6. BACKGROUND 6.1 Significance and Overall Rationale Malignant brain tumors now represent the second most frequent cause of cancer death in children. Despite aggressive and highly toxic multi -modality therapy including surgery, craniospinal radiation, and HDC + PBSCT, almost half of the children diagnosed with medulloblastoma and primitive neuroectodermal tumors (MB/PNETs), still die from recurrent dis ease. Furthermore, survivors are often left with severe and lifelong treatment- associated cognitive and motor deficits. The development of more effective and less toxic tumor -specific therapies is paramount in improving clinical outcomes for children affe cted by MB/PNETs. Immunotherapy targeting tumor -specific antigens expressed within brain tumors is a treatment modality potentially capable of meeting this clear and urgent need. Despite considerable advancements and promising clinical results observed in immunotherapy trials at our center and others directed against adult malignant brain tumors, efforts in the immunologic treatment of pediatric brain tumors have been limited to relatively few notable studies. This is often due, at least in part, to the limited amount of viable tumor tissue available for tumor cell- based vaccine preparations, and the lack of identification of consistently expressed tumor -specific antigens within these cancers. The use of total tumor RNA -loaded dendritic cells (DCs) was pioneered by Drs. Nair and Gilboa at Duke University , as a novel platform for inducing potent immunologic responses against the variety of uncharacterized and patient -specific antigens present within malignant tumor cells. We have demonstrated that sufficient RNA for clinical vaccine preparations can be amplified with high fidelity using existing molecular technologies from as few as 500 isolated pediatric and adult brain tumor cells, thus allowing vaccine preparation from surgical biopsies and even microdisse cted archival tumor specimens. We are currently exploring adoptive cellular therapy using amplified tumor RNA -pulsed DCs and autologous lymphocyte transfer (DC + xALT therapy) in adult patients with recurrent GBM (FDA IND BB -13630; Duke IRB protocol 6677; PI: Duane A. Mitchell, M.D., Ph.D.). In this proposal, we aim to extend evaluation of this novel platform to the treatment of recurrent MB/PNETs (reMB/PNETs) during hematopoietic recovery from HDC + PBSCT and NMA salvage chemotherapy . Immunotherapy adminis tered during recovery from HDC and NMA salvage therapy may have tremendous advantages, as adoptive cellular therapy following lymphodepletive conditioning regimens has emerged as the most effective treatment strategy for advanced and refractory melanoma . Remarkable objective clinical responses in up to 75% of treated refractory melanoma patients have been observed, including durable complete regressions of metastatic lesions within the CNS . Although the mechanisms by which lymphodepletion enhances immunotherapy in humans are not well elucidated, elegant studies in murine tumor models have highlighted some key catalysts for induction of potent anti -tumor immunity. D epletion of immunosuppressive T regs, increased bioavailability of inflammatory cytokin es and homeostatic proliferative cytokines (most notably IL -7 and IL -15) after removal of host lymphocytes and NK cells that Re-MATC H Protocol Page 38 of 106 Version: 20180628 compete with transferred tumor -specific T cells, and increased toll -receptor agonistic signals during myeloablative therapy play si gnificant roles . Our HYPOTHESIS is that DC + xALT therapy targeting reMB/PNETs during recovery from myeloablative (MA) and non- myeloablative (NMA) chemotherapy will induce potent tumor -specific immune responses that mediate the safe eradication of invasi ve malignant disease. In this effort, we aim to leverage the myelosuppressive toxicity of standard- of-care HDC and NMA salvage therapy in order to enhance the potential efficacy of a novel immunotherapy employing the use of amplified tumor RNA -pulsed DCs. Toward that end, our SPECIFIC AIMS are to: 1. Conduct a Phase II clinical trial of DC + xALT therapy during hematopoietic recovery from NMA salvage chemotherapy and HDC + PBSCT in pediatric patients with reMB/PNETs with the following sub -aims: a. Determine the impact of DC +xALT therapy on progression- free survival (PFS) and overall survival (OS) of pediatric patients with reMB/PNETs compared to historical cohort ; b. Investigate the correlation between clinical outcome and the frequency and persistence of tumor - specific T cells in the peripheral blood of treated patients; c. Determine the impact of induction therapy and HDC + PBSCT (Group A) or salvage chemotherapy (Group B) on circulating levels of homeostatic and inflammatory cytokines, toll -like receptor activation status, and on the functional recovery of lymphocyte and NK cellular subsets in children with re - MB/PNETs receiving DC + xALT therapy; d. Characterize the frequency of expression of the TAA -40 identified in common malignancies in first an d second recurrent MB/PNETs in order to identify potential candidates for subsequent evaluation in antigen -specific immunotherapy approaches . 6.2 Immunotherapy for Pediatric Brain Tumors Despite brain tumors being the leading cause of cancer deaths in children and the considerable advances in immunologic targeting of adult brain tumors, studies examining immunotherapy for MB/PNETs are markedly lacking except for a few notable reports. Efforts in targeting pediatric brain tumors are hindered somewhat by the rel atively small number of clinical cases compared to adult cancers, the lack of syngeneic murine models of common pediatric brain tumors in which immunotherapy efforts can be evaluated in preclinical studies, and the significant regulatory hurdles in conduct ing experimental therapies in the pediatric setting. Despite these limitations, the use of HDC + PBSCT and NMA chemotherapy in pediatric patients with reMB/PNETs affords an ideal setting in which to leverage the recent findings by our laboratory and other s in the benefits of treatment- induced lymphopenia in enhancing immune responses to tumor immunotherapy. Three notable studies have demonstrated the safety and potential efficacy of immunotherapy in children with malignant brain tumors. Ashley and collea gues conducted a phase I study of 9 pediatric patients with recurrent brain tumors using monocyte -derived DCs pulsed with tumor RNA isolated from resected tumor specimens [1]. DCs were derived from monocytes after 7 days of culture with IL -4 and granulocyte -macrophage colony -stimulating factor (GM - CSF), incubated with RNA for 45 minutes in serum -free media, washed and then cyropreserved until administration. DC vaccines were administered intradermally and intravenously in treated patients. The feasibility and safety of tumor RNA pulsed DC vaccines was demonstrated in this study with 7 of the 9 enrolled patients receiving RNA -pulsed DC vaccines. 1 patient demons trated a partial response to therapy as assessed by MRI and 2 patients showed disease stabilization. Tumor -specific immune responses were demonstrated after vaccination in 2 of 7 patients. A subsequent study by the same group in 11 pediatric patients wi th newly -diagnosed, Stage 4 neuroblastoma, evaluated tumor RNA -pulsed DC vaccines after HDC+PBSCT [2]. The patients received t wo courses of carboplatin induction therapy followed by standard chemotherapy, surgery, radiation, and HDC + stem cell rescue. DC vaccines were administered intravenously and intradermally beginning six weeks after stem cell transplantation and were prepared as described in the previous study. The investigators noted immunologic Re-MATC H Protocol Page 39 of 106 Version: 20180628 impairment in recall responses an d proliferative responses in patients at 6 weeks post HDC+PBCT. 7 of 11 enrolled patients were successfully treated during this study. The safety and feasibility of the total tumor RNA DC vaccines after HDC + PBSCT was demonstrated by this study but no cl inical or radiographic responses were observed. Importantly, Ashley and colleagues demonstrated the safety and feasibility of total tumor RNA -pulsed DCs in pediatric patients with recurrent and primary brain tumors in these studies. Recent advances, how ever, in our understanding of DC biology and their use in anti -tumor immunotherapy have made even the limited clinical responses observed in the first study somewhat encouraging. These significant differences in the previously published reports and the me thods we will use in this study are as follows: • Ashley and colleagues used immature DCs in their study which have now been shown to be vastly inferior in the induction of immunologic responses. Our DCs are matured with a combination of IL - 1β, TNF α, and IL -6. • Route of injection comparisons have shown that intravenous injection of DCs in humans is in many cases tolerigenic and the preferred route for induction of immunity is intradermal. Our vaccines will be administered through intradermal injection only. • We have shown electroporation of RNA into DCs to be 100 to 1000 times more efficient than co- incubation of DCs with “naked RNA”. The labile nature of RNA results in degradation with a half - life of seconds to minutes, thus electroporation to deliver RNA dir ectly into DCs is far superior in mediating gene expression and anti -tumor immunity. • We and others have shown that the early recovery period after lymphodepletion is the most effective period for enhancing immunity to vaccination and adoptive T cell transfer and thus we will administer DC+ALT therapy during the peri -transplant period (24 hrs after PBSCT) as has been done in studies at the NCI using myeloablative therapy and adoptive cellular therapy in patients with melanoma. • Ashley and colleagues administered 3 to5 vaccines to their patients, while we will continually vaccinate patients throughout the recovery period of lymphopenia and normalization of T cell function (up to 1 year post HDC + PBSCT) as we have shown capacity to increase immunologic responses dramatically over time in vaccinated patients with newly -diagnosed GBM. In fact, significant anti - tumor immune responses have often occurred in our patients only after repetitive monthly vaccinations (Figs.