Michael E. Phelps, Ph.D. - US grants

The long term objective of this program is to develop the methods for using PET to measure biochemical, neurochemical and biological mechanisms of disease, providing emical markers for highly specific differential diagnosis and treatment. In one part of is objective, we focus our effort on a single neurochemical (dopaminergic) system to study its regulatory mechanisms at an ultrastructural level and develop in vivo probes and assay methods of the pre- and postsynaptic chemical processes that underlie neurotransmitter function. The validity and value of these methods will be assessed under altered states of pharmacologic and electrical stimulation, as well as MPTP induced degeneration. Initially, selected diseases of this system: Parkinsons and umans exposed to MPTP - (presynaptic); Huntington's disease (HD) - (postsynaptic); progressive supranuclear palsy - (pre- and postsynaptic) will be used as models of system failure. Subsequently, these studies will evolve into the use of the methods to study the mechanisms of these diseases and their treatment. Another approach will be to use combinant DNA markers to identify HD gene carriers, while using PET to isolate and follow disease progression in target sites of HD gene expression in the brain. Studies of the metabolic aspects of maturation provide a unique model to investigate structure/function relationships that occur during periods of neuronal plasticity, in normal development or during compensatory responses to injury. Metabolic, anatomical and behavioral studies will be used to assess promotion of compensatory responses (i.e., novel growth and sparing of neuronal processes) by physical and chemical stimuli. These studies will combine specific findings in humans and rigorous detailed investigations only possible in animals. Intractable childhood seizures, Down Syndrome and infantile hemiplegia will be used as model disorders to investigate the metabolic basis of altered development and neuronal plasticity, with improved clinical management with PET expected to be an early outcome. A 3-D system for merging structure (MRI) and function (PET) will provide a systematic, accurate and efficient way iiot only process and interpret imaging data, but also to provide a new way to organize, describe and display human macroscopic functional anatomy of the brain.

We propose to develop and apply emission tomography (PET) as an analytical biochemical assay technique to study normal human cerebral function and affective disorders. The objectives are: 1.) Chemistry/Biochemistry. Rapid synthetic techniques will be developed and/or improved for preparation of (F-18)-fluoro and (C-11) deoxyglucose for measurement of glucose metabolism, C-11 and N-13 labeled amino acids for measurement of protein synthesis rates and F-18 ligands for receptor assays (principally spiroperidol but also development of high specific activity F-18 labeling techniques to be applied to other ligands). Tissue biochemical assay and autoradiographic studies in rats and monkeys will be used to structure and define kinetic models and select among different tracers for protein synthesis and receptor assay measurements. 2.) Tomography. The NeuroECAT will be upgraded to provide unprecedented level of spatial resolution, image quality and quantification necessary to measure the detailed neuroanatomical components of cerebral function required by this proposal. 3.) Tracer kinetic modeling. Operational equations for measuring cerebral blood flow, protein synthesis, oxygen metabolism and receptor assays will be developed and validated. 4.) Normal Subjects. Studies will be performed to validate methods, and to determine norms and variations in local glucose and oxygen metabolism, blood flow and protein synthesis rates. Studies will be carried out to determine metabolic and blood flow responses to specific sensory stimulations to activate visual, auditory, linguistic and cognitive functions and the use of these stimulation-response tasks as provocative tests for affective disorder patients. 5.) Patients. Local glucose metabolism will be measured in patients with bipolar and unipolar affective disorders to determine if there are measurable local biochemical alterations, to establish the neuroanatomical distribution and magnitude of the functional changes and to objectively evaluate the metabolic consequences of drugs administered diagnostically (Ritalin - unipolar, depressed and also normal volunteers as controls) and therapeutically (Lithium - bipolar). Success in basic studies of F-18 spiroperidol will allow studies in these patients to examine the dopamine hypothesis of manic/depressive disorders.

By means of emission computed tomography of patients, we will quantify and localize cerebral biochemical patterns in order to explore the mechanisms underlying human epilepsy, behavior disorders, and other neurological abnormalities. Local cerebral glucose metabolism will be measured by scanning after intravenous injection of F-18-fluorodeoxyglucose. The feasibility will be tested for in vivo receptor assays in man based on emission computed tomography of positron emitter labeled radioligands. Parallel studies will be carried out using quantitative autoradiography of C-14-deoxyglucose and other agents in suitable animal models.

The overall goal of this proposal is to develop and apply methods for the tracer kinetic radioassay of dopaminergic systems in the mammalian brain. This approach will combine animal experimentation and human studies with positron emission tomography (PET). The proposed studies will build upon our presently funded work to extend the availability of biochemical probes (e.g., substrate analogs, receptor binding ligands, and enzyme suicide inhibitors) to the study of dopaminergic function in health and disease. Tracer kinetic modeling approaches will be used to determine efficient ways of monitoring brain energy metabolism during behavioral tasks and of enhancing the specificity, delivery and localization of dopamine specific tracers in the central nervous system. A new human PET instrument, that we have developed, will be applied to these studies; it will provide unprecedented spatial resolution and sampling abilities. An animal PET device will be developed (funded from other sources: DOE) and applied in this proposal; it will provide spatial resolution approaching the theoretical limit for PET instruments. Anatomical methods for data analysis will optimize the localization of neurochemical sites in both human and animal brains. Validated biochemical probes of dopaminergic function will be tested during perturbations of dopamine networks in the brain (e.g., electrical stimulation, chemical stimulation, degenerative states (MPTP), lesions, behavioral tasks, and neuropsychiatric disorders). The distribution of dopaminergic sites and their quantitative function both pre- and post-synaptically will be evaluated in normal states, during motor tasks, under the influence of dopamine stimulant drugs, and in patients with spontaneous (depression and obsessive- compulsive disorder) and drug-induced (cocaine and amphetamine addiction) alterations in mood.

The overall goal of this proposal is to develop and apply methods for the tracer kinetic radioassay of dopaminergic systems in the mammalian brain. This approach will combine animal experimentation and human studies with positron emission tomography (PET). The proposed studies will build upon our presently funded work to extend the availability of biochemical probes (e.g., substrate analogs, receptor binding ligands, and enzyme suicide inhibitors) to the study of dopaminergic function in health and disease. Tracer kinetic modeling approaches will be used to determine efficient ways of monitoring brain energy metabolism during behavioral tasks and of enhancing the specificity, delivery and localization of dopamine specific tracers in the central nervous system. A new human PET instrument, that we have developed, will be applied to these studies; it will provide unprecedented spatial resolution and sampling abilities. An animal PET device will be developed (funded from other sources: DOE) and applied in this proposal; it will provide spatial resolution approaching the theoretical limit for PET instruments. Anatomical methods for data analysis will optimize the localization of neurochemical sites in both human and animal brains. Validated biochemical probes of dopaminergic function will be tested during perturbations of dopamine networks in the brain (e.g., electrical stimulation, chemical stimulation, degenerative states (MPTP), lesions, behavioral tasks, and neuropsychiatric disorders). The distribution of dopaminergic sites and their quantitative function both pre- and post-synaptically will be evaluated in normal states, during motor tasks, under the influence of dopamine stimulant drugs, and in patients with spontaneous (depression and obsessive- compulsive disorder) and drug-induced (cocaine and amphetamine addiction) alterations in mood.

This proposal is for matching funds to purchase a new cyclotron to replace our existing 21 year old biomedical cyclotron that is failing due to its advanced age and obsolete technology. The cyclotron provides all the necessary positron labeled compounds for our funded research studies of the biochemical and biological mechanisms of normal human brain development and neuronal plasticity, including the compensatory reorganization of the brain following injury, disease or surgery. It is critical to our studies of developmental disorders, pediatric. and adult epilepsy, Alzheimer's, Parkinson's, Huntington's, depression/mania, drug abuse, coronary artery disease, cardiomyopathies and cancer of the breast, lung, bone, brain, liver and lymphic system. Without the cyclotron, it would be impossible for us to continue any of these funded research projects. These projects involve approximately $3.2 million per year of NIH and $3.4 Million of DoE funded research all of which are completely dependent upon the cyclotron facility. We have raised 88.6 % of the required funding by obtaining a significant discount on the equipment from the vendor, obtaining a supplementary grant from DoE and soliciting private donations. Since DoE funds a significant fraction of the research projects with the cyclotron, we requested and received a $908,000. In addition, we also obtained $455,000 from the Ahmanson Foundation to apply to this purchase. The existing research grants will provide the necessary maintenance and operating support for the cyclotron. The administration of the cyclotron is carried out by a Resource Allocation Committee (RAC) that consists of the PIs of the major grants and representatives of the technology components (i.e., cyclotron chemistry, PET scanners and animal laboratories). This administrative configuration has been in effect for 15 years and has efficiently allocated the resources of the cyclotron and other major instruments.

The objectives and specific aims of this application are to educate graduate students, postdoctoral fellows and residents in molecular imaging. The annual meeting of the Academy of Molecular Imaging (AMI) brings together a unique group of physicians, scientist and students from academic institutions, pharmaceutical companies, hospitals and industry to create a forum for sharing the latest research and developments in all areas of molecular imaging. The annual conference features parallel tracks specific to each council of the AMI, the Institute for Molecular Imaging, the basic science and instrumentation development council; Society of Non-lnvasive Imaging in Drug Development, the molecular therapeutics councils; the Institute for Clinical PET, the clinical council, and; the Institute for Molecular Technologies, the industry council. We are fortunate to have confirmed a number of esteemed and engaging speakers, including keynote speakers Dr. Andrew von Eschenbach, Dr. Nora Volkow and Dr. Mark Ellisman. Some of the topics that will be discussed are: PET/CT applications; Assay Development; Multi-modality Imaging; New targets for Imaging; Instrumentation/Image Analysis; Drug Development in Oncology; NIH Molecular Libraries and Imaging Roadmap plus selected abstracts of the latest basic science, drug development and clinical findings. The meeting will be held from March 27 - 31, 2004 at the Gaylord Palms in Orlando, Florida.

DESCRIPTION (provided by applicant): UCLA has a mature small animal imaging program based on digital whole body autoradiography (DWBA) and micro positron emission tomography (microPET). We have significant investments in the study of small animal cancer models using PET reporter gene technology. Reporter genes in combination with DWBA and microPET have provided us the ability to study cancer biology, cell trafficking, and pre-clinical models for gene therapy. It is through these studies that we have better understood the limitations of our current technologies and therefore propose the acquisition of micro computed tomography (microCAT) and optical charge coupled device (CCD) imaging systems. We will also acquire a critically needed computer server and data archiving system for the large amounts of data generated through this work. We will initially allow six seasoned cancer investigators to use the resource and then grow by adding up to three investigators per year. MicroCAT will allow us to image the underlying anatomy in our mouse cancer models. This will be critical in helping us to understand the location(s) of various cellular events without the need to sacrifice the animals. Furthermore through direct research proposed in this work we will register the microPET and microCAT information to provide our researchers with maximal information on function and anatomy. The optical CCD system will also be critical in helping to accelerate our cancer related research. The use of the firefly luciferase (FLUC) reporter gene will allow us to more rapidly study small animal models without radioactive probes. This system has the capability of imaging low levels of light from within a living small animal. We will use reporter systems that couple FLUC and PET reporter genes in order to have the flexibility to image in either an optical CCD system or the microPET. This will help to accelerate the development of various models that are dependent on reporter gene technology. Quantitation of data from all modalities will always be stressed throughout the SAIRP. We will also develop a strong training program that will help investigators and their students to become independent and confident in using the available resources. Use of the entire resource will be coordinated by intemet based scheduling software and an oversight committee. We are confident that the new resources with microCAT and optical CCD technologies will help to provide UCLA investigators with state-of-art tools for non-invasively imaging mouse cancer models.

DESCRIPTION (provided by applicant): The UCLA scholars in Oncologic Molecular Imaging (SOMI) is a diverse training program bringing together 9 Departments, predominantly from the UCLA Life and Health Sciences, in order to train the next generation of molecular imaging scientist. Oncologic molecular imaging is a rapidly growing area which combines the disciplines of cell/molecular biology, chemistry, biomedical physics, biomathematics, pharmacology, imaging sciences, and clinical medicine to advance cancer research, diagnosis and management. The UCLA Department of Molecular & Medical Pharmacology (DMMP) and the Crump Institute for Molecular Imaging (CIMI) are key players in helping to move the field of molecular molecular imaging forward. With initial critical seed support from the UCLA Jonson Comprehensive Cancer Center (JCCC), UCLA has helped to rapidly grow the field of molecular imaging. Funding sources for the molecular imaging program at UCLA now include the Department of Energy, NCI (P50) In Vivo Cellular and Molecular Imigaing Center (ICMIC) grant, Several NCI R01'a, and NCI Small Animal Imaging Resource Program (SAIRP) grant. The time is ripe for training the next generation of scientists that can bridge the various fields needed to study cancer biology/therapeutics in intact living organisms. In the current proposal, our goals are to train 19 post-doctoral fellows through a diverse group of 29 basic science and clinical faculty mentors representing 7 program areas, 6 formal courses in molecular imaging, pharmacology, cancer biology, cancer immunology, virology, gene therapy, and clinical hematology/oncology rounds. Fellows will be recruited into a three year program and will complete coursework and research with at least two mentors. Furthermore, they will do a mock grant proposal in their third year to help them gain experience and confidence in the grant application process. Special attention will be given towards recruiting minority applicants. A Training Committee will carefully monitor the process of all SOMI trainees, and an Advisory Committee will monitor the entire SOMI program. Graduates of SOMI will be uniquely trained to study cancer in an organism context with state-of-the-art technologies. We are evolving area of the oncologic molecular imaging.

DESCRIPTION (provided by applicant): UCLA has a mature small animal imaging program based on micro-positron emission tomography, x-ray micro computed tomography, in-vivo bioluminescence and digital whole body autoradiography imaging. Central to this program, is our small animal imaging resource (SAIR), which provides service and support through a state of the art facility to more than 24 independent Principal Investigators funded through the NIH and other agencies. Most of the research projects of these investigators are focused in cancer diagnosis and therapy. In addition to this service component, the roles of the SAIR within the UCLA and the US environments are to: (a) educate students, post-doctoral scholars, physicians and other biology researchers from within and outside UCLA in the tools, technologies and applications of imaging, and (b) foster collaborations and develop new technologies and methodologies that will improve the quantitative capabilities of non-invasive imaging. These goals will hopefully lead to better understanding of human disease and might lead to better methods for diagnosis and treatment of cancer. As part of this SAIR proposal, besides the research support and education, two developmental projects are included, that should improve the quality and quantitative accuracy of the acquired data, while they reduce the impact from radiation exposure on the studied subjects. The first project will seek to standardize the animal handling and care part of the imaging protocol prior to, during and after the procedure, such that the animal's response is as uniform as possible. The second project seeks to estimate at first, secondly optimize and thirdly track the radiation exposure to the animal subjects throughout sequences of multiple imaging experiments that can last several months. Both these projects will greatly benefit not only the research experiments carried through the UCLA SAIR, but all preclinical research in the US.

DESCRIPTION (provided by applicant): The UCLA Scholars in Oncologic Molecular Imaging (SOMI) is an integrated, cross-disciplinary postdoctoral training program bringing together faculty mentors from 7 departments and 6 divisions in the David Geffen School of Medicine, the College of Letters and Sciences, and the Henry Samueli School of Engineering and Applied Science. Molecular imaging, the non-invasive monitoring of specific molecules and biochemical process in living organisms, continues to expand its applications in the detection and management of cancer. SOMI faculty mentors provide a diverse training environment spanning mathematics, physics, engineering, chemistry, biochemistry, cancer biology, immunology, and medical sciences. The centerpiece of the SOMI program is the opportunity for trainees (Ph.D. or M.D.) to conduct innovative molecular imaging research co-mentored by faculty in complementary disciplines. SOMI trainees also engage in specialized coursework, seminars, a clinical tutorial program, and participate in a course on ethics and responsible research. The three-year program culminates in preparation of a mock grant, as trainees transition to independent careers in cancer molecular imaging. During the initial funding period, 19 trainees entered the SOMI program, and nine SOMI fellows have subsequently been appointed to faculty positions. In the renewal period, we propose to continue and strengthen the SOMI program through addition of a new Program Area (Systems Biology), strategic additions to our faculty mentors, and continuous improvement of the other training and career development components of the program. The goal of the SOMI program is to continue to provide talented young investigators with the scientific and professional training needed to become leaders in the field of molecular imaging of cancer.

Three Core Resources facilities that support NSBCC Projects are described. The IMED Core #1, is located at UCLA, and is designed to provide project support and capability for the translation of our more advanced technology platforms to the greater (local) oncology community, thus expanding the influence of our most robust nanotechnologies The IMED core is wholly integrated into the UCLA Institute for Molecular Medicine (IMED). IMED is designed to support translational medicine projects both within and outside of the central NSBCC effort. This core also contains our key in vivo molecular imaging infrastructure and provides training for imaging and training on NSBCC technologies for translation. A devoted space with the larger IMED facility has been equipped with a suite of tools designed to customize NSBCC in vitro diagnostic technologies for specific translational medicine projects, including tools for the preparation and purification of key NSBCC nano/bio materials, the development of custom panels of protein biomarkers, and tools for surface customization. That NSBCC core facility is designed to fully leverage off of the much larger UCLA IMED translational medicine facility as well as the community of cancer researchers and clinicians that utilize that facility. The other two core resources are devoted to technology development. The NSBCC projects are driven by fundamental and clinical problems in cancer biology and the treatment of cancer patients. As such, our ultimate deliverables are, by and large, technology platforms (e.g. an in wfro diagnostic chip) that are combined with biological content (e.g. a panel of protein biomarkers). The nanofabrication and nanomatenals characterization core #2 at Caltech is designed to support NSBCC activities that range from rapid prototyping of nanotech and microfluidics platforms, nanomaterials synthesis and characterization, surface science, and technology platform scale-up. The systems biology core #3 at the Institute for Systems Biology is designed to support the genomics, transcriptomics, and proteomics work, as well as the computational biology and data base mining and development that provides the base support for much of the biological content within our deliverables.

? DESCRIPTION (provided by applicant): We propose the NanoSystems Biology Cancer Center (NSBCC) as a collaboration between Caltech and the UCLA Geffen School of Medicine, to develop nanotechnologies for addressing challenges in combinatorial cancer therapies. Four scientific Projects are supported by two Core Resources and an Administrative structure designed to promote cross-university interactions at the frontiers of cancer biology, clinical oncology, and the basic and engineering sciences. By leveraging strong support from our respective institutions, the Jonsson Comprehensive Cancer Center, and commercial partners, we integrate world-class physical, biological and engineering sciences at Caltech with cutting edge cancer biology and cancer clinical care at UCLA. The NSBCC faculty include four clinical researchers, 5 assistant professors, and several senior researchers from both campuses, and is led by led by Jim Heath (Caltech) and co-led by Michael Phelps (UCLA). Heath and Phelps have track records of building leading cancer research programs that draw across disciplines, with effective translation into the clinic and marketplace. Our Projects balance discovery with translation. Two Projects involve nanotherapies, and two involve the development of nanotech tools for guiding the selection of combination both cancer immunotherapy and targeted therapy treatments. We focus on brain cancers and melanoma, which allows us to take advantage of momentum from the current funding cycle. However, we seek broadly applicable technologies. This holds especially for the case of immunotherapy, where the challenge is to bring the remarkable recent successes in the field (partly driven by our investigator's melanoma trials) to larger patient populations. In Proj. 1, we propose nanoparticle (NP) vehicles designed to deliver therapies and therapy combinations to fully engage a tumor that lies across an intact blood brain barrier (BBB). This project builds upon initial promising results, and from an NSBCC history of delivering NP therapeutics into Phase I and Phase II (and soon Phase III) trials. Proj. 1 takes guidance (as well as a novel panel of human-derived intracranial tumor models) from Proj. 4, where we propose nanotech and microtech tools to quantitatively assay for >200 proteins and metabolites from single cancer cells separated from a GBM tumor, with the goal of understanding the dynamical responses of those cells to targeted monotherapies. Those responses invariably lead to some form of resistance, and we seek to decode those responses to identify effective therapy combinations. For Project 2 we propose to integrate 3 Caltech inventions. The first are epitope targeted PCC Agents (Heath), which are synthetic ligands that can be developed to target oncoproteins containing single activating point mutations. We target the oncoproteins AktE17K and KrasG12D. KrasG12D is the most dominant oncoprotein in human cancer, and also considered undruggable. These targeting ligands are combined with proteolysis-targeting chimeric molecules (protacs; Deshaies) that exploit the natural cellular machinery to label a protein for destruction. The PCC Agent-protacs are delivered into cells by adapting NP chemistries that were first developed and clinically translated by Davis. Targeting just the mutant protein can open up the therapeutic window for targeted inhibitors, thus enabling new therapy combinations. In Project 3, we turn to cancer immunotherapy by evolving our powerful suite of immune monitoring tools into platforms for understanding immune cell/tumor cell interactions within the tumor microenvironment. In particular, guided by exome analysis of the tumor, we construct nanotechnology-based libraries for a ~60-plex sorting of tumor neoantigen specific T cell populations that can be applied directly to fresh biopsied tumors. This helps identify those T cells that have clonally expande within the tumor, and permits us to identify the tumor antigen, the T Cell receptor ?/? sequence (for cloning), and the functional activity of the T cell. This technology is applied a set of matched patient tumor biopsies from recent immunotherapy trials run by Project 3 PI Ribas, and should provide guidance for treating those patient groups that currently exhibit transient, positive responses to PD-1 blockade, as well as helping to frame treatment ideas for patients that do not exhibit even transient responses. In Project 3, we also propose a novel in vivo imaging nanotechnology for the kinetic tracking of T cell infusions in tumor models. The technology draws from the genetic ability of certain microorganisms to generate gas- filled nanovesicles, and yields an image contrast mechanism that will be adapted to TCR- or chimeric antigen receptor (CAR)-engineered T cells for imaging T cell infiltrates into mouse tumor models, using the high resolution imaging modalities of ultrasound or hyperpolarized 129Xe MRI. This provides us with the ability to test, in vivo, hypotheses generated from the in vitro assays. For all projects, significant preliminary data is provided.