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

Abstract

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.

• Authors: Gaoxin Lei, Menglong Xu, Zhipan Xu, Lili Gu, Chenchen Lu, Zhengli Bai, Yue Wang, Yongbo Zhang, Huajing Hu, Yiwei Jiang, Wenfeng Zhao, Shuhua Tan
• Journal: International journal of molecular sciences (Int J Mol Sci) Vol. 18 Issue 10 (Sep 27 2017) ISSN: 1422-0067 [Electronic] Switzerland
• PMID: 28953230 (Publication Type: Journal Article)

Chemical References

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

Topics

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