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A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0002-9969-0317
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2014 (English)In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 102, no 3, 252-259 p.Article in journal (Refereed) Published
Abstract [en]

Glucagon-like peptide 1 (GLP-1) and related peptide agonists have been extensively investigated for glycaemic control in Type 2 diabetes, and may also have therapeutic applications for other diseases. Due to the short half-life (t1/2<2 min) of the endogenous peptide, caused by proteolytic degradation and renal clearance, different strategies for half-life extension and sustained release have been explored. In the present study, conjugates between a GLP-1 analogue and a 5 kDa albumin-binding domain (ABD) derived from streptococcal protein G have been chemically synthesized and evaluated. ABD binds with high affinity to human serum albumin, which is highly abundant in plasma and functions as a drug carrier in the circulation. Three different GLP-1-ABD conjugates, with the two peptides connected by linkers of two, four, and six PEG units, respectively, were synthesized and tested in mouse pancreatic islets at high (11 mM) and low (3 mM) glucose concentration. Insulin release upon stimulation was shown to be glucose-dependent, showing no significant difference between the three different GLP-1-ABD conjugates and unconjugated GLP-1 analogue. The biological activity, in combination with the high affinity binding to albumin, make the GLP-1-ABD conjugates promising GLP-1 receptor agonists expected to show extended in vivo half-life.

Place, publisher, year, edition, pages
2014. Vol. 102, no 3, 252-259 p.
Keyword [en]
glucagon-like peptide 1, albumin-binding domain, half-life extension, peptide conjugate, insulin release
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-141009DOI: 10.1002/bip.22474ISI: 000337513400004Scopus ID: 2-s2.0-84922005237OAI: oai:DiVA.org:kth-141009DiVA: diva2:693804
Note

Updated from Manuscript to Journal. QC 20140711

Available from: 2014-02-05 Created: 2014-02-05 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Chemical Engineering of Small Affinity Proteins
Open this publication in new window or tab >>Chemical Engineering of Small Affinity Proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS).

In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I).

In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III).

In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V).

Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. viii, 79 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:3
Keyword
Affibody molecules, albumin binding domain, ligation, protein synthesis, solid phase peptide synthesis
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-141014 (URN)978-91-7595-004-4 (ISBN)
Public defence
2014-03-07, FR4 (Oskar Klein), AlbaNova Universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20140207

Available from: 2014-02-07 Created: 2014-02-05 Last updated: 2014-02-07Bibliographically approved

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Lindgren, JoelEriksson Karlström, Amelie

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