TY - JOUR ID - 7820 TI - Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3) JO - Iranian Journal of Basic Medical Sciences JA - IJBMS LA - en SN - 2008-3866 AU - Homayouni, Vida AU - Ganjalikhani-hakemi, Mazdak AU - Rezaei, Abbas AU - Khanahmad, Hossein AU - Behdani, Mahdi AU - Kazemi Lomedasht, Fatemeh AD - Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran AD - Genetic Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran AD - Biotechnology Research Center, Biotechnology Department, Venom & Bio-therapeutics Molecules Lab, Pasteur Institute of Iran, Tehran, Iran Y1 - 2016 PY - 2016 VL - 19 IS - 11 SP - 1201 EP - 1208 KW - antibody KW - Heavy chain antibody KW - Nanobody KW - Phage display KW - T-cell immunoglobulin and mucin domain 3 DO - 10.22038/ijbms.2016.7820 N2 - 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. UR - https://ijbms.mums.ac.ir/article_7820.html L1 - https://ijbms.mums.ac.ir/article_7820_62b85dc2cb571a9f88fd37dc253dffa0.pdf ER -