Zhao Shuai, Zeng Wanting, Yu Fang, Xu Pingping, Chen Chin-Yu, Chen Wanping, Dong Yanming, Wang Fei, Ma Lixin
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China.
Biomolecules. 2025 Jan 12;15(1):111. doi: 10.3390/biom15010111.
Nanobodies have gained attention as potential therapeutic and diagnostic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their ability to bind and neutralize the virus. However, rapid, scalable, and robust production of nanobodies for SARS-CoV-2 remains a crucial challenge. In this study, we developed a visual and high-efficiency biomanufacturing method for nanobodies with by fusing the super-folder green fluorescent protein (sfGFP) to the N-terminus or C-terminus of the nanobody. Several receptor-binding domain (RBD)-specific nanobodies of the SARS-CoV-2 spike protein (S) were secreted onto the surface of cells and even into the culture medium, including Fu2, ANTE, mNb6, MR3-MR3, and n3113.1. The nanobodies secreted by retained equal activity as prior research, regardless of whether sfGFP was removed. Since some of the nanobodies bound to different regions of the RBD, we combined two nanobodies to improve the affinity. Fu2-sfGFP-ANTE was constructed to be bispecific for the RBD, and the bispecific nanobody exhibited significantly higher affinity than Fu2 (35.0-fold), ANTE (7.3-fold), and the combination of the two nanobodies (3.3-fold). Notably, Fu2-sfGFP-ANTE can be normally secreted into the culture medium and outer membrane. The novel nanobody production system enhances the efficiency of nanobody expression and streamlines the downstream purification process, enabling large-scale, cost-effective nanobody production. In addition, cells secreting the nanobodies on their surface facilitates screening and characterization of antigen-binding clones.
纳米抗体因其结合和中和严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的能力而成为潜在的治疗和诊断试剂，受到了广泛关注。然而，快速、可扩展且稳健地生产用于SARS-CoV-2的纳米抗体仍然是一个关键挑战。在本研究中，我们通过将超折叠绿色荧光蛋白（sfGFP）融合到纳米抗体的N端或C端，开发了一种可视化且高效的纳米抗体生物制造方法。几种SARS-CoV-2刺突蛋白（S）的受体结合域（RBD）特异性纳米抗体被分泌到细胞表面甚至培养基中，包括Fu2、ANTE、mNb6、MR3-MR3和n3113.1。无论sfGFP是否被去除，由细胞分泌的纳米抗体都保留了与先前研究相同的活性。由于一些纳米抗体与RBD的不同区域结合，我们将两种纳米抗体组合以提高亲和力。构建了对RBD具有双特异性的Fu2-sfGFP-ANTE，该双特异性纳米抗体表现出比Fu2（35.0倍）、ANTE（7.3倍）以及两种纳米抗体组合（3.3倍）显著更高的亲和力。值得注意的是，Fu2-sfGFP-ANTE可以正常分泌到培养基和外膜中。这种新型纳米抗体生产系统提高了纳米抗体的表达效率，简化了下游纯化过程，实现了大规模、具有成本效益的纳米抗体生产。此外，在其表面分泌纳米抗体的细胞便于对抗原结合克隆进行筛选和表征。
