Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide

Abstract

The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long serum half-life and can be used to extend the serum half-lives of therapeutic proteins and peptides. We used glucagon-like-peptide 1 (GLP-1) as a model peptide in the present study. A “clickable” non-natural amino acid—p-azido-l-phenylalanine (AzF)—was incorporated into three specific sites (V16, Y19, and F28) of a GLP-1 variant, followed by conjugation with HSA through strain-promoted azide–alkyne cycloaddition. All three HSA-conjugated GLP-1 variants (GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA) exhibited comparable serum half-lives in vivo. However, the three GLP1_HSA variants had different in vitro biological activities and in vivo glucose-lowering effects, demonstrating the importance of site-specific HSA conjugation. The platform described herein could be used to develop other therapeutic peptides with extended serum half-lives.

Effect of C-terminus Conjugation via Different Conjugation Chemistries on In Vivo Activity of Albumin-Conjugated Recombinant GLP-1

“…The half-lives of these variants were comparable with those of other GLP1-HSA conjugates as previously reported (8.4, 7.4, and 8.0 h for GLP1_8G16AzF-HSA, GLP1_8G19AzF-HSA, and GLP1_8G28AzF-HSA, respectively) [24]. As the site-specific HSA conjugation at positions 16,19, and 28 of GLP1 led to very similar plasma half-lives [24], we speculated that the terminal plasma half-life of GLP1_8G37AzF-HSA would also be comparable. These results indicated the conjugation of HSA at the C-terminus of GLP-1 successfully extended the plasma half-life of GLP-1.…”

“…We previously reported the site-specific HSA conjugation to GLP-1 by expressing recombinant GLP-1 variants bearing an NNAA as an albumin conjugation site fused to superfolder green fluorescent protein (sfGFP) in genetically engineered Escherichia coli [ 24 ]. Two of the resulting GLP-1 variants with human serum albumin (HSA) conjugated at positions 16 and 28, respectively, exhibited significantly prolonged plasma half-lives in mice (~8 h) [ 24 ].…”

“…We previously reported the site-specific HSA conjugation to GLP-1 by expressing recombinant GLP-1 variants bearing an NNAA as an albumin conjugation site fused to superfolder green fluorescent protein (sfGFP) in genetically engineered Escherichia coli [ 24 ]. Two of the resulting GLP-1 variants with human serum albumin (HSA) conjugated at positions 16 and 28, respectively, exhibited significantly prolonged plasma half-lives in mice (~8 h) [ 24 ]. Although we carefully selected the albumin conjugation site on GLP-1 in order for it not to interfere with the interactions of position 16 of GLP-1with GLP-1R ( Figure 1 A), in vitro and in vivo glucose-lowering activities of the resulting albumin conjugates were only moderate [ 24 ].…”

“…Two of the resulting GLP-1 variants with human serum albumin (HSA) conjugated at positions 16 and 28, respectively, exhibited significantly prolonged plasma half-lives in mice (~8 h) [ 24 ]. Although we carefully selected the albumin conjugation site on GLP-1 in order for it not to interfere with the interactions of position 16 of GLP-1with GLP-1R ( Figure 1 A), in vitro and in vivo glucose-lowering activities of the resulting albumin conjugates were only moderate [ 24 ]. Enhancing the biological potency of a drug can help reduce the amount of injected drug needed for efficacy, thereby contributing to a reduction in production cost.…”

“…In the current study, we investigated the effects of albumin conjugation sites and conjugation chemistry on in vivo blood-lowering activities with the ultimate goal of designing albumin-GLP1 conjugates with enhanced therapeutic potency. Based on mutation studies, an albumin conjugate site at GLP1 position 16 was selected in a previous study to minimize the perturbation of interactions between the GLP-1 variant and GLP-1R [ 24 ]. However, considering the bulky structure of albumin, we hypothesize that the albumin conjugated to GLP-1 may to some extent block the binding of the conjugate to GLP-1R.…”