Freezing-assisted synthesis of covalent C–C linked bivalent and bispecific nanobodies. Issue 2 (24th October 2018)

• Record Type: Journal Article
• Authors: Zang, Berlin; Ren, Jun; Li, Da; Huang, Chundong; Ma, Hao; Peng, Qiang; Ji, Fangling; Han, Lulu; Jia, Lingyun
• Is Part Of: Organic & biomolecular chemistry. Volume 17:Issue 2(2018)
• Page Start: 257
• Page End: 263
• Publication Date: 2018-10-24
• Subjects: Chemistry, Organic -- Periodicals; Bioorganic chemistry -- Periodicals; Chemistry, Physical organic -- Periodicals; 547
• DOI: 10.1039/c8ob02323a
• Languages: English
• ISSNs: 1477-0520

Abstract

Abstract : C–N linked bivalent nanobody (upper) and C–C linked nanobody (bottom) presented as cartoon. (The elephant represents the nanobody; the banana represents the antigen). Abstract : 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.