The Key Laboratory of Environment and Genes Related to Disease of Ministry of Education, Health Science Center, Xi'an Jiaotong University, Xi'an, China.
Research Group of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.
Front Immunol. 2021 Feb 22;12:641819. doi: 10.3389/fimmu.2021.641819. eCollection 2021.
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 competitive epitope binning. By analysis of the radioactivity in each organ after injection of Tc 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.
从骆驼科单链抗体衍生而来的纳米抗体已成为诊断、治疗和研究膜受体结构和功能的有力工具。在这项研究中，我们通过对用重组小鼠库普弗细胞受体 Clec4F 免疫的羊驼的淋巴细胞进行噬菌体展示筛选，开发了一系列纳米抗体，该受体通过与半乳糖和 N-乙酰半乳糖胺结合参与病原体识别。生物淘选从 Clec4F 中回收了 14 种不同的纳米抗体，其亲和力范围为 0.2 到 2 nM，如 SPR 所测定。这些纳米抗体主要识别 4 种不同的表位，如竞争性表位 binning 分析所示。通过分析在无经验小鼠中注射 Tc 标记的 Clec4F 纳米抗体后每个器官的放射性，我们发现这些纳米抗体靶向肝脏。此外，我们对来自不同表位组的两个 Clec4F 纳米抗体进行了原子分辨率的结构表征，这揭示了 CDR2 和 CDR3 区域内的独特特征。总之，我们开发了一系列针对库普弗细胞特异性受体 Clec4F 的多个不同识别表位的纳米抗体，这可能对其结构和功能研究以及作为分子成像和治疗剂有用。
