Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-۳ (TIM-۳)

Objective(s): As T-cell immunoglobulin and mucin domain ۳ (TIM-۳) 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-۳ nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. Materials and Methods:We immunized a camel with TIM-۳ antigen and then, synthesized a VHH phagemid library from its B cell’s transcriptome using nested PCR. Library selection against TIM-۳antigen 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-۳ specific nanobody with TIM-۳ expressing HL-۶۰ and HEK cell lines. Results:Specific ۱۵kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-۳ with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-۳ expressing HL-۶۰ cell line. Conclusion: Finally, we successfully prepared a functional anti-humanTIM-۳ 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 ۳