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.