Development and Characterization of Nanobodies Targeting the Kupffer Cell

Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of ^99mTc labeled Clec4F nanobodies in naïve mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.