Development and characterization of an exendin-4 variant with extended plasma half-life by PASylation®

Glucagon-like peptide-1 (GLP-1) is an incretin hormone known to stimulate glucose-dependent insulin secretion. The GLP-1 receptor agonist, exendin-4, has similar properties as GLP-1 and is currently in clinical use for the treatment of type 2 diabetes mellitus (Byetta®). However, the 38 amino acid exendin-4 peptide suffers from rapid kidney clearance with a short plasma half-life (t1/2 = 2.4 h) due to its small molecular size. To achieve a therapeutic effect, the peptide has to be injected twice daily. To extend the half-life of the exendin-4 molecule in vivo, we genetically engineered an exendin-PAS® fusion protein. The PAS sequence is a conformationally disordered polypeptide composed of the amino acids Pro, Ala, and Ser. By way of genetic fusion, a highly soluble biological PAS polymer with large hydrodynamic volume - having similar properties as the chemical polymer PEG - is attached to exendin-4, which can be expected to extend circulation in blood by a factor 10 to 100, depending on the length of the PAS tag. The PAS200, PAS400 and PAS600 fusion proteins, as well as the unfused exendin-4, were successfully produced both as MBP fusion proteins in the cytoplasm and, mediated by the MalE signal peptide, in the periplasm of Escherichia coli. Homogeneous preparations were obtained from the periplasmic cell extract after one-step affinity chromatography via the Strep-tag II®. Alternatively, in the case of the MBP fusion proteins the (PASylated) exendin-4 moiety was successfully liberated via TEV protease cleavage. When compared with the unfused exendin-4 molecule, a significantly increased hydrodynamic volume was observed during size exclusion chromatography. Circular dichroism difference measurements revealed a characteristic random coil spectrum for the PAS moieties. Receptor binding experiments are underway. Our results so far indicate that PASylation of exendin-4 should yield pharmacokinetic properties similar to PEGylation without need of in vitro modification steps.