A Novel Fully Human Agonistic Single Chain Fragment Variable Antibody Targeting Death Receptor 5 with Potent Antitumor Activity In Vitro and In Vivo

Int J Mol Sci. 2017 Sep 27;18(10):2064. doi: 10.3390/ijms18102064.


Agonistic antibodies, which bind specifically to death receptor 5 (DR5), can trigger apoptosis in tumor cells through the extrinsic pathway. In this present study, we describe the use of a phage display to isolate a novel fully human agonistic single chain fragment variable (scFv) antibody, which targets DR5. After five rounds of panning a large (1.2 × 10⁸ clones) phage display library on DR5, a total of over 4000 scFv clones were screened by the phage ELISA. After screening for agonism in a cell-viability assay in vitro, a novel DR5-specific scFv antibody TR2-3 was isolated, which inhibited COLO205 and MDA-MB-231 tumor cell growth without any cross-linking agents. The activity of TR2-3 in inducing apoptosis in cancer cells was evaluated by using an Annexin V-PE apoptosis detection kit in combination with flow cytometry and the Hoechst 33342 and propidium iodide double staining analysis. In addition, the activation of caspase-dependent apoptosis was evaluated by Western blot assays. The results indicated that TR2-3 induced robust apoptosis of the COLO205 and MDA-MB-231 cells in a dose-dependent and time-dependent manner, while it remarkably upregulated the cleavage of caspase-3 and caspase-8. Furthermore, TR2-3 suppressed the tumor growth significantly in the xenograft model. Taken together, these data suggest that TR2-3 exhibited potent antitumor activity both in vitro and in vivo. This work provides a novel human antibody, which might be a promising candidate for cancer therapy by targeting DR5.

Keywords: apoptosis; death receptor 5 (DR5); human antibody; phage display; single chain fragment variable (scFv).

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Antineoplastic Agents, Immunological / pharmacology*
  • Apoptosis / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Complementarity Determining Regions / chemistry
  • Complementarity Determining Regions / genetics
  • Disease Models, Animal
  • Female
  • Humans
  • Mice
  • Neoplasms / drug therapy
  • Neoplasms / metabolism*
  • Neoplasms / pathology*
  • Peptide Library
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Receptors, TNF-Related Apoptosis-Inducing Ligand / agonists*
  • Receptors, TNF-Related Apoptosis-Inducing Ligand / chemistry
  • Receptors, TNF-Related Apoptosis-Inducing Ligand / metabolism
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology
  • Single-Chain Antibodies / pharmacology*
  • Xenograft Model Antitumor Assays


  • Antineoplastic Agents, Immunological
  • Complementarity Determining Regions
  • Peptide Library
  • Receptors, TNF-Related Apoptosis-Inducing Ligand
  • Single-Chain Antibodies
  • TNFRSF10B protein, human