Early Translational II
$5 055 248
Glioblastoma multiforme is the most prevalent and aggressive type of brain tumor, and devastating to any patient unfortunate enough to receive its diagnosis. As one of the most populous states in the nation, more Californians are diagnosed with glioblastoma multiforme than nearly every other state. Over the past 20 years surgery, radiation therapy and chemotherapy have been utilized with frustrating results. Surgery is very risky and radiation and chemotherapy kill both cancer and healthy cells. Today, even with the most advanced treatments available, survival rates have improved by an average of only 9 months – from 3 months without treatment to 12 months with treatment. Our proposed research focuses on a new theory that brain tumor cells are initiated and maintained by a small fraction of cells with stem cell properties. This “cancer stem cell” hypothesis states that if this small subset of cancer stem cells could be inactivated, the tumor would cease to grow. Therefore, we reasoned that cancer-specific genetic alterations in a glioblastoma tumor could be a potential marker for cancer stem cells and zeroing in on these cells could result in targeted therapeutics. What we have already done is to develop an antibody that recognizes two known potential cancer stem cell markers, CD133 and a tumor-specific variant of the epidermal growth factor receptor, or EGFRvIII. CD133 is a marker for normal neural and hematopoietic stem cells and EGFRvlll is a receptor that is genetically altered in glioblastoma tumors. Although CD133 is found in normal cells and the EGFRvIII is found in cancer cells (and rarely present in normal tissues), they are both tightly associated in glioblastoma tumors. We have now shown that tumors that express both CD133 and EGFRvIII grow much more quickly. We have already developed a “bispecific” antibody that recognizes both of these markers and we have shown that this antibody selectively kills the cancer cells in glioblastoma tumors that express both CD133 and EGFRvIII, but not normal stem cells. When we injected glioblastoma cells pre-treated with BsAb into mice, tumor formation was severely inhibited. These results are very promising. To move this research forward we plan to identify high yield methods for BsAb production for use in animal testing. We will establish the most effective dose and route of delivery that reproducibly results in tumor regression. Once determined, we will evaluate any side-effects of the treatment. One goal of this treatment is to increase treatment specificity, so that the tumor cells are eradicated but not at the expense of healthy cells. Our goal is to ready this antibody for the investigational new drug phase and to ultimately generate a human therapeutic effective against glioblastoma multiforme.
Statement of Benefit to California:
Glioblastoma is a devastating diagnosis. The most common and malignant form of brain cancer, the most aggressive treatments available yield an average survival of only 12-14 months. As the most populous state in the nation, more Californians are diagnosed with glioblastoma each year than any other state, with a consequent significant economic toll to the state as well as its emotional toll. As the leader in cutting edge biomedical research, California through CIRM has recognized the unmet need to provide a roadmap for the translation of stem cell research to clinical applications. Through CIRM there is an unparalleled opportunity to foster clearly-defined discovery that will not only benefit Californians with glioblastomas, but potentially those with many other cancers, and ultimately all Californians, through healthier citizens, increased employment opportunities, and reduced economic burdens. We have previously shown that two markers of cancer stem cells, CD133 and EGFRvIII, are tightly associated in glioblastoma tumors. We created a recombinant bispecific antibody (BsAb) selectively targeting CD133 and EGFRvIII. This antibody selectively kills glioblastoma tumor cells but not healthy cells. When glioblastoma cells pre-treated with BsAb were injected into mice, tumor formation was significantly reduced, strongly suggesting that targeting of the EGFRvIII/CD133 cancer stem cell population can inhibit glioblastoma formation. The key objective of our project is to identify efficient and high yield methods for BsAb production, identify an effective dose and route of delivery for the treatment of brain tumors, and evaluate any potential effects on cells/tissues that express CD133. Our goal is to ready the BsAb for investigational new drug-related development. Californians will benefit from this research project in several significant ways. 1) Most importantly, this research has the promise to dramatically extend the long-term survival rates for Californians with glioblastomas, with potential applications to multiple other human cancers. 2) The research will take place in California with direct benefit to the California economy through the hiring of employees and purchase of supplies and reagents. 3) With successful completion of the proposed project, an investigational new drug application will be the direct next step, requiring employing a local contract research organization to generate clinical grade antibody, requiring additional employees along with associated expenditures. 4) If the therapeutic BsAb generated is commercialized, profits derived from the production of the BsAbs by CIRM policy will result in improved treatments to insured patients and lower cost treatments to the uninsured, thus ultimately benefiting all Californians. 5) Finally, funding this research will help raise awareness of California’s prominence as a national and international leader in stem cell research with the potential to benefit all glioblastoma patients.
This Development Candidate (DC) Award application focuses on the development of a bispecific antibody (BsAb) that targets Glioblastoma Multiforme (GBM) cancer stem cells (CSCs), as a therapeutic candidate for treatment of GBM. The applicant hypothesizes that targeting of CSCs is essential to controlling tumor growth but notes that safe targeting of CSCs is problematic as CD133, one of the essential markers of CSCs, is expressed by many normal stem cells. In order to overcome this obstacle, the applicant has generated a BsAb targeting both CD133 and a tumor variant of Epidermal Growth Factor Receptor (EGFR), designated EGFRvIII, which is frequently expressed in GBM tumors. The applicant provides preliminary data that these two markers are co-expressed in GBM tumor cells and that the BsAb targets these CSCs for lysis. The application consists of 4 aims. Experiments proposed in Aim 1 are designed to identify high yield methods for BsAb production and purification. In Aim 2, the effects of the BsAb on EGFRvIII in a cell line expressing EGFRvIII will be investigated. In Aim 3, the applicant plans to identify the appropriate BsAb dose and route of delivery for treatment of GBM tumors using a mouse model of human GBM. Finally, in Aim 4, the applicant will examine the potential side effects of the BsAb on normal human stem cells, which commonly express CD133, and determine organ toxicity and distribution of the BsAb in the mouse. This application is logical, built on interesting data, and the objectives and rationale for this project are well described. Reviewers noted that the stem cell connection in this project is not overly strong. CD133 targeting is justified only if the BsAb has a higher affinity for EGFRvIII expressing GBM cells than an EGFRvIII monospecific antibody and, should this not be the case, stem cells are not necessary for this project. However, reviewers recognized that the applicant provides preliminary data suggesting that the BsAb would have an increased affinity and includes experiments in the research plan to definitively determine whether the BsAb would have greater affinity than a monospecific antibody. Reviewers universally agreed that GBM represents a tremendous unmet medical need. However, reviewers were not convinced that the proposed therapeutic would impact treatment of GBM and were therefore uncertain of the potential impact of this proposal. The application includes relevant preliminary data and a strength of the proposal is that the antibody has already been generated. One reviewer noted that the preclinical model was an excellent choice. Aims 1, 2, and 4 were generally considered strong but reviewers provided several comments. In Aim 1, reviewers thought that biodistribution to the brain should be demonstrated before optimizing production and purification methods. Reviewers questioned the use of the proposed cell line in Aim 2 and were not convinced by the arguments provided that the proposed cell line should be used instead of the more therapeutically relevant primary neurospheres. The primary concern in Aim 4, also noted by the applicant in the proposal, was the limitations of analyzing organ toxicity and distribution in the mouse system since the human antibody does not recognize mouse CD133. Reviewers expressed more substantial criticisms related to Aim 3. The applicant provides preliminary data demonstrating efficacy, however, tumors are pretreated with the BsAb before implanting. This is not very clinically relevant and ignores the blood-brain barrier (BBB). Reviewers were disappointed that the applicant proposes to do pharmacokinetic studies only in blood and not in brain. Reviewers considered this lack of attention to the BBB a major weakness of this proposal. The applicant recognizes that it is necessary for the BsAb to cross the blood-brain barrier (BBB) but does not consider this a major concern as the BBB is disrupted in GBM and has not posed a problem for other antibody treatments, such as treatment with the anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody, Avastin. However, reviewers noted that the argument that the BBB is leaky in GBM is not convincing as penetration of biologics across the BBB is unpredictable even with small molecules and work done with antibody fragments as imaging agents have shown variable penetration. Further, Avastin is not a good comparison as it targets the vascular endothelium and does not have to cross the BBB. Reviewers commented that bulk disease is amenable to surgical resection and/or radiotherapy and more efficacious therapies for GBM will need to target the leading edge of the disease which infiltrates the normal brain where the BBB is intact. Reviewers commented that it is not feasible to bring forward a candidate for brain tumors without knowing if it will cross the BBB, and agreed that it is essential for a DC application targeting GBM to comprehensively address this important point. Reviewers considered the Principal Investigator (PI) to be excellent and appropriately committed to the project. The PI has assembled a strong team of investigators and is collaborating with reputable companies. The collaborators collectively have the appropriate expertise to perform the proposed studies, although the exact role of all collaborators is not clearly defined. The environment is excellent. Reviewers noted that the budget is quite large and there are a large number of FTEs. Overall, reviewers agreed that although the proposed therapeutic candidate targets a disease with tremendous unmet medical need, the research plan does not address key elements required for this therapeutic to impact treatment of GBM and did not recommend this application for funding.