Site-specific conjugation of human serum albumin to recombinant Glucagon like peptide-1 (GLP-1) for effective treatment of type 2 diabetes mellitus

Abstract

Glucagon like peptide-1 (GLP-1) is a peptide hormone and has a tremendous therapeutic potential for treating type 2 Diabetes Meliitus (T2DM). However, its short half-life is a significant drawback to be utilized in its native form. To address this issue, various ways to extend half-life of GLP-1 have been explored. Human serum albumin (HSA) has a very long in vivo half-life (about 20 days). Therefore, HSA has been utilized to extend the in vivo half-life of other proteins. In this dissertation, we used the site-specific conjugation of HSA to extend the in vivo half-life of GLP-1 to minimize a loss in the activity caused by the disturbance between GLP-1 receptor and GLP-1. The dissertation details two investigations. First, we used the site-specific incorporation technique of a “clickable” p-azido-l-phenylalanine (AzF) for conjugation of HSA. Superfolder green fluorescent protein (sfGFP) was used as a fusion tag to enable expression of GLP-1 in E. coli. The AzF-incorporated GLP-1 variants were successfully conjugated to HSA (GLP1-HSA variants) through strain-promoted azide-alkyne cycloaddition. The resulting conjugates (GLP1_16AzF-HSA, GLP1_19AzF-HSA, GLP1_28AzF-HSA) exhibited the extended half-lives in vivo. However, the in vitro biological activities and in vivo glucose-lowering activities varied among GLP1-HSA variants. Furthermore, the AzF-incorporated GLP1_HSA variants exhibited moderately reduced biological activity. Secondly, we hypothesized that three factors lead to the activity loss upon HSA conjugation of GLP-1, conjugation site of HSA, conjugation chemistry, and geometry of GLP-1 backbone. Therefore, we designed and evaluated three new GLP1-HSA conjugate variants (GLP1_8G37C-HSA, GLP1_8A37C-HSA, and GLP1_8G37AzF-HSA) and two of them exhibited improved in vivo glucose lowering activity. The significance of this dissertation research lies in 1) investigating the extension of in vivo half-life of GLP-1 via site-specific conjugation of HSA and 2) identifying factors affecting the activity loss of GLP-1 upon HSA conjugation.