Nanobody, a unique type of antibody only have the 1/10 size of the conventional antibody, have been well developed for various applications in the past 20 years. Bi/Multi-valent or multispecific nanobodies have better performance on antigen binding and are able to target various epitopes. In this regard, most of the nanobody drugs in clinical trials are multivalent or multispecific. Bi/Multi-valent or multi-specific nanobodies are usually prepared by technology of recombinant expression. However, since the antigen-binding region of nanobody is close to the N-terminus, the resulting linking of C-terminal with N-terminal could easily affect the activity of the subsequent nanobody. In this work, we generated aldehyde group at specific site in C-terminal of nanobody via formylglycine-generating enzyme (FGE) catalysis, and then added bis-hydrazide/aminooxy- PEG molecules to the aldehyde-nanobodies to prepare the covalent C-C linked nanobodies. We observed that the dimerization-efficiency was increased to about 50% under freezing condition. Then we found the K<sub>D</sub> of the bivalent nanobody was increased by 20 times which proved that the bivalent nanobodies with natural structures are able to maintain the activity and function of the antibody to a large extent. More importantly, the method provides a facile way for the synthesis of bivalent or multivalent nanobody.

Facile preparation of covalent C-to-C linked nanobody fusions by freezing

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.