Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata, 992-8510, Japan.
Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 183-8538, Japan.
Biochem Biophys Res Commun. 2020 Feb 26;523(1):72-77. doi: 10.1016/j.bbrc.2019.12.018. Epub 2019 Dec 9.
A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.
双特异性抗体（bsAb）是一类新兴的下一代生物治疗药物。BsAbs 是一种工程抗体，在一个分子中具有两个抗原结合表位。尽管已经提出了大量的双特异性构建体，但这种环状骨架拓扑结构从未得到过证明。环状拓扑结构对于固定两个表位的方向和防止外肽酶消化具有潜在的益处。我们使用肽和蛋白质的分裂内含子环化连接构建了一种环状连接的双特异性 VHH，称为环体。构建的环体具有抗蛋白水解的保留双特异性。抗 EGFR×抗 GFP 环体可特异性染色 EGFR 阳性细胞与 GFP。抗 EGFR×抗 CD16 环体对 EGFR 阳性癌细胞表现出细胞毒性活性，与串联 VHH 构建体的活性相当。双特异性抗体新拓扑结构的成功证明将扩展基于抗体的药物和试剂的开发构建策略。
