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

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. [ABSTRACT FROM AUTHOR]