Radovan Komel
University of Ljubljana, Slovenia
Title: Potential human glioblastoma cancer markers identified with nanobody-based reverse proteomic approach
Biography
Biography: Radovan Komel
Abstract
Glioblastoma multiforme (GBM) is a highly lethal form of cancer where the standard therapies of surgery followed by radiation and chemotherapy cannot significantly prolong the life expectancy of the patients. Tumor recurrence shows even more aggressive form compared to the primary tumors and cancer stem cells, resistant to conventional therapy, seem responsible for early relapse. There is a lack of GBM and GBM stem cell biomarkers specific enough to provide early diagnosis of the disease and efficient targeting therapy. The discovery of heavy-chain only antibodies (HCAbs) in camelids appears to have opened a new opportunity of searching for cancer markers and developing targeted treatment approaches. HCAb-derived nanobodies (Nbs) are small and stable single-domain antigen-binding fragments with a high degree of sequence identity to the human heavy chain variable domain which offer them advantages over classical antibodies. We immunized an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. A nanobody library was constructed in a phage display vector and using phage display technology, we selected specific GBM stem-like cell binders through a number of affinity selections. The selected nanobody clones were recloned, expressed in E. coli and purified by IMAC and size-exclusion chromatography. Specific nanobody-antigen pairs were obtained and MS analysis revealed ten proteins that were up-regulated in the GBM stem-like cells compared to the controls. Following application of Nb234 (anti-TRIM28) and Nb206 (anti-TufM) on different cell lines, variable distribution of TufM during the cell cycle and much higher toxic effect of both Nbs was observed on GBM compared to the control.