@article { author = {Homayouni, Vida and Ganjalikhani-hakemi, Mazdak and Rezaei, Abbas and Khanahmad, Hossein and Behdani, Mahdi and Kazemi Lomedasht, Fatemeh}, title = {Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3)}, journal = {Iranian Journal of Basic Medical Sciences}, volume = {19}, number = {11}, pages = {1201-1208}, year = {2016}, publisher = {Mashhad University of Medical Sciences}, issn = {2008-3866}, eissn = {2008-3874}, doi = {10.22038/ijbms.2016.7820}, 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.}, keywords = {antibody,Heavy chain antibody,Nanobody,Phage display,T-cell immunoglobulin and mucin domain 3}, url = {https://ijbms.mums.ac.ir/article_7820.html}, eprint = {https://ijbms.mums.ac.ir/article_7820_62b85dc2cb571a9f88fd37dc253dffa0.pdf} }