University of SaskatchewanHARVEST
  • Login
  • Submit Your Work
  • About
    • About HARVEST
    • Guidelines
    • Browse
      • All of HARVEST
      • Communities & Collections
      • By Issue Date
      • Authors
      • Titles
      • Subjects
      • This Collection
      • By Issue Date
      • Authors
      • Titles
      • Subjects
    • My Account
      • Login
      JavaScript is disabled for your browser. Some features of this site may not work without it.
      View Item 
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item

      Selection and Characterization of Synthetic Antibodies Against Human Rad51

      Thumbnail
      View/Open
      FU-THESIS-2017.pdf (4.712Mb)
      Date
      2017-02-23
      Author
      Fu, Yongpeng 1990-
      Type
      Thesis
      Degree Level
      Masters
      Metadata
      Show full item record
      Abstract
      Chemotherapy is the predominant approach for treating cancer. However, disease relapse frequently occurs because chemotherapy often fails to eliminate all tumor cells due to intrinsic or acquired drug resistance. The failure of conventional chemotherapeutics regimes for cancer highlights the need for novel therapeutic interventions. Recent studies have shown that human Rad51, an evolutionarily conserved DNA recombinase required for homologous recombination, exhibits elevated expression in many cancer cells and is implicated in drug resistance after chemotherapy. Targeted inhibition of human Rad51 has been explored as a way to sensitize cancer cells to chemotherapy. Given the properties of antibodies and their fragments as high-affinity inhibitors, we used antibody phage display to generate antigen-binding fragments (Fabs) against human Rad51. We first isolated human Rad51 specific Fabs by screening a synthetic Fab phage display library against recombinant human Rad51. We isolated a human Rad51 Fab, referred to as Fab F2, which bound human Rad51 with a KD of 8.1 nM. Fab F2 inhibited the DNA binding activity of human Rad51 but did not inhibit human Rad51 ATP hydrolysis activity. We converted Fab F2 into an scFv-Fc fragment (scFv: single-chain variable fragment; Fc: glycosylated crystallizable fragment) for expression in human embryonic kidney 293T cells. Overexpression of scFv-Fc fragment in human embryonic kidney 293T cells increased 4.48-fold more sensitivity to the DNA-damaging agent methyl methanesulfonate in clonogenic survival assays. To enable the delivery of Fab F2 into cells we fused it to a cell membrane import tag (FabItag I2) based on a patent (WO 2014005219 A1) from iProgen Biotech Inc. We labeled FabItag I2 with an 800CW fluorophore and showed that FabItag I2 permeated human embryonic kidney 293T cells using fluorescence microscopy and flow cytometry. FabItag I2 increased the sensitivity of human embryonic kidney 293T cells to methyl methanesulfonate by 2 folds in clonogenic survival assays.
      Degree
      Master of Science (M.Sc.)
      Department
      Biochemistry
      Program
      Biochemistry
      Supervisor
      Geyer, Clarence R
      Committee
      Xiao, Wei; Napper, Scott; Stone, Scot
      Copyright Date
      February 2017
      URI
      http://hdl.handle.net/10388/7766
      Subject
      Phage display
      Cancer
      Rad51
      Fab
      Intracellular antibody
      Collections
      • Graduate Theses and Dissertations
      University of Saskatchewan

      University Library

      © University of Saskatchewan
      Contact Us | Disclaimer | Privacy