Beckman Research Institute of City of Hope
Stephen J. Forman, M.D.
The overall goal of the City of Hope Lymphoma SPORE is to develop translational studies to improve the treatment of Hodgkin and non-Hodgkin lymphoma. This application, consisting of four translational research projects and four cores, will develop novel approaches that are derived from molecular and immunologic studies in lymphoma and T-cell and antibody-based therapies. An important theme of the translational studies in this grant is to develop effective therapeutics for lymphoma that will reduce toxicities associated with current treatment regimens for Hodgkin and non-Hodgkin Lymphoma which can be utilized for treatment of older patients. Project 1 will study the effectiveness of cellular immunotherapy for B cell lymphoma utilizing engineered central memory derived CD19-specific T-cells. Investigators in this project have developed a T-cell genetic modification platform for expressing chimeric immunoreceptors that redirect antigen specificity and effector function of central memory T-cells towards cell surface epitopes on B cell lymphomas and will test the persistence and expansion of these cells after infusion. Because epidemiologic studies indicate that stem cell damage from pretransplant therapeutic exposures may play a role in the subsequent development of myelodysplasia, Project 2 will longitudinally study a population of patients with Hodgkin and non-Hodgkin Lymphoma. The investigators will continue their studies of the cellular and molecular factors that are predictive for development of myelodysplasia, and to determine the molecular sequence of events that lead to this complication of treatment. In Project 3 investigators will test a molecularly engineered novel anti-CD20 IL-2 immunocytokine for the treatment of patients with CD20+ lymphoma. An important component of this project will be to delineate the immunologic effector mechanisms operative in immunocytokine-mediated anti-lymphoma in vivo activity. Project 4 will develop siRNA based therapeutics against transcription factors (STAT3) important in lymphoma behavior using an antibody directed cyclodextrin nanoparticle for treatment of patients with B cell lymphoma. The projects in this Lymphoma SPORE will be supported by four cores including: Administration, Biostatistics and Research Informatics, Tissue Bank for Molecular and Cellular Studies, and Biological Manufacturing. This Lymphoma SPORE will also support a Developmental Research Program and a Career Development Program to foster the advancement of pilot translational research projects and young investigators focused on lymphoma.
PROJECT 1: CD19-SPECIFIC CENTRAL MEMORY T CELL ADOPTIVE IMMUNOTHERAPY FOR NON-HODGKIN LYMPHOMA
Michael Jensen, M.D. (basic)
Leslie Popplewell, M.D. (clinical)
David Maloney, M.D. (clinical)
Disease relapse is a leading etiology of morbidity and mortality for patients with non-Hodgkin lymphoma (NHL) and autologous hematopoietic stem cell transplant following a myeloablative preparative regimen (auto-HSCT) can salvage only a subset of these patients. This Lymphoma SPORE Project 1 renewal resubmission seeks to develop targeted cellular immunotherapy capable of eradicating lymphoma post transplant minimal residual disease (MRD) using CD19-specific T cell adoptive immunotherapy (AIT). Over the last funding cycle, we developed a genetic engineering approach to equip T cells with the ability to recognize and lyse tumor cells of B-cell lineage NHL using chimeric antigen receptors (CARs) specific for CD19 and initiated a first-in-human pilot clinical trial. Our initial clinical experience revealed a significant obstacle to achieving robust therapeutic activity; namely, that infused T cells survived only transiently in the tumor-bearing recipient. To address this critical issue, we elected to study the fate of adoptively transferred T cells using a non-human primate model that closely recapitulates clinical therapy in humans. These studies have revealed a key insight-- that a rare population of T cells present in the circulation, the central memory T cell (TCM) is capable of high-level sustained engraftment following adoptive transfer. Accordingly, we have reconfigured our clinical T cell production platform to immunomagnetically purify CD8+ TCM from peripheral blood mononuclear cell preparations. These CD8+ Tcm then undergo polyclonal activation, lentiviral vector transduction to express a CD19-specific scFvFc:zetaCAR, and are expanded over a brief 21-day interval in IL- 2/IL-15 prior to cryopreservation. The first specific aim of the project seeks to demonstrate that CD19R-CAR+CD8+ Tcm-derived effector cells from NHL patients are phenotypically and functionally similar to those derived from healthy donors. The second aim seeks to define the maximum tolerated dose (MTD) of CD19R-CAR+coa+ Tcm-derived effector cells when infused shortly following auto-HSCT (Day +2} when patients with recurrent diffuse large cell lymphoma (DLCL) have minimal disease burdens and are profoundly lymphopenic from the transplant myeloablative preparative regimen. The third specific aim seeks to determine the magnitude and duration of persistence of transferred T cells in treated patients, the ability of transferred effector cells to revert to TMEM and populate memory T cell niches, and the anti-CD19 effector functioning of these cells in patients as determined by tracking the kinetics of CD19+ B cell reconstitution. Lastly, we will seek to demonstrate the exportability of the approach by opening the trial to patient accrual both at the FHCRC in Seattle as well as City of Hope. Each of these Specific Aims focuses on key translational research objectives essential for moving the field of lymphoma AIT forward, including the conduct of a first-in-human trial using central memory T cell adoptive therapy. As such, Project 1 is fully aligned with the SPORE mandate for innovative translational research.
PROJECT 2: THERAPY-RELATED LEUKEMIA FOLLOWING AUTOLOGOUS TRANSPLANTATION FOR LYMPHOMA
Ravi Bhatia, M.D. (basic)
Timothy R. O’Connor, Ph.D. (basic)
Smita Bhatia, M.D., M.P.H. (clinical)
Therapy-related myelodysplasia or acute myelogenous leukemia (t-MDS/AML) is a dreaded complication and the most common cause of non-relapse mortality in patients undergoing autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Previous studies indicate that t-MDS/AML results from damage to hematopoietic stem cells from genotoxic cancer treatment. However, the sequential cellular and molecular changes leading to development of t-MDS/AML are not known. We are conducting a prospective, longitudinal study of patients with HL and NHL undergoing aHCT, in order to gain insight into the pathogenesis and identify predictors of susceptibility to t-MDS/AML. Patients are being followed longitudinally from pre-aHCT to five years post-aHCT, with serial banking of peripheral blood (PB) and bone marrow (BM) samples at pre-determined time-points. The known timing and nature of genotoxic exposures for management of HL or NHL, together with banking of sequentially obtained samples, provides a unique opportunity to study sequential molecular and cellular changes in this high-risk population. We are exploring the hypothesis that defects in DNA repair, DNA damage response and increased proliferative demand on HSC that have already acquired DNA damage may contribute to genomic instability and emergence of a malignant clone. Studies conducted during the previous grant period have indicated a role for accelerated telomere shortening and abnormal regulation of hematopoietic stem cell proliferation in the pathogenesis of t-MDS/AML and support this hypothesis. Our previous studies have also resulted in development of methodologies to evaluate DNA repair and acquisition of chromosomal aberrations in primary hematopoietic cells. We propose to build upon our previous findings and systematically investigate the role of these mechanisms in the pathogenesis of t-MDS/AML, with an overarching goal of developing biomarkers of enhanced risk of t-MDS/AML. We aim to understand the mechanisms and significance of DNA repair defects, altered telomere regulation and acquisition of cytogenetic lesions in the pathogenesis of t- MDS/AML; and to understand the interaction of these abnormalities, therapeutic exposures and demographic variables in determining risk of t-MDS/AML. Successful completion of these studies will provide insights into the pathogenesis of t-MDS/AML and allow accurate assessment of risk factors associated with the development of t-MDS/AML. Early identification of high-risk populations may aid in modification of treatment regimens to reduce risk of this complication.
PROJECT 3: DE-IMMUNIZED ANTI-DC20-IL2 FOR THE TREATMENT OF FOLLICULAR LYMPHOMA
Andrew Raubitschek, M.D. (basic)
Ryotaro Nakamura, M.D. (clinical)
Project 3 seeks to address the high relapse rate of non-Hodgkin follicular lymphoma (FL) using immunocytokine (ICK) therapy combined with rituximab and single fraction radiation. We have developed and produced a clinical-trial grade immunocytokine composed of de-immunized anti-CD20 mouse monoclonal antibody (Leu16) fused with two IL2 molecules. DI-Leu16-IL2 (IND100885) has been administered to two patients with B-cell NHL and exhibits low toxicity with good immune activation. This project proposes three specific aims to assess the effectiveness and safety of DI-Leu16-IL2 therapy.
Specific Aim 1: Perform a Pilot-Phase I study of DI-Leu16-IL2 combined with rituximab and single fraction involved-field radiation for treatment of follicular NHL. The primary objective of the trial is to select the schedule and dose of DI-Leu16-IL2, when given in combination with rituximab and single fraction radiation (XRT) that yields the more favorable immunologic response while maintaining safety. The secondary objective is to describe the safety, tolerability, and toxicity profile of DI-Leu16-IL2, given in combination with rituximab and XRT.
Specific Aim 2: Perform a Phase II study of DI-Leu16-IL2 combined with rituximab and single fraction involved-field radiation for treatment of follicular NHL. In this aim, we will conduct a two-center Phase II trial of patients with FL who have relapsed or progressed after at least one prior regimen. The primary objective is to determine the anti-tumor activity of DI-Leu16-IL2 when combined with rituximab and single fraction radiation as assessed by overall response rate (CR+PR).
Specific Aim 3: Evaluate the Biodistribution of DI-Leu16-IL2 using 111In labeled DI-Leu16-IL2. The primary goal of this study is to assess the biodistribution of DI-Leu16-IL2 using 111In DI-Leu16-IL2. The secondary goal is to obtain preliminary data that explores the possible association between DI-Leu16-IL2 uptake and FDG images (pre- and post- therapy) and tumor response.
Significance: This non-transplant ICK-based therapy, if able to improve response duration for patients with follicular lymphoma, would have a significant clinical impact due to the increasing prevalence of this disease. The low toxicity profile of the combined treatment would be especially beneficial in older patients.
Innovation: Our approach to targeted eradication of follicular lymphoma via the DI-Leu16-IL2 immunocytokine in concert with single fraction local irradiation is novel in both its design and implementation. We have manufactured our own DI-Leu16-IL2, obtained FDA approval and commenced preliminary testing of this promising reagent. The addition of low-dose irradiation to this immunotherapy is hypothesized to enhance the immunologic response to DI-Leu16-IL2 treatment without significant additional toxicity.
PROJECT 4: ANTIBODY MEDIATED NANOPARTICLE TARGETING OF siRNA FOR THE TREATMENT OF LYMPHOMA
David Colcher, Ph.D. (basic)
Mark E. Davis, Ph.D. (basic)
John Rossi, Ph.D. (basic)
Hua Yu, Ph.D. (basic)
Stephen J. Forman, M.D. (clinical)
RNA interference is a natural mechanism by which small interfering RNAs (siRNA) down-regulate the expression of targeted genes at the level of messenger RNA via post-transcriptional gene silencing. This proposal pioneers a new approach in cancer therapeutics: silencing of critical yet difficult-to-drug molecular targets affecting lymphoma behavior and treatment response. A major limitation in the use of siRNA for therapy has been the means of delivering a targeted nucleic acid-based therapeutic to tumor cells. Recent developmental studies conducted in collaboration with Caltech have shown that a cyclodextrin polymer, developed by an integrated materials science approach, can address some of the problems in siRNA delivery. Thus, in Project 4 of this SPORE application, we will conduct experiments to optimize the design and delivery of therapeutic siRNA targeted to STAT3, a transcription factor central to the up-regulation of many genes involved in lymphoma cell survival, proliferation and resistance to therapy. Based on the concept that simultaneous targeting of multiple genes could maximize the antitumor effect, we will conduct experiments utilizing a bifunctional siRNA against STAT3, along with candidate genes from preclinical models of activated B cell lymphoma. We will use monoclonal antibodies against cell surface targets to facilitate internalization of the nanoparticle-packaged siRNA to target transcription factors in the lymphoma cell nucleus. Thus, we propose to: 1) Optimize the design and function of single and bifunctional siRNA against STAT3 and Edg1 in B cell lymphoma and compare their efficacy in vitro and in vivo; 2) Evaluate the anti-lymphoma effects of the antibody-cyclodextrin nanoparticle siRNAs against STAT3/Edg1 using cell culture and preclinical models of activated B cell lymphoma to study the impact of inhibition mediated by the antibody-polymer-siRNA construct in vitro and in vivo; and 3) Given that STAT3 is also a transducer molecule for a variety of extracellular signals mediating crosstalk between tumor-resistant and host-immune cells, particularly in the microenvironment, we will determine the effects of the antibody nanoparticle siRNA on tumor angiogenesis and the local immunologic microenvironment.
Thus, in lymphoma models we will evaluate the impact of STAT-1 pathway inhibition as it relates to tumor growth and apoptosis, as well as its effects on angiogenesis and the local immunosuppression that accompanies malignancy. The proposed experiments will optimize the design of a new therapeutic agent that could lead to a clinical trial for treatment of patients with recurrent activated B cell lymphoma. In addition, the ability to generalize this approach utilizing internalizing antibody-directed siRNA-containing nanoparticles can be used and tested in other tumor systems based on the specificity of the siRNA.
CORE A: ADMINISTRATIVE CORE
Stephen J. Forman, M.D.
David Colcher, Ph.D.
The Administrative Core will provide for the administration of the Lymphoma SPORE including administrative and budget support for all the SPORE investigators. In addition, this Core will provide communication with the National SPORE Program staff, coordination with the City of Hope Comprehensive Cancer Center Administrative Office, preparation of progress reports, management of financial and reporting obligations, organization of the weekly research meetings of the basic science and clinical research staff, coordination of intra- and inter-institutional and inter-SPORE projects, as well as organizing the attendance and presentations at the annual City of Hope Lymphoma SPORE retreat and SPORE workshops at the National Cancer Institute. Importantly, this core will be responsible for the oversight of the Developmental Research and Career Development Programs in the City of Hope Lymphoma SPORE. Publications, grant submissions and INDs will also be coordinated and supported through this Core. In addition, the Administrative Core will arrange the Executive and Steering Committee meetings that will review the progress of the work and organize the annual External Advisory Board meetings.
CORE B: BIOSTATISTICS AND RESEARCH INFORMATIC CORE (BRIC)
Joyce C. Niland, Ph.D.
Joycelynne M. Palmer, Ph.D.
Core B, the Biostatistics and Research Informatics Core (BRIC) of the City of Hope lymphoma SPORE, provides statistical expertise across all SPORE research activities, including study design, safety monitoring, data collection, data quality assurance, data analysis, and multi-center study site coordination. BRIC will ensure that the proposed research hypotheses will be addressed with appropriate measures, tests, and interpretation, whether the data come from epidemiological studies, basic science, translational, immunologic studies, imaging, or clinical trials. The centralized, comprehensive framework of BRIC assures each SPORE investigator access to statistical experts who have appropriate experience, interests and time to engage in the collaborative development of study designs, analysis plans, data analysis, interpretation, and abstract /manuscript preparation.
The BRIC will also provide infrastructure for the management and integration of both existing and newly collected data through consistent and compatible data handling. The Core plays an integral role in the scientific development, execution, and analysis of all projects in the SPORE, including the clinical trials. Core investigators have extensive experience in quantitative methods for biomedical applications, including clinical, basic, and translational science studies, particularly in hematologic malignancies. BRIC is committed to collaboration, to help ensure the scientific integrity of the SPORE investigations, by participating in regular project and program meetings, and providing rigorous and innovative input on all statistical and information management matters arising within the projects.
The specific aims of BRIC are as follows:
Specific Aim 1: To support high quality data management through state-of-the-art information technologies, ensuring standardization, quality assurance, security, training, monitoring, and data integration, and multicenter coordination when appropriate.
Specific Aim 2: To provide biostatistical support to the Projects, Cores and Programs, by consulting on the design, data monitoring, analysis, statistical modeling, visualization, interpretation and publication of the data generated by SPORE activities.
CORE C: TISSUE BANK FOR CELLULAR AND MOLECULAR STUDIES
Lawrence Weiss, M.D.
Ravi Bhatia, M.D.
Smita Bhatia, M.D., M.P.H.
The purpose of the tissue core is to facilitate research on lymphoid malignancies by members of the City of Hope Lymphoma Spore. The core provides services in the following areas: (1) acquisition and banking of fresh and paraffin-fixed tissues of lymphoma patients at City of Hope, including specimens obtained prospectively and retrospectively; (2) comprehensive work-up of lymphoma specimens to ensure correct diagnosis, classification, and detection of minimal residual disease, including immunohistochemical, flow cytometric, molecular pathologic, and cytogenetic studies; (3) performance and assistance in routine histologic processing and immunohistochemical staining of lymphoma tissues, including animal tissue, and preparations from cell lines, as well as specialized histologic services such as preparation of multitumor blocks or tissue microarrays to the specifications of researchers; (4) performance and assistance in Laser Capture microdissections from paraffin or frozen sections to obtain enrichment of specific cell types including isolation of malignant cells for analysis of proteins, RNA and/or DNA content; (5) flow cytometry studies, including complex characterization of lymphoma and host cell populations; (6) cytogenetic studies, including identification of vector-carrying cells and (7} consultation services to other SPORE investigators. In addition a Hematopoietic cell tissue bank has been established to prospectively collect and store fresh bone marrow, peripheral blood and peripheral blood stem cell samples from lymphoma patients undergoing autologous peripheral stem cell transplant (aPBSCT). This bank will support studies in Project 2 evaluating pathogenesis and predictors for t-MDS/AML in lymphoma patients post-aHCT. Samples are obtained pre-transplant and post-transplant at 100 days, 6 months, 1 year, and then annually through 5 years after transplant. Aliquots of the PBSC autograft are also banked. The core also collects and enters follow up outcome information for all patients. The core uses an IRB approved protocol for sample and data collection (see Appendix). The core also follows patients who have received their initial transplant treatment but who are not under current follow up at City of Hope, by contacting the treating physician, and if necessary, recalling the patient to the City of Hope for clinical evaluation, blood draw and bone marrow biopsy.
CORE D: BIOLOGICS MANUFACTURING CORE
David Colcher, Ph.D.
David DiGiusto, Ph.D.
David Hsu, Ph.D.
The Biologics Manufacturing core for this SPORE application is dedicated to the production of clinical trial materials according to programmatic requirements outlines in the individual projects and all applicable state, local and Federal regulations for investigational products. The core is divided into three production groups; cell products, monoclonal antibodies and viral vectors. Each group is led by a Director with extensive experience in the production of clinical trials materials and Good Manufacturing Practices (GMP). Each group is supported by a Quality Systems Team (Quality Assurance and Quality Control) that ensures that testing and documentation of investigational product manufacturing is performed according to the applicable regulations. Manufacturing will occur at two sites, The Center for Biomedicine and Genetics (CBG) and the Laboratory for Cellular Medicine (LCM) manufacturing facility in the new Arnold and Mabel Beckman Center for Cancer lmmunotherapeutics and Tumor Immunology completed in 2009. Monoclonal antibodies and constructs for Projects 1, 3 and 4 and Viral vectors for Project 1 will be manufactured in the CBG while cell products for Project 1 will be manufactured in the LCM. Over the past five years monoclonal antibody products have been produced for the previous SPORE Projects 1, 2, 4 and 5, some of which have been used in clinical trials. Extensive development of manufacturing, conjugating and vialing procedures for MAb products has taken place. A new method for the conjugation of MAbs at clinical scale quantities has been developed and recently published. The Laboratory for Cellular Medicine (LCM) has manufactured products for the previous iteration of SPORE Project 2 (Adoptive Immunotherapy for Follicular Lymphoma), is currently manufacturing products for Mantle Cell/DLBC Lymphoma (Project 1). Extensive process development has taken place in the areas of closed system cell processing, cell selection, lentiviral-based genetic modification and expansion in preparation for manufacturing investigational cell products for Lymphoma and other hematological malignancies. Plasmid DNA and Viral vectors have been manufactured for Follicular Lymphoma and Mantle Cell/DLBC Lymphoma protocols and are currently being developed for treating NHL patients for SPORE Project 1.