Evaluation and improvement of the pharmacokinetics, biodistribution and stability of fibronectin-derived binding proteins (monobodies)

In my work, I have provided primary insight into the pharmacological properties and stability of monobodies and demonstrated a facile way to improve not only their in vivo-properties but also plasma stability in vitro through fusion to an albumin binding domain. The resulting albumin binding domain-monobodies (ABD-Mb) retained binding capability to their primary target at low nanomolar affinity and bound effectively to human and mouse albumin. Additionally, ABD-monobodies exhibited prolonged in vitro-plasma stability and the in vivo-circulation time of ABD-AS25 in mice was significantly increased by 92-fold, compared to wildtype AS25, with the majority of the ABD-monobody remaining in the bloodstream, while the wildtype monobody was cleared through the kidneys within minutes after application. By addition of a third binding moiety against surface targets like PD-L1, EGFR and HER2, whose deregulation is commonly associated with cancer, I created trifunctional monobodies with the ability to specifically bind cells expressing the respective target at high levels, without perturbing the binding of the initial monobody or the albumin binding domain. Since our laboratory has developed monobodies to target intracellular oncogenes, I further investigated the degradation kinetics and mechanism of monobodies upon their Doxycycline-induced intracellular expression, showing that intracellular levels of HA4-YA monobody in HeLa, A549 or Baf3 p210 cells declined rapidly within the first 24 hours after Doxycycline was withdrawn. This decline was partially prevented by inhibition of the ubiquitin proteasome pathway by Bortezomib, indicating this pathway to play a major role in the degradation of intracellular monobodies. Consequently, I have exchanged the tags and mutated lysine-residues of intracellular monobodies to prevent ubiquitination, the first critical step in the course of proteasomal degradation.