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Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
KTH, School of Biotechnology (BIO), Protein Technology. (Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface. 

The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection. 

In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future. 

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 80
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:17
Keywords [en]
directed evolution, microbial display, E. coli, Affibody molecule, autotransporter, medical imaging, HER receptor family, Sortase A
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-213451ISBN: 978-91-7729-504-4 (print)OAI: oai:DiVA.org:kth-213451DiVA, id: diva2:1137375
Public defence
2017-10-06, F3, Lindstedtsvägen 26, Sing-Sing, floor 2, KTH Campus, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20170904

Available from: 2017-09-05 Created: 2017-08-31 Last updated: 2017-09-05Bibliographically approved
List of papers
1. An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
Open this publication in new window or tab >>An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
2014 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 13, p. 179-Article in journal (Refereed) Published
Abstract [en]

Background: Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. Results: The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection using FACS from a 1: 100,000 background yielded around 20,000-fold enrichment in a single round and high viability of the isolated bacteria after sorting. Conclusions: The new expression vectors are promising for combinatorial engineering of Affibody molecules and the strategy for small-scale production of soluble recombinant proteins has the potential to increase throughput of the entire discovery process.

Keywords
Affibody molecule, Bacterial display, Directed evolution, Combinatorial protein engineering, AIDA-I, Autotransporter, FACS, Secreted protein production, E. coli, Phage display
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-156531 (URN)10.1186/s12934-014-0179-z (DOI)000349043900001 ()2-s2.0-84924087937 (Scopus ID)
Funder
Swedish Research Council, 621-2012-5336
Note

QC 20150303

Available from: 2014-11-28 Created: 2014-11-28 Last updated: 2017-12-05Bibliographically approved
2. Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase A
Open this publication in new window or tab >>Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase A
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Combinatorial protein engineering using libraries displayed on various microorganisms is a powerful method forgeneration of new affinity proteins. Successful efforts often result in broad panels of isolated binders, which are thentypically subcloned, produced, purified and characterized in various assays. Many such assays also require conjugation tofor example reporters or other functional molecules and the downstream production and modification thus tends to be verylaborious and limits the number of candidates that can be screened. Staphylococcal sortase A is a natural transpeptidasethat catalyzes the ligation between a LPXTG motif and N-terminal glycines and is today used in a variety of applicationsfor site-specific conjugation of different molecules to recombinant proteins. We have previously developed a surfacedisplay method for combinatorial protein engineering of Affibody molecules on the outer membrane of E. coli usingautodisplay. Here, we introduced a sortase-A recognition motif into the displayed recombinant proteins and evaluatedsortase-mediated release and specific conjugation of various reporters to Affibody molecules. The approach has potentialto significantly increase the flexibility and throughput of downstream characterization of affinity proteins after directedevolution using cell display and FACS.

Keywords
AIDA-I, Affibody molecules, autodisplay, autotransporter, bioconjugation, combinatorial protein engineering, directed evolution, protein ligation, sortase A
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-213450 (URN)
Note

QC 20170904

Available from: 2017-08-31 Created: 2017-08-31 Last updated: 2017-09-04Bibliographically approved
3. 111In-labeled NOTA-conjugated Affibody molecules for visualization of HER3 expression in malignant tumors
Open this publication in new window or tab >>111In-labeled NOTA-conjugated Affibody molecules for visualization of HER3 expression in malignant tumors
Show others...
2014 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, p. S311-S311Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-161572 (URN)000348841900497 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 18-22, 2014, Gothenburg, SWEDEN
Note

QC 20150326

Available from: 2015-03-26 Created: 2015-03-13 Last updated: 2017-12-04Bibliographically approved
4. Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coli
Open this publication in new window or tab >>Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coli
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is the most frequently used method, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli have several properties that are valuable for library applications and then in particular the high transformation efficiency. Although the first studies on display of recombinant peptides and proteins on E. coli were reported over 25 years ago, the method is still not fully established for directed evolution of affinity proteins. More recently, the use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and in particular for directed evolution of different enzymes. Here, we report on display of a large naïve Affibody library on the outer membrane of E. coli using the autotransporter AIDA-I. The expression cassette was first engineered by removing non-essential sequences, followed by introduction of an Affibody library, comprising more than 109 variants, into the new display vector. Selections by FACS against five different target molecules resulted in a panel of binders with down to nanomolar affinities.

Keywords
Bacterial display, affibody library, AIDA-I, autodisplay, autotransporter, directed evolution, FACS
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-213448 (URN)
Note

QC 20170904

Available from: 2017-08-31 Created: 2017-08-31 Last updated: 2017-12-18Bibliographically approved
5. PET Imaging of HER3-Expression in Tumours Using a 68Ga-Labeled Affibody Molecule
Open this publication in new window or tab >>PET Imaging of HER3-Expression in Tumours Using a 68Ga-Labeled Affibody Molecule
Show others...
2014 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, p. S310-S310Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-161575 (URN)000348841900493 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 18-22, 2014, Gothenburg, SWEDEN
Note

QC 20150326

Available from: 2015-03-26 Created: 2015-03-13 Last updated: 2017-12-04Bibliographically approved
6. PET imaging of epidermal growth factor receptor expression in tumours using Zr-89-labelled ZEGFR:2377 affibody molecules
Open this publication in new window or tab >>PET imaging of epidermal growth factor receptor expression in tumours using Zr-89-labelled ZEGFR:2377 affibody molecules
Show others...
2016 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 48, no 4, p. 1325-1332Article in journal (Refereed) Published
Abstract [en]

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a radionuclide Zr-89. The Zr-89-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160 +/- 60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, Zr-89-DFO-ZEGFR: 2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6 +/- 0.5% ID/g and tumour-to-blood ratio of 3.7 +/- 0.6 at 24 h after injection. Zr-89-DFO-ZEGFR: 2377 provides higher tumour-to-organ ratios than anti-EGFR antibody Zr-89-DFO-cetuximab at 48 h after injection. EGFR-expressing tumours were clearly visualized by microPET using Zr-89-DFO-ZEGFR: 2377 at both 3 and 24 h after injection. In conclusion, Zr-89-DFO-ZEGFR: 2377 is a potential probe for PET imaging of EGFR-expression in vivo.

Place, publisher, year, edition, pages
Spandidos, 2016
Keywords
epidermal growth factor receptor, affibody molecules, radionuclide imaging, positron emission tomography, zirconium-89, xenografts
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-185347 (URN)10.3892/ijo.2016.3369 (DOI)000372567100002 ()2-s2.0-84959020465 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer Society
Note

QC 20160421

Available from: 2016-04-21 Created: 2016-04-18 Last updated: 2017-11-30Bibliographically approved

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