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

Abstract

Glucagon-like peptide-1 (GLP-1) is a peptide hormone with tremendous therapeutic potential for treating type 2 diabetes mellitus. However, the short half-life of its native form is a significant drawback. We previously prolonged the plasma half-life of GLP-1 via site-specific conjugation of human serum albumin (HSA) at position 16 of recombinant GLP-1 using site-specific incorporation of p-azido-phenylalanine (AzF) and strain-promoted azide-alkyne cycloaddition (SPAAC). However, the resulting conjugate GLP1_8G16AzF-HSA showed only moderate in vivo glucose-lowering activity, probably due to perturbed interactions with GLP-1 receptor (GLP-1R) caused by the albumin-linker. To identify albumin-conjugated GLP-1 variants with enhanced in vivo glucose-lowering activity, we investigated the conjugation of HSA to a C-terminal region of GLP-1 to reduce steric hindrance by the albumin-linker using two different conjugation chemistries. GLP-1 variants GLP1_8G37AzF-HSA and GLP1_8G37C-HSA were prepared using SPAAC and Michael addition, respectively. GLP1_8G37C-HSA exhibited a higher glucose-lowering activity in vivo than GLP1_8G16AzF-HSA, while GLP1_8G37AzF-HSA did not. Another GLP-1 variant, GLP1_8A37C-HSA, had a glycine to alanine mutation at position 8 and albumin at its C-terminus and exhibited in vivo glucose-lowering activity comparable to that of GLP1_8G37C-HSA, despite a moderately shorter plasma half-life. These results showed that site-specific HSA conjugation to the C-terminus of GLP-1 via Michael addition could be used to generate GLP-1 variants with enhanced glucose-lowering activity and prolonged plasma half-life in vivo.

Keywords: albumin conjugation; glucagon-like peptide-1; in vivo glucose-lowering activity; plasma half-life extension.

Figure 1

Structures of three glucagon-like peptide-1 (GLP-1)-human serum albumin (HSA) variants. GLP-1 variants conjugated to HSA at position 16 (A) and position 37 (B) using strain-promoted azide-alkyne cycloaddition and Michael addition. (C) GLP-1 variants conjugated to HSA at position 37 using inverse-electron demand Diels-Alder reaction and Michael addition.

Figure 2

Construction of the GLP-1 and superfolder green fluorescent protein (sfGFP)-fused GLP-1 (sfGFP-GLP1) variants. (A) The amino acid sequences of six GLP-1 variants. The green-colored X notated at GLP1_8G16AzF and GLP1_8G37AzF was the incorporation site of the non-natural amino acid, AzF. (B) The amino acid sequence and (C) protein features of sfGFP-GLP1_C with the polyhistidine-tag (purple), sfGFP (green), linker (brown), factor Xa cleavage site (orange), and GLP1_C (red).

Figure 3

Expression, purification, and confirmation of C-terminal cysteine-substituted sfGFP-GLP-1_8G37C. (A) SDS-PAGE protein gel images of cell lysates before induction (BI) and after induction (AI). A prominent band expected to be sfGFP-GLP1_8G37C is observed (arrow). (B) Purified sfGFP-GLP1_8G37C in an SDS-PAGE gel after Ni-NTA affinity chromatography.

Figure 4

Monoisotopic mass confirmation of GLP1_8G37AzF, GLP1_8G37C, and GLP1_8A37C by matrix-assisted laser desorption/ionization time-of-light mass spectroscopy (MALDI-TOF MS). The monoisotopic mass to charge ratio for GLP1_8G37AzF, GLP1_8G37C, and GLP1_8A37C were 3474.0, 3414.9, and 3428.7 m/z, respectively.

Figure 5

Purification and confirmation of GLP1_8G37C-HSA and their intermediates. (A) Protein gel image after Coomassie blue staining of the intermediates during GLP1_8G37C-HSA production. Molecular weight standards (lane M), purified HSA (lane 1), sfGFP-GLP1_8G37C-HSA purified with unreacted sfGFP-GLP1_8G37C (lane 2), GLP1_8G37C-HSA with sfGFP-GLP1_8G37C after factor Xa cleavage (lane 3), and purified GLP1_8G37C-HSA (lane 4). In lane 3, the band for sfGFP-GLP1_8G37C is observed at a lower position compared to the band in lane 2, which is expected to be the result of factor Xa cleavage. (B) Protein gel image after Coomassie blue staining of the final products of GLP1_8G37C-HSA (lane 1) and GLP1_8A37CHSA (lane 2). (C) MALDI-TOF MS spectrum of GLP1_8G37AzF-HSA, GLP1_8G37C-HSA, and GLP1_8A37C-HSA.

Figure 6

Pharmacokinetic profiles of intravenously injected GLP1_8G37C-HSA and GLP1_8A37C-HSA conjugates in BALB/c mice. Data in the graph indicate the mean ± standard deviation (n = 4/group). The plasma concentrations of the samples were plotted using a logarithmic scale.

Figure 7

Blood glucose levels of PBS (negative control), GLP1_C (positive control), and the GLP1-HSA variants. (A) PBS or 30 nmol/kg doses of GLP1_C or the GLP1-HSA variants were subcutaneously injected into C57BL/6J mice (n = 3/group) 20 min prior to the intraperitoneal injection of glucose (1.5 g/kg, 0 min). Data in the graph indicate the mean value ± standard deviation (n = 3/group). (B) The area under curves (AUC) calculated from 0 to 120 min were compared. Mean value ± standard deviation is presented (n = 3/group), * p-value < 0.01; NS: not significant.

Figure 8

In vitro biological activity of GLP1_C, GLP1_8GWT, GLP1_8G16AzF-HSA, GLP1_8G37AzF-HSA, GLP1_8G37C-HSA, and GLP1_8A37C-HSA evaluated in GLP-1R-expressing HEK-293 cells. The Y-axis denotes the percent activity calculated as a percentage of average cyclic adenosine monophosphate (cAMP) production by 1 μM GLP1_C. Each point in the graph indicates the mean value ± standard deviation (n = 3/group).