Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3)

Homayouni V., Ganjalikhani-Hakemi M., Rezaei A., Khanahmad H., Behdani M., Lomedasht F. K.

Iranian Journal of Basic Medical Sciences, vol.19, no.11, pp.1201-1208, 2016 (SCI-Expanded) identifier

  • Publication Type: Article / Article
  • Volume: 19 Issue: 11
  • Publication Date: 2016
  • Doi Number: 10.22038/ijbms.2016.7820
  • Journal Name: Iranian Journal of Basic Medical Sciences
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.1201-1208
  • Keywords: Antibody, Heavy chain antibody, Nanobody, Phage display, T-cell immunoglobulin and -mucin domain 3
  • Istanbul Medipol University Affiliated: No


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.