点击次数：

论文类型：期刊论文

发表时间：2019-01-14

发表刊物：ORGANIC & BIOMOLECULAR CHEMISTRY

收录刊物：SCIE、PubMed、Scopus

卷号：17

期号：2

页面范围：257-263

ISSN号：1477-0520

摘要：Bi-valent/specific antibodies are coming to the forefront of therapeutic and diagnostic applications for extending the functions of conventional antibodies. Nanobodies as building blocks, due to their small sizes, are prone to synthesizing these homo/hetero-dimers. However, the classical C-terminus to N-terminus (C-N) ligation manner for generating the dimer results in the inhibition of the antigen-binding capacity of the bivalent/specific antibodies. In this study, we designed and constructed several C-terminus to C-terminus (C-C) linked bivalent and bispecific nanobodies against the human beta 2-microglobulin via freezing, overcoming the biological function-disrupt raised by the C-N ligation. The nanobody modified by the formylglycine generating enzyme was ligated to a hydrazide or aminooxy bi-functionalized linker. During the process, we discovered that freezing significantly improved the efficiency of hydrazone or oxime formation between the linker and nanobodies, which could not take place at room temperature. By freezing from -10 to -20 degrees C, up to 50% yield of bivalent nanobodies was achieved within 24 h. The C-C linked nanobody-fusions maintained almost all of its binding activity and exhibited an increase by two orders of magnitudes in affinity kinetics, demonstrating the superiority of C-C over the C-N linking approach.