Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

hESC as tools to investigate the neural crest origin of Ewing's sarcoma

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$675 001
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESC) hold great promise as sources of tissue for regenerative medicine and therapeutics. In addition, their utility as tools to study the origins and biology of human disease must not be underestimated. hESC give rise to tissue-specific adult stem cells and, ultimately, to all mature tissues in the body. As such, disruptions to normal stem cell function can have catastrophic consequences and result in life-threatening or debilitating disease. Understanding how such diseases arise will afford novel insights into how we can better prevent and treat them. Laboratory based studies with hESC therefore stand to make extraordinary contributions to human health. Human tumors, and in particular the cancers that affect children, often look like tissues that have not developed normally and whose growth has gone unchecked. In fact, recent studies have shown that, in many cases, tumors arise because genetic mutations in the DNA of normal stem cells lead to disordered development, resulting in formation of malignant rather than normal tissues. For example, leukemia can arise when a mutation occurs in a normal blood stem cell, thus inducing formation of cancerous rather than normal blood. Analogous situations exist in other human tissues and their respective tumors. However, because of the relative rarity of normal stem cells in other parts of the body and our inability to effectively isolate them, very little is yet known about how these stem cells go awry and create cancer. hESC, therefore, represent an invaluable resource for the generation of tissue-specific stem cells and for studies of the genesis of human, and in particular, pediatric cancer. Several different human cancers are believed to arise either directly or indirectly from stem cells called neural crest stem cells (NCSC). NCSC exist in small numbers throughout the body and contribute to the formation of multiple different tissues including the peripheral nervous system and the pigment cells of our skin. It is our central hypothesis that NCSC-derived tumors arise because genetic mutations in NCSC lead to disordered tissue development and the initiation of cancer. Ewing’s sarcoma family tumors (ESFT) are highly aggressive tumors that primarily affect children and young adults. ESFT have a specific mutation in their DNA and this mutation leads to the creation of a cancer-causing gene. We believe that expression of this abnormal gene in NCSC disrupts normal stem cell differentiation and development and, ultimately, leads to ESFT formation. In this proposal we will use hESC as tools to prove or disprove this theory. Unfortunately, despite highly toxic and aggressive treatment, the survival rate for patients diagnosed with ESFT remains poor. By creating novel hESC-based models to study the origin and biology of ESFT we aim to gain novel insights into the origin and biology of these tumors that will aid in the development of more effective, less toxic therapies.
Statement of Benefit to California: 
Human embryonic stem cells (hESC) represent a tremendous resource as tools to study numerous human diseases, including cancer. Cancer claims the lives of over 50,000 Californians, including over 300 children, annually. Laboratory based studies using hESC, such as those proposed in this application, stand to make extraordinary and unique contributions to our understanding of the origin and biology of human cancer. These contributions will ultimately aid in the development of novel therapeutic strategies designed to improve survival and quality of life of cancer patients. In this proposal we will exploit the power of hESC to study the cellular origins of sarcomas. Sarcomas arise in the bones and soft tissues and primarily affect children and young adults. Despite intensive therapy, the survival rate of patients diagnosed with sarcoma remains poor. The proposed research will provide much needed insight into sarcoma biology and will enable development of novel sarcoma-targeted therapies. In addition, the hESC-derived models that we establish will be readily adaptable to and available for studies of other human cancers.
Progress Report: 
  • Recent studies have shown that mutations in the DNA of adult stem cells can lead to the formation of cancerous rather than normal tissues. However, with the exception of blood, adult stem cells are rare and not readily accessible for isolation or study. Thus, very little is yet known about how these stem cells are hijacked to cause cancer.
  • Our laboratory is studying how mutations in stem cells give rise to Ewing sarcoma. Ewing sarcoma family tumors (ESFT) are highly aggressive tumors that primarily affect children and young adults. ESFT have a specific mutation in their DNA that leads to the creation of a cancer-causing gene called EWS-FLI1. It is our hypothesis that expression of EWS-FLI1 in adult stem cells generates ESFT. In particular, we are interested in a very rare population of adult stem cells called neural crest stem cells (NCSC) and these cells have been the focus of our CIRM-funded grant.
  • We initially proposed that human embryonic stem cells (hESC) could be used to generate NCSC and that these cells would be invaluable tools with which to study the origin of ESFT. In the first year of the grant we successfully achieved this goal and the work has been published. In the second year of the grant we have studied the consequences of activating the EWS-FLI1 on these cells. Importantly, our work shows that NCSC that express EWS-FLI1 do not differentiate normally. Instead they acquire properties of cancer stem cells. Thus, we propose that ESFT arise from NCSC that acquire a genetic mutation that prevents them from developing normally. These abnormal stem cells then go on to develop into full blown tumors.
  • By creating novel stem cell models to study the origin of ESFT we are gaining new insights into how these tumors arise in children. These insights will ultimately aid in the development of more effective therapies that can be designed to destroy abnormal cancer-causing stem cells whilst sparing normal stem cells.

Derivation and Characterization of Cancer Stem Cells from Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$642 500
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Cancer is the leading cause of death for people younger than 85 (1). High cancer mortality rates underscore the need for more sensitive diagnostic techniques as well as therapies that selectively target cells responsible for cancer propagation (1) Compelling studies suggest that human cancer stem cells (CSC) arise from aberrantly self-renewing tissue specific stem or progenitor cells and are responsible for cancer propagation and therapeutic resistance (2-9). Although the majority of current cancer therapies eradicate rapidly dividing cells within the tumor, the rare CSC population may be quiescent and then reactivate resulting in disease progression and relapse (2-9). We recently demonstrated that CSC are involved in progression of chronic phase chronic myelogenous leukemia (CML), a disease that has been the subject of many landmark discoveries in cancer research(19-30), to a more aggressive and therapeutically recalcitrant myeloid blast crisis (BC) phase. These CSC share the same cell surface markers as granulocyte-macrophage progenitors (GMP) but have aberrantly gained the capacity to self-renew as a result of activation of the Wnt/-catenin stem cell self-renewal pathway (4). Because human embryonic stem cells (hESC) have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells in vitro, they represent an ideal model system for generating and characterizing human CSC (10-18). Thus, hESC cell research harbors tremendous potential for developing life-saving therapy for patients with cancer by providing a platform to rapidly and rationally test new therapies that specifically target CSC (2-18). To provide a robust model system for screening novel anti-CSC therapies, we propose to generate and characterize CSC from hESC (10-18). We will investigate the role of genes that are essential for initiation of CML such as BCR-ABL and additional mutations such as b-catenin implicated in CSC propagation (19-30). The efficacy of specific Wnt/b-catenin antagonists at inhibiting BCR-ABL+ human ES cell self-renewal, survival and proliferation alone and in combination with potent BCR-ABL antagonists will be assessed in sensitive in vitro and in vivo assays with the ultimate aim of developing highly active anti-CSC therapy that may halt cancer progression and obviate therapeutic resistance (4,31).
Statement of Benefit to California: 
The research outlined in this proposal represents a unique opportunity for collaborations between investigators from disparate disciplines to use human embryonic stem cells to challenge an existing paradigm namely that leukemic blasts are responsible for progression of chronic myelogenous leukemia (CML) rather than leukemic stem cells (LSC). Current clinical diagnostic tests are not sufficiently sensitive to predict timing of progression for all patients with CML nor are they adequate for determining the type of therapeutic intervention required. Moreover, the primary therapy for CML, Abl kinase inhibition, was shown to be cardiotoxic when given long-term at high doses. Furthermore, amplification of BCR-ABL is not the sole event that occurs during CML progression to blast crisis. Identification and inhibition of molecular mutations responsible for the generation of LSC in CML blood and/or marrow may prevent progression to blast crisis (BC) and would represent an innovative, effective form of CML therapy. Modeling of LSC responsible for CML progression in human embryonic stem cells could have a significant impact on our understanding of the pathophysiology of CML, provide novel diagnostic and therapeutic modalities and improve the quality and possibly quantity of life of patients with CML. By using BCR-ABL transduced human embryonic stem cells, we will rigorously evaluate the LSC hypothesis and as a consequence, the additional molecular events required for progression to blast crisis CML. The ultimate aims of this grant are to develop more sensitive methods to predict leukemic progression and to identify novel molecular therapeutic targets through the development of LSC models using human embryonic stem cells. We aim to provide a robust, reproducible system for testing novel anti-LSC compounds alone and in combination in order to expedite the development of novel therapeutic agents for anti-LSC clinical trials at {REDACTED}. Not only may the translational research performed in the context of this grant speed the delivery of innovative anti-LSC therapies for Californians with leukemia, it will help to train California’s future R&D workforce in addition to developing leaders in translational medicine. This grant will provide the personnel working on the project with a clear view of the importance of their research to cancer therapy and a better perspective on future career opportunities in California.
Progress Report: 
  • SEED Grant Research Summary
  • Compelling studies suggest that cancer stem cells (CSC) arise from primitive self-renewing progenitor cells. Although many cancer therapies target rapidly dividing cells, CSC may be quiescent i.e. asleep resulting in therapeutic resistance. Recently, we demonstrated that CSC drive progression of chronic phase (CP) chronic myeloid leukemia (CML), a subject of many landmark cancer research discoveries, to a therapeutically recalcitrant myeloid blast crisis (BC) phase. CML CSC share cell surface markers with granulocyte-macrophage progenitors (GMP) and have amplified expression of the CML fusion gene, BCR-ABL. In addition, they aberrantly gain self-renewal capacity, in part, as a result Wnt/β-catenin activation. Because human embryonic stem cells (hESC) have robust regenerative capacity and can provide a potentially limitless source of tissue specific progenitor cells in vitro, they represent an ideal model system for generating and characterizing human CSC. The main goals of this research were to generate CSC from hESC to provide an experimentally amenable platform to expedite the development of sensitive diagnostics that predict progression and combined modality anti-CSC therapy.
  • To this end, we tested whether BCR-ABL expression in hESC is sufficient to induce changes characteristic of CML stem cells. Unlike mouse ESC, introduction of a novel lentiviral BCR-ABL vector into hESC did not drive myeloid differentiation nor did it induce stromal independence in vitro underscoring key differences between mouse and human hESC and the importance of in vivo models. Notably, Hues16 cells had a higher propensity to differentiate into CD34+ cells than other hESC lines particularly in AGM co-cultures and thus, were used in subsequent in vivo experiments. Moreover, this SEED grant funded Yosuke Minami in Professor Jean Wang’s lab to create a unique CML blast crisis mouse model typified by GMP expansion and resistance to a BCR-ABL inhibitor, imatinib (Minami et al, PNAS 2008;105:17967-72). In addition, a bioluminescent humanized model of blast crisis CML was created based on transplantation of GMP from patient blood into immune deficient mice (RAG2-/-gc-/-). Cells were tagged with firefly luciferase that emits a bioluminescent signal so that leukemic transplantation efficiency could be tracked in vivo (IVIS). As few as 1,000 human blast crisis CML GMP could transplant leukemia in immune deficient mice thereby providing an important model for studying the molecular events that contribute to leukemic transformation (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the second aim, we hypothesized that BCR-ABL is sufficient for generating CML from self-renewing stem cells. In these studies, Hues16 cells differentiated into CD34+ cells were lentivirally transduced with BCR-ABL leading to sustained BCR-ABL engraftment in 50% of transplanted mice. Chronic phase CD34+ cells derived from CML blood were less efficient at sustaining CML engraftment (7%) suggesting that hESC derived CD34+ cells have higher self-renewal potential and are similar to advanced phase CML progenitors.
  • Thirdly, we hypothesized that BCR-ABL was necessary but not sufficient for progression to blast crisis. Introduction of lentiviral activated beta-catenin or shRNA to GSK3beta, together with BCR-ABL did not enhance BCR-ABL engraftment compared with BCR-ABL transduction of hESC alone. These studies suggested that hESC may already have sufficient self-renewal capacity to sustain the malignant CML clone and are molecularly comparable to advanced CML progenitors that behave like CSC. In addition, through extensive cDNA sequencing of human blast crisis CML progenitors, we found that 57% of samples harbored a misspliced form of GSK3beta that promoted tumor production and could serve as a novel prognostic marker in CML clinical trials (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the final aim, we hypothesized that CML CSC are not eliminated by BCR-ABL inhibitors alone and that combined modality therapy will be required. In collaborative research involving in vitro analysis of imatinib resistant CML progenitors and more recently in a humanized mouse model of blast crisis CML, we found that dasatinib, a potent BCR-ABL inhibitor, is necessary but not sufficient for CSC eradication. Discovery of a GSK3beta deregulation, a negative regulator of both beta-catenin and sonic hedgehog (Shh) pathways (Zhang et al, Nature 2009), led us to disover that Shh combined with BCR-ABL inhibition abrogated CSC driven tumor formation (manuscript in preparation) providing the impetus for an upcoming Pfizer sponsored Shh inhibitor clinical trial for refractory hematologic malignancies.

The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$777 467
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The successful use of human embryonic stem cells (hESCs) as novel regenerative therapies for a spectrum of currently incurable diseases critically depends upon the safety of such cell transfers. hESCs contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of their genetic material. While many of these retroelements have been permanently silenced during evolution by crippling mutations, many remain active and capable of moving to new chromosomal locations potentially producing disease-causing mutations or cancer. More mature differentiated cells control retroelement movement (retrotransposition) by methylating the DNA comprising these elements. Strikingly, such DNA methylation is largely absent in hESCs because these cells must be able to develop into a wide spectrum of different tissues and organs. Thus, in order to protect the integrity of their genomes, hESCs must deploy an additional defense to limit retroelement retrotransposition. Recent studies of HIV and other exogenous retroviruses have identified the APOBEC3 family of genes (A3A-A3H) as powerful anti-retroviral factors. These APOBEC3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also used by retroelements for their successful retrotransposition. We hypothesize that one or more of the APOBECs function as guardians of genome integrity in hESCs. We propose to compare and contrast which APOBEC3s are expressed in one federally approved and nine nonapproved hESC lines and to assess the natural level of retroelement RNA expression occurring in each of these lines. Next we will test whether the knockdown of expression of these APOBEC3s in the hESCS lines by RNA interference leads to a higher frequency of retrolement retrotransposition. Finally, if higher levels of retrotransposition are detected, we will examine whether these cells display an impaired ability to differentiate into specific tissue types corresponding to the three germ cell layers (ectoderm, mesoderm, and endoderm) and whether increased retrotransposition is associated with a higher frequency of malignant transformation within the hESC cultures. These studies promise to provide important new insights into how genomic stability in is maintained in hESCs and could lead to the identification of specific GMP culture conditions that minimize the chances of such unwanted retrotransposition events in cells destined for infusion into patients. These studies are directly responsive to the CIRM request for application. If funded, these studies would allow the entry of my laboratory with extensive APOBEC experience, into the exciting field of stem cell biology.
Statement of Benefit to California: 
Harnessing the exciting potential of embryonic stem cells as therapies for a wide range of diseases like diabetes, Alzheimer’s disease, myocardial infarction among others first requires ensuring that the infusion of these cells into patients can be performed safely. Of note, human embryonic stem cells contain up to 3 million “jumping genes” or mobile genetic retroelements that can potentially move from location to another in the genome. Great harm could occur if the movement of these retroelements in human embryonic stem cells results in the mutation of key genes or the inactivation of tumor suppressor genes, the latter could facilitate the development of cancer in recipients of these cells. The safety of stem cell therapy thus depends on the rigorous maintenance of genomic integrity and stability within the embryonic stem cell during its manipulation. Strikingly, the major cellular defense against the movement of the retroelements to new genetic locations, DNA methylation, is greatly reduced in human embryonic stem cells. A general state of hypomethylation is likely required to permit these pluripotent cells to differentiate into multiple cell types. With DNA methylation no longer able to constrain the activity of these retroelements, we believe a second natural defense springs into action to protect these stem cells. We proposeto identify and characterize this defensive network. These studies could lead to new approaches for maintaining or even enhancing this defense when embryotic stem cells are manipulated in culture, thereby helping to ensure the safety of embryonic stem cells destined for therapeutic transfer. Thus, the results of these studies will have both scientific and practical value. As such, we believe these studies will benefit the citizens of California certainly at a societal level and potentially at a personal level.
Progress Report: 
  • Human embryonic stem cells contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of human genome. While many of these retroelements have been silenced during evolution by crippling mutations, many remain active and capable of jumping to new chromosomal locations potentially producing disease-causing mutations or cancer. In tissues, mobility of these elements is suppressed by DNA methylation, which inactivates expression of the retroelement RNAs. In sharp contrast, embryonic stem cells exhibit very dynamic changes in DNA methylation, where the methylation patterns are gained and lost at high rates. During periods of low DNA methylation, retroelement RNA expression likely increases. Accordingly, hESCs must deploy other defensive strategies in order to maintain genomic integrity. Recent studies have identified the APOBEC3 family of genes (A3A-A3H) as powerful antiviral factors. These A3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also employed by retroelements for their successful retrotransposition. We hypothesized that one or more of the APOBECs function as guardians of genome integrity in hESCs. In the last two years we have found that six out of the seven human A3 genes located in a tandem array on chromosome 22 are expressed in hESCs. A3A, which in prior studies was suggested to exert the greatest anti-retroelement effects, surprisingly is not expressed in hESCs. Further, we find that the A3 proteins decrease when pluripotent cells differentiate into somatic cells suggesting an important function of these A3 proteins in pluripotent hESCs. We established a LINE1 retrotransposition assay in hESCs that allows us to visualize genetic jumping of this class of “marked” retroelements via flow cytometry. Using this assay we have found that LINE1 elements effectively jump in hESCs. To test our central hypothesis, namely that A3 proteins guard the genome in hESCs, we have established experimental conditions for RNAi knock-down of all expressed A3 genes. By combining the knock-down and the retrotransposition assay we demonstrated that the knock-down of one member of the A3 protein family leads to a 3.5-fold increase in LINE1 retrotranspositon. This finding highlights a protective role for the A3 family of cytidine deaminases that helps safeguard the genome integrity of hESCs.

Genetic Enhancement of the Immune Response to Melanoma via hESC-derived T cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00203
ICOC Funds Committed: 
$642 501
Disease Focus: 
Melanoma
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The overall goal of the proposed studies is to utilize human gene therapy approach using human embryonic stem cells to direct our body’s defenses to specifically attack melanoma tumor cells. Current technologies try to accomplish this by genetically manipulating certain circulating T lymphocytes, such that they will target tumor cells. T lymphocytes are the major cell type of our body’s immune system. However it is likely that this type of approach will not result in the presence of stable, lifelong genetically modified T cells. In contrast, a potentially more long-lasting approach would be to genetically modify human embryonic stem cells with the same therapeutic gene. Stem cells have the ability to form any type of blood cell, including T cells. Importantly, stem cells can persist for the life of the individual, and thus have the potential to produce genetically modified T cells for many years. In addition, these new tumor specific cells should expand in the body in response to the presence of the tumor, thus a large supply of tumor-fighting cells should be available as long as needed. This project proposes to develop novel means to introduce the anti-cancer gene into human embryonic stem cells. These stem cells will then be differentiated to generate tumor specific T cells utilizing animal model systems. We will then use several laboratory and mouse models to determine if the T cells derived from these genetically modified stem cells have anti-tumor activity. If successful, we will have provided proof-of principle that long-lived stem cells have the potential be utilized as a means of producing anti-cancer T cells. In the long run, these results could provide important information for design of future clinical trials designed to produce life-long improved anti-cancer immune responses.
Statement of Benefit to California: 
We propose to use human embryonic stem cells to develop a novel, yet potentially very effective method to treat invasive melanoma. Melanoma is a serious type of skin cancer which, if not removed early, spreads internally and is usually fatal. Overall melanoma is the 6th most common cancer in males and 7th in females and the incidence of this form of cancer is currently increasing at an epidemic rate. Although melanomas may occur in areas of skin that are not normally exposed to sunlight, sun exposure is believed to be a factor in about 70% of new cases. California’s mild winters and high number of sunny days provide opportunities for a number of occupational and recreational outdoor activities, and people in California are exposed to more than average levels of solar radiation. Consequently, there is a higher risk of developing this disease. As a matter of fact, California is one of the five US states with the highest predicted incidence of new cases of melanoma. According to the California Cancer Registry, each year 4,700 new cases of invasive melanoma and over 800 deaths related to this disease are reported in California, with the incidence rate increasing by 15% over the last decade. While the white population is at the greatest risk of developing this disease, it was recently reported that the rates of invasive melanoma have risen substantially in Hispanic people living in California as well. If our proposal is successful, our work could pave the way to the development of a new and effective form of melanoma therapy, one which would clearly benefit all the people of California affected by this disease.
Progress Report: 
  • In this grant we proposed to genetically engineer human embryonic stem cells (hESC) and hematopoietic stem cells (HSC) and to use them to produce T cells with enhanced ability to kill melanoma cells. Our proposal consists of several steps. In the first year of the grant, we completed the first step and introduced the genes for a melanoma specific T cell receptor (TCR) into hESC and HSC. In this, second year of funding we were able to generate genetically modified T cells from hESC and HSC and to characterize the HSC-derived cells in more details. We found that HSC-derived T cells carrying the new TCR are indistinguishable from normal T cells, based on the cell surface expression of other T cell specific proteins. Also, we found that they can kill human melanoma cells in a Petri dish. We are currently evaluating their ability to destroy tumors in experimental animals transplanted with human melanoma cells. This is a more relevant approach as it mimics the potential treatment of melanoma patients. We are also trying to obtain larger numbers of the genetically modified hESC-derived T cells and analyze them in the same types of assays. Our data are encouraging and suggestive of possible clinical application of these cells in future.

Development of Therapeutic Antibodies Targeting Human Acute Myeloid Leukemia Stem Cells

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01485
ICOC Funds Committed: 
$19 999 996
Disease Focus: 
Blood Cancer
Cancer
Solid Tumor
Collaborative Funder: 
UK
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow that is rapidly fatal within months if untreated. Even with aggressive treatment, including chemotherapy and bone marrow transplantation, five-year overall survival rates range between 30-40%. Evidence indicates that not all cells in this cancer are the same, and that there is a rare population of leukemia stem cells (LSC) that are responsible for maintaining the disease. Thus, in order to cure this cancer, all LSC must be eliminated, while at the same time sparing the normal blood forming stem cells in the bone marrow. We propose to develop therapeutic antibodies directed against surface markers present in much larger amounts on LSC than on the surface of normal blood forming stem cells. We recently identified and validated several such protein markers including CD47, which we determined contributes to leukemia development by blocking the ingestion and removal of leukemia cells by immune system cells called macrophages. In this way, CD47 acts as a “don’t eat me” signal on LSC. Moreover, we determined that monoclonal antibodies (mAbs) directed against CD47, able to block its interaction with macrophages, mask the “don’t eat me” signal resulting in ingestion and elimination of leukemia in mouse pre-clinical models. We propose a combination of clinical studies, basic research, and pre-clinical development to prepare a therapeutic antibody directed against CD47 and/or other LSC-specific proteins for Initial New Drug (IND) filing with the FDA, and then a Phase I clinical trial to be conducted at {REDACTED} and in the Collaborative Funding Partner country. In collaboration with the pioneering Collaborative Funding Partner country AML Working Group, we will track expression of the LSC proteins in patient samples and correlate with clinical outcomes. This will allow us to identify particular LSC proteins that must be targeted to achieve cure, thereby prioritizing candidate therapeutic antibodies for clinical development. Concurrently, we will conduct basic research and pre-clinical development to prepare these candidates. Basic research during years 1 and 2 will focus on the characterization of anti-CD47 mAb efficacy, investigation of mAb targeting of additional LSC molecules, and determination of efficacy in combinations with anti-CD47. Pre-clinical development during years 1 and 2 will focus on blocking anti-CD47 mAbs, including antibody humanization and large animal model pharmacologic and toxicity studies. Similar studies will be conducted with the most promising antibodies resulting from our basic research. During years 3-4, we will proceed with GMP grade production of the best candidate, followed by efficacy testing in mouse models and large animal models. Finally, in year 4, we will prepare an IND filing with the FDA/MHRA and develop a Phase I clinical trial with this antibody for the treatment of AML. Ultimately, therapeutic antibodies specifically targeting AML LSC offer the possibility of less toxicity with the potential for cure.
Statement of Benefit to California: 
Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with nearly 13,000 new diagnoses annually in the US and 2,200 in the Collaborative Funding Partner country. Current standard of care for medically fit patients consists of several cycles of high dose chemotherapy, and often includes allogeneic hematopoietic cell transplantation. Even with these aggressive treatments, which cause significant morbidity and mortality, relapse is common and the five-year overall survival is 30-40%, but <10% in patients with relapsed or refractory disease or in the majority of AML patients who are over age 65. The goal of this research proposal is to prepare therapeutic antibodies directed against AML stem cell-specific antigens for IND filing with the FDA and a Phase I clinical trial. There are several potential benefits of this research for California: (1) most importantly, this research has the potential to revolutionize current clinical practice and provide a targeted therapy for AML that offers the possibility of less toxicity with the potential for cure; (2) this research will directly contribute to the California economy by funding a contract manufacturing organization to generate and produce GMP-grade clinical antibody, by employing several individuals who will be essential for the conduct of these studies, and through the purchase of equipment and reagents from California vendors; (3) additional clinical and economic benefits for California will derive from the potential application of clinical agents developed here to a number of other human cancers and cancer stem cells; (4) our animal models indicate that a significant fraction of patients with fatal AML can be cured, resulting in savings on their clinical care plus their return as productive contributors to the California economy; (5) if our therapeutic antibodies show clinical benefit in AML, they will be commercialized, and under CIRM policy, profits derived from treating insured patients and lower cost therapies for uninsured patients, would enrich the state and the lives of its citizens; (6) finally, this research has the potential to maintain California as the national and world-wide leader in stem cell technology.
Progress Report: 
  • Our program is focused on producing new therapeutic candidates to prolong remission and potentially cure highly lethal cancers where patients have few alternative treatment options. We have selected Acute Myelogenous Leukemia (AML) as the initial clinical indication for evaluating our novel therapeutics, but anticipate a full development program encompassing many other types of solid tumor cancers.
  • Our strategy is to develop an antibody that binds to and eliminates the cancer-forming stem cells in leukemia and other solid tumors. While current cancer treatments (e.g. surgery, chemotherapy, radiation) will frequently get rid of the bulk of the tumor, they rarely touch the tiny number of cancer stem cells that actually re-generate the masses of cancer cells that have been eliminated. When the latter occurs, the patient is described as having a relapse, leading to a disease recurrence with poor prognosis. Our strategy is to eliminate the small number of cancer-regenerating stem cells by targeting cell membrane proteins expressed by these cells.
  • We have discovered that many cancer cells coat themselves with a protein called CD47 that prevents them from being eaten and disposed of by the patient’s blood cells. In this context, CD47 can be considered a ‘don’t eat me’ signal that protects the cancer cells from being phagocytosed i.e. ‘eaten’. The antibody we are developing binds to and covers the ‘don’t eat me’ CD47 protein, so that the patient’s blood cells are now able to ‘eat’ the cancer cells by standard physiological responses, and eliminate them from the body.
  • Developing an antibody such as this for use in humans requires many steps to evaluate it is safe, while at the same ensuring it targets and eliminates the cancer forming stem cells. The antibody must also ‘look’ like a human antibody, or else the patient will ‘see’ it as a foreign protein and reject it. To achieve these criteria, we have made humanized antibodies that bind to human CD47. We have shown that the antibodies eliminate cancer cells in two ways: (i) blood cells from healthy humans rapidly “ate” and killed leukemia cells collected from separate cancer patients when the anti-human CD47 antibody was added to a mixture of both cell types in a research laboratory test tube; (ii) the anti-human CD47 antibody eliminates human leukemia cells collected from patients, then transferred into special immunodeficient mice which are unable to eliminate the human tumor cells themselves. In these experiments, the treated mice remained free of the human leukemia cells for many weeks post-treatment, and could be regarded as being cured of malignancy.
  • To show the antibodies were safe, we administered to regular mice large amounts of a comparable anti-mouse CD47 antibody on a daily basis for a period of many months. No adverse effects were noted. Unfortunately our antibody to human CD47 did not bind to mouse CD47, so it’s safety could not be evaluated directly in mice. Since the anti-human CD47 antibody does bind to non-human primate CD47, safety studies for our candidate therapeutic need to be conducted in non-human primates. These studies have been initiated and are in progress. Following administration of the anti-human CD47 antibodies, the non-human primates will be monitored for clinical blood pathology, which, as in humans, provides information about major organ function as well as blood cell function in these animals.
  • The next step after identifying an antibody with strong anti-cancer activity, but one that can be safely administered to non-human primates without causing any toxic effects, is to make large amounts of the antibody for use in humans. Any therapeutic candidate that will be administered to humans must be made according to highly regulated procedures that produce an agent that is extremely “clean”, meaning free of viruses, other infectious agents, bacterial products, and other contaminating proteins. This type of production work can only be performed in special facilities that have the equipment and experience for this type of clinical manufacturing. We have contracted such an organization to manufacture clinical grade anti-human CD47 antibodies. This organization has commenced the lengthy process of making anti-CD47 antibody that can be administered to humans with cancer. It will take another 18 months to complete the process of manufacturing clinical grade material in sufficient quantities to run a Phase I clinical trial in patients with Acute Myelogenous Leukemia.
  • Our program is focused on producing new therapeutic candidates to prolong remission and potentially cure highly lethal cancers where patients have few alternative treatment options. Our strategy is to develop an antibody that will eliminate the cancer stem cells which are the source of the disease, and responsible for the disease recurrence that can occur months-to-years following the remission achieved with initial clinical treatment. The cancer stem cells are a small proportion of the total cancer cell burden, and they appear to be resistant to the standard treatments of chemotherapy and radiation therapy. Therefore new therapeutic approaches are needed to eliminate them.
  • In year 2 of the CIRM award, we have continued to develop a clinical-grade antibody that will eliminate the cancer stem cells in Acute Myelogenous Leukemia (AML). We have identified several antibodies that cause human leukemia cells to be eaten and destroyed by healthy human white blood cells when tested in cell culture experiments. These antibodies bind to a protein called CD47 that is present on the outer surface of human leukemia cells. The anti-CD47 antibodies can eliminate leukemia growing in mice injected with AML cells obtained from patients. We have now extensively characterized the properties of our panel of anti-CD47 antibodies, and have identified the lead candidate to progress though the process of drug development. There are several steps in this process, which takes 18-24 months to fully execute. In the last 12 months, we have focused on the following steps:
  • (i) ‘Humanization’ of the antibody: The antibody needs to be optimized so that it looks like a normal human protein that the patient’s immune system will not eliminate because it appears ‘foreign’ to them.
  • (ii) Large scale production of the antibody: To make sufficient quantities of the antibody to complete the culture and animal model experiments required to progress to clinical safety trials with patients, we have contracted with a highly experienced manufacturing facility capable of such large-scale production. We have successfully transferred our antibody to them, and they have inserted it into a proprietary expression cell that will produce large amounts of the protein. This process is managed through weekly interactions with this contract lab. They send us small amounts of the material from each step of their manufacturing process and we test it in our models to ensure the antibody they are preparing retains its anti-cancer properties throughout production.
  • (iii) Pre-clinical safety studies: The antibody must be tested extensively in animals to ensure it does not cause serious limiting damage to any of the normal healthy tissues in the recipient. We have spent much of the last 12 months performing these types of safety experiments. The antibody has been administered to both mice and non-human primates and we have evaluated their overall health status, as well as analyzing their blood cells, blood enzyme levels, and urine, for up to 28 days. We have also collected samples of their organs and tissues to evaluate for abnormalities. Thus far, these assessments have appeared normal except for the development of a mild anemia a few days after the initial antibody injection. Subsequent experiments indicate that this anemia can be managed with existing approved clinical strategies
  • (iv) Determination of optimal dose: We have used mice injected with human cancer cells from AML patients, and determined how much antibody must be injected into these mice to produce a blood level that destroys the leukemia cells. This relationship between antibody dose and anti-cancer activity in the mouse cancer model enables us to estimate the dose to administer to patients.
  • Hematologic tumors and many solid tumors are propagated by a subset of cells called cancer stem cells. These cells appear to be resistant to the standard cancer treatments of chemotherapy and radiation therapy, and therefore new therapeutic approaches are needed to eliminate them. We have developed a monoclonal antibody (anti-CD47 antibody) that recognizes and causes elimination of these cancer stem cells and other cells in the cancer, but not normal blood-forming stem cells or blood cells. Cancer stem cells regularly produce a cell surface ‘invisibility cloak’ called CD47, a ‘don’t eat me signal’ for cells of the native immune system. Anti-CD47 antibody counters the ‘cloak, allowing the patient’s natural immune system eating cells, called macrophages, to eliminate the cancer stem cells.
  • As discussed in our two-year report, we optimized our anti-CD47 antibody so that it looks like a normal human protein that the patient’s immune system will not eliminate because it appears ‘foreign’. In this third year of the grant, we initiated the pre-clinical development of this humanized antibody, and assigned the antibody the development name of Hu5F9. Our major accomplishments in the third year of our grant are as follows:
  • (i) In addition to the hematological malignancies we have studied in previous years, we have now demonstrated the Hu5F9 is effective at inhibiting the growth and spread throughout the body [metastasis] of a large panel of human solid tumors, including breast, bladder, colon, ovarian, glioblastoma [a very aggressive brain cancer], leiomyosarcoma, head & neck squamous cell carcinoma, and multiple myeloma.
  • (ii) We have performed extensive studies optimizing the production and purification of Hu5F9 to standards compatible with use in humans, including that it is sterile, free of contaminating viruses, microorganisms, and bacterial products. We will commence manufacturing of Hu5F under highly regulated sterile conditions to produce what is known as GMP material, suitable for use in humans.
  • (iii) Another step to show Hu5F9 is safe to administer to humans is to administer it to experimental animals and observe its effects. We have demonstrated that Hu5F9 is safe and well tolerated when administered to experimental animals. Notably, no major abnormalities are detected when blood levels of the drug are maintained in the potentially therapeutic range for an extended duration of time.
  • (iv) We have initiated discussions with the FDA regarding the readiness of our program for initiating clinical trials, which we anticipate to start in the first quarter of 2014. To prepare for these trials we have established a collaboration between the Stanford Cancer Institute and the University of Oxford in the United Kingdom, currently our partners in this CIRM-funded program.
  • To our knowledge, CD47 is the first common target in all human cancers, one which has a known function that enables cancers to grow and spread, and one which we have successfully targeted for cancer therapy. Our studies show that Hu5F9 is a first-in-class therapeutic candidate that offers cancer treatment a totally new mechanism of enabling the patient’s immune system to remove cancer stem cells and their metastases.
  • Hematologic tumors and many solid tumors are driven by a subset of cells called cancer stem cells. These cancer stem cells must be eliminated for cures, however, they have been found to be resistant to the standard cancer treatments of chemotherapy and radiation therapy. Therefore, new therapeutic approaches are needed to target these abnormal stem cells. Previously, we found that cancer stem cells have developed a clever way to hide from the patient’s immune system. They display a protein called CD47 on their surface that signals to the immune system “don’t eat me”, thereby preventing their elimination. We have developed a monoclonal antibody (anti-CD47 antibody) that blocks this signal leading to elimination of these cancer stem cells, but not normal most normal cells, by the natural immune system. In our pre-clinical studies, we showed that anti-CD47 antibodies eliminates cancer cells and cancer stem cells from many different types of human cancer including: leukemia, breast cancer, colon cancer, prostate cancer, ovarian cancer, and others. In addition, anti-CD47 antibodies are effective at preventing and even eliminating metastases in animal models. These results indicate that anti-CD47 antibodies have great potential for the treatment of human cancer.
  • In order to develop this approach into a clinical therapeutic, we first optimized our anti-CD47 antibody so that it looks like a normal human protein that the patient’s immune system will not reject. Over the course of this grant project, we have conducted the pre-clinical development of this humanized antibody, termed Hu5F9-G4.
  • (1) Hu5F9-G4 has been manufactured according to Good Manufacturing Practices (GMP) as required by the United States Food and Drug Administration (FDA) for administration to humans. The drug product was manufactured and tested to be free of contaminants and is now ready for clinical use.
  • (2) Hu5F9-G4 has undergone extensive testing to investigate potential toxic effects in humans. According to FDA regulatory guidelines, Hu5F9-G4 was tested in experimental animals where it was given in various increasing doses. In all studies, Hu5F9-G4 was well-tolerated and caused no serious side effects.
  • (3) We have developed a phase 1 first-in-human clinical trial protocol for the investigation of Hu5F9-G4 in patients with solid tumors. In addition, we have prepared all the necessary documentation and clinical operations plans necessary to execute this clinical trial.
  • (4) We have submitted the necessary information on anti-cancer activity, manufacturing, safety, and clinical trial plans to the FDA in an Investigational New Drug (IND) application. This application was approved by FDA for the clinical trial in patients with solid tumors.
  • (5) We continue to develop parallel clinical trial plans for a phase 1 study in patients with acute myeloid leukemia (AML), and anticipate submitting our regulatory filing in 2015.
  • In summary, our studies show that Hu5F9-G4 is a first-in-class therapeutic candidate that offers cancer treatment through a totally new mechanism of enabling the patient’s immune system to remove cancer stem cells and prevent their metastases.

Development of Highly Active Anti-Leukemia Stem Cell Therapy (HALT)

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01430
ICOC Funds Committed: 
$19 999 826
Disease Focus: 
Blood Cancer
Cancer
Collaborative Funder: 
Canada
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Leukemias are cancers of the blood forming cells that afflict both children and adults. Many drugs have been developed to treat leukemias and related diseases. These drugs are often effective when first given, but in many cases of adult leukemia, the disease returns in a form that is not curable, causing disability and eventual death. During the last few years, scientists have discovered that some leukemia cells possess stem cell properties that make them more potent in promoting leukemia growth and resistance to common types of treatment. These are called leukemia stem cells (LSC). More than in other cancers, scientists also understand the exact molecular changes in the blood forming cells that cause leukemias, but it has been very difficult to translate the scientific results into new and effective treatments. The main difficulty has been the failure of existing drugs to eliminate the small numbers of LSC that persist in patients, despite therapy, and that continue to grow, spread, invade and kill normal cells. In fact, the models used for drug development in the pharmaceutical industry have not been designed to detect drugs or drug combinations capable of destroying the LSC. Drugs against LSC may already exist, or could be simple to make, but there has not been an easy way to identify these drugs. Recently, physicians and scientists at universities and research institutes have developed tools to isolate and to analyze LSC donated by patients. By studying the LSC, the physicians and scientists have identified the molecules that these cells need to survive. The experimental results strongly suggest that it will eventually be possible to destroy LSC with drugs or drug combinations, with minimal damage to most normal cells. Now we need to translate the new knowledge into practical treatments. The CIRM Leukemia Team is composed of highly experienced scientists and physicians who first discovered LSC for many types of leukemia and who have developed the LSC systems to test drugs. The investigators in the Team have identified drug candidates from the vigorous California pharmaceutical industry, who have already performed expensive pharmacology and toxicology studies, but who lack the cells and model systems to assess a drug’s ability to eliminate leukemia stem cells. This Team includes experts in drug development, who have previously been successful in quickly bringing a new leukemia drug to clinical trials. The supported interactive group of physicians and scientists in California and the Collaborative Funding Partner country has the resources to introduce into the clinic, within four years, new drugs for leukemias that may also represent more effective therapies for other cancers for the benefit of our citizens.
Statement of Benefit to California: 
Thousands of adults and children in California are afflicted with leukemia and related diseases. Although tremendous gains have been made in the treatment of childhood leukemia, 50% of adults diagnosed with leukemia will die of their disease. Current therapies can cost tens of thousands of dollars per year per patient, and do not cure the disease. For the health of the citizens of California, both physical and financial, we need to find a cure for these devastating illnesses. What has held up progress toward a cure? Compelling evidence indicates that the leukemias are not curable because available drugs do not destroy small numbers of multi-drug resistant leukemia stem cells. A team approach is necessary to find a cure for leukemia, which leverages the expertise in academia and industry. Pharmaceutical and biotech companies have developed drugs that inhibit pathways known to be involved in leukemia stem cell survival and growth, but are using them for unrelated indications. In addition, they do not have the expertise to determine whether the inhibitors will kill leukemia stem cells. The Leukemia Team possesses stem cell expertise and has developed state of the art systems to determine whether drugs will eradicate leukemia stem cells. They have also have access to technologies that may allow them to identify patients who will respond to the treatment. The development plan established by the Leukemia Disease Team will also serve as a model for the clinical development of drugs against solid tumor stem cells, which are not as well understood. In summary, the benefits to the citizens of California from the CIRM disease specific grant in leukemia are: (1) direct benefit to the thousands of leukemia patients (2) financial savings due to definitive treatments that eliminate the need for costly maintenance therapies
Progress Report: 
  • Development of Highly Active Leukemia Therapy (HALT)
  • Leukemias are cancers of the blood forming cells that affect both children and adults. Although major advances have been made in the treatment of leukemias, many patients still succumb to the disease. In these patients, the leukemias may progress despite therapy because they harbor primitive malignant stem-like cells that are resistant to most drugs. This CIRM disease specific grant aims to develop a combination of highly active anti-leukemic therapy (HALT) that can destroy the drug-resistant cancer stem-like cells, without severely harming normal cells.
  • During the current year of support, substantial progress has been made in achieving this goal. The CIRM investigators have shown that two different drugs that inhibit different proteins in leukemia stem cells can sensitize them to chemotherapeutic agents, and block their ability to self-renew. The CIRM investigators have also demonstrated that two different antibodies against molecules on the surface of the leukemia cells can inhibit their survival in both test tube experiments and in mouse models.
  • Extensive experiments are underway to confirm these promising results. The results will enable the planning and implementation of potentially transforming clinical trials in leukemia patients, during the period of CIRM grant support.
  • During the past 12 months, our disease team has made further progress in
  • the development of stem cell targeted treatment for chronic lymphocytic
  • leukemias and other leukemias. Stem cells express some molecules on the
  • surface that are different from the corresponding molecules on adult
  • cells. The ROR1 molecule is highly expressed by malignant cells from
  • patients with chronic lymphocytic leukemia, as well as by progenitor cells
  • from other forms of leukemia and lymphoma. It is not expressed by normal
  • adult cells. With the support of the CIRM Disease Team grant, the
  • cooperating investigators have prepared monoclonal antibodies against the
  • ROR1 molecule, that are potent and specific. In animal models, the
  • antibodies can retard leukemia growth and spread. Unlike other anti-cancer
  • drugs, the new antibodies are not toxic for normal bone marrow cells.
  • Thus, they can potentiate the action of other agents used for the
  • treatment of leukemia.
  • The disease team is now focused on the pre-clinical development, safety
  • testing, and scale-up manufacturing of our new, promising agents, in
  • preparation for their introduction into the clinic.
  • During the past 12 months, our disease team has made further progress in
  • the development of stem cell targeted treatment for chronic lymphocytic
  • leukemias and other leukemias. Stem cells express some molecules on the
  • surface that are different from the corresponding molecules on adult
  • cells. The ROR1 molecule is highly expressed by malignant cells from
  • patients with chronic lymphocytic leukemia, as well as by progenitor cells
  • from other forms of leukemia and lymphoma. It is not expressed by normal
  • adult cells. With the support of the CIRM Disease Team grant, the
  • cooperating investigators have prepared a humanized monoclonal antibody against the
  • ROR1 molecule, that is potent and specific. In animal models, the
  • antibodies can retard leukemia growth and spread. Unlike other anti-cancer
  • drugs, the new antibodies are not toxic for normal bone marrow cells.
  • Thus, they can potentiate the action of other agents used for the
  • treatment of leukemia.
  • The disease team is now focused on the pre-clinical development, safety
  • testing, and scale-up manufacturing of our new, promising agents, in
  • preparation for their introduction into the clinic.
  • During the past 12 months, our disease team has made further progress in
  • the development of stem cell targeted treatment for chronic lymphocytic
  • leukemias and other leukemias. Stem cells express some molecules on the
  • surface that are different from the corresponding molecules on adult
  • cells. The ROR1 molecule is highly expressed by malignant cells from
  • patients with chronic lymphocytic leukemia, as well as by progenitor cells
  • from other forms of leukemia and lymphoma. It is not expressed by normal
  • adult cells. With the support of the CIRM Disease Team grant, the
  • cooperating investigators have prepared a humanized monoclonal antibody against the
  • ROR1 molecule, that is potent and specific. In animal models, the
  • antibodies can retard leukemia growth and spread.
  • The disease team has now finalized the pre-clinical development, safety
  • testing, and scale-up manufacturing of our new, promising agent, in
  • preparation for their introduction into the clinic.

Stem Cell-Mediated Oncocidal Gene Therapy of Glioblastoma (GBM)

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01426
ICOC Funds Committed: 
$19 162 435
Disease Focus: 
Brain Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
Brain tumors (BTs) are incurable, whether they start in the brain or spread there from other sites. Despite advances in surgical, radiation, pharmacologic, and gene therapies, survival with a BT remains dismal. Current therapies are limited by their inability to reach widely disseminated tumor cells that become dispersed within normal brain structures. Interestingly, the therapeutic property that is needed to overcome this major obstacle to effective treatment of BTs matches well with one of the better accepted attributes of neural stem cells (NSCs): an attraction for sites of pathology in the adult brain, including primary & metastatic cancer. If armed with a proper tumor-killing gene, NSCs (whether administered into the brain or into the bloodstream), that are drawn to cancers, will dramatically reduce tumor burden, and will track after even single migrating tumor cells. The NSCs perform this action without themselves becoming tumorigenic or augmenting the pre-existing tumor, and this can be assured by having NSCs express a suicide gene that can be activated and cause NSCs to die. The tumor homing phenomenon of NSCs was first revealed by researchers on this proposed team and, in fact, the central concepts presented here have since been extended to many other kinds of disease. In this proposal, we will use a number of authentic mouse models of primary BTs to pre-clinically test therapeutic NSCs. Human NSCs (hNSCs) will be derived from 3 distinct sources, with each having been proffered as therapeutic, but never having been compared head-to-head in treating tumors. Each of these hNSCs will be modified using two therapeutic genes: TRAIL, which is a protein that specifically kills tumor cells, but does not harm normal cells and tissues, and cytosine deaminase which converts a non-toxic chemical into a toxic chemotherapeutic. We expect our research to identify the best hNSC + therapeutic gene combination to advance for clinical trial in patients with BTs, following our obtaining regulatory approval for using hNSC therapy at the end of this project. Because immunocompatibility of the hNSCs with recipient patients is not a concern in BT therapy, a limited number of hNSC lines can be used for treating all prospective patients. Furthermore, BT treatment does not require long-term NSC survival and can be combined with commonly used BT therapies. Finally, NSCs can be imaged in patients and therefore monitored after administration. Developing this approach for treatment of BT patients offers an ideal setting and opportunity for achieving dramatic results from stem cell therapy, and the results of this project will likely be applicable to the treatment of other cancers.
Statement of Benefit to California: 
Brain tumors (BTs) are incurable, whether they start in the brain or spread there from other sites. Despite advances in surgical, radiation, drug, & gene therapies, survival with a BT is extremely short, because current therapies are limited by their inability to reach tumor cells that spread widely to normal brain structures. Interestingly, the therapeutic property that is needed to overcome this major treatment obstacle matches well with one of the better accepted attributes of neural stem cells (NSCs): an attraction for sites of disease in the adult brain, including primary & metastatic cancer. If engineered to be armed with a tumor-killing gene, NSCs (whether administered into the brain or into the bloodstream), that are attracted to cancers, could dramatically reduce patient tumor burden, and track after even single migrating tumor cells, in a manner that has never been achieved. The NSCs would perform this action without themselves causing tumors or increasing growth of the patient’s tumor, and this would be assured by engineering the NSCs to self-destruct. The tumor homing phenomenon of NSCs was first revealed by researchers on this proposed team and, in fact, the central concepts presented here have since been extended to many other kinds of disease. In this proposal, we will use a number of authentic mouse models of primary BTs to test therapeutic NSCs before testing them in humans. Human NSCs (hNSCs) will be derived from 3 distinct sources, with each having been proposed as therapeutic, but never having been compared head-to-head in treating cancer. Each of these stem cells will be modified using two different therapeutic genes: TRAIL, a protein that specifically kills tumor cells, but does not harm normal cells and tissues, and cytosine deaminase, which converts a non-toxic chemical into a chemotherapy drug that kills the tumor. We expect our research to identify the best hNSC + therapeutic gene combination to advance for evaluation in clinical trials in patients with intracranial BTs, after we have performed all necessary animal safety testing and submitted a complete plan for review by the US FDA and NIH. Members of this proposed team have experience in bringing cancer therapies to clinical trial, hold the IP surrounding the use of stem cells against cancer, have begun discussions with the FDA and NIH, and have enlisted a GMP facility. Because immune system compatibility between donor and recipient of the hNSCs with the recipient is not a concern in BT therapy, a small number of donors could be used to produce genetically modified hNSCs to treat all prospective patients. Developing this approach for treatment of BTs offers an ideal setting and opportunity for achieving dramatic results from stem cell therapy, and accomplishing substantial improvements in quantity and quality of life for BT patients would no doubt increase California's worldwide visibility in offering the best possible medical care for cancer patients.
Progress Report: 
  • During the first year of this project we have made substantial progress toward achieving the ultimate goal of developing a stem cell (SC) therapy for treating patients with recurrent glioblastoma (GBM). At the outset, we began investigating three SC candidates as the cellular vehicle to carry a therapeutic payload and disperse within the tumor of GBM patients: mesenchymal stem cells (MSCs); fetal neural stem cells (fNSCs); and adult neural stem cells (aNSCs). In addition, we were considering two therapeutic genes as the payload, cytosine deaminase (CD) and tumor necrosis factor related apoptosis-inducing ligand (TRAIL), and two routes of therapeutic SC administration for treating brain tumor patients, intravascular and direct intratumoral. Thus, at the start of the project, there were twelve possibilities (3 stem cell vehicles x 2 therapeutic genes x 2 routes of administration) to investigate and compare prior to determining the best combination to develop for a GBM clinical trial. From this starting point we have been able to rapidly eliminate the aNSCs from consideration due to their slow rate of proliferation that would limit their expansion to sufficient number for use in a clinical product for patients. Next we determined that SC access to intracranial tumor through intravascular injection was negligible, and that it is highly unlikely that SC administration by this route would result in a sufficient number of SCs reaching intracranial tumor for achieving therapeutic benefit in treating patients with recurrent GBM. Thus, our work to date has resulted in the narrowing options for SC + therapeutic gene + route of delivery to four: two cellular vehicle candidates (fNSCs and MSCs) and two therapeutic gene payloads (CD and TRAIL). During the first year of this project, each of the four combinations has been tested and have demonstrated anti-tumor activity. During early year 2 research we will determine the most effective combination based on preclinical testing results using multiple human GBM models. The decision regarding most effective therapeutic gene + stem cell vehicle will be achieved within six months, and from that point, in going forward, project emphasis will focus on the development of a specific product candidate, including manufacturing process and assay development, GLP/GMP product manufacturing, further preclinical animal studies to demonstrate efficacy and safety, and development of a clinical protocol. In association with the research accomplished to date we have developed and applied several approaches that will prove useful for SC research and clinical application in general. Foremost among these is the use of micron-sized particles of iron oxide (MPIOs) for labeling SCs prior to their injection into animal subjects, and then monitoring the movement of labeled SCs using magnetic resonance imaging (MRI). This is a powerful technique with implications for understanding the distribution and persistence of SCs in patients receiving SC therapies. For our project, this method is informing us about the distribution of labeled SCs within and around brain tumors, as well as with regard to how long the SCs remain in animal subjects. In addition to the MRI detection of iron particle labeled SCs, we have developed and refined a technique for determining the amount of human SC DNA in animal subject tissues, which has a sensitivity of detecting one human cell among more than a million host cells. Similar to the MRI detection of labeled SCs, the DNA detection method provides us a very sensitive approach for monitoring SC biodistribution and persistence in animal subjects, and it is broadly applicable to all SC research in which rodent models are used for pre-clinical investigation of SCs for treating disease. We are also developing novel approaches for the use of optical imaging to visualize stem cells labeled with fluorescent reporters, and for monitoring the anti-tumor activity of therapeutic stem cells administered to animal subjects. These novel approaches are contributing to the repertoire of techniques available to expedite the identification and application of therapeutic SCs in clinical settings. This project is a collaboration among outstanding scientists and clinicians at five of California’s leading medical research institutions: the Sanford-Burnham and Salk Institutes in La Jolla, and the San Francisco, Los Angeles, and San Diego campuses of the University of California (including Ludwig Institute at UCSD). By leveraging complementary expertise of these investigators, we have made rapid progress in the preclinical animal studies, design of the clinical trial protocol, and the product development studies that will lead to preparation of a gene-modified SC product for the clinical trial. These activities will culminate in an IND application to FDA that will allow us to test the safety and efficacy of our SC product in patients with this devastating illness.
  • This project was initiated in April of 2010, and was for comparing
  • • three types of stem cells
  • • two distinct therapeutic gene modifications of stem cells, and
  • • intravascular administration vs. direct tumor injection of stem cells
  • in order to identify the most efficacious stem cell + therapeutic gene + route of administration for treating patients with recurrent glioblastoma (GBM), a brain tumor that has a dismal prognosis, and that badly needs innovative approaches for improving treatment outcomes.
  • Major conclusions from this project, as concerns the objectives indicated above, are:
  • 1. Stem cells administered by the vascular route do not reach brain tumors established in rodent subjects, to an extent which demonstrable therapeutic stem cell anti-tumor activity should be anticipated. In most instances, intravascular administration results in no detectable stem cells in intracranial tumor in rodent models. Therefore, therapeutic stem cells need to be administered direct into brain tumors in order to achieve a sufficient number and concentration of stem cells for observing anti-tumor effect.
  • 2. Neural stem cells and mesenchymal stem cells delivered directly into intracranial tumor display similar extents of dispersion in tumor, indicating these stem cell types should perform comparably as concerns their ability to disseminate within, and deliver therapy to tumor.
  • 3. However, unmodified (non-immortalized) neural stem cells, derived from single adult or fetal sources, have insufficient proliferative capacity for production as therapeutic stem cells to be used in clinical trials that enroll multiple patients. Because of the ready availability of mesenchymal stem cells (MSCs), from many donors, combined with the proliferative capacity of MSCs, MSCs were determined as the preferred candidate for developing therapeutic stem cells to treat patients with recurrent GBM.
  • 4. Studies conducted with therapeutic stem cell + tumor cell mixtures indicated superior anti-tumor activity of cytosine deaminase modified stem cells + 5-fluorocytosine (FC), relative to secretable TRAIL modified stem cells, when anti-tumor activity is examined in liquid media (cell culture). The two types of therapeutic stem cells showed comparable anti-tumor activities when administered directly into brain tumor in animal (rodent) subjects.
  • 5. In relation to other types of therapies (e.g., chemotherapeutics, antibodies, liposomal drugs) being tested by members of this disease team, manufactured therapeutic stem cells displayed low (modest) anti-tumor activity in animal subjects with brain tumor.
  • Technical advances, discovery, and products developed in association this project, and that can be shared/transferred in support of other CIRM funded research, include:
  • • Development of approaches for delivering stem cells through distinct routes of administration in rodent subjects.
  • • Development of a method, based on the use of polymerase chain reaction, for detecting human cells in rodent tissues, with a sensitivity of detection of one human cell per 100,000 mouse cells.
  • • Development of a cell labeling approach that enables tracking of stem cell migration in rodent subjects.
  • • Development of a histochemical method for detection of labeled human cells in rodent tissues.
  • • Development and characterization of multiple, tumorigenic human glioblastoma xenograft models for use in therapeutic testing.

Stem Cell-mediated Therapy for High-grade Glioma: Toward Phase I-II Clinical Trials

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01421
ICOC Funds Committed: 
$18 015 429
Disease Focus: 
Brain Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Despite aggressive multimodal therapy and advances in imaging, surgical and radiation techniques, malignant brain tumors (high-grade gliomas) remain incurable, with survival often measured in months. Treatment failure is largely attributable to the diffuse and invasive nature of these brain tumor cells, ineffective delivery of chemotherapeutic agents to tumor sites, and toxic side-effects to the body, which limits the dose of drug that can be given. Therefore, new tumor-selective therapies are critically needed. Neural stem cells (NSCs) offer an unprecedented advantage over conventional treatment approaches because of their unique ability to target tumor cells throughout the brain. This ability allows NSCs to be used to deliver prodrug-activating enzymes to tumors, where these enzymes will generate high concentrations of powerful anti-cancer agents selectively at tumor sites. We will use an established human NSC line to develop a novel NSC-based product to deliver the enzyme carboxylesterase (CE), which will activate a systemically administered prodrug, CPT-11, to a powerful chemotherapeutic agent, SN-38, selectively at tumor sites, destroying invasive glioma cells while sparing normal tissues. Based on our preliminary data, we hypothesize that CE-expressing NSCs will home to tumor sites in the brain, and, in combination with CPT-11, will generate high concentrations of SN-38 specifically at tumor sites. Thus, in addition to potentially improving lifespan by concentrating the powerful chemotherapeutic agent selectively at tumor sites, this NSC-mediated treatment strategy should significantly decrease toxic side-effects to normal tissues, thus preserving or improving the patient’s quality of life. Our research, regulatory and clinical teams have the collective expertise and experience to conduct the preclinical studies necessary to optimize the efficacy of this innovative treatment approach. Specifically, we will determine the optimal dose and route of NSC administration; the optimal prodrug dosing regimen; and assess the safety of this treatment approach. We will perform these studies and analyses, generate clinical grade products, and file and obtain all appropriate regulatory documents and approvals. Completion of these activities will lead to the filing of a new Investigational New Drug (IND) proposal to the FDA, for a first-in-human Phase I clinical trial of this pioneering NSC-mediated treatment in patients with recurrent high-grade gliomas. Importantly, our NSC line can be further modified for tumor-localized delivery of a variety of therapeutic agents, and can be given serially or in combination to maximize therapeutic benefit. Thus, the potential medical impact of this innovative NSC-mediated therapeutic approach may be very far-reaching, as it can be developed for application to other types of malignant brain tumors, as well as for metastatic cancers.
Statement of Benefit to California: 
Despite aggressive multimodality therapy and advances in imaging, surgical and radiation techniques, high-grade gliomas remain incurable, with survival often measured in months. Approximately, 22,500 malignant brain tumors are diagnosed annually in the U.S., of which more than 2,600 cases are in California. New therapies are desperately needed to improve both the survival and quality of life of these brain tumor patients and to reduce the economic impact of billions of dollars in related healthcare costs. We propose to develop a novel neural stem cell (NSC)-based treatment method that will selectively target glioma cells with a potent chemotherapy agent, locally activated by the NSCs at tumor sites to destroy neighboring tumor cells. Our tumor-selective approach also has the advantage of minimizing toxicity to normal tissues, thereby decreasing systemic side effects and damage to normal brain. This new therapeutic strategy, therefore, not only has the potential to improve survival, but, by preserving cognitive function and quality of life, it could also enable adult Californians diagnosed with brain tumors to continue making societal contributions that would benefit all Californians. Important for clinical translation of this novel therapeutic approach, we have established the NSC line to be used in this study as a fully characterized cGMP Master Cell Bank. The NSC line is thus expandable, easily distributed to other medical centers, and cost-effective, which will allow this therapeutic approach to be quickly adopted. Importantly, this NSC line can be further modified for tumor-localized delivery of a variety of therapeutic agents, which may be given serially or in combination to maximize therapeutic benefit. There is tremendous potential for developing NSC-mediated treatment applications for other types of malignant brain tumors, as well as for metastatic solid tumors throughout the body. Therefore, the impact of these proposed studies to advance NSC-mediated treatment of glioma may be very far-reaching and may significantly contribute to reducing healthcare costs. Finally, the combined strengths and experience of our research team will enable us to advance this NSC-meditated therapeutic approach in a timely, streamlined, and cost-effective manner to submit a new IND application for initiating first-in-human clinical trials in California, providing benefit to state taxpayers by efficient use of tax dollars and initial access to this novel therapy. In addition, our CIRM Disease Team NSC-mediated cancer treatment studies would stimulate and advance collaborative partnerships and alliances with other cancer centers and affiliates, pharmaceutical companies, academic institutions, and philanthropic societies within California, which would further enhance local and state economies.
Progress Report: 
  • Primary brain tumors are among the most difficult cancers to treat. High-grade gliomas, the most common primary brain tumors in adults, remain incurable with current therapies. These devastating tumors present significant treatment challenges for several reasons: 1) surgical removal runs the risk of causing permanent neurologic damage and does not eliminate cancer cells that have migrated throughout the brain; 2) most anti-cancer drugs are prevented from entering the brain because of the presence of the blood-brain barrier, which often does not allow enough chemotherapy into the brain to kill the cancer cells; and 3) typically, the amount of chemotherapy that can be given to cancer patients is limited by intolerable or harmful side effects from these agents. If concentrated cancer therapies could be specifically localized to sites of tumor, damage to healthy tissues would be avoided.
  • The long-range goal of this research project is to develop a neural stem cell (NSC)-based treatment strategy that produces a potent, localized anti-tumor effect while minimizing toxic side effects. NSCs hold the promise of improved treatment for brain cancers because they have the natural ability to distribute themselves within a tumor, as well as seek out other sites of tumor in the brain. Because they can home to the tumor cells, NSCs may offer a new way to bring more chemotherapy selectively to brain tumor sites. After modifying the NSCs by transferring a therapeutic gene into them, NSCs can serve as vehicles to deliver anti-cancer treatment directly to the primary tumor, as well as potentially to malignant cells that have spread away from the original tumor site. With funding from CIRM, we are studying the ability of NSCs, that carry an activating protein called carboxylesterase (CE) to convert the chemotherapy agent CPT-11 (irinotecan) to its more potent form, SN-38, at sites of tumor in the brain.
  • During the first year of funding we have determined that 1) when administered directly into the brain or into a peripheral vein (intravenous injection) of mice with brain tumors, NSCs will travel to several different subtypes of gliomas; 2) we can engineer the NSCs to consistently produce high levels of more powerful forms of CE: rCE and hCE1m6; 3) glioma cells die when they are exposed to very low (nanomolar) concentrations of SN-38, and 4) although glioma cells survive when exposed to a relatively high concentration of CPT-11 alone, they do die when the same concentration of CPT-11 is administered in combination with either rCE or hCE1m6. These results suggest that the engineered NSCs are expressing relatively high levels of CE enzymes and that the CE enzymes are converting CPT-11 into SN-38. We have also been able to label our NSCs with iron particles, so that we can track their movement in real-time by magnetic resonance imaging (MRI), and follow their location and distribution in relation to the tumor.
  • All of our data thus far support the original hypothesis that effective, tumor-specific therapy for glioma patients can be developed using NSCs that express rCE or hCE1 and the prodrug CPT-11. During the second year of CIRM funding, we will further analyze our data to make a final determination regarding the best form of CE to develop towards clinical trials, and the best dose range and route of delivery of NSCs to achieve maximal tumor coverage. We will then begin our therapeutic studies and start discussions with the Food and Drug Administration, to define the safety studies necessary to obtain approval for testing this new treatment strategy in patients with brain tumors.
  • High-grade gliomas, the most common primary brain tumors in adults, have a poor prognosis and remain incurable with current therapies. These devastating tumors present significant treatment challenges: 1) surgery may cause permanent neurologic damage; 2) surgery misses cancer cells that have invaded beyond the edge of the tumor or to other sites in the brain; 3) many, if not most, chemotherapy drugs cannot enter the brain because of the blood-brain barrier; and 4) due to the highly toxic nature of chemotherapy agents the therapeutic window (the difference between the dose that kills the tumor and the dose that causes toxic side effects) is very small, resulting in undesirable side-effects. Therefore, if therapeutic agents could be localized and concentrated selectively to the tumor sites, treatment efficacy may be improved while toxic side effects are minimized.
  • The overarching goal of this project is to develop a human Neural Stem Cell (NSC)-based treatment strategy that produces potent localized anti-tumor effects while minimizing toxic side effects. NSCs hold the promise of improved treatment for brain cancers because they have an innate ability to distribute within and around a tumor mass and to seek out tumor cells that have invaded further into surrounding brain tissue. By homing to cancer cells, NSCs offer a way to selectively deliver concentrated chemotherapy to brain tumor sites. We are modifying NSCs to make the protein carboxylesterase (CE), which will convert a systemically administered prodrug, CPT-11 (irinotecan) to an active, potent anti-cancer drug, SN38 at the tumor sites.
  • Our second year of funding was highly productive and informative. We validated key elements of our system, successfully negotiating Go/No Go milestones, yielding substantial progress:
  • (1) We have selected the optimal genetically modified human CE to efficiently convert CPT-11 to SN-38. This CE is being developed for clinical grade use.
  • (2) We have determined the volume of tumor coverage by NSCs injected directly into the brain versus injecting them intravenously. We found that we achieve more tumor coverage with direct injection of the NSCs into the brain, and will focus on developing this approach for initial NSC.CE/CPT-11 clinical trials. However, following intravenous injections we found the NSCs localize prominently at the invasive tumor edges, which may prove therapeutically efficacious as well. Due to the significant clinical and commercial advantages that intravenous administration presents, this approach will also be developed toward patient trials. We have determined the starting NSC dose range for both approaches.
  • (3) We have shown that CPT-11 + CE is1,000 fold more toxic to glioma cells than CPT-11 alone. Importantly, microdialysis studies in our preclinical models have confirmed the conversion of CPT-11 to SN-38 by our CE-secreting NSCs in the brain.
  • (4) We have completed studies labeling our NSCs with iron (Feraheme) nanoparticles, which allows for non-invasive cell tracking by Magnetic Resonance Imaging (MRI). Safety studies for clinical use of this iron-labeling method were completed and submitted to the FDA, for consideration of use in brain tumor patients enrolled in our current NSC.CD/5-FC recurrent glioma clinical trial. This would be the first-in-human use of Feraheme-labeled stem cells for MRI tracking.
  • Our results to date robustly support the original hypothesis that an effective, glioma-specific therapy can be developed using NSCs that home to tumors and express CE to convert CPT-11 to the potent anti-cancer agent SN-38. Pre-clinical therapeutic efficacy studies to optimize CPT-11 regimens are now in progress.
  • High-grade gliomas, the most common primary brain tumors in adults, have a poor prognosis and remain incurable with current therapies. These devastating tumors present significant treatment challenges; 1) surgery may cause permanent neurologic damage; 2) surgery misses cancer cells that have invaded beyond the edge of the tumor or disseminated to other sites in the brain; 3) many, if not most, chemotherapy drugs cannot enter the brain because of the blood-brain barrier; and 4) due to the highly toxic nature of chemotherapy agents the therapeutic window (the difference between the dose that kills the tumor and the dose that causes toxic side effects) is very small. Therefore, if therapeutic agents could be concentrated and localized to the tumor sites, treatment efficacy may be improved while toxic side effects are minimized.
  • The overarching goal of this project is to develop a human Neural Stem Cell (NSC)-based treatment strategy that produces potent localized anti-tumor effects while minimizing toxic side effects. NSCs hold the promise of improved treatment for brain cancers because they have an innate ability to distribute within and around a tumor mass and to seek out other, secondary and smaller tumor nodules in the brain. By homing to cancer cells, NSCs offer a way to selectively deliver concentrated chemotherapy to brain tumor sites. After modifying NSCs by adding the gene to make the protein carboxylesterase (CE), NSCs deliver CE to convert the drug CPT-11 (irinotecan) to its more potent form, SN-38 at primary and secondary brain tumor sites.
  • The major milestone in our third year of funding was that we completed our pre-IND package and held our pre-IND meeting with the FDA. To this end, we validated the following:
  • (1) NSCs can potentiate the in vivo efficacy of irinotecan (CPT-11) using a low dose (7.5 mg/kg) daily x 5 schedule. Both real time Xenogen and integrated morphometric analysis of immunohistochemically stained sections of tumor were used to determine tumor volumes.
  • (2) In vivo pharmacokinetics demonstrated increased accumulation of SN-38 in tumor over that of tumor interstitium. The concentrations of tumor SN-38 were approximately 3-fold higher in tumor-bearing brain tissue than in corresponding normal tissue supporting the hypothesis that NSCs can direct toxic chemotherapy in a tumor localized manner.
  • (3) Following FDA approval of the incorporation of iron (Feraheme) into NSCs, three patients were treated with FeHe-labeled HB1.F3.CD, the first generation NSCs undergoing clinical trial. There were no adverse effects from the treatment demonstrating relative safety and lack of toxicity of this method.
  • Our results to date robustly support the original hypothesis that an effective, glioma-specific therapy can be developed using NSCs that home to tumors and express CE to convert CPT-11 to SN-38. During the fourth and coming year of CIRM funding, we will conduct experiments to determine the optimal schedule for NSC/CPT-11 therapy and demonstrate the safety and lack of toxicity of the treatment schema in rodents to fulfill requirements for IND submission and clinical trial in humans.
  • High-grade gliomas, the most common primary brain tumors in adults, have a poor prognosis and remain incurable with current therapies. These devastating tumors present significant treatment challenges; 1) surgery may cause permanent neurologic damage; 2) surgery misses cancer cells that have invaded beyond the tumor edge to other sites in the brain; 3) many, if not most, chemotherapy drugs cannot enter the brain because of the blood-brain barrier; and 4) chemotherapy drugs are toxic to normal tissues as well as tumor, causing undesirable side effects. Therefore, if therapeutic agents could be concentrated and localized to the tumor sites, treatment efficacy may improve while side effects are minimized.
  • Our goal is to bring to the clinic a human Neural Stem Cell (NSC)-based treatment strategy that produces potent localized anti-tumor effects while minimizing toxic side effects. NSCs have a natural ability to home to invasive brain tumor cells throughout the brain. NSCs, used as a delivery vehicle, offer a novel way to selectively target chemotherapy to brain tumor sites. NSCs are modified to express a certain enzyme (carboxylesterase; CE), that converts systemically administered prodrug (irinotecan) to a much more potent form (SN-38), that is up to 1000 times more effective at killing brain tumor cells.
  • Milestones reached in our fourth year include:
  • (1) receiving regulatory approval from the NIH/OBA following a public form in September, 2013.
  • (2) determining the dose and timing of NSC and irinotecan administration for optimal therapeutic efficacy in pre-clinical brain tumor models.
  • (3) demonstrating that the CE-expressing NSCs can increase concentrations of the toxic drug SN-38 by > 6-fold compared to giving irinotecan alone. Furthermore, SN-38 concentrations were dose proportional to administered irinotecan concentrations.
  • (4) Safety-toxicity studies required by the FDA for Investigational New Drug (IND) approval were completed. These studies demonstrated no significant toxicities and safety of our NSC treatment protocol in preclinical brain tumor models.
  • Our results to date support our hypothesis that a safe and effective NSC-mediated therapy can be developed for clinical use in patients with high-grade glioma, with potential application to other types of brain tumor and brain tumor metastases. We hope to initiate clinical trials with our CE-expressing NSCs and irinotecan by the end of 2014.

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