Darell D. Bigner, M.D., Ph.D. - US grants

This is a Program Project Grant on human malignant gliomas and medulloblastoma. As detailed in the body of this application, these two nervous system neoplasms remain as significant health problems. Despite intensive research efforts by many investigators, there has been little progress made in the etiology, early diagnosis, or treatment of malignant primary brain tumors during the last 20 years. This Program Project Grant will solidify and intensify the efforts of an outstanding group of internationally recognized investigators with a long history of effective collaboration. They will approach the malignant brain tumor problem in areas of etiology, diagnosis, pre-clinical, and clinical treatment. Projects range from mechanistic studies on promotion and progression of brain tumors in experimental animals (Project 4) to development and characterization of operationally specific glioma antigens defined my murine and primate monoclonal antibodies (Project 1); analysis and manipulation of control of blood flow and permeability in gliomas and medulloblastoma (Project 2); new methods of treatment of brain tumors in experimental animal with antigenic modification of tumor cells and utilization of specialized drug and radiation delivery systems (Project 3); and clinical investigation (Project 5), extending an immunotherapeutic trial which has been shown non-toxic and highly promising in phase I trials and initiating a randomized phase III evaluation of a new drup (AZQ) which has been show to be highly promising phase II trials.

Our objectives are to investigate basic aspects of brain tumors (especially gliomas) in etiological, immunological, tumor biological, and experimental chemotherapeutic areas. In etiological studies, we will seek the association of retroviruses and human papovaviruses with spontaneous human or animal gliomas, establish the transplacental neuro-oncogenicity of acrylonitrile, study genetic susceptibility of mice to neuro-oncogenetic viruses and chemical carcinogens, and use banding and nonbanding cytogenetic techniques to guide etiological studies of animal and human gliomas. In immunological studies, we will purify and characterize antigens associated with spontaneous human and mouse gliomas and determine if quantitation of these antigens can be used to monitor tumor size and as an adjunct in diagnosis. In tumor biological studies, we will establish whether or not the phenotypic heterogeneity of neoplastic cells in human gliomas with respect to morphological, biochemical, and immunological differentiation extends to chemotherapeutic sensitivity as tested in athymic mice and whether endothelial cells of human and experimental gliomas are neoplastic. We will use a human glioma animal model to test intracarotid chemotherapy and radioimmunotherapy with radioiodinated monoclonal antibodies against human gliomas in rats. (TA)

We are seeking support for the Sixth International Conference on Brain Tumor Research and Therapy. This meeting, which will be held between October 20 and October 23, 1985, has been held biannually since 1975. Its purpose is to exchange information and expand communication and existing knowledge in basic and clinical research on central nervous system (CNS) neoplasms. An Advisory Council will oversee the general organization of the meeting, and a Program Committee will select specific topics and speakers. No attempt will be made to be comprehensive; rather, we will emphasize areas of particular interest and in which imminent progress is anticipated. The meeting will be open for abstract submission, although participation will be limited to 150 individuals. The tentative agenda includes clinical and experimental therapy, tumor biology (including oncogenes and growth factors), CNS carcinogenesis, quantitative CNS tumor imaging, and immunobiology of primary CNS tumors. Proceedings of the Conference will be published in the Journal of Neuro-Oncology.

The central hypothesis of this proposal is that there is an abnormal qualitative and quantitative distribution of gangliosides in malignant gliomas and medulloblastomas, perhaps similar to fetal brain, but different from normal adult tissues, especially normal adult neurons and glia. Biochemical analyses will first characterize and quantitate the types of gangliosides present in a large series of malignant human glioma biopsies, permanent glioma derived cell lines, and athymic mouse and rat-human glioma xenografts and detemine if unique gangliosides are present which are characteristics of gliomas. This methodology will then be utilized to similarly characterize and quantitate the types of gangliosides present in our models of human medulloblastoma. Our specific aims are: a) To produce monospecific monoclonal antibodies directed against the unique D-54 MG mono- and disialogangliosides 3'-iso-LM1, LD1, and GM3, GD3, GM3, GD2 and additional unique gangliosides defined in the course of this study; b) to define the distribution in human glioma tissue of these gangliosides qualitatively at the cellular level using immunocytochemistry with monoclonal antibodies directed against 3'-iso-LM1, LD1, GM3, GD3, GM2, GD2 and each new unique ganglioside and quantitatively at the tissue level using analytic techniques and thin layer chromatography on multiple human gliomas with varying cytomorphologies and degrees of malignancy; c) to use these monoclonal antibodies directed against 3'-iso-LM1, LD1, GM3, GD3, GM2, GD2 and each new unique ganglioside to examine the function of the defined gangliosides in cell-cell interactions, clonogenicity, and invasion utilizing in vitro and in vivo assays of cell growth, adhesion, and invasiveness; d) to use those monoclonal antibodies directed against 3'-iso-LM1, LD1, GM3, GD3, GM2, GD2 or any of the new unique gangliosides exhibiting broad reactivity within and among human gliomas and minimal normal tissue reactivity to demonstrate radioimaging and radiotherapeutic efficacies in the nude mouse - human glioma xenograft model; e) to define similarly the potentially abnormal qualitative and quantitative distribution of gangliosides in medulloblastoma, using the most complete and rigorously characterized models for this tumor extant and to execute similar studies as with gliomas. These studies will, therefore define the biodistribution of the altered gangliosides in human glioma and medulloblastoma, contribute to the understanding of the functional significance of gangliosides in adhesion, growth, and invasion, and establish pre-clinical efficacy of radiolabeled anti-ganglioside antibodies in glioma and medulloblastoma radioimaging and radiotherapy to aid in selecting monoclonal antibodies for clinical trials.

This is a Specialized Center of Research on human malignant gliomas and medulloblastomas. As detailed in the body of this application, these two nervous system neoplasms remain as significant health problems. Despite intensive research efforts by many investigators, there has been little progress made in the etiology, early diagnosis, or treatment of malignant primary brain tumors during the last 20 years. This Specialized Center of Research will continue the solidification of and intensification of the efforts of an outstanding group of internationally recognized investigators with a long history of effective collaboration. They will approach the malignant brain tumor problem in areas of etiology, mechanisms of altered growth control, diagnosis, pre-clinical, and clinical therapy. Projects range from: mechanistic studies on etiology, potential suppressor genes, and amplified and activated oncogenes in childhood brain tumors (Project IVa); to selection of glioma and medulloblastoma associated molecules for targeted therapy and growth inhibition (Project I); specifically production of anti- ganglioside and glycolipid monoclonal antibodies (Project Ia); analysis of new gangliosides and glycolipids and specificity analysis of anti-ganglioside mono-clonal antibodies (Ia1); development of operationally specific monoclonal antibodies against receptors and other proteins associated with glioblastomas and medulloblastomas (Project Ib); detailed study of the structure of the normal and abnormal epidermal growth factor receptor and its role in altered growth control in gliomas and medulloblastomas (Project Ic); protein biochemistry support for sequencing the proteins and epitopes under study in Projects Ib and Ic (Project Ib1, Ic1); to delivery of macromolecules and therapy of human glioma and medulloblastoma xenografts, specifically mechanisms of control of blood flow and permeability to normal brain and brain tumors (Project IIa); development of radiolabeled monoclonal antibodies for radioimmunotherapy of ana-plastic human glioma, medulloblastoma, and neoplastic meningitis (Project IIb); and increased delivery and therapy of human gliomas and medulloblastomas with radiolabeled monoclonal antibodies and drugs (Project IIc); to human therapeutic trials including intrathecal therapy of patients with neoplastic meningitis with radiolabeled monoclonal antibodies (Project III, IIIa, and IIIb); bifunctional alkylator therapy of medulloblastomas and childhood gliomas (Project IVb); to multiple modality clinical therapy trials with brain tumor patients in Projects Va, Vb, Vc, and Vd, including Phase I, Phase II, and randomized Phase III trials.

radionuclides; meningitis; neoplasm /cancer radionuclide therapy; melanoma; neoplasm /cancer immunotherapy; iodine; monoclonal antibody; nervous system neoplasms; human therapy evaluation; radiobiology; neoplasm /cancer radionuclide diagnosis; clinical trials; drug administration routes; hybrid antibody; medulloblastoma; glioma; breast neoplasms; metastasis; meninges; radiation therapy dosage; scintillation cameras; human subject; laboratory mouse;

DESCRIPTION: (Applicant's Abstract) Primary and metastatic brain tumors and neoplastic meningitis (NM) are resistant to present nonspecific therapies which often result in significant toxicity to normal brain. Targeted therapy with radiolabelled monoclonal (MAbs) and immunotoxins is an attractive approach for increasing efficacy for brain tumors and decreasing toxicity to normal CNS but it needs refinement. Anti- epidermal growth factor receptor variant III (anti-EGFRvIII) MAbs react with glioblastoma multiforme, breast and lung carcinoma, but not normal tissue. The applicant's hypothesis is that 1) tumor specific MAbs such as anti-EGFRvIII MAbs, 2) genetically engineered MAb fragments with appropriate affinity, half-life, and tumor penetration and 3) high linear energy transfer radionuclides such as the alpha-emitter 211At and Pseudomonas exotoxin MAb constructs will improve MAb therapy. The Specific Aims are: 1) To develop additional murine MAbs against different conformational or linear sequence epitopes of cell-surface expressed EGFRvIII; to select from their three characterized and new anti-EGFRvIII MAbs the best localizing radiolabelled MAb and the most toxic immunotoxin-conjugated MAb; and to prepare from the best anti-EGFRvIII murine MAb human/mouse chimeric and humanized MAbs, genetically engineered CH2-domain deleted "F(ab')2," Fab, and monovalent (sFv) and divalent (sFv(2)) single-chain fragments with a range of affinities. 2) To construct potent immunoconjugates from the best EGFRvIII MAbs and fragments of Aim 1 by labeling them with 131I or 211At using approaches that maximize tumor retention of radioactivity after internalization and by preparing immunotoxins via chemical or genetic incorporation of regions of Pseudomonas exotoxin A. 3) To select the best MAb or fragment construct on the basis of in vitro localization as determined by projected radiation dosimetry from paired-label biodistribution and quantitative autoradiography; and on the basis of survival prolongation in human intracerebral and NM xenograft models following therapeutic trial of equitoxic doses of radiolabelled MAbs and immunotoxins. To determine the least toxic and most effective route of administration for the radiolabelled MAbs and fragments and immunotoxins comparing intravenous, intratumoral, intraresection cavity, and intrathecal administration. 4) To perform all quality control, toxicologic, pharmacologic, and pathologic evaluation required to obtain FDA approval for Investigational New Drug permits for the best immunotoxin and 131I- and 211At-labeled anti-EGFRvIII constructs; and to pursue clinical trials with support from the NINDS-Duke Specialized Research Brain Tumor Center (NS20023, D. Bigner, Director).

This is the only fully funded Specialized Research Center of the National Institute of Neurological Disease and Stroke on primary and metastatic brain tumors of adults and children. It is presently in its 10th year of funding and this application represents its third submission for competitive review and renewal. As detailed in the body of this application, both primary and metastatic tumors to the CNS remain as significant health problems and are actually increasing in magnitude. Despite intensive research efforts by many investigators there has been little progress made in uncovering etiology, or improving treatment of primary or metastatic brain tumors during the last 30 years. This Specialized Research Center on Primary and Malignant Tumors of the CNS will continue intensification of the brain tumor research efforts of an outstanding group of internationally recognized investigators with a long history of effective collaboration. They will approach primary and metastatic brain tumors in adults and children in areas of etiology, mechanisms of transformation and altered growth control, improved diagnosis, preclinical and clinical therapy. Projects range from basic mechanistic studies on etiology such as tumor suppressor genes in human medulloblastoma, discovery of new drugs and mechanisms of drug resistance, to selection of molecular targets against adult and childhood brain tumors for targeted therapy and growth inhibition. Specific development of targeted therapy with radiolabeled and toxin-conjugated; preclinical and clinical trials of regional or compartmental therapy with radiolabeled MAbs, toxin-conjugated MAbs, and new drugs will ultimately be combined in years 3-5 with Phase 2 and 3 studies of systemic therapy with regional and compartmental therapy to reach tumor cells in the entire neuraxis. We believe that during this five year grant period better control of local and distant recurrence of primary and metastatic brain tumors can be achieved, improvement can be made in therapy of neoplastic meningitis, and quality and quantity of survival of both groups of patients will be improved. Moreover, our basic studies should define new molecular targets for future approach in 5-10 years with additional targeted therapy such as gene therapy replacement of putative suppressor genes and gene therapy of other molecular targets involved in transformation and altered growth control.

Core support is requested for a Staff Assistant, a Laboratory Assistant, and a Research Technician responsible for respectively; 1) budgetary, administrative, and clerical activities; 20 maintenance of medium solutions, instrument and glassware preparation; and 3) maintenance of a barrier-sustained isolator nuclear colony of athymic mice and rats and a breeding colony of athymic mice and rats derived from that nuclear colony. This Research Technician also is responsible for passage of human brain tumor xenografts and growth and supply of those xenografts to individual projects which then grow them for studies as they are needed.

human therapy evaluation; meningitis; neoplasm /cancer radionuclide therapy; neoplasm /cancer chemotherapy; brain neoplasms; combination cancer therapy; metastasis; neoplasm /cancer immunotherapy; infection related neoplasm /cancer; clinical trial phase I; biotin; radiation therapy dosage; exotoxins; immunoconjugates; drug administration routes; camptothecin; melphalan; monoclonal antibody; medulloblastoma; glioma; clinical research; human subject;

isolation /quarantine; neoplasm /cancer

DESCRIPTION (provided by applicant): There will be more than 17,000 new cases of primary malignant brain tumors diagnosed in 2002 and more than 13,100 deaths. There has been little progress in the treatment of malignant gliomas in the last 30 years. Unarmed and armed MAbs are now being approved by the FDA for treatment of systemic cancers such as breast carcinoma, non-Hodgkin's lymphoma and hairy cell leukemia. Our hypothesis is that poor drug delivery and widespread migration of GBM cells will be overcome by using intracranial microdiffusion [(convection-enhanced delivery (CED)] of MAbs or their fragments as unarmed MAbs, toxin conjugates, or radiolabeled conjugates. Genotypic and phenotypic heterogeneity and cellular resistance to chemotherapy will be overcome by targeting multiple cell-surface expressed molecular targets of GBMs, namely EGFRvIII, MRP3, GPNMBwt and GPNMBsv, and 3'-isoLM1 and 3'6'-isoLD1. Anti-EGFRvIII MAbs have been developed in the last period of this grant and an scFv-PE38 KDEL single fragment chain Pseudomonas exotoxin construct will enter clinical trial in late 2002. We propose raising three additional MAbs, one reactive with GPNMBwt and GPNMBsv, another specific for 3'-isoLM1 and 3'6'-isoLD1, and anti-MRP3 MAbs. All of these molecules are involved in the malignant phenotype of GBM. Elimination of cells expressing these four molecules should result in significant survival increases in GBM patients. Our specific aims are: 1) To prepare high affinity MAbs reactive with GPNMBwt and GPNMBsv, MRP3, and 3'-isoLM1 and 3'6'-isoLD1.2) To use the MAbs from Specific Aim 1 and anti-EGFRvIII MAbs from the previous grant period to determine the true incidence of expression, cell and tissue localization and heterogeneity of expression of GPNMB, MRP3, 3'-isoLM1 and 3'6'-isoLD1, and EGFRvIII in malignant gliomas. 3) To determine in vitro whether unarmed MAbs reactive with GPNMBwt and GPNMBsv, MRP3, and 3'-isoLM1 and 3'6'-isoLD1 have anti-proliferative and/or apoptosis-initiating activity. 4) To prepare scFv-Pseudomonas toxin constructs and 131I and 211Atlabeled divalent minibodies reactive with GPNMBwt and GPNMBsv, MRP3, and 3'-isoLM1, and 3'6'- isoLD1, and to compare their cystostatic and cytocidal activity in vitro and in vivo. 5) Under D. Bigner's Brain Tumor Center grant, perform FDA-required toxicity and efficacy of three best toxin and three best radiolabeled constructs and submit IND and carry out clinical studies in glioma patients.

DESCRIPTION (provided by applicant): The Duke Brain Tumor SPORE grant consists of 5 projects, 5 cores, a developmental Research Program and 2 Career Development Program. Each project is led by a basic and clinical principal investigator. Project 1: Development of monoclonal antibody reagents against targets identified in Project 2 by serial analysis of gent expression. Project 2 will provide new peptide-based targets 'for vaccine trials in Project 3 and identify new cell-surface targets for Project 1. It also will identify small molecule inhibitors of signal transduction pathway: Activated in gliomas. Project 4 will investigate DNA repair-mediated BCNU resistance and new treatments to welcome that resistance. Project 5 is a case-control study of environmental risk factors and polymorphisms in metabolic genes and the risk and outcome of primary brain tumors. The cores include a tissue procurement core, an investigational new drug permit core, a Phase 1/11 clinical trials core, a biostatistics and information systems core and an administrative core. The Developmental Research Program will consist of pilot project. Identified by seeking applications from the entire cancer center membership. The two initial projects are treatment of central nervous system malignancies with radiohalogenated therapeutics undergoing DNA incorporation by Dr. Michael Zalutsky and convection-enhanced delivery for brain tumors by Dr. Dennis Dr. Groothuis. The two initial Career Developmental awardees are Dr. Matthias Gromeier for non-pathogenic onocolytic poliovirus recombinants for the treatment of malignant brain tumors and delineation of molecular determinants of responsiveness to EGFR signal transduction inhibitors by Dr. Jeremy Rich.

During the last five years the Duke Comprehensive Cancer Center has continued to fulfill its mission, maturing as a "matrix-plus" organization. Five young investigators have been recruited directly into the Section of Cell Growth, Regulation and Oncogenesis. Overall, recruitment throughout the Duke University Medical Center has facilitated growth of the Cancer Center to include 255 members who participate in 12 Programs. The Cancer Center's Programs have been reorganized to reflect more accurately interactions between members, as well s to facilitate more effectively their collaboration. New Programs have been established in Structural Biology, Host Resistance and Leukocyte Biology, Multidisciplinary Clinical Trials and Cancer Prevention, Detection and Control Research. Shared Resources now include 23 facilities that facilitate both laboratory and clinical cancer research. New facilities have been established in protein crystallization, 2-D gel analysis, immunocytochemistry and somatic cell hybridization. Resources have been substantially strengthened in peptide sequencing, molecular graphics, nuclear magnetic resonance, confocal microscopy, in-patient clinical research, bone marrow transplantation and biostatistics. The Cancer Center Isolation Facility has been doubled in size. New construction has been initiated that will provide 40,000 NSF of additional space for Cancer Center investigators in fundamental and bridging sciences. Use of development funds has permitted recruitment into the Section of Cell Growth, Regulation and Oncogenesis, a dramatic expansion of biostatistics, initiation of the Cancer Prevention, Detection and Control Research Program, recruitment in psychosocial investigation, strengthening of Shared Resources, provision of interim funding and the support of pilot projects. Facilitated by the Core Grant, Duke investigators have published greater than 4,500 papers during the last five years that have contributed substantially to the fields of cell regulation, DNA replication, structural biology, viral oncology, leukocyte biology, neurooncology, autologous bone marrow transplantation, gynecologic oncology, multidisciplinary treatment, clinical application of hematopoietic growth factors and cancer screening. Support is requested for Senior Leaders, Program Leaders, Administration, Shared Resources and Development to implement fully the strategic plan for the Duke Comprehensive Cancer Center.

DESCRIPTION (provided by applicant): This Duke Brain Tumor SPORE grant consists of four projects, five cores, a Developmental Research Program, and a Career Development Program. The goal of the proposed research is to advance knowledge of brain tumor biology and etiology and to translate these into clinical protocols that represent significant and novel advances in the management of these therapeutically intractable tumors. The grant, as a whole, and each project and core, are led by basic and clinical Principal Investigators. In Project 1, novel therapeutics that target the GSTP1 protein, overexpressed and a poor prognostic indicator in malignant gliomas, will be rationally developed and evaluated for clinical efficacy. Project 2 will investigate novel molecular mechanisms of resistance of CNS tumors to Temodar, a clinically active brain tumor agent and evaluate the clinical efficacy of a PARP inhibitor in reversing Temodar resistance. Project 3 will investigate dendritic cell-based vaccination against CMV as a therapeutic modality in malignant glioma and the underlying mechanisms of the anti-tumor response. In Project 4, molecular and epidemiologic studies will examine the etiology of brain tumors in a case-control study. Exposure to putative neurocarcinogens will be examined and polymorphisms in metabolism and DNA repair genes will be examined as a determinant of brain tumor risk and treatment-associated toxicity. The cores include a Tissue Procurement and Genetics Core, an Investigational New Drug Permit Core, a Phase I/II Clinical Trials Core, a Biostatistics and Information Systems Core, and an Administrative Core which will provide critical infrastructural and service functions. The Developmental Research Program will consist of pilot projects identified from the entire Cancer Center membership at Duke as well as from selected, outside institutions. Emphasis is on investigators who have not previously worked in neuro-oncology. Nine examples of pilot projects from senior investigators at Duke who have not previously worked on brain tumors are included.

neoplasm /cancer therapy; brain neoplasms; health science research potential; health science research support; biomedical facility;

Seeinstructions): Core A. IND, Regulatory, and Bioinformatics Core. Darell D. Signer, M.D., Ph.D., Core Leader Core A integrates the development, certification, pre-clinical evaluation, biostatistical and regulatory aspects of reagents that will be evaluated in the SRC and separately funded grants. Specifically, Core A provides: 1) continued development and certification of new reagents directed against targets identified by gene mining approaches in separately funded grants and the establishment and characterization of genetically modified antibodies, immunotoxins, and vaccine antigens in the form of peptides or RNA for clinical application;2) preparation of IND applications and maintenance of regulatory compliance for all reagents generated in this SRC or in separately funded grants;and 3) coordinated informatic and statistical oversight and analysis during the design and conduct of the pre-clinical and clinical studies outlined in the SRC and in separately funded grants. This Core will focus on two areas: 1) chimerization, fragment engineering, biochemical and genomic production and pre-clinical evaluation of monoclonal antibody and immunotoxin constructs in athymic rodent models for intracerebal and intrathecal compartmental therapy, and 2) in vitro characterization and pre-clinical toxicity evaluation of vaccine strategies consisting of conjugated peptides and RNA-loaded dendritic cells in immunocompetent syngeic murine astrocytoma models. Reagents will be developed, refined, or advanced to clinical trial that target EGFR/EGFRvlll, GP240/hmwCSPG, GPNMB, MRP3, and gangliosides 3'-isoLM1 and 3',6'-isoLD1. The requisite methodology for the generation of scFv diabodies, minibodies, and CH2 domain-deleted F(ab')2 MAbs for improved penetration, blood clearance, and lack of glomerular trapping is available in this Core. A large repertoire of large and small animal models that mimic human CMS disease and available administration routes (e.g. i.t. and i.e.) for preclinical evaluation of stability, localizing ability, immunologic, and tumoristatic or tumoricidal effect of developed agents is also available in this Core. We have multiple agents currently ready for testing and several others that should matriculate during the first year. This level of familiarity with these agents makes Core A a natural hub for IND preparation, statistical oversight, and the development and application of Nautilus and Oracle databases as well. RELEVANCE (Seeinstructions): This Core provides the statistical analysis to ensure proper design and interpretation of all experiments performed in this SRC. Experiments performed in this Core will ensure the quality and safety of all reagents produced within this SRC before clinical use. Finally, this Core provides the infrastructure to collect clinical data and to properly catalog, store and retrieve valuable clinical specimens.

Core C. Administrative Core. Darell D. Signer, M.D., Ph.D., Core Leader. Support is requested for an Administrative Core including 0.6 calendar months effort for Dr. Bigner as SRC Program Director/Principal Investigator and for John H. Sampson, MD, PhD, MHScto assist Dr. Bigner and to serve as co-chair of the SRC Operations Committee. Drs. Bigner and Sampson will provide oversight of the scientific and administrative activities of the Specialized Research Center. Dr. Bigner will continue as he has for the past 25 years to chair, along with Dr. Sampson as co-chair, the Operations Committee for the SRC which includes all project and core leaders (Drs. Zalutsky, Reardon, and McLendon). Together they will foster communication and collaboration between the projects and cores and monitor and assess progress. Drs. Bigner and Sampson will meet weekly with project and core leaders and will serve as the editorial review team for all publications resulting from SRC activities. Drs. Bigner and Sampson will plan and coordinate regular seminars with the SRC, which will include internal and external speakers, and which are a part of the monthly Neuro-Oncology Program Seminar Series. Drs. Bigner and Sampson will coordinate the participation of SRC investigators in national and international workshops, conferences, and meetings on brain tumor research. They also will coordinate all the activities of the SRC Internal and External Advisory Committees and will serve as SRC liaisons with other appropriate academic units within Duke. Core support is requested for a Staff Specialist, as in previous years of this grant, who will be responsible for scheduling all internal and external meetings, budgetary monitoring, and general administrative assistance. Also, support is requested for a Laboratory Technician, as in previous years of this grant, for maintenance of media solutions, instrument, and glassware preparation. Core C provides support for all three research projects and two cores. Core C functions, for which support is requested in this application, are identical to those of the last five years with the addition of the effort for Dr. Sampson as co-chair of the Operations Committee. RELEVANCE (See instructions): The Administrative Core is necessary to coordinate all of the activities in the projects, all of which are designed to develop new and more effective and less toxic malignant brain tumor treatments. In addition, a specially trained technician is necessary to provide the necessary solutions and ultraclean glassware for the experiments proposed.

This Center grant was the first Specialized Research Center on brain tumors funded by the National Institute of Neurological Diseases and Stroke. It has focused on primary and metastatic tumors of the CNS of adults and children over its 25 year history. This application represents its sixth submission for competitive review and renewal. Primary and metastatic tumors of the CNS remain significant health problems in both adults and children, and this has been the research focus of this grant in the past. However, the focus of this new competitive renewal has turned to primary CNS tumors in adults only. Research on metastatic tumors and tumors of the CNS in children will continue at the Preston Robert Tisch Brain Tumor Center at Duke, but under separate funding mechanisms. Despite intensive research efforts by many investigators, there has been little progress in uncovering etiology or improving treatment of primary brain tumors during the last 30 years. This Specialized Research Center will continue brain tumor research efforts by including an outstanding group of internationally recognized investigators with a long history of effective collaboration. In this application, primary tumors in adults, both solid intracranial tumors and neoplastic meningitis, will be approached with novel therapeutic modalities. The three research projects include Project 1, a targeted radiotherapeutic approach utilizing modular recombinant transporters (MRT) labeled with Auger electron-emitting radionuclides;Project 2 utilizes an immunological approach with temozolomide-induced lymphopenia and a multivalent vaccine;and Project 3 includes multiple translational clinical trials for primary CNS tumors. Projects 1 and 3 are continuations of projects in the current grant. Project 2 is new to this SRC. Three Cores will support these research projects. They include an IND, Regulatory, and Bioinformatics Core (Core A);a Brain Tumor Biorepository, Histopathology, and Immunologic Monitoring Core (Core B), and an Administrative Core (Core C). Cores A and C are currently funded;Core B is a continuation from earlier grant periods.

The common interests of the Neuro-oncology Program are primary malignant brain tumors of adults and children. The scientific goals are: Aim 1: to conduct epidemiological and molecular epidemiological studies to investigate etiology and to identify populations at greater and lesser risk for development of malignant brain tumors in adults and children; Aim 2: to determine molecular mechanisms of transformation, altered growth control, and invasion of malignant brain tumors of adults and children; Aim 3: to identify new drugs active against primary brain tumors of adults and children, to determine mechanisms of drug resistance in primary brain tumors, and to institute methods to overcome drug resistance; Aim 4: to develop monoclonal antibodies and recombinant DNA antibody fragments reactive with molecular targets, primary brain tumors, and to develop immunoconjugates for brain tumor treatment; Aim 5: to develop new radiolabeling technology for peptides and monoclonal antibodies and their fragments that will facilitate the investigation of promising radionuclides, including the a-emitter [211]Astatine and the B-emitter[177] Lutetium, in targeted radiotherapy clinical trial for brain tumor patients; Aim 6: to develop cell-mediated immunotherapy and dendritic-based vaccine trials for brain tumors; Aim 7: to develop oncolytic poliovirus with no neurovirulence, but retention of oncolytic capacity for gliomas, into a reagent that can be used for therapy of malignant gliomas and neoplastic meningitis from breast cancer; Aim 8: to develop imaging capabilities with Positron Emission Tomography (PET) for improved brain tumor diagnosis, monitoring of therapeutic response, and determination of patient-specific radiafion dosimetry in radiolabeled antibody, chemotherapy and small molecular inhibitor clinical trials in brain tumor patients; Aim 9: to design and execute Phase I, Phase 11, and Phase 111 clinical trials in primary and metastatic brain tumors in adults and children, based on laboratory discoveries within the Program, and to execute clinical trials for improvement of quality of life in brain tumor patients. The Program includes 26 members from 12 basic and clinical departments within Duke University. Total funding for program members is $18,693,484, of which $10,458,928 is from peer- reviewed sources. A cancer focus is illustrated by $5,092,026 or 48.7% of funding from the NCI, the American Cancer Society or the Department of Defense. From 2004-2008, program members published 612 papers in peer-reviewed journals cited in PubMed. Of these publications, 20% are the result of intra-programmatic collaborations and 27% due to inter-programmatic collaborations.

PROJECT SUMMARY (See instructions): Project 3: EGFRwt and EGFRvlll Dual-Specific Immunotoxin for Glioblastoma Multiforme Darell D. Bigner, M.D., PhD., Project Leader Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumor. Median survival remains at 15 to 18 months and recurrence is almost universal despite extensive surgery, external beam radiotherapy and chemotherapy. Basic studies by us and TCGA have shown that EGFRwt and EGFRvlll are the most commonly amplified genes in GBM. We have developed a duals specific high affinity immunotoxin D2C7-(scdsFv)-PE38KDEL (D2C7IT) that avidly kills GBM cells expressing EGFRwt and/or EGFRvlll in vitro and in vivo. Our hypothesis is that treatment of GBM reactive with our dual-specific EGFRwt and EGFRvlll D2C71T construct delivered by CED with catheter placement determined by computer algorithm and delivery monitored by imaging will result in efficacious and minimally toxic treatment of GBM. Our Specific Aims are: 1) to prepare a GLP clinical batch of at least 250 mgm of D2C7IT; to perform all efficacy, toxicity, potency, sterility and stability studies; and to obtain an FDA IND for D2C7IT; 2) to conduct a Phase I dose-escalation study of D2C71T of recurrent GBM patients whose tumors react with D2C7. EGFRwt and EGFRvlll amplification and expression will be conducted by fluorescence in situ hybridization (FISH) quantitative PCR and immunohistochemistry. CED with computer algorithm catheter placement and delivery imaging by [124] l-labeled albumin PET and co- CED-infused gadolinium-DTPA MRI will be performed; 3) at the Phase 1 MTD, to conduct a single arm Phase 11 study of recurrent GBM patients who have failed standard of care and bevacizumab compared to a similar historical control arm.

Shared Resource Core C. Administration. Darell D. Bigner, M.D., Ph.D., Core Director Support is requested for an Administrative Core including effort for Dr. Darell Bigner as P01 Program Director and Principal Investigator and Director of the Administrative Core. Dr. Bigner will provide oversight of the scientific and administrative activities of the program project grant. Dr. Bigner will chair the P01 Executive Committee which includes all research project leaders and shared resource core directors (Drs. Michael Zaiutsky, John Sampson, James Vredenburgh, and Daniel Barboriak). Together they will foster communication and collaboration between the research projects and shared resource cores and monitor and assess progress. Dr. Bigner will meet weekly with research project leaders and shared resource core directors, and he and his staff will serve as the editorial review/clearinghouse for all publications resulting from P01 activities. Dr. Bigner will plan and coordinate regular seminars which will include the P01 project leaders, core directors and investigators. These seminars will be a part of the monthly Neuro-Oncology Program Seminar Series coordinated by Dr. Bigner. Dr. Bigner will coordinate the participation of P01 investigators in national and international workshops, conferences, and meetings on brain tumor research. Also he will coordinate all the activities of the P01Internal and External Advisory Committees and will serve as the P01 liaison with other appropriate academic units within Duke University and with outside entities. Core support is requested for a Staff Specialist who will be responsible for scheduling all internal and external meetings, budgetary monitoring, and general administrative assistance. Core C will provide administrative support for all three research projects and the two shared resource cores.

DESCRIPTION (provided by applicant): This is an application to the NCI from a group of senior scientists and physician/scientists with a long history of collaboration who proposes novel approaches for treatment of two of the conditions in neuro-oncology which are most refractory to treatment, neoplastic meningitis (NM) and glioblastoma multiforme (GBM). NM is characterized by the dissemination of malignant tumor cells within the leptomeningeal space and metastatic spread through the cerebrospinal fluid along the brain and spine. NM from carcinoma of the breast and lung are the most common subtypes. Prognosis of NM is grim with median survival of 2 to 6 months. Current therapy is ineffective and limited by toxicity to the CNS. GBM is the most common and most malignant primary brain tumor. Despite hundreds of clinical trials, only two agents, temozolomide and bevacizumab, have been approved for treatment by the FDA in the last decade. Less than half of patients treated with these two agents respond. Recurrence after treatment is almost universal and median survival is 15 to 18 months with few long-term survivors. Targeted immunotherapy with monoclonal antibodies (MAbs) or their fragments armed with Astatine 211 or Pseudomonas exotoxin for the treatment of NM or GBM is proposed in Projects 1 and 3. In Project 2 vaccine approaches against the ubiquitous antigens from CMV in GBM are proposed to be improved by decreasing immunosuppressive regulatory T-cells or TReg in the setting of temozolomide-induced lymphopenia by administering a specific humanized MAb reactive with the IL-2Ra receptor. Project 1, led by Michael Zaiutsky, is entitled Targeted Radiotherapy of Neoplastic Meningitis using Monoclonal Antibodies Labeled with Alpha Particle Emitting At. Project 2, led by John Sampson, and is entitled, Targeting Immunosuppression Pathways to Enhance Brain Tumor Immunotherapy. Project 3, led by Darell Bigner, and is entitled, EGFRwt and EGFRvlll Dual-Specific Immunotoxin for Glioblastoma Multiforme. These three projects will be supported by an Imaging Core, Daniel Barboriak, Core Director; a Clinical Support Core for biostatistics, informatics, immune monitoring, and a brain tumor tissue biorepository, James Vredenburgh, Core Director; and an Administrative Core, Darell Bigner, Core Director.

? DESCRIPTION (provided by applicant): The central hypothesis of the project described in this Outstanding Investigator Award application is that regional tumor-targeted cytotoxic therapies, such as oncolytic poliovirus (PVS-RIPO) and the D2C7 PE38 immunotoxin (D2C7-IT), not only specifically target and destroy tumor cells, but in the process initiate immune events that promote an in situ vaccine effect, which can be amplified by immune checkpoint blockade to engender a long-term systemic immune response that effectively eliminates recurrent and disseminated glioblastoma multiforme cells (GBM). Ultimately, both agents may be used together providing two different antigenic targets and cytotoxicity mechanisms along with immune checkpoint blockade. In the initial years of the grant, transgenic mice carrying the human poliovirus gene will allow orthotopic growth of 3 astrocytic murine tumor cells that have been transfected with the human poliovirus receptor gene. The resultant tumors will be treated with PVS-RIPO and the nature and mechanism of the immune response will be characterized. Following that, the astrocytic animal tumors will be grown orthotopically, treated with PVS-RIPO, and then treated with anti-CTLA-4 and PD1 checkpoint inhibitors, both individually and together. A similar set of studies will be carried out with the D2C7 immunotoxin in 3 astrocytic murine tumor models that have been transfected with the murine version of the human epidermal growth factor receptor gene, which contains the murine epitope reactive with the D2C7-IT. A similar series of studies with orthotopic murine astrocytic tumors transfected with the D2C7 EGFR epitope will be treated with D2C7-IT, and individually and together, with the checkpoint inhibitors anti-CTLA-4 and PD1. The immune response will be characterized in depth, before and after treatment, with the checkpoint inhibitors. Following these animal studies, a similar series of human clinical trials in GBM patients will be carried out with treatment with either PVS-RIPO or D2C7-IT and the checkpoint inhibitors anti-CTLA-4 and PD1, both individually, and ultimately together. I believe the outcome of these studies will represent paradigm shifts in GBM treatment resulting in significant increases in high quality of life and overall survival.

PROJECT SUMMARY ? Project 3 The notoriously immunosuppressive tumor microenvironment (TME) is a major detriment to effective cancer immunotherapy. Pre-clinical evidence in immunocompetent rodent tumor models show that recombinant, non- pathogenic poliovirus (PVSRIPO) can elicit tumor antigen-specific antitumor immunity, in part through reversing immunosuppression and generating an immune-engaged TME. PVSRIPO targets tumor cells and antigen presenting cells (APCs; macrophages/microglia, dendritic cells) via its receptor, the immune checkpoint molecule CD155. PVSRIPO exhibits potent cytotoxic properties in malignant cells, largely due to unhinged PKC-Raf-ERK1/2-MNK signals that provide an enormous advantage to viral protein synthesis. Intriguingly, infection of APCs has a very different outcome. In macrophages or dendritic cells, PVSRIPO infection leads to chronic, non-cytopathogenic viral propagation that produces durable induction of type 1 interferon-dominant stimulation. PVSRIPO-infected APCs exhibit enhanced antigen-presentation and T cell co- stimulation capacity. Viral tumor cell lysis, combined with pro-inflammatory APC stimulation occurring in a context of rampant immune cell invasion into the tumor, set the stage for initiation of antitumor immunity. PVSRIPO has demonstrated promise in an ongoing Phase 1 clinical trial against recurrent WHO stage IV malignant glioma (glioblastoma, gliosarcoma) and was granted Breakthrough Therapy Designation in May, 2016. In the course of this trial, we observed intriguing responses to temozolomide or lomustine in patients that experienced biopsy-proven or imaging-suggested evidence for tumor recurrence post PVSRIPO. We are pursuing the following Aims: 1) Conduct a Phase 2 randomized clinical trial of PVSRIPO alone or in combination with lomustine in patients with recurrent WHO Grade IV malignant glioma. We propose a single-institution, two-arm randomized Phase 2 study that examines the survival of recurrent GBM patients treated with PVSRIPO alone and PVSRIPO + lomustine chemotherapy. Study objectives are: a) to assess the efficacy of a single dose of PVSRIPO with or without a single dose of lomustine among adults with recurrent GBM relative to the survival observed in a (FDA sanctioned) historical control group; b) to assess the safety of PVSRIPO treatment with lomustine; and c) to define changes visualized on imaging due to intratumoral inoculation of PVSRIPO alone or in combination with lomustine. 2) Perform immune monitoring to mechanistically unravel PVSRIPO immunotherapy synergy with lomustine. We will use immune cell profiling and T Cell Receptor (TCR) sequencing to assess: a) global (whole blood) T cell populations; b) effector anti-polio memory T cell populations; c) T cells responding to autologous dendritic cell (DC) stimulus with defined GBM antigens (EGFR, HER2, survivin, hTERT).