Developing GLP-1 Derivative with an Extended Half-life via Site-specific Conjugation of Albumin for Long Acting Drug Delivery (SCALADE) Technique

Abstract

GLP-1 is a potent therapeutics for Type 2 diabetes mellitus. GLP-1 induces insulin-secretion depending on the glucose level and proliferates beta-cell while it has an extremely short half-life due to its small size and rapid protease cleavage by DPPⅣ. In general, albumin conjugation is advantageous regarding half-life extension. However, this strategy results in remarkably decreased potency for GLP-1R agonists. Both terminal sides of GLP-1 are important for GLP-1R interaction given two studies – Ala substitution and structure. Indeed, it is required to search and compare residues in the middle part of GLP-1 for the conjugation site. Therefore, we produced DPPⅣ-resistant HSA-GLP-1 conjugates with three candidates – V16, Y19, and F28 as a control – using site-specific conjugation of albumin for long acting drug delivery (SCALADE) technique, which is HSA is site-specifically conjugated to GLP-1 via biorthogonal SPAAC reaction. GLP-1 was expressed as a form of the fusion protein with sfGFP and p-azido-phenylalanine (AzF) was incorporated into each candidate site in the E. coli expression system. In vitro activity of three candidates supported the structure-activity relationship of GLP-1: F28 drastically decreased in EC50 value as expected, but V16 and Y19 variants were more potent. Especially, the V16 variant had a significant acute effect on IPGTT in vivo and remarkably extended the serum half-life 168 times compared to wild type GLP-1 and even 1.3 times longer than DPPⅣ-vulnerable variant. In conclusion, three HSA-GLP-1 conjugates were compared and it is confirmed that V16, the most potent variant, has significant activity in vivo with remarkably extended serum half-life.