDLNs) . Regions of interest will represent the quantity of 111In signal and be gated on VDLNs with calculations corrected for isotope decay. The quantity of DC uptake in VDLNs will serve as the migration efficiency of the cellular vaccine, and these migration rates will be analyzed for correlation with OS in these patients. This will serve to address the secondary objective if migration rates of DC vaccines given after Td pre- conditioning correlate with OS of patient s receiving this therapy. Beginning with Untreated Temozolomide I-ATTAC Version: 20220204 18 protocol version 20220204, 111Indium- labeling of c ells has been discontinued as the objective has already been met in a previously completed study entitled ELEVATE (Pro00054740). 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 adsorbe d are readily available as several approved administrations [i.e. Daptacel ( DTaP), Infanrix (DTap), Tenivac ( Td adult), Boostrix (Tdap)] [100, 101]. 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 [102]. 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 enzyme -linked immunosorbent assay (ELISA ) is regarded as protective. Diphtheria antitoxin levels ≥1.0 IU/mL by ELISA have been associated with long- term protection [103]. Following deep subcutaneous/intramuscular ( s.c./i.m. ) administration of the tetanus toxoid vaccine, toxoid molecules a re 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. This cellular processing is similarly thought to occur for native immature DCs at the skin site and for vaccinated DCs. Upon encounter with tetanus toxoid, the now activated mature DC at the vaccine site migrates along lymph channels to the VDLN where they encounter naive T H2 cells, each with their own unique T cell r eceptor (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 pass 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 [104]. 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 T H2 whose receptors are specific for this complex. The process, t ermed linked recognition, results in the T H2 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 cells[104]. 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 I-ATTAC Version: 20220204 19 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 (NCT0 0639639) 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 malignant brain tumors. 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 pre -conditioning at a single vaccine site increased the migration of a bilater al DC vaccine to both inguinal lymph nodes . Regardless of the side of the Td intradermal skin prep, DC migration to bilateral inguinal VDLNs was increased to similar magnitudes , 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 ass ociation between levels of DC migration and survival. In our preclinical model, Td pre -conditioning prior to vaccination with tumor antigen -specific DCs 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- conditioning. 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. 7.1.1 Pre-Clinical Experience In our laboratories and those of others, systemic immunization using DCs co- cultured with uncharacterized tumor homogenate [105], whole tumor RNA [106], unidentified peptides eluted from tumor cells by gentle acid washing [107], or a distinct peptide encompassing the tumor - specific epidermal growth f actor receptor variant type III (EGFRvIII ) mutation [108], have been shown to induce humoral and cell -mediated systemic immune responses and to prolong the survival of rodents with brain tumors. In our laboratory [105], inbred VM/Dk mice received three or four weekly intraperitoneal injections of autologous bone marrow -derived DCs transiently co -cultured with tumor homogenat e. The homogenate was derived from a syngeneic murine astrocytoma cell line derived from a spontaneously occurring astrocytoma in the inbred VM/Dk mouse strain. Splenocytes from I-ATTAC Version: 20220204 20 mice immunized in this way were able, in vitro, to lyse the astrocytoma cell line that was used to generate the tumor homogenate. They were also able to lyse other astrocytoma cell lines derived from the same inbred mouse strain, but they had no effect against syngeneic fibroblasts. Similarly, these immunized mice also demonstrated a significantly increased antibody titer against the astrocytoma cell line used to generate the homogenate. In addition, mice immunized with DCs transiently co -cultured with tumor homogenate that were subsequently challenged with a lethal dose of this astrocytoma cell line intracerebrally were found to have a median survival >160% longer than those immunized with DCs cultured without tumor homogenate ( P = 0.016). In addition, 50% of the mice treated with the tumor homogenate -supplemented DCs survived long - term without any evidence of tumor growth and also survived a rechallenge of tumor cells indicating that a sustained antitumor immune response had been established. These findings are especially significant in light of the fact that the astrocytoma cell lin e used is known to secrete the immunosuppressive agent t ransforming growth f actor -β (TGF -β) which is secreted by most human gliomas [109-113] . In another report from our laboratory [106], C57BL/6 mice received three weekly intraperitoneal injections of autologous bone -marrow derived DCs co -cultured with tumor homogenate or whole tumor RNA derived from the poorly immunogenic B16F10 melanoma cell line. Standard in vitro cytotoxicity assays again revealed that splenocytes harvested from mice immunized with DCs transiently co -cultured with either tumor -derived homogenate or whole tumor RNA were able to lyse B16F10 melanoma cells but not unrelated tumor cells from the same MHCbackground. In this experiment, mice immunized with autologous bone -marrow derived DCs co -cultured with tumor homogenate or whole tumor RNA increased median survival by > 233% ( P = 0.0006) and 48% ( P = 0.0001), respectively, rel ative to mice immunized with DCs co -cultured with tumor homogenate or whole tumor RNA derived from an unrelated tumor with the same MHC background. In addition, 8/13 (61.5%) in the specific homogenate group and 4/10 (40%) in the specific RNA group survived beyond the endpoint of the study without evidence of tumor. Immunization of mice with pre -existing tumors with specific tumor homogenate also demonstrated the potency of this immunization approach by increasing survival by 62.5% relative to controls. In t hese mice, an inflammatory infiltrate composed of mononuclear cells and polymorphonuclear leukocytes was identified , only in mice treated with DCs co -cultured with tumor homogenate that matched the intracerebral tumor challenge. In a recent report from an other laboratory [107], the survival of tumor -bearing rats injected subcutaneously with autologous bone marrow- derived DCs co -cultured with peptides eluted fr om tumor cells with a gentle acid wash was significantly prolonged compared to tumor -bearing rats receiving equivalent numbers of DCs co- cultured with peptides acid- eluted from normal astrocytes (P < 0.05). Median survivals in these groups were 35 and 22 days , respectively. In addition, three of the twelve rats (25%) treated with DCs co -cultured with acid -eluted tumor peptides remained alive at the end of the experiment. In addition, immunohistochemical analysis of five animals from each group in this expe riment documented an increased peritumoral and intratumoral infiltration of CD8+ T-cells, and to a lesser extent CD4+ T cells and macrophages, in the group treated with DCs co -cultured with peptides acid-eluted from tumor cells when compared to controls. 7.1.2 Clinical Experience Prior Experience in Patients with GBM Receiving GM- CSF -Containing DC Vaccines I-ATTAC Version: 20220204 21 In two previous studies conducted under Investigational New Drug (IND)-12839 and one under IND-13240, we have vaccinated patients with newly -diagnosed GBM with DCs loaded with mRNA encoding the human CMV matrix protein pp65. T o date , a total of 59 patients have received 545 vaccinations with pp65-loaded DCs. One patient in the ATTAC -GM trial ( NCT00639639, IND -12839) , which utilized pp65- specific DC vaccines admixed with GM -CSF, had a severe G rade 3 immunologic reaction after receiving vaccine #8 [114]. The patient developed flushing of the face, reddening of the eyes, nausea, tunnel vision, headache, raised red bumps on the chest, back, antecubital spaces of both arms, and large red swollen areas in both injection sites. Alle rgy and Immunology was consulted and recommended testing the individual components of the vaccine for hypersensitivity. The components were separated into two parts : GM-CSF and DCs pulsed with pp65 RNA . Immunologic workup for this patient revealed sensitization to the GM -CSF component of the vaccine and the production of high levels of anti-GM-CSF au toantibodies during vaccination (Figure 3, Figure 4). Removal of GM-CSF from the DC vaccine allowed continue d vaccination (total of 10 vaccines) without incident for this patient. No other study drug AEs were detected in this study . The allergic reaction is detailed in the MEDWATCH submitted May 16, 2011, IND 12839 serial number 34. Figure 3: Polyclonal A ntibody Median F luorescence I ndex (MFI) A gainst GM -CSF is P lotted Over T ime Coinciding With Vaccine and A pheresis Administrations in One subject . Anti -GM-CSF Antibodies (IgG +IgM) Increases Over Time With Repeated Vaccinations Using Recombinant GM -CSF and Begin to Decrease A fter the Adjuvant Was Removed. Phereesis 1Vaccine 1 Vaccine 2 Vaccine 3 Pheresis 2 Vaccine 4 Vaccine 5 Vaccine 6 Vaccine 7 Vaccine 8 Vaccine 9Vaccine 10 Post-vaccines010203040 IgG+gM (poly)MFI Anti-GMCSF (IgG + IgM) Antibodies I-ATTAC Version: 20220204 22 Figure 4: IgE A ntibody Median Fluorescence Index (MFI) A gainst GM -CSF is Plotted O ver Time C oinciding With Vaccine and Apheresis A dministrations in On e Subject. IgE A ntibody Fluorescence Against GM -CSF Peaks at V accine#8, Consistent With the S ubject’s Anaphylactic Episode at T his Time Point Before S lowly Decreasing Back to Baseline After Adjuvant R emoval. Intradermal, intravenous and intralymphatic migration studies using 111In labeled DCs have been successfully done in both animal models and patients with no adverse effects [ 99, 115-117] . Under IND-12839, we have used labeled DCs with 111In and used single -photo emission computerized tomography (SPECT) imaging to track the migration to the VDLNs . This work was published in Nature [ 118]. There is a vast amount of experience with both the safety and efficacy of immunotherapy targeting human CMV [37]. Vaccination specifically against CMV has effectively and without toxicity, reduced the risk of viral infection and transmission to fetuses in clinical trials [61, 65, 69-72 ]. Adoptive T -cell therapy has been used to safely and successfully protect against CMV reactivation in myelodepleted bone marrow transplant patients [43, 44, 46, 119]. In addition, T cell-mediated immunotherapy has proven highly effective in the treatment of CMV -associated disease within the central nervous system [47] and in the treatment of acute CMV infections [44, 48]. Tumors associated with other human Herpesviruses, such as Epstein- Barr virus -associated lymphoma, including tumors within the CNS, have also been effectively treated and even large tumors have been cured by immunotherapy [49-52, 54, 120]. The pp65 minimal epitope peptide (495-503) was shown to be safe in doses up to 10mg in hematopoietic stem cell transplant patients, with grade 1 adverse events being most commonly reported in approximately 10- 15% of the participants and was success ful in expanding CMV reactive T- cells [121]. Phereesis 1Vaccine 1 Vaccine 2 Vaccine 3 Pheresis 2 Vaccine 4 Vaccine 5 Vaccine 6 Vaccine 7 Vaccine 8 Vaccine 9Vaccine 10 Post-vaccines345678 IgEMFI Anti-GMCSF (IgE Isotype) I-ATTAC Version: 20220204 23 7.2 Study Purpose/Rationale Our Overall Goal in this project is to extend our substantial pre -clinical and clinical findings and verify these in a larger cohort. We hypothesize that CMV pp65- loaded DCs given with Td and GM-CSF to a larger group of patients with TMZ -induced lymphodepletion will confirm our previous findings by inducing significant DC migration to draining lymph nodes, producing high levels of Chemokine (C -C motif) ligand 3 (CCL3 ) and polyfunctional CMV pp65- specific T cells systemically, and confirming extra -ordinary PFS and OS. We will conduct a larger Phase 2 trial of CMV pp65- loaded DC vaccination in patients with GBM. We will confirm our observations from smaller studies , in order to determine if a Phase 3 trial is warranted and to estimate patient numbers that might be required. Patients with CMV positive, newly -diagnosed GBM will receive serial vacci nes with CMV pp65- loaded DCs with Td and GM - CSF vaccine site pre- conditioning after standard of care treatment with concurrent external beam radiation and TMZ chemotherapy. Only patients that are methylguanine -DNA methyltransferase (MGMT) unmethylated will be enrolled because adjuvant TMZ shows very little benefit in that population so it can be avoided after the initial treatment that induces transient lymphodepletion. Next , we will also confirm predictive biomarkers of survival. In our prior studies , DC migration to draining lymph nodes and systemic CCL3 and CMV pp65- specific polyfunctional T cells predicted PFS and OS. Here we will collect samples to confirm these predictive biomarkers prospectively. Strikingly, Td- treated patients had systemically en hanced DC migration to VDLNs and significantly improved PFS and OS compared to controls ( median PFS 18.5 vs 10.8 months, OS 25.7 vs 18.5 months ) with half the Td cohort living > 44.1 months from diagnosis . Our murine studies corroborated that Td pre-condit ioning systemically enhanced DC migrati on and suppressed tumor growth. In the proposed clinical stud y, we aim to enhance the migration of CMV pp65 RNA - pulsed DCs with Td and adjuvant GM -CSF and will assess if increased migration using these strategies can be predictive of patient survival outcomes . I-ATTAC Version: 20220204 24 8 OBJECTIVES AND ENDPO INTS Table 1. Objectives and Endpoints Objective Endpoint Analysis Primary Determine if Td preconditioning of pp65 loaded DC vaccination with GM-CSF extends over all survival. Median survival from initiation of dose intensive adjuvant temozolomide . See Section 15.5 Secondary Assess the association between migration of CMV pp65 RNA - pulsed DCs with GM -CSF to site - draining inguinal lymph nodes after Td pre -conditioning and subsequent survival . (PLEASE NOTE: With Protocol v.20220204 , this objective has been removed as it was already addressed in a previous study.) For survival after vacc ine 4, the hazard ratio associated with a 1 unit increase in migration, defined as t he maximum percentage of 111In- labeled DCs reaching inguinal nodes during the 48 hours after the 4 th vaccination . See Section 15.6.1 Secondary Assess the association between CCL3 measured post -Td pre - conditioning and subsequent survival . For survival after vaccine 4, the hazard ratio associated with a 1 -unit increase in CCL3 measured post-Td pre -conditioning at vaccine 4. See Section 15.6.2 Secondary Assess the association between fold changes in T cell polyfunctionality and subsequent survival . Mean fold change from baseline in the frequency of pp65 antigen-specific CD8+ T cells producing three or more cytokines (IFNγ, CCL3, IL -2, TNFα, CD107a) at leukapheresis 2 14± (4) days after vaccine 3 . Mean fold change from baseline in the frequency of pp65 antigen -specific CD8 + T cells producing three or more cytokines (IFNγ, CCL3, IL -2, TNFα, CD107a) at vaccine 6. For survival after vaccine 4, the hazard ratio associated with a 1 -fold increase in the frequency of pp65 antigen-specific CD8+ T cells produci ng three or more cytokines (IFNγ, CCL3, IL -2, TNFα, CD107a). See Section 15.6.3 Secondary Assess whether T Reg levels remain low without adjuvant temozolomide . Maximum mean peak increase from vaccine 1 in percent T Reg of CD4+ T cells . See Section 15.6.4 Secondary Assess the safety of DI-TMZ treatment followed by DC vaccination . Proportion of patients who experience unacceptable toxicity . See Section 15.6.5 Exploratory Assess the association CCL3 levels and migration . Mean fold change in CCL3 b etween pre - and post-Td pre -conditioning. Correlation between CCL3 change from fold change in CCL3 , and migration . See Section 15.7.1 Exploratory Assess the assoc iation between pp65-specific polyfunctional CD8+ T cells and increased DC migration . Correlation between fold change from baseline (pre-vaccine 1) in polyfunctional CD8+ T cells and migration . See Section 15.7.2 Exploratory Assess the association between pp65-specific polyfunctional CD8+ T cells and increased serum CCL3 . Correlation between fold change in polyfunctionality and fold change in CCL3 See Section 15.7.3 I-ATTAC Version: 20220204 25 9 INVESTIGATIONAL PLAN 9.1 Study D esign Approximately 64 patients with resected, newly -diagnosed WHO Grade IV glioma who are CMV+ and in which the MGMT is not methylated will be accrued to this study before standard of care radiation therapy (RT) and concurrent TMZ, with the goal of treating 48 patients with dose - intensified temozolomide and pp65 loaded dendritic cell vaccine after completion of standard RT and TMZ. All enrolled patients will undergo a leukapheresis after resection for harvest of peripheral blood lymphocytes (PBLs ) for generation of DCs. Patients will then receive approximately 6 weeks of standard of care radiation therapy ( RT) and concurrent TMZ at a standard targeted dose of 75 mg/m 2/d (please see Package Insert uploaded in e lectronic IRB for standard therapy) . Patients should start RT within approximately 7 weeks of surgery. [PI discretion can be used in this situation to allow patients outside of this window to enroll.] Patients who either experience progressive disease during radiation, are dependent on steroid supplements above 2 mg/day at time of first vaccination, are unable to tolerate TMZ, or whose DCs fail to meet release criteria will be withdrawn from the study and replaced . For patients whose initial leukapheresis yields less than 3 vaccines, repeat leukapheresis may be obtained a minimum of 2 weeks from the previous leukapheresis (and may be repeated as needed) if pre -apheresis blood work is within the Apheresis Center’s parameters and as long as this does not cause a significant delay in treatment for the patient . At th e post-RT clinic visit the patient’s MRI and blood work is evaluated before starting a single post -RT cycle of dose inten sified TMZ (100 mg/m 2/day for 21 days). On day 23 (± 2 days ) of the cycle, patients will receive the first of 3 pp65 DC vaccines . DC vaccines #1 -3 will be given every two weeks (± 2 days). All patients will receive up to a total of 10 DC vaccines , with vac cines administered every 35 days (± 7 days) after the third vaccine, given bilaterally at the groin site unless progression occurs with no further cycles of TMZ . DC vaccines will be given intradermally (i.d.) and divided equally to both inguinal regions. B efore the first DC vaccination, patients will receive 0.5 mL of Td (tetanus and diphtheria toxoids adsorbed) intramuscularly into the deltoid muscle to ensure adequate immunity to the tetanus antigen. The Td vaccine will be obtained through Duke Investigat ional Chemotherapy Services (ICS) Pharmacy. Patients will undergo leukapheresis again for immunologic monitoring with specific assessment of baseline antigen-specific cellular and humoral immune responses if needed for further DC generations 14 (± 4) days after vaccine #3. Prior to pp65 DC vaccination #4, (3±1) weeks after leukapheresis 2 the vaccine site will receive a pre -conditioning intradermal injection of Td (1 flocculation unit (Lf), in 0.3 mL of saline for a total of 0.4 mL ). As part of standard care for these patients, upon tumor progression, participants may undergo stereotactic biopsy or resection. As this is not a research procedure consent will be obtained separately. However, if tissue is obtained, it will be used to confirm tumor progression histologically and to assess immunologic cell infiltration and pp65 antigen escape at the tumor site. No other treatment intervention is allowable while on study, unless the patient needs bevacizumab at 7.5 mg/kg IV approximately every 3 weeks per the “ Special Considerations” in Section 9.1.3. 9.1.1 Definition of Unacceptable Toxicit ies I-ATTAC Version: 20220204 26 Toxicities will be graded according to the National Cancer Institute Common Toxicity Criteria of Adverse Events (NCI CTCAE ) version 5 criteria. An unacceptable toxicity is defined as any Grade 3 or greater toxicity that is possibly, probably, or definitely attributed to the pre-conditioning agent Td or pp65 DC vaccine that does not resolve to baseline in 2- 3 weeks ; any Grade 3 hypersensitivity react ions or autoimmune toxicity requiring steroids or hormone replacement ; and is not due to progressive disease , or any life –threatening event not attributable to concomitant medication, co- morbid event, or disease progression. Rules for monitoring unacceptable toxicity are provided in Section 15.6.5. 9.1.2 Dose Modification TMZ will be administered concomitantly with standard external beam RT under the direction of the oncologist. Please see TMZ package insert uploaded in e lectronic IRB for standard of care treatment guidelines. The dose of the concomitant TMZ with RT may be adjusted at the discretion of the treating oncologist. The study drug ( CMV pp65 RNA -pulsed DCs with GM -CSF) dose will not be modified in this trial. Those subjects whose cells fail to meet release criteria will be removed and replaced. 9.1.3 Safety Considerations Management of Toxicities If a Grade 3 NCI CTC or greater toxicity is seen that is not attributable to a concomita nt medication, co -morbid event, or disease progression that has been documented radiographically or clinically, the next immunization for that patient will be withheld for up to 2 months or until the NCI CTC toxicity improves to a Grade 1 or less. However, planned procedures requiring hospitalization, or long -term clinical decline that is now seen in patients years from whole brain radiation therapy ( WBRT ), which are clearly not related to study drug, but are the natural development common in this patient population, will not be considered a n unacceptable toxicity nor will have immunizations withheld . Special Consideration Due to the impact of high dose steroids on the development of an optimal immune response, if a patient demonstrates neurologic or cerebral radiographic signs suggestive of a localized inflammatory reaction, secondary to the immune response triggered by DC vaccination, that requires an increase in dexamethasone dose, every effort should be made to not increase the dose above 4 mg per day at any time. Instead, patients should be treated with bevacizumab at the reduced dose of 7.5 mg/kg IV approximately every 3 weeks. If a patien t requires planned treatment for their tumor with bevacizumab > 7.5 mg/kg approximately every 3 weeks, they will be considered off study and enter the follow -up phase. Neuroimaging (MRI) will be performed according to protocol schedule and, at that time, it will be assessed whether further treatment with bevacizumab is needed to control the cerebral inflammation. Bevacizumab will not be provided by the study. Every attempt should be made to reduce or discontinue dexamethasone, when clinically possible, so as not to mitigate immune response. If there are AEs or other circumstances prohibiting the use of bevacizumab, cor ticosteroids , surgery, or other interventions deemed more appropriate for the patient by the treating physician will be used, if needed, to treat any localized inflammatory reaction secondary to DC vaccination. I-ATTAC Version: 20220204 27 Adverse Event Reporting and Documentation An “Adverse Event” will be defined as any adverse change from the subject’s pre -treatment baseline condition (which is assessed at the post -RT/TMZ clinic visit), including any clinical or laboratory test abnormality that occurs during the course of research . Adverse events will be categorized and graded in accordance with the NCI CTC AE (Version 5). A “Serious Adverse Event” will be defined as an undesirable sign, symptom or medical condition which : 1) is fatal or life threatening; 2) requires inpatient hospi talization for > 24 hours or a prolongation of existing hospitalization; 3) results in persistent or significant disability/incapacity; 4) constitutes a congenital anomaly or a birth defect and/or; 5) medically significant such that it may jeopardize the subject, and may require medical or surgical intervention to prevent one of the outcomes listed above. A summary of all adverse events (not just those considered related to study drug ) that occur following RT/TMZ will be kept which will categorize the event by organ system, relationship to treatment, its grade of severity, and resolution. Periodic review by the primary investigator ( PI) and weekly review at the Duke Preston Robert Tisch Brain Tumor Center ( PRTBTC ) Adverse Event (AE) meeting of the collectiv e adverse events will occur with the intention of identifying any trends or patterns in toxicity. If any such trends are identified, depending on their severity and frequency, a protocol amendment will be considered. All adverse events which are serious and unexpected should be reported immediately to Dr. Mustafa Khasraw M.D. at 919-684- 5301 during regular business hours or at 919-2060493 after hours and to the Federal Drug Administration ( FDA ). Fatal or life -threatening, unexpected adverse events will be reported to the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 7 calendar days after first knowledge by the sponsor followed by as complete a report as possible within 8 additional calendar days . Serious, unexpected adver se events that are not fatal or life -threatening will be reported to the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 15 calendar days after first knowledge by the sponsor. All adverse events that are considered seriou s, unanticipated, and related or possibly related to the research ( as defined by 21CRF312.32[a]) will be reported to the Duke University Medical Center Institutional Review Board ( IRB) using the appropriate serious adverse event ( SAE ) report form . At the time of the annual progress report to the Duke University Medical Center IRB, a summary of the overall toxicity experience will be provided. 9.1.4 Missed Doses To ensure that repetitive DC vaccines will be given to patients, the initial 3 will be given every 2 weeks (± 2 days), and the monthly DCs will be given every 35 days (± 7 days ). Therefore, with a margin of 2 days all patients will receive up to 10 DC vaccines or until progression, whichever comes first . At the discretion of the study PI, patients who mis s vaccine administration for whatever reason will have their appointment re -scheduled to administer the vaccine as soon as possible and the study PI will assess whether this deviation needs to be filed with the IRB . For continued non- compliance with the sc heduled vaccine appointments, the subject may be removed from the trial at the discretion of the study PI. 9.1.5 Concomitant Medications I-ATTAC Version: 20220204 28 Concomitant medications will be managed by the treating oncologist and recorded at each study visit by the study coordinator. 9.2 Rationale for Selection of Dose, Regimen, and Treatment Duration In our previous randomized clinical trial of 12 patients with newly -diagnosed GBM, we evaluated the impact of vaccine site pre-conditioning with Td toxoid [118]. Patients randomized to Td showed increased dendritic cell migration bilaterally and significantly improved progression- free and overall survival. Furthermore, we observed a modest assoc iation between effective DC migration to VDLNs and clinical outcomes. Based on the small sample size of the initial study, we would like to conduct a validation study with Td pre -conditioning that is powered sufficiently to evaluate the impact of DC migration on clinical outcomes. 9.3 Rationale for Correlative Studies Please see Section 12.7.4 111Indium- labeling of Cells for in vivo Trafficking Studies . PLEASE NOTE: With approval of Protocol v.20220204, this correlative study will no longer be conducted. 9.4 Definition of Evaluable Subjects , On Study, and End of Study Subjects evaluable for the analysis of the primary endpoint of overall survival will include all patients who initiate DI -TMZ . Once the patient signs an informed consent form ( ICF), that subject will be considered “on study .” Rationale for taking patient off protocol treatment will be documented. 9.5 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 12.6, which describes procedures and process for prematurely withdrawn patients. 10 STUDY DRUG 10.1 Names, Classification, and Mechanism of Action Tetanus -Diphtheria Toxoid (Td Toxoid adsorbed) Td is i ndicated for active booster immunization against tetanus, diphtheria, and pertussis as a single dose; substitute 1- time dose of Tdap for Td booster, then standardly boost with Td every 10 y ears. Please refer to S ection 9.1 on the use of Td in this protocol. DC V accin e Human CMV pp65- LAMP mRNA- pulsed autologous DCs with GM -CSF is the name of the study drug given with every vaccine . This vaccination will then consist of administration of 2 x 107 CMV pp65- LAMP mRNA loaded mature DCs as described above . In up to 16 pati ents, the fourth vaccine will be labeled with 111In (50 μCi / 5 x 107 DCs) (Nycomed -Amersham, Chicago, IL) prior to injection . The class of action for all study drugs is a biological with the exception of the tetanus toxoid, which is an antitoxin. I-ATTAC Version: 20220204 29 GM-CSF (LEUKINE®; S argramostim) GM-CSF w ill be obtaine d from commercial supply and stored in the ICS , as a sterile, white, preservative- free powder lyophilized powder in a vial containing 250 mcg to be reconstituted in 0.5 mL of sterile water for injection . Leukapheresis and D endritic C ell V accine Generatio n At least two leukaphereses may be performed on each patient enrolled on this protocol. The leukapheresis will be used for DC generation and immunologic monitoring. If the subjects were part of the companion study (Pro00102797) and have already undergon e the leukapheresis procedure the DCs from that study will be used for the current study and any unused vaccine products will be handled as indicated by the patient in the companion study. All leukaphereses will be a pproximately a 4-hour leukapheresis. It is estimated that 10 -12 L of blood will be processed during this leukapheresis. An additional 4-hour leukapheresis will be requested from patients with positive immunological responses to the therapy for additional immunologic monitoring studies or for pat ients who need additional DCs generated to ensure a total of 10 vaccines . DCs will be generated from the first leukapheresis in vitro by 7-day culture with GM -CSF and IL - 4. Peripheral blood mononuclear c ells ( PBMC ) for in vitro generation of DCs will be o btained by leukapheresis at the Duke Apheresis Unit and transported to the Cell Processing facility . For patients without sufficient venous access for leukapheresis a temporary intravenous catheter may be inserted. At the end of the 7 day incubation for g eneration of DC , a sample of the media is taken for mycoplasma testing, the cells are then harvested and electroporated with pp65 -LAMP mRNA. The DCs are placed in a flask with AIM V media GM -CSF + IL -4 + TNF -α + IL -6 + IL -1β at 37OC, 5% CO 2 for 18 -20 hours for maturation . The cells are washed twice with PBS and frozen at 2-4 x 107cells/mL in 90% autologous human AB serum (Valley Biomedical, Winchester, VA 22602), 10% DMSO and 5% dextrose. The DCs are then stored until needed at –135 oC. After freezing, an aliquot of cells is thawed for QA/QC. This testing will look at viability, (>70%) endotoxin content, (<5 E.U. /Kg B.W.) mycoplasma contamination (negative) and sterility testing for aerobic and anaerobic bacterial cultures (1 x 106 DCs) and fungal cultures (1 x 106 DCs). For each vaccination, cells that have passed quality assurance/quality control ( QA/QC ) will be rapidly thawed at 37 oC, washed three times with phosphate buffered saline (PBS) and counted. The cell concentration will be adjusted to 5 x 107 cells/mL and DCs will be resuspended in preservative free saline and GM -CSF and placed into a sterile tuberculin syringe with a 25- gauge needle. For all DC preparations, and 111In-labeled DCs, from the final preparation a sample of cells will be sent fo r Gram stain and endotoxin testing prior to administration. DC vaccination will not be given until endotoxin testing has been passed (< 5.0 E.U/Kg) and the Gram stain has been found I-ATTAC Version: 20220204 30 to be negative . An aliquot of cells will also be sent for aerobic and anae robic bacterial cultures (1 x 106 DCs) and fungal cultures (1 x 106 DCs). The SOP MPACT -QA-0017 Responding to Sterility Test Results describes the roles, responsibilities, and actions that should be taken i n the event of a positive, delayed, incomplete, or invalid sterility test result for a product manufactured in the MPACT facility after it has been administered to a study subject. Briefly, personnel informed of the result will notify MPACT Management who is responsible for notifying all relevant study personnel. T he Principal Investigator or treating Neuro -Oncologist ( or his or her design ee) will notify the patient. The patient will be asked to be evaluated by a physician within 48 business hours . Based on the route of administration of the investigational product, appropriate cultures (aerobic, anaerobic, and fungal) will be ordered for the subject (blood cultures or wound cultures) along with CBC and CMP tests. If the patient has or develops a temperature ≥38.5°C or clinical evidence of infection, the patient will be treated expectantly with antibiotics based on the sensitivities of the organisms identified from the immunization product , as medically necessary . An independent infectious disease consultation will be obtained to guide further therapy if medically necessary . MPACT management and MPACT QA will perform an investigation into the sterility tst result. If there are remaining vials from the affected batch, a vial will be sent for repeat sterility testing, and the remainder of the batch will b e quarantined until re -testing is complete with negative results. Administration of subsequent products from the affected batch will be held, and upcoming study visits should be rescheduled as necessary. I mmunizations will proceed only if the patient full y recovers and subsequent samples are found to be sterile. 10.2 Packaging and Labeling of Study Agents • For CMV pp65- LAMP mRNA-pulsed DCs and CMV pp65- LAMP mRNA loaded mature DCs l abeled with 111In (50 μCi / 5 x 107 DCs) : Vaccine Label Patient Name / MRN DOB / ALPS # Trial Name and Number Date FOR AUTOLOGOUS USE ONLY NOT EVALULATED FOR INFECTIOUS SUBSTANCES Caution: New Drug Limited by Federal Law to Investigational Use • For t etanus diphtheria toxoid used in pre-conditioning Name MRN DOB Drug: Td • Td™ (Tetanus diphtheria toxoid adsorbed); Stored at 4oC used as Td booster vaccine Drug: Td Lot # Expiration Date 10.3 Supply, Receipt, and Storage The DCs will be stored in a locked liquid nitrogen freezer in the Molecular Products and Cellular Therapies (MPACT) c urrent Good Manufacturing Practice (cGMP ) facility . The Nautilus I-ATTAC Version: 20220204 31 Laboratory Information Management System (LIMS ) database will track receipt and storage location. Table 2. Storage of Study Agents MPACT Facility Duke ICS Pharmacy Duke Radiopharmacy CMV pp65 -LAMP mRNA -pulsed DCs Td used for booster and for pre-conditioning CMV pp65 -LAMP mRNA loaded mature DCs labeled with 111In 10.4 Dispensing and Preparation The pp65 DC vaccine s will be delivered from the MPACT facility directly to the clinic under the supervision of the trained research staff . Each labeled patient vaccine will be transported to the clinic in a separate cooler. DCs will be administered according to protocol . The patient’s name, Study identification ( ID), date of birth ( DOB ), and Duke history number will be double verified prior to DC administration as is standard Duke transfusion procedure. The radiolabeled DC products used with the migration studies will be p repared from the pp65 DC vaccines sent from the MPACT facility to the Radiopharmacy and dispensed radiolabeled from the Duke Radiopharmacy. Table 3. Dispensing of Study Agents MPACT Facility Duke ICS Pharmacy Duke Radiopharmacy CMV pp65 -LAMP mRNA -pulsed DCs Td used for booster and for pre-conditioning CMV pp65 -LAMP mRNA loaded mature DCs labeled with 111In 10.5 Compliance and Accountability All DC vaccines will be stored in the Molecular Produc ts and Cellular Therapies (MPACT) cGMP Facility in a temperature controlled, locked access controlled storage unit . A drug log sheet will be used to track and document the drug . The products will be signed out and distributed by the MPACT personnel. The MPACT personnel use safe medication pract ices to reduce the risk of medication errors and adverse events when setting up study drug procedures . Investigational drugs are ordered, received, stored, and dispensed for MPACT protocols that are approved by the Duke University Health System ( DUHS ) IRB. Investigational drugs are stored separately from other drugs in an area of limited access and in accordance with special storage requirements . They are clearly labeled with the identity of the study drug and other control numbers . All drug transfers, receipts, and disposal are recorded in the Duke Nautilus system . 10.6 Disposal and Destruction Radioactively labeled drug will be disposed of according to standard Duke radiation safety practices . Un-labeled drug will be autoclaved. 11 SUBJECT ELIGIBILITY Inclusion C riteria I-ATTAC Version: 20220204 32 • Age ≥ 18 years of age. • Newly diagnosed World Health Organization ( WHO) Grade IV Glioma with definitive resection prior to consent , with residual radiographic contrast enhanc ing disease on the post-operative computed tomography ( CT) or Magnetic Resonance Imaging (MRI ) of <1 cm in maximal diameter in any plane. • Able to receive standard of care radiation and chemotherapy for approximately 6 weeks duration and of more than 54GY • MRI post RT does not show progressive disease outside the radiation field • Enough tumor tissue available for determination of MGMT gene promoter status (must be unmethylated) or prior pathology report available confirming MGMT gene promoter status. • CMV Seropositive. • KPS of ≥ 70% . • Hemoglobin ≥ 9.0 g/dl, absolute neutrophil count ( ANC ) ≥ 1,000 cells/µl, platelets ≥ 100,000 cells/µl prior to starting TMZ cycle 1 (patient must meet these criteria within 4 weeks after the end of XRT/TMZ to be eligible) . • Serum creatinine ≤ 3 times institutional upper limit of normal for age , serum aspartate aminotransferase ( AST ) ≤ 3 times institutional upper limit of normal for age and • Bilirubin ≤ 1.5 times upper limit of normal prior to starting TMZ cycle 1 (Exception: Patient has known Gilbert’ s Syndrome or patient has suspected Gilbert’s Syndrome, for which additional lab testing of direct and/or indirect bilirubin supports this diagnosis. In these instances, a total bilirubin of ≤ 3.0 x ULN is acceptable. ). • Signed informed consent approved by the Institutional Review Board. • Female patients must not be pregnant or breast -feeding. Female patients of childbearing potential (defined as < 2 years after last menstruation or not surgically sterile) must use a highly effective contraceptive method (a llowed methods of birth control, [i.e. with a failure rate of < 1% per year] are implants, injectables, combined oral contraceptives, intra- uterine device [IUD; only hormonal], sexual abstinence or vasectomized partner) during the trial and for a period of > 6 months following the last administration of trial drug(s). Female patients with an intact uterus (unless amenorrhea for the last 24 months) must have a negative serum pregnancy test within 48 hours prior to first study procedure (leukapheresis) . • Ferti le male patients must agree to use a highly effective contraceptive method (allowed methods of birth control [i.e. with a failure rate of < 1% per year] include a female partner using implants, injectables, combined oral contraceptives, IUDs [only hormonal ], sexual abstinence or prior vasectomy) during the trial and for a period of > 6 months following the last administration of trial drugs. Exclusion Criteria: • Pregnant or breast -feeding . • Women of childbearing potential and men who are sexually active and not willing/able to use medically acceptable forms of contraception . • Patients with known potentially anaphylactic allergic reactions to gadolinium- diethylenetriamine penta -acetic acid (DTPA) . I-ATTAC Version: 20220204 33 • Patients who cannot undergo MRI due to obesity or to having cer tain metal in their bodies (specifically non-MRI compatible pacemakers, infusion pumps, metal aneurysm clips, metal prostheses, joints, rods, or plates). • Patients with evidence of tumor in the brainstem, cerebellum, or spinal cord, radiological evidence of multifocal disease, or leptomeningeal disease. • Severe, active comorbidity, including any of the following: - Unstable angina and/or congestive heart failure requiring hospitalization; - Transmural myocardial infarction within the last 6 months; - Acute bacterial or fungal infection requiring intravenous antibiotics at the time of study initiation; - Chronic obstructive pulmonary disease exacerbation or other respiratory illness requiring hospitalization or precluding study therapy; - Known hepatic insufficiency resulting in clinical jaundice and/or coagulation defects; - Known Human Immunodeficiency Virus (HIV) and Hepatitis C positive status; - Major medical illnesses or psychiatric impairments that, in the investigator's opinion, will prevent administration or completi on of protocol therapy; - Active connective tissue disorders, such as lupus or scleroderma that, in the opinion of the treating physician, may put the patient at high risk for radiation toxicity . • Co-medication that may interfere with study results; e.g. immu no-suppressive agents other than corticosteroids . • Prior, unrelated malignancy requiring current active treatment with the exception of cervical carcinoma in situ and adequately treated basal cell or squamous cell carcinoma of the skin. (Treatment with tamo xifen or aromatase inhibitors or other hormonal therapy that may be indicated in prevention of prior cancer disease recurrence, are not considered current active treatment.) • Patients are not permitted to have had any other conventional therapeutic interven tion other than steroids prior to enrollment outside of standard of care chemotherapy and radiation therapy . Patients who receive previous inguinal lymph node dissection, radiosurgery, brachytherapy, or radiolabeled monoclonal antibodies will be excluded. • Current, recent (within 4 weeks of the administration of this study agent), or planned participation in an experimental anti -cancer drug study . • Known history of autoimmune disease (with the exceptions of medically -controlled hypothyroidism and Type I Diabetes Mellitus). 12 SCREENING AND ON- STU DY TESTS AND PROCEDURES 12.1 Screening Examination The screening examination will take place at the Duke PRTBTC clinic visit. An informed consent must be signed by the patient before any screening procedure takes place. Tumor MGMT promotor methylation status will be reviewed and only patients with non -methylated tumors will be enrolled. MGMT gene promoter methylation status is obtained at Duke using validated testing from LabCorp by PCR and/or Caris by pyrosequencing . If MGMT gene promoter methylation status has already been conducted outside of Duke, the results will be used as long as the testing was performed by a validated method/test. I-ATTAC Version: 20220204 34 Patients whose methylation status is inconclusive will not be eligible to participate in the study. The baseline physical and neurologic examination with KPS score along with standard of care blood work and CMV immune screen will be performed and documented by the neuro- oncology team and verified by the study team during this PRTBTC clinic visit. All subject data is standard of care evaluation that occurs for all patients being seen in the PRTBTC. If the subject is considered a screen failure prior to vaccine treatment, the source documents for electronic data entry will be obtained from the Duke electronic medical record . Initial clinical evaluations will also include a baseline and study eligibility MRI (with and without contrast ) of the brain per standard of care for comparison to subsequent MRI images. Mini -mental status examination (MM SE) will be performed at the post -RT/TMZ visit. The patient must have had a definitive resection. Residual radiographic contrast enhancement on post-resection CT or MRI must not exceed 1 cm in diameter in any planes at time of consent . Patients with pro gressive disease after radiation will not be a candidate for the vaccine despite being previously consent ed and will be removed from the study and replaced (please see Section 12.7.4 for radiologic evaluations) . During the initial intake process within the Preston Robert Tisch Brain Tumor Center, all patients’ tissue is tested for diagnosis and IDH-1/2 mutation analysis through either the Duke Pathology Department or CARIS. The study will collect this information in order to ensure baseline molecular diagnostics of the tumor for each patient is known. At the discretion of the study investigator, for surgical blocks containing initial resection and biopsy at recurrence (if applicable) , formalin -fixed paraffin -embedded slides may be requested for immunohistochemistry assays for the detection of CMV . These slides for CMV detection will not exceed 10% of the original specimen block, so as to preserve adequate tissue should patients prefer future diagnostic studie s. Patient consent will be obtained for obtaining these slides in either scenario if a surgery/biopsy was performed at Duke University or at an external institution. If resection is done outside of Duke University Hospital, formalin -fixed paraffin -embedd ed slides sectioned from outside blocks , not to exceed 10% of surgical specimen block tissue, prepared on Fischer Plus glass or Histostix coated slides when available will be mailed to: Mustafa Khasraw M.D. , Duke Brain Tumor Immunotherapy Program Departm ent of Neurosurgery The Preston Robert Tisch Brain Tumor Center at Duke DUMC Box 3624 047 Baker House, Trent Drive Duke University Medical Center Durham, North Carolina 27710, USA After patients have been consented, they will be entered into the e lectron ic Research system . 12.2 Treatment Period All enrolled patients will then undergo a leukapheresis for generation of DCs and for baseline immunological monitoring . If the patients were part of the companion study (Pro00102797) and I-ATTAC Version: 20220204 35 have already undergone the leu kapheresis procedure the relevant DCs from that study will be used for the current study and any unused vaccine products will be handled as indicated by the patient in the companion study. Within 48 hours of leukapheresis, patients will have blood samples taken for the following tests as required by the Duke Apheresis Center: CBC with auto differential, CMP, ionized Calcium, and Beta-Human Chorionic Gonadotropin ( β-HCG) (for females of child -bearing potential) . Total estimated blood volume required for these evaluations is 12- 15 mL. For patients without sufficient venous access for leukapheresis, a temporary central intravenous catheter may be inserted . To prevent the development of hypocalcemia from the citrate used for leukapheresis, all patients will be instructed to take oral Tums, 2 tablets three times a day and at bedtime the day before and the day of the leukapheresis procedure . Patients who have lower levels of calcium will be treated per Apheresis lab standard protocols under the direction of apheresis attending physician. This first leukapheresis will be a pproximately a 4-hour leukapheresis, and it is estimated that 10- 12 L of blood will be processed during this leukapheresis. One red top tube 10 mL draw tube will be obtained prior to leukapheresis #1 for immune monitoring. Nine yellow ACD 8.5 mL tubes and 2 red top 10 mL draw tubes will be obtained prior to leukapheresis #2. I-ATTAC Version: 20220204 36 Table 4. Schedule of Events CMV Screening Main Consent11 Leukapheresis #17 RT/TMZ Post-RT DI TMZ Cycle 1 Td Booster & Vaccine #1 Vaccine #2 Vaccine #3 Leukapheresis #27 Td Pre - conditioning8 Vaccine #4 Vaccines #5 -10 Progression9 Time and Range Within 7 weeks of resection Within 4 ( -2) weeks of RT Day 2 3 (+/-2 days ) Td booster given before vaccine #1 2 weeks post Vaccine #1 (+/-2 days ) 2 weeks post Vaccine #2 (+/-2 days) 14 (+/ -4) days after vaccine #3 3 (+/ -1) week after leukapheresis #2, 12-24 hours prior to vaccine #4 3 (+/ -1) week after leukapheresis #2 Every 35 (+/-7) days after previous vaccine CMV Screening Consent X Main Consent10 X X Medical History/ Baseline Symptoms X Tumor Pathol ogy2 X1 X Physical Exam, Neurological Exam, KPS , Vitals3 X1 X X X X X X X MMSE3 X MRI4 X1 X X X X CMV IgG X CBC (with auto differential)5 X X X X X X X X X CMP5 X X X X X X X X Beta HCG quantitative (WOCBP only) X X X Ionized Calcium X X Immune Monitoring Blood6 X (1R) X (9Y, 2R) X (9Y, 2R) X (9Y, 2R) X (9Y, 2R) X (2R) X (9Y, 2R) X (9Y, 2R) X (9Y, 2R) SOC XRT/TMZ X Dendritic Cell Vaccine X X X X X I-ATTAC Version: 20220204 37 Con meds and AEs X X X X X X X X X X X X 1Can be performed prior to any ICF being signed as part of standard of care evaluation. 2Patients must have a WHO Grade IV Glioma with definitive resection prior to enrollment, with residual radio graphic contrast enhancement on most recent CT or MRI of <1 cm in maximal diameter in any plane. Methylation status must be checked per SOC practices (via LabCorp, CARIS, etc.) and patient m ust be unmethylated in order to be eligible to proceed with signi ng consent to undergo leukapheresis. 3Clinical evaluations with each vaccine will include a general physical examination, complete neurologic examination, KPS rati ng, and vitals (prior to vaccine and 30 mins post vaccine). MMSE will be performed at the Po st-RT/TMZ visit only. 4A baseline and study eligibility MRI (with and without gadolinium enhancement) of the brain per standard of care for comparis on to subsequent MRI images will also be obtained. MRI or contrasted CT, every 8 ± 4 weeks per PRTBTC stand ard of care (may be adjusted by the treating neuro -oncologist). 5CBC (with auto differential) and CMP to confirm eligibility, as per standard of care, and as per this schedule of events. Whenever blood for immune monitoring is obtained CBC with auto differ ential and CMP will be done to assess absolute number of T Regs. 6Blood work for immunologic monitoring will be drawn before leukapheresis #1 (1 red top 10mL tube prior to leukapheresis), on the day of Post -RT/TMZ visit (9 yellow ACD 8.5mL tubes and 2 red t op 10mL tubes), on the day of but prior to vaccine #1 and vaccine #3 (9 yellow ACD 8.5mL tubes and 2 red top 10mL tubes), on the day of Td preconditioning but before the administration of Td (2 red top 10mL tubes), at leukapheresis #2 (9 yellow ACD 8.5mL t ubes and 2 red top 10mL tubes) when leukapheresis is performed 14 (+/ -4) days post vaccine #3, at the time of vaccine 4 (9 yellow ACD 8.5mL tubes and 2 red top 10mL tubes), and then at vaccines 5 through 10 (9 yellow ACD 8.5mL tubes and 2 red top 10mL tube s) and/or at progression (whichever comes first). If an additional leukapheresis must occur earlier than 14 (± 4) days after vaccine 3, then no immune monitoring will be drawn. All patients will undergo at least 2 leukapheresis procedures – prior to radia tion (1 red top 10mL tube) and approximately 2 weeks post vaccine #3 (9 yellow ACD 8.5mL tubes, 2 red top 10mL tubes) See footnote 7. For subjects who complete all 10 study vaccines without progressing, an attempt will be made to obtain blood for immunol ogic monitoring 2- 3 times a year at standard Duke Preston Robert Tisch Brain Tumor Center visits and at progression (whichever comes first). 7Leukapheresis #1 will be scheduled following consent. Per standard Duke Apheresis Protocol, all subjects will ha ve CBC with auto differential, CMP, ionized Calcium, and βHCG (WOCBP only) within 48 hours prior to the procedure(s). Leukapheresis #2 will occur approximately 14 (± 4) days after third i mmunization to obtain PBMCs for Immunologic Monitoring and generation of additional DCs for continued vaccinations. Additional leukapheresis can be obtained up to every 2 weeks if needed accordi ng to the Duke Apheresis Center protocol, but these will likely be needed less than every 2 months throughout the study to generat e enough DCs to continue vaccinations. No immune monitoring blood will be drawn at the additional leukapheresis procedures; only at leukapheresis #1 prior to radiation and at leukapheresis #2 appr oximately 2 weeks post vaccine #3. Additional leukapheresis following leukapheresis #1 will be represented as: Leukapheresis #1a, Leukapheresis #1b, etc. Additional leukapheresis following Leukapheresis #2 will be represented as: Leukapheresis #2a, Leukapheresis #2b, etc. 8On the day before the fourth DC vaccine , patients will receive vaccine site pre-conditioning strategy. A single dose of Td toxoid (1 flocculation unit, Lf) will be administered to a single side of the groin 12- 24 hours prior to the fourth DC vaccine, which is always given bilaterally at the gro in site. Standard of care blood for CBC (with auto differential) and CMP will be drawn during the pre -conditioning visit. 2 red top 10 mL draw tubes for immune monitoring will be obtained on the same day of the pre -conditioning visit but prior to patients receiving Td pre-conditioning. 9As part of standard care for these patients, upon tumor progression, participants may undergo stereotactic biopsy or resection. As this is not a research procedure consent will be obtained separately. However, if tissue is o btained, it will be used to confirm tumor progression histologically and to assess immunologic cell infiltration and pp65 ant igen escape at the tumor site. 10When CMV and/or methylation status is unknown, patients may sign CMV and/or leukapheresis consent for the Umbrella study (Pro00102797). This allows patients to undergo CMV screening and/or leukapheresis prior to signing main consent for this study. If patients sign the leukapheresis consent for the Umbrella study prior to leukapheresis, they will sign main consent for this study at the Post -RT/TMZ visit. I-ATTAC Version: 20220204 38 12.3 End of Treatment Vaccines are given as described above for a total of 10 or until progression (whichever comes first). O nce the vaccinations are complete, the treatment phase of the study will be over and the follow -up period will begin. 12.4 Follow -up Period Patients will be followed for survival , progression, and subsequent therapies only, and this data will be recorded by the research team . For recording of subsequent therapies, the type of therapy (ies) that the subject receives will be recorded with approximate start and stop date , if possible. For subjects who complete all 10 study vaccines without progressing , an attempt will be made to obtain blood for immunologic monitoring 2- 3 times a year at sta ndard Duke Preston Robert Tisch Brain Tumor Center visits . If the subject progresses in the follow -up period, blood for immunological monitoring will be drawn at the time of progression, if possible. 12.5 End of Study Rationale for taking a patient off study will be documented (see S ection 12.6.1). In the Follow -up Period, patients will be followed for survival , progression, and subsequent therapies only , and this data will be recorded by t he research team . 12.6 Early Withdrawal of Subject(s) 12.6.1 Criteria for Early Withdrawal Subjects may voluntarily withdraw from the study at any time . Subjects may be withdrawn by the PI and considered screen failures prior to vaccine therapy and the PI may also wi thdraw a subject from the study at any time based on his/her discretion. Reasons for PI -initiated withdrawal may include, but are not limited to the following: • Inability to complete appro ximately >4 weeks of RT with TMZ as per standard of care. • Inability to tolerate TMZ • Patients with an active infection requiring treatment or having an unexplained febrile illness (Tmax > 99.5° F) . • Patients requiring an increase in corticosteroids, with the exception of nasal or inhaled steroid, such that at the time of fir st vaccination they require a dose above 2 mg of dexamethasone / day. O nce vaccinations have been initiated, if patients subsequently require increased steroids, they will still be permitted to remain on the study, but every effort will be made to minimize steroid requirements. • Adverse events • Abnormal laboratory values • Abnormal test procedure results (DCs fail to meet release criteria; inability to tolerate leukapheresis ) • Protocol deviation • Administrative issues • Disease progression • Pregnancy 12.6.2 Follow -up Requirements for Early Withdrawal I-ATTAC Version: 20220204 39 Consented s ubjects that are withdrawn prior to cycle 1 of post -RT DI TMZ for any of the aforementioned reasons will be considered eligibility failures and thus will not be followed for survival. All other subjects will be followed by the study coordinator until death or are lost to follow -up. 12.6.3 Replacement of Early Withdrawal (s) As noted in Section 12.6.2, s ubjects who voluntarily withdraw prior to cycle 1 of post -RT DI TMZ will not be followed for survival, and will be replaced. 12.7 Study Assessments 12.7.1 Medical History Medical history will be obtained from the Duke electronic system and from the subject and/or family at the screening visit and reviewed at each study visit . This data may in clude the following: - All past medical and surgical history ; - Current medications ; - Changes in physical or neurologic symptoms ; - Any adverse events. 12.7.2 Physical Exam Vital signs and physical and neurologic examinations will be assessed and recorded along with a KPS score prior to enrollment and at each visit. 12.7.3 Use of Antihistamines Subjects will be advised to avoid antihistamine use 48 hours prior to each vaccine administration, the day of vaccine administration, and for 48 hours following each vaccine administ ration. If the subject has a pre- existing condition that requires antihistamine usage, the PI and the treating oncologist will decide if it is safe and appropriate for the subject’s antihistamines to be held before and following vaccine administrations. 12.7.4 Radiologic Evaluations Patients with newly diagnosed GBM will be imaged by MRI as per standard of care for eligibility and baseline measurements , and to assess progression prior to vaccine therapy; although the purpose of this study is not to detect tumor responses, any evidence of tumor response will be determined according to the Duke PRTBTC standard operating procedure ( SOP). Immunotherapy Response Assessment in Neuro- Oncology (iRANO) criteria [122, 123] established by consensus conference, will be used for assessment of response and pseudoprogression following immunotherapy . If pseudoprogression is suspected, the subject will continue with vaccine therapy for a minimum of 2-3 subse quent months so long as subject remains clinically and radiographically stable compared to the MRI showing suspected pseudoprogression. If the subject continues to do well beyond the 3 months, they will continue on study as planned. Subjects demonstrating definitive progression will be removed from study . Tumor progression will need to be documented histologically, unless there are clinical contraindications, to exclude inflammatory res ponses presenting as radiographic or clinical changes, which could indicate potentially toxic or therapeutic responses and not tumor progression. If tissue is obtained through the Duke Brain Tumor Biorepository , it will be used to confirm tumor progression histologically and to assess immunologic cell infiltration and examine pp65 expression to evaluate antigen loss at the tumor I-ATTAC Version: 20220204 40 site. Upon progression, patients may be treated on other therapies as directed by the treating Oncologist. 12.7.5 Immunologic Assessmen ts Immunological response evaluations will be conducted as described in Table 4. All patients will undergo at least 2 leukapheresis procedures – prior to radiation (1 red top 10mL tube) and approximately 2 weeks post vaccine #3 (9 yellow ACD 8.5mL tubes, 2 red top 10mL tubes). If an additional leukapheresis must occur earlier than 14 (± 4) days after vaccine 3, then no immune monitoring will be drawn at this additional leukapheresis. For subjects who complete all 1 0 study vaccines without progressing , an attempt will be made to obtain blood for immunologic monitoring 2-3 times a year at standard Duke neuro -oncology visits and at progression (whichever comes first). The immune monitoring blood that is collected prior to the initiation of post RT -TMZ cycle 1 (on the same day) will be the baseline for T reg analysis. The immune monitoring blood drawn at pre-vaccine #1 will be the baseline for polyfunctional T cell analysis. The blood drawn on the same day as (but prior to) Td preconditioning will be analyzed for the baseline CCL3. A comparison of pre-therapy lymphocyte functions to those at intervals after each immunization will be made . These tests may provide evidence for the development of immune responses following D Cs immunization and will play an important role in the design of future DC -based clinical trials. Cellular immune responses to pp65 will be measured by polyfunctional T cell assays using 10-12 color panels previously optimized in our laboratory , which includes detection of CCL3 on T cells . The effect of treatment on cytokines and other soluble factors will be assayed in the serum/plasma. Serum/plasma will be measured to determine levels of CCL3 from the blood drawn at the Td preconditioning visit, and at t he vaccine #4 visit . T Reg kinetics will be measured by using a constellation of markers specific for T Regs such as foxp3, CD25 and CD4. Whenever blood for immune monitoring is obtained, we will submit a CBC with auto differential to assess absolute number of T Regs. These will be drawn with SOC blood draws whenever possible. In addition, blood and tumor samples will be stored for possible future research in the Duke BTIP Laboratory where all other samples from this study are stored. The samples being stor ed are ONLY for this study . Our mouse studies being done in the BTIP Lab are identifying markers that may influence migration, therefore, as other markers are identified, we would like to investigate whether these same markers are identified in human subj ects enrolled on this trial. Polyfunctional Flow Cytometry Peripheral blood will be drawn into 9 yellow (ACD) tubes containing acid citrate dextrose and 2 red top tube s for serum. PBMC s will be separated by density gradient centrifugation using Leukosep tubes. Polyfunctional T cell analysis will be done in Dr. Sanchez -Perez’s immunology laboratory Dr. Sanchez -Perez ’s immunology laboratory conducts all the clinical immune monitoring for the Duke Brain Tumor Center . Polyfunctional T cell panels have been developed in our laboratory and optimized for detection of CCL3 in T cells . This process involves the rapid early detection and analysis of the production of Tumor Necrosis Factor -α (TNF -a), CCL3, IFN-γ, interleukin -2 (IL- 2), and CD107a (a marker of cell cytotoxicity) prior to cellular secretion following antigen -specific stimulation in vitro . The functional CD4 and CD8 immune response of the patients will be monitored using a 10- 12-color assay. To detect an increase in polyfunctional T cells we will analyze the fold change of T cells secreting TNF -a, CCL3, IFN-γ, IL-2 and CD107a from the blood I-ATTAC Version: 20220204 41 samples from pre -vaccine 1, and second leukapheresis (or blood draw) occurring 14 ± ( 4) days after vaccine #3. Isolated PBMC s will be stimulated for 6 hou rs with the pool of 138 peptide s spanning the entire pp65 gene . The cells will be stained for the surface markers CD3 (to identify it as a T cell), CD4 (to define as specific helper cell), CD8 (to define antigen specific cytotoxic T cells) and then submitted for intracellular cytokine staining. This procedure allows for the highly sensitive detection of TNF -a, CCL3, IFN-γ, IL-2 and CD107a secreting cells , which are assumed to be specifically responding to the stimulating antigen . The maturation state and the activation status of samples will be detected by polychromatic flow cytometry using optimized panels which will include CD3, CD4, CD8, CCR7, CD45RA, HLA - DR, CD69 and HLA -DR. Levels of naïve (TN – CD45RA+CCR7+), central memory (TCM – CD45RA- CCR7+), effector memory (TEM – CD45RA- CCR7 -), or terminally differentiated effector memory (TEMRA – CD45RA+CCR7+) T cells . Serology/ Multiplex Platform CCL3 and levels of other inflammatory soluble factors may be assayed by multiplex platform. The effect of treatment on CCL3 Serum/plasma will be assayed ( 2 red top 10 mL tube s) to determine if levels of CCL3 are impacted by Td pre-conditioning and will be drawn prior to the Td pre - conditioning as described above, and at the vaccine #4 visit (9 yellow ACD 8.5 mL tubes, , 2 red top 10 mL tubes) 24 hours after Td pre -conditioning. Antibody levels to the immunizing antigen may be measured. TReg Kinetics T Regs will be analyzed by fl ow cytometry using a constellation of markers specific for T Regs included CD4, CD25 and foxp3. For T Reg analysis , the time points will be leukapheresis 1, before DI TMZ in the Post -RT TMZ , pre- vaccine 1, leukapheresis 2, pre- vaccine 4, pre-vaccine 5, & pre- vaccine 6. We will analyze the percent of Foxp3, CD25, CD4 T cells over these time points. 13 SAFETY MONITORING AN D REPORTING 13.1 Adverse Events An AE is any untoward medical occurrence in a subject receiving pre-conditioning or the study vaccine and which does not necessarily have a causal relationship with this treatment . For this protocol, the definition of AE also includes worsening of any pre -existing medical condition . An AE can therefore be any unfavorable and unintended or worsening sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of the DCs, whether or not related to use of the DCs . Abnormal laboratory findings without clinical significance (based on the PI’s judgment) should not be recorded as AEs . But, laboratory value changes that require therapy or adjustment in prior therapy are considered adverse events. I-ATTAC Version: 20220204 42 Adverse events will be collected from the time of administration of the first DC vaccine through 1 month after the subject’s last vaccine, all AEs must be recorded in the subject ’s medical record and adverse events case report form. AEs will be assessed according to the CTCAE version 5. If CTCAE grading does not exist for an AE, the severity of the AE will be graded as mild (1), moderate ( 2), severe (3), life -threatening (4), or fatal (5). Attribution of AEs will be indicated as follows: - Definite: The AE is clearly related to the study drug - Probably: The AE is likely related to the study drug - Possible: The AE may be related to the stud y drug - Unlikely: The AE is doubtfully related to the study drug - Unrelated: The AE is clearly NOT related to the study drug Attribution of AEs will be determined for each study intervention individually (pp65 DC vaccine, pre-conditioning agent [Td], TMZ, bevacizumab). 13.1.1 Reporting of AEs A summary of all adverse events ( not just those considered related to the study drug) will be kept which will categorize the event by organ system, relationship to which treatment, its grade of severity, and resolution. Periodic review by the PI and monthly review at the PRTBTC Adverse Event meeting of the collective adverse events will occur with the intention of identifying any trends or patterns in toxicity. If any such trends are identified, depending on their severi ty and frequency, a protocol amendment will be considered. 13.2 Adverse Events of Special Interest Adverse events will not be collected until the patient has received the DC vaccine, unless they are considered an Adverse Event of Special Interest related to the leukapheresis procedure. Only these special interest adverse events will be collected prior to the DC vaccine, all other events occurring prior to DC vaccine administration will not be recorded or monitored. Special Interest Adverse Events that may occur during the leukapheresis procedure include: • Allergic Reaction • Anaphylaxis • Pre-syncope • Syncope • Vasovagal reaction Special Interest Adverse Events that may occur after the procedure, but may still be related to leukapheresis include: • Vascular Access Complications • Venous Injury 13.3 Serious Adverse Events I-ATTAC Version: 20220204 43 An AE is considered “serious” if in the opinion of the investigator it is one of the following outcomes: • Fatal • Life-threatening • Constitutes a congenital anomaly or birth defect • A medically sign ificant condition ( defined as an event that compromises subject safety or may require • medical or surgical intervention to prevent one of the three outcomes above). • Requires inpatient hospitalization or prolongation of existing hospitalization • Results in p ersistent or significant incapacity or substantial disruption to conduct normal life functions. 13.3.1 Reporting of SAEs All SAEs should be reported immediately to Dr. Mustafa Khasraw M.D. at 919- 684-5301 during regular business hours or at (Pager: 919-206-0493) after hours and to the FDA . Fatal or life - threatening, unexpected adverse events will be reported to the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 7 calendar days after first knowledge by the sponsor followed by as complete a report as possible within 8 additional calendar days . Serious, unexpected adverse events that are not fatal or life- threatening will be reported to the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 15 calendar days after first knowledge by the sponsor. All adverse events that are considered serious, unanticipated, and related or possibly related to the research (as defined by 21CRF312.32[a]) will be reported to the Duke University Medical Center IRB and the FD A using the appropriate SAE report ing process . At the time of the annual progress report to the Duke University Medical Center IRB and the FDA , a summary of the overall toxicity experience will be provided. Safety Oversight Committee (SOC OMM ) The Duke Ca ncer Institute (DCI) SOC OMM is responsible for annual data and safety monitoring of DUHS sponsor -investigator phase I and II, therapeutic interventional studies that do not have an independent data safety monitoring board ( DSMB ). The primary focus of the S OC is review of safety data, toxicities and new information that may affect subject safety or efficacy. Annual safety reviews includes but may not be limited to review of safety data, enrollment status, stopping rules if applicable, accrual, toxicities, re ference literature, and interim analyses as provided by the sponsor -investigator. The SOC in concert with the DCI Monitoring Team (see Section 14.1 for Monitoring Team description) oversees the conduct of DUHS c ancer -related, sponsor -investigator therapeutic intervention and prevention intervention studies that do not have an external monitoring plan, ensuring subject safety and that the protocol is conducted, recorded and reported in accordance with the protocol , SOPs, Good Clinical Practice (GCP ), and applicable regulatory requirements . 13.4 External Data and Safety Monitoring Board The Principal Investigator and Sub- Investigators must comply with applicable federal, state, and local regulations regarding reporting and disclosure of conflict of interest. Conflicts of interest (COI) may arise from situations in which financial or other personal considerations have the potential to compromise or bias professional judgment and objectivity. Conflicts of interest include I-ATTAC Version: 20220204 44 but are not limited to royalty or consulting fees, speaking honoraria, advisory board appointments, publicly -traded or privately -held equities, stock options, intellectual property, and gifts. The Duke University School of Medicine’s Research Integrity O ffice (RIO ) reviews and manages research -related conflicts of interest. The Principal Investigator and Sub- Investigators must report conflicts of interest annually and within 10 days of a change in status, and when applicable, must have a documented management plan that is developed in conjunction with the Duke RIO and approved by the IRB/ Institutional Ethics Committee ( IEC). Due to potential for COI in relation to proprietary interest in the pp65CMV DC vaccine, a Data Safety and Monitoring Board (DSMBplus ) has been established. The Duke PRTBTC DSMBplus Charter is available upon request . 14 QUALITY CONTROL AND QUALITY ASSURANCE 14.1 Monitoring The DCI Monitoring Team will conduct monitoring visits to ensure subject safety and to ensure that the protocol is conduct ed, recorded, and reported in accordance with the protocol, standard operating procedures, good clinical practice, and applicable regulatory requirements . As specified in the DCI Data and Safety Monitoring Plan, the DCI Monitoring Team will conduct routine monitoring after the third subject is enrolled, followed by annual monitoring of 1 – 3 subjects until the study is closed to enrollment and subjects are no longer receiving study interventions that are more than minimal risk . Additional monitoring may b e prompted by findings from monitoring visits, unexpected frequency of serious and/or unexpected toxicities, or other concerns and may be initiated upon request of DUHS and DCI leadership, the DCI Cancer Protocol Committee, the SOC, the sponsor, the Princi pal Investigator, or the IRB . All study documents must be made available upon request to the DCI Monitoring Team and other authorized regulatory authorities, including but not limited to the National Institute of Health, National Cancer Institute, and the FDA . Every reasonable effort will be made to maintain confidentiality during study monitoring. 14.2 Audits The Duke University Office of Audit, Risk and Compliance ( OARC ) may conduct audits to evaluate compliance with the protocol and the principles of GCP. T he PI agrees to allow the auditor(s) direct access to all relevant documents and to allocate his/her time and the time of the study team to the auditor(s) in order to discuss findings and any relevant issues. OARC audits are designed to protect the rights and well -being of human research subjects. OARC audits may be routine or directed (for cause). Routine audits are selected based upon risk metrics generally geared towards high subject enrollment, studies with limited oversight or monitoring, Investigator initiated Investigational Drugs or Devices, federally -funded studies, high degree of risk (based upon adverse events, type of study, or vulnerable populations), Phase I studies, or studies that involve Medicare populations. Directed audits occur at the di rective of the IRB or an authorized Institutional Official. I-ATTAC Version: 20220204 45 OARC audits examine research studies/clinical trials methodology, processes and systems to assess whether the research is conducted according to the protocol approved by the DUHS IRB. The primary purpose of the audit/review is to verify that the standards for safety of human subjects in clinical trials and the quality of data produced by the clinical trial research are met. The audit/review will serve as a quality assurance measure, internal to th e institution. Additional goals of such audits are to detect both random and systemic errors occurring during the conduct of clinical research and to emphasize “best practices” in the research/clinical trials environment . 14.3 Data Management and Processing 14.3.1 Study Documentation Study documentation includes but is not limited to source documents, case report forms, monitoring logs, appointment schedules, study team correspondence with sponsors or regulatory bodies/committees, and regulatory documents that can be found in the DCI -mandated “Regulatory Binder”, which includes but is not limited to signed protocol and amendments, approved and signed informed consent forms, FDA Form 1572, College of American Pathologists ( CAP ) and Clinical Laboratory Improvement Act (CLIA) laboratory certifications, and clinical supplies receipts and distribution records. Source documents are original records that contain source data, which is all information in original records of clinical findings, observations, or other activities i n a clinical trial necessary for the reconstruction and evaluation of the trial . Source documents include but are not limited to hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing records, recorded data from automated instruments, copies or transcriptions certified after verification as being accurate copies, microfiches, photographic negatives, microfilm or magnetic media, x -rays, subject files, and records kept at the pharmacy, at the laboratories and at medico -technical departments involved in the clinical trial. When possible, the original record should be retained as the source document . However, a photocopy is acceptable provided that it is a clear, legible, and an exact duplication of the original document. 14.3.2 Data Management The subject’s medical records will be the primary source document for the study. Source documents include all information in original records and certified copies of original records of clinical findings, observations, or other activities in a clinical investigation used for reconstructing and evaluating the investigation. 1 Source documentations may also include paper eligibility checklists, data flowsheets, patient reported outcomes and other paper documents. The PI, study coordinator, study research nurse, data management team and all associated study key personnel, are permitted to make entries, changes, or corrections in the source documents or database per the study delegation of authority log . Errors on the source documents will be crossed out with a single line, and this line will not obscure the original entry. Changes or corrections will be dated, signed, initialed, and explained (if necessary). Database changes will be tracked via electro nic trail automatically . 1 In 21 CFR 312.62(b), reference is made to records that are part of case histories as “supporting data;” the ICH guidance for industry E6 Good Clinical Practice: Consolidated Guidanc e (the ICH E6 guidance) (available at http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm) uses the term “source data/documents.” For the purpose of this guidance, these terms describe the same information and have been used interchangeably. I-ATTAC Version: 20220204 46 14.3.3 Data Management Procedures and Data Verification The DCI Information Technology (IT) Shared Resource has developed Title 21 CFR Part 11 compliant databases for cancer clinical trials. DCI IT has extensive expertise in database quality assurance, data standards, and use of the C ancer Biomedical Informatics Grid ( caBIG ) tools to support cancer researchers. Data queries will be generated automatically by the e lectronic case report form (eCRF ) system. These data queries signify the p resence of data inconsistencies. The study and data management team will cross -reference the data to verify accuracy. Missing or implausible data will be highlighted for the PI requiring appropriate responses (i.e., confirmation of data, correction of data , completion or confirmation that data is not available, etc.). The database will be reviewed and discussed prior to database closure, and will be closed only after resolution of all remaining queries . 14.3.4 Coding All medical terms will be coded using CTCAE ( version 5) . 14.3.5 Study Closure Following completion of the studies, the PI will be responsible for ensuring the following activities: • Data clarification and/or resolution ; • Accounting, reconciliation, and destruction/return of used and unused study drugs ; • Revi ew of site study records for completeness ; • Shipment of all remaining laboratory samples to the designated laboratories . 15 STATISTICAL METHODS AND DATA ANALYSIS All statistical analysis will be performed under the direction of the statistician designated in key personnel.