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Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0002-9969-0317
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0002-0695-5188
2017 (engelsk)Inngår i: ChemBioChem, ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, nr 20, s. 2056-2062Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Protein therapeutics suffer from low oral bioavailability, mainly due to poor membrane permeability and digestion by gastrointestinal proteases. To improve proteolytic stability, intramolecular thioether crosslinks were introduced into a three-helix affibody molecule binding the human epidermal growth factor receptor (EGFR). Solid-phase peptide synthesis was used to produce an unmodified control protein domain and three different crosslinked protein domain variants: one with a thioether crosslink between the N-terminal lysine residue and a cysteine residue in the second loop region (denoted K4), a second with a crosslink between the C-terminal lysine residue and a cysteine residue in the first loop region (denoted K58), and a third with crosslinks in both positions (denoted K4K58). Circular dichroism (CD) and surface-plasmon-resonance-based (SPR-based) biosensor studies of the protein domains showed that the three-helix structure and high-affinity binding to EGFR were preserved in the crosslinked protein domains. In vitro digestion by gastrointestinal proteases demonstrated that the crosslinked protein domains showed increased stability towards pepsin and towards a combination of trypsin and chymotrypsin.

sted, utgiver, år, opplag, sider
WILEY-V C H VERLAG GMBH , 2017. Vol. 18, nr 20, s. 2056-2062
Emneord [en]
affibodies, crosslinking, protein engineering, protein modifications, thioethers
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-217433DOI: 10.1002/cbic.201700350ISI: 000413353800015PubMedID: 28836374Scopus ID: 2-s2.0-85030102142OAI: oai:DiVA.org:kth-217433DiVA, id: diva2:1158160
Merknad

QC 20171117

Tilgjengelig fra: 2017-11-17 Laget: 2017-11-17 Sist oppdatert: 2024-07-04bibliografisk kontrollert
Inngår i avhandling
1. Investigations of chemical and enzymatic functionalization of affinity proteins
Åpne denne publikasjonen i ny fane eller vindu >>Investigations of chemical and enzymatic functionalization of affinity proteins
2020 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Abstract

Affinity proteins are important reagents in research, diagnostics and therapeutic settings. The focus of this thesis has been on investigating different chemical and enzymatic strategies for engineering of affinity proteins to generate affinity reagents with improved or changed functionality. The modifications introduced in affibodies, representing a class of small, three-helix engineered scaffold proteins, and antibodies were selected and implemented through rational design, using a combination of solid phase peptide synthesis, genetic engineering and enzymatic conjugation, depending on the case.

In a first study, thioether crosslinks were introduced between internally positioned lysines and cysteines of the human epidermal growth factor receptor (hEGFR)-targeting affibody ZEGFR:1907, to test the possibility to increase the proteolytic stability of the affibody scaffold. Three different variants of crosslinked affibodies were produced, containing one or two crosslinks. All three variants showed similar affinities to EFGR, and secondary structure contents, as the unmodified control protein. The crosslinked affibodies were challenged with the endopeptidases pepsin, found in the stomach, and trypsin and chymotrypsin, found in the gut. All affibodies showed improved stability towards at least one of the proteases, but the largest improvement was seen for the affibody harboring two crosslinks, which displayed the greatest stability in both assays.

Improvement in proteolytic stability of affibodies was further explored. In another study a sortase A-catalyzed intramolecular head-to-tail conjugation of the dimeric human epidermal growth factor 2 (HER2)-targeting affibody (ZHER2:342)2 was performed. Analysis showed no change in α-helicity for the cyclic dimer compared to the linear control, and a slight increase in melting temperature. Interestingly, in contrast to the linear variant, the cyclic dimer showed no signs of proteolytic degradation after 60 min exposure to the exopeptidase carboxypeptidase A.

The ability to change protein functionality by chemical modification was explored in two studies. The immunoglobulin-binding Z domain, from which the affibody scaffold is derived, was used as a model protein in one study, where light-induced affinity modulation was investigated. An azobenzene switch that isomerizes from a trans to a cis state was introduced end-to-end to one of the helices in three different designs of the Z domain. The conformational change induced by isomerization was hypothesized to be large enough to cause a loss in binding affinity in the conjugated affibody, which was tested in an affinity chromatography assay in which one of the affibodies captured to an IgG-sepharose column showed loss of affinity during illumination.

Peptide nucleic acid (PNA) probes have previously successfully been used for selective hybridization between the primary, tumor-targeting agent and the secondary agent in a pretargeting set-up for in vivo tumor imaging or directed therapy. In a last study, a Z domain-PNA conjugate produced via sortase A-mediated conjugation was photoconjugated to a lactosaminated antibody for possible use as an in vivo clearing agent for clearance of excess of primary probes via an hepatic route. The clearing agent showed partial success in a mouse model but the concept needs further work.

The work in this thesis shows the diverse possibilities available for changing the functionality of affinity proteins through chemical and enzymatic methods for different applications, and provides a framework for potential further improvement of both affibody and antibody functionality.

Abstract [sv]

Affinitetsproteiner är viktiga reagenser inom forskning, diagnostik och i terapeutiska sammanhang. Fokus för denna avhandling har varit att undersöka olika kemiska och enzymatiska strategier för konstruktion av affinitetsproteiner för att generera affinitetsreagens med förbättrad eller förändrad funktionalitet. Modifieringarna som introducerades i affibody-molekyler (en typ av små, designade helix-proteiner) och i antikroppar valdes och implementerades med rationell design, genom en kombination av fastfas-peptidsyntes, gentekniska modifieringar och enzymatisk konjugering, beroende på situationen.

I en första studie introducerades intramolekylära tioeter-korslänkar mellan lysiner och cysteiner i den human epidermal growth factor receptor (hEGFR)-bindande affibodyn ZEGFR:1907, för att testa möjligheten att öka affibody-strukturens proteolytiska stabilitet. Tre olika varianter av korslänkad affibody framställdes med antingen en eller två korslänkar. Alla tre varianterna visade liknande affinitet till EFGR och hade samma sekundärstrukturinnehåll som det omodifierade kontrollproteinet. I ett stabilitetstest utsattes de korslänkade affibody-molekylerna för endopeptidaserna pepsin som finns i magen, samt trypsin och kymotrypsin som finns i tarmen. Alla tre affibody-molekylerna visade förbättrad stabilitet gentemot åtminstone ett av proteaserna, men den största förbättringen observerades hos den affibody-molekyl som innehöll två tvärbindningar, som hade bäst stabilitet i båda testerna.

Förbättring av proteolytisk stabilitet hos affibody-molekyler undersöktes ytterligare i en annan studie där sortas A-katalyserad intramolekylär ringslutningskonjugering av den dimeriska human epidermal growth factor receptor 2 (HER2)-bindande affibody-molekylen (ZHER2:342)2. Den cykliska dimeren uppvisade en liten ökning i smälttemperatur jämfört med den linjära dimeren samt oförändrad α-helicitet. Intressant nog visade den cykliska dimeren, i motsats till den linjära, inga tecken på proteolytisk nedbrytning efter 60 minuters exponering med exopeptidaset karboxypeptidas A.

Förmågan att ändra proteinfunktionalitet genom kemisk modifiering undersöktes i två studier. Den immunglobulinbindande Z-domänen, från vilken affibody-strukturen har utvecklats, användes som ett modellprotein i en studie där ljusinducerad affinitetsmodulering undersöktes. En azobensenswitch som isomeriserar från ett trans- till ett cis-tillstånd kopplades till en av helixarna i tre olika designer av Z-domänen. Den konformationella förändring som induceras genom isomerisering antogs vara tillräckligt stor för att orsaka en förlust i bindningsaffinitet för konjugerad affibody. Denna hypotes testades i en affinitetskromatografianalys, i vilken det visade sig att en av de modifierade affibody-molekylerna som bundit till en IgG-sepharose-kolonn tappade affinitet vid belysning.

Peptidnukleinsyra (PNA)-prober har tidigare framgångsrikt använts för selektiv hybridisering mellan tumörbindande primärt reagens och sekundärt reagens i ett pretargeting-system för in vivo avbildning av tumörer eller riktad terapi. I en sista studie fotokonjugerades ett Z-domän-PNAkonjugat producerat via sortas A-medierad konjugering till en laktosaminerad antikropp för möjlig användning som ett in vivo clearing agent (CA) för att avlägsna överskott av primärt reagens genom nedbrytning via levern. CA visade delvis framgång i en musmodell men konceptet behöver utvecklas vidare.

Arbetet i denna avhandling visar de olika möjligheter som finns tillgängliga för att ändra affinitetsproteiners funktionalitet genom kemiska och enzymatiska metoder för olika tillämpningar och ger ett ramverk för potentiell ytterligare förbättring av både affibody- och antikroppsfunktionalitet.

sted, utgiver, år, opplag, sider
KTH Royal Institute of Technology, 2020. s. 86
Serie
TRITA-CBH-FOU ; 2020:35
Emneord
Affinity protein, affibody, conjugation, solid phase peptide synthesis, proteolytic stability, peptide nucleic acid (PNA).
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-279052 (URN)978-91-7873-618-8 (ISBN)
Disputas
2020-09-11, https://kth-se.zoom.us/webinar/register/WN_wkqf-V1QTmq4Czc22i7IMQ, Stockholm, 09:00 (engelsk)
Opponent
Veileder
Merknad

QC 2020-08-17

Tilgjengelig fra: 2020-08-17 Laget: 2020-08-12 Sist oppdatert: 2022-06-26bibliografisk kontrollert

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