title: Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3).
abstract: OBJECTIVE
  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.
  
  
  METHODS
  We immunized a camel with TIM-3 antigen and then, synthesized a VHH phage: mid 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Vida
authors.initials: V
authors.lastname: Homayouni
authors.affiliation: Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Mazdak
authors.initials: M
authors.lastname: Ganjalikhani-Hakemi
authors.affiliation: Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Abbas
authors.initials: A
authors.lastname: Rezaei
authors.affiliation: Genetic Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Khanahmad
authors.affiliation: Biotechnology Research Center, Biotechnology Department, Venom & Bio-therapeutics Molecules Lab, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Behdani
authors.affiliation: Biotechnology Research Center, Biotechnology Department, Venom & Bio-therapeutics Molecules Lab, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Fatemeh Kazemi
authors.initials: FK
authors.lastname: Lomedasht
keywords: Antibody; Heavy chain antibody; Nanobody; Phage display; T-cell immunoglobulin and -mucin domain 3