Freezing-assisted synthesis of covalent C-C inked bivalent and bispecific nanobodies
Freezing-assisted synthesis of covalent C-C inked bivalent and bispecific nanobodies
复制标题
共价 C-C 墨水二价和双特异性纳米抗体的冷冻辅助合成
DOI：
10.1039/c8ob02323a
复制
发表时间：
2019
影响因子：
3.2
通讯作者：
Jia Lingyun
中科院分区：
文献类型：
--
作者：
Zang Berlin;Ren Jun;Li Da;Huang Chundong;Ma Ha;Peng Qiang;Ji Fangling;Han Lulu;Jia Lingyun
双价/特异性抗体扩展了常规抗体的功能，成为治疗和诊断应用的前沿。纳米体作为构建单元，由于其体积小，易于合成这些同/异二聚体。然而，产生二聚体的经典c端到n端（C-N）连接方式会抑制二价/特异性抗体的抗原结合能力。在这项研究中，我们设计并构建了几个c端到c端（C-C）连接的二价和双特异性纳米体，通过冷冻来对抗人β2-微球蛋白，克服了C-N连接引起的生物功能破坏。由甲酰基甘氨酸生成酶修饰的纳米体被连接到一个肼或氨基双功能化的连接体上。在此过程中，我们发现冷冻显著提高了连接剂和纳米体之间形成腙或肟的效率，而这在室温下是无法发生的。在−10至−20°C的低温条件下，24小时内二价纳米体的产率可达50%。碳- C连接的纳米体融合体几乎保持了所有的结合活性，亲和动力学提高了两个数量级，证明了碳- C比碳- n连接方法的优越性。
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 β2-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 °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.