Stability of llama heavy chain antibody fragments under extreme conditions

Abstract

Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The applications in which these VHHs are to be used, require functionality in non-physiological environments. High temperature, anionic and non-ionic surfactants in shampoo, and the low pH and digestive enzymes of the gastrointestinal tract put high demands on the stability of the VHHs. Regardless of these harsh conditions, the VHHs seem to exceed conventional antibody fragments and are known for their high stability. Stability in the previous sentence must be regarded as functionality under these conditions. In chapter 2 we showed that VHHs can be functional at high temperatures, despite of the fact that they were already unfolded at high temperatures. Unfolding of the VHH population started at 60 ºC, and most of the population of VHHs is unfolded at 80 ºC. However, after addition of the antigen, complete refolding and complex formation was observed. This shows the fast and highly efficient refolding of the VHHs, which can be of use in a wide range of applications. The single domain of the VHHs is the logical cause of the ability of refolding and explains why refolding is not observed in more complex antibodies, being composed of several domains, which are not able to refold properly. This refolding property of VHHs, together with the low degree of aggregation, implies that these VHHs can be functional beyond their intrinsic stability. The adaptations to the single domain status shown by VHHs, reside predominantly in the region where in conventional antibodies the VL is attached. Four substitutions are hallmarks for VHHs, making the ‘former’ VL interface highly hydrophilic. Furthermore, chapter five describes that more substitutions can be observed from VH to VHH. Most of these substitutions are from large hydrophobic residue to hydrophilic (charged) residues. These substitutions are not only involved in making the VHH more soluble, in chapter three and four two mutational studies are described which indicate that the residues in this so-called ‘former’ VH-VL interface could also have a prominent role in the stability of the VHHs. Chapter three describes the easy and rapid selection of VHHs able to bind in shampoo by addition of shampoo to the selection protocol. The VHHs obtained from this selection were investigated and the protein sequences show that selection is driven towards a small subclass of VHHs. This subclass exceed other VHHs for stability in shampoo, indicating that selection was directed towards stability in shampoo. This technique was used in a second study to ensure stability of VHHs in the environment of the gut. VHHs against rotavirus were selected at low pH and in the presence of pepsin. To further enhance the stability of the selected subclass of VHHs, protein engineering was used to ensure stability in the intestine. A mutational study was performed to remove trypsin cleavage sites from the VHH. Mutant R27A showed a reduced trypsin susceptibility. Furthermore, this VHH also showed increased production levels, increased thermostability and equal affinity compard to the wildtype VHH1. These results show that VHHs are ideal candidates for a wide range of applications. As shown in this thesis, the high stability of these VHHs, together with the rapid and easy selection methods, and subsequent improvement by protein engineering, results in exquisite opportunities for application in harsh conditions.