Background: T cell immunoglobulin and mucin domain 3 (TIM3) is one of the major inhibitory immune checkpoints expressed on T cells. Blocking the intraction between TIM3 and its inhibitory ligand galectin‑9 may potentiate the effects of immunotherapy or overcome the adaptive resistance . 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.
Methods: We immunized a camel with TIM-3 antigen and then, synthesized a VHH phage: prepared library from its B cell’s transcriptome using nested PCR. Library. selection against TIM-3 antigen 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 linesAn explanation of the study design and experimental method.
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: 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.
