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

Homayouni, Vida and Ganjalikhani-hakemi, Mazdak and Rezaei, Abbas and Khanahmad, Hossein and Behdani, Mahdi and Kazemi Lomedasht, Fatemeh (2016) Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3). Iranian Journal of Basic Medical Sciences, 19 (11). pp. 1201-1208.

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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.

Item Type: Article
Subjects: QW Microbiology and Immunology
Divisions: Journals > Iranian J Basic Medical Sciences
Depositing User: ijbms ijbms
Date Deposited: 26 Sep 2017 18:03
Last Modified: 26 Sep 2017 18:03
URI: http://eprints.mums.ac.ir/id/eprint/4892

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