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Preparation and Characterization of a Novel Nanobody Against T-Cell Immunoglobulin and Mucin-3 (Tim-3) Publisher



Homayouni V1 ; Ganjalikhanihakemi M1 ; Rezaei A1 ; Khanahmad H2 ; Behdani M3 ; Lomedasht FK3
Authors
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Authors Affiliations
  1. 1. Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  2. 2. Genetic Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Biotechnology Research Center, Biotechnology Department, Venom & Bio-therapeutics Molecules Lab Pasteur Institute of Iran, Tehran, Iran

Source: Iranian Journal of Basic Medical Sciences Published:2016


Abstract

Objective(s): As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. Materials and Methods: We immunized a camel with TIM-3 antigen and then, synthesized a VHH phagemid library from its B cell’s transcriptome using nested PCR. Library selection against TIM-3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA) column, SDS-PAGE and Western blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines. Results: Specific 15kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3 with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-3 expressing HL-60 cell line. Conclusion: Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody with a high affinity and an anti-proliferative activity on an AML cell line in vitro. © 2016, Mashhad University of Medical Sciences. All rights reserved.
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