J. S. Kuo1, M. Zorniak1, E. V. Shusta2, J. S. Kuo1 1University Of Wisconsin,Neurological Surgery,Madison, WI, USA 2University Of Wisconsin,Chemical And Biological Engineering,Madison, WI, USA
Introduction: Glioblastoma multiforme (GBM) is a poorly treated human brain cancer with few tools available for detection and isolation of their therapeutic-resistant, glioblastoma cancer stem-like cell (GSC) populations. A non-immune, human single-chain antibody (scFv) yeast display library was mined for human GSC-specific antibodies via biopanning.
Methods: Standard molecular biology techniques were employed, along with novel yeast biopanning strategy involving positive and negative selections (Wang et al, Nature Methods. 2007 Feb;4(2):143-5) followed by screening identified clones against patient-derived GSC and control normal neural stem cell lines. Briefly, GSC-binding scFv candidates were isolated after combining nine rounds of enrichment via positive screening with several rounds of negative screening against normal human astrocytes, neural stem cells, and serum-cultured GBM tumor.
Results: Clonal scFv assessment by restriction enzyme fingerprinting revealed 62 unique scFv clones. Each yeast-displayed scFv clone was characterized for qualitative binding selectivity against 12 distinct human lines of normal, GSC, and patient-matched GBM cells. Clone scFv-9.7, in particular, demonstrated substantial binding specificity for five GSC lines representing three different classes of tumor invasiveness and various neural progenitor lineages. GSC-specificity was further verified using secreted and purified scFv-9.7, which detected highly infiltrative GSCs from tumor xenografts via flow cytometry, and successfully targeted and fluorescently visualized tumor xenografts in vivo when conjugated with a near-infrared dye.
Conclusion: In summary, rapid screening via yeast antibody library biopanning identified human GSC-specific antibodies for potential development into immunotargeted diagnostics and therapeutics in brain cancer.
