Nanobodies are the antigen recognizing part of single chain antibodies. They represent a novel class of therapeutic proteins that due to their small size (12-15 kDa) and unique structure make them ideal as novel inhibitors.1 Nanobodies against intracellular targets have been reported as intra-bodies, i.e. expressed in the cell where activity is desired.
The aim of this project is to develop techniques for chemical synthesis of nanobodies. A synthetic route would expand the possibilities to design tailored nanobodies with a range of chemical modifications such as tags, linkers, reporter groups, etc... In particular, the addition of unnatural amino acids would give the ability to attach chemical probes that can be used to internalize the antibody, for example.
For this purpose, Nanobody80 (Nb80) was chosen as model. This nanobody binds to the intracellular part of the β2-adrenenergic receptors and modulates its signalling.2 From a peptide synthesis point of view, nanobodies are challenging due to the relatively long sequence (circa 120 amino acids) and the tendency to form secondary structures during the synthesis leading to aggregation during synthesis. Several attempts to assemble the Nb80 sequence synthetically have been made and two major synthetic strategies are being explored; Fmoc based solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The Fmoc SPPS approach has so far only yielded partial Nb80 sequences with poor purity and it is currently facing some limitations. The NCL strategy is advantageous for synthesis of larger peptide sequences as the nanobody can be divided in fragments (four, three and two in this case) and then they can be assembled together. After optimization of the peptide synthesis conditions, we have now achieved the synthesis of all fragments needed to investigate the NCL strategies.