Cancer stem cells are hypothesized to be the primary basis for tumor initiation, maintenance and relapse in high grade brain tumors. CD133+ cells have been identified as a major tumor initiating population in gliomas. Unfortunately, therapies directed at CD133 alone also target normal neural and hematopoietic stem cells. To specifically target brain tumor stem cells (BTSCs) we sought to identify marker(s) that are specific for tumor initiating cells rather than those which are shared by normal stem cells. An EGF receptor mutant, EGFRvIII, is frequently present in many solid tumors, such as glioblastoma multiforme (GBM), but not in normal tissue making it ideal for targeted therapies. In this study, we investigated the possibility that EGFRvIII is expressed in BTSCs. Freshly resected human brain tumors were analyzed for CD133 and EGFRvIII co-expression by flow cytometry. EGFRvIII was expressed in a significant fraction (70±8%, n=15) of the CD133+ population, an ~10 fold higher incidence of co-expression than if expression was random. Limiting dilution assays showed that the frequency of self-renewing cells was 1/25 for EGFRvIII+CD133+ cells, 1/40 for EGFRvIII+CD133- cells and 1/60 for EGFRvIII-CD133+ cells. Furthermore, EGFRvIII expression was maintained in tumor spheres derived from 12/17 GBM, 1/1 gliosarcoma, 2/3 medulloblastoma, 2/4 ependymoma and 1/1 AT/RT. However, EGFRvIII expression was lost when tumor spheres were exposed to differentiation conditions, suggesting a preferential and constrained expression of EGFRvIII in tumor progenitors. We are currently comparing the tumorigenic potential of EGFRvIII+CD133+ cells with that of the CD133+ population. Since the cancer stem cell population of a tumor is hypothesized to be responsible for tumor initiation and maintenance, we tested the hypothesis that specific removal of only the cancer stem cell population will prevent tumor formation. To specifically target EGFRvIII+CD133+ cells, we engineered a bispecific recombinant antibody that recognizes both CD133 and EGFRvIII. Single chain recombinant antibodies (scFv) against CD133 and EGFRvIII were generated and cloned into a bicistronic expression vector, which allowed us to produce a bispecific-tribody in mammalian cells. ELISA and live cell pull down assays show that the BsAb preferentially and competitively bound to only those cells which expressed both antigens. We also showed that the Fc portion of this tribody is sufficient to induce antibody dependent cytotoxicity by human NK cells. Importantly, this BsAb did not bind to or target normal neural stem cells. Sphere forming assays following ADCC showed that EGFRvIII+CD133+ cells are required for tumor sphere formation in EGFRvIII+ GBM tumors giving strong credence to the hypothesis that EGFRvIII+CD133+ may be the most tumorigenic population in GBM tumors which express this oncogenic receptor.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4801.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009