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Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.

 

In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.

 

Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , viii, 84 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:22
Keyword [en]
Affibody, peptide synthesis, protein detection, molecular imaging
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-9626ISBN: 978-91-7415-152-7 (print)OAI: oai:DiVA.org:kth-9626DiVA: diva2:126762
Public defence
2008-12-12, D2, Lindstedtsv 5, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100719Available from: 2008-11-21 Created: 2008-11-21 Last updated: 2010-07-19Bibliographically approved
List of papers
1. Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein
Open this publication in new window or tab >>Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein
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2005 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, 1043-1050 p.Article in journal (Refereed) Published
Abstract [en]

Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

Keyword
affinity proteins; biosensors; FRET; peptides; protein modifications; solid-phase synthesis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5646 (URN)10.1002/cbic.200400388 (DOI)000229730000015 ()2-s2.0-20444507608 (Scopus ID)
Note
QC 20100715Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved
2. Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules
Open this publication in new window or tab >>Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules
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2005 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 341, no 2, 334-343 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, ZTaq and ZIgA, binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.

Keyword
Affibody; Protein microarray; Immobilization; Biosensor
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5647 (URN)10.1016/j.ab.2005.03.039 (DOI)000229460800017 ()2-s2.0-19444365959 (Scopus ID)
Note
QC 20100715Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved
3. Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence
Open this publication in new window or tab >>Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence
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2007 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 34, no 5, 722-733 p.Article in journal (Refereed) Published
Abstract [en]

Purpose  Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Non-invasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule ZHER2:342, which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2-expressing xenografts using radioiodinated ZHER2:342. The goal of this study was to develop a method for 99mTc labelling of ZHER2:342 using the MAG3 chelator, which was incorporated into ZHER2:342 using peptide synthesis, and evaluate the targeting properties of the labelled conjugate. Methods  MAG3-ZHER2:342 was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for 99mTc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of 99mTc-MAG3-ZHER2:342 and 125I-para-iodobenzoate -ZHER2:342 was compared 6 h p.i. Results  Synthetic MAG3-ZHER2:342 possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with 99mTc with an efficiency of about 75–80%. Labelled 99mTc-MAG3-ZHER2:342 retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. 99mTc-MAG3-ZHER2:342 showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression. Conclusion  Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled site-specific 99mTc labelling of the ZHER2:342 Affibody molecule with preserved targeting capacity.

Keyword
NEGATIVE BREAST-CANCER; DISSEMINATED PERITONEAL DISEASE; IN-VITRO CHARACTERIZATION; HER-2/NEU ONCOPROTEIN; HER2 EXPRESSION; CORRESPONDING METASTASES; COMBINATORIAL LIBRARIES; C-ERBB-2 EXPRESSION; DIFFERENT CHELATORS; ANTI-HER2 AFFIBODY
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-9721 (URN)10.1007/s00259-006-0266-4 (DOI)000246095900013 ()2-s2.0-34047191078 (Scopus ID)
Note
QC 20100715Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved
4. 99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody molecule
Open this publication in new window or tab >>99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody molecule
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2007 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 34, no 11, 1843-1853 p.Article in journal (Refereed) Published
Abstract [en]

Purpose  Monitoring HER2 expression is crucial for selection of breast cancer patients amenable to HER2-targeting therapy. The Affibody molecule ZHER2:342 binds to HER2 with picomolar affinity and enables specific imaging of HER2 expression. Previously, ZHER2:342 with the additional N-terminal mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) sequence was labelled with 99mTc and demonstrated specific targeting of HER2-expressing xenografts. However, hepatobiliary excretion caused high radioactivity accumulation in the abdomen. We investigated whether the biodistribution of ZHER2:342 can be improved by substituting glycyl residues in the chelating sequence with more hydrophilic seryl residues.

Methods  The Affibody molecule ZHER2:342, carrying the chelators mercaptoacetyl-glycyl-seryl-glycyl (maGSG), mercaptoacetyl-glycyl-D-seryl-glycyl [maG(D-S)G] and mercaptoacetyl-seryl-seryl-seryl (maSSS), were prepared by peptide synthesis and labelled with 99mTc. The differences in the excretion pathways were evaluated in normal mice. The tumour targeting capacity of 99mTc-maSSS-ZHER2:342 was studied in nude mice bearing SKOV-3 xenografts and compared with the capacity of radioiodinated ZHER2:342.

Results  A shift towards renal excretion was obtained when glycine was substituted with serine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced threefold for the maSSS conjugate in comparison with the maGGG conjugate 4 h post injection (p.i.). The tumour uptake of 99mTc-maSSS-ZHER2:342 was 11.5 ± 0.5% IA/g 4 h p.i., and the tumour-to-blood ratio was 76. The pharmacokinetics and uptake characteristics of technetium-labelled ZHER2:342 were better than those of radioiodinated ZHER2:342.

Conclusion  The introduction of serine residues in the chelator results in better tumour imaging properties of the Affibody molecule ZHER2:342 compared with glycyl-containing chelators and is favourable for imaging of tumours and metastases in the abdominal area.

Keyword
affibody molecule; HER2; peptide synthesis; technetium-99m; tumour targeting
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-9726 (URN)10.1007/s00259-007-0474-6 (DOI)000250205400017 ()2-s2.0-35348984582 (Scopus ID)
Note
QC 20100719Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved
5.  99mTc-maEEE-ZHER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors
Open this publication in new window or tab >> 99mTc-maEEE-ZHER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors
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2007 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 18, no 6, 1956-1964 p.Article in journal (Refereed) Published
Abstract [en]

Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of 99mTc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule ZHER2:342 with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of 99mTc-maGEG-ZHER2:342 and 99mTc-maEEE-ZHER2:342 was compared with 99mTc-maGGG-ZHER2:342 in normal mice, and the tumor targeting properties of 99mTc-maEEE-ZHER2:342 were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for 99mTc-labeling of ZHER2:342 reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. 99mTc-maEEE-ZHER2:342 showed a receptor-specific tumor uptake of 7.9 ± 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with 99mTc-maEEE-ZHER2:342 could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for 99mTc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making 99mTc-maEEE-ZHER2:342 a promising tracer for clinical imaging of HER2 overexpression in tumors.

Keyword
SINGLE-CHAIN FV; HUMAN-BREAST-CANCER; FRAGMENTS; PEPTIDES; ANTIBODY; THERAPY; RECOMMENDATIONS; MECHANISMS; GUIDELINES; HERCEPTIN
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-9723 (URN)10.1021/bc7002617 (DOI)000251166400035 ()2-s2.0-36849020126 (Scopus ID)
Note
QC 20100716Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved
6. Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake
Open this publication in new window or tab >>Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake
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2008 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 35, no 12, 2245-2255 p.Article in journal (Refereed) Published
Abstract [en]

Purpose  Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, 99mTc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering.

Materials and methods  Anti-HER2 ZHER2:342 Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with 99mTc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, 99mTc-maEEE-ZHER2:342, in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, 99mTc-maESE-ZHER2:342, was compared with radioiodinated ZHER2:342. Results  All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 ± 5, 68 ± 21 and 71 ± 10%IA/g, for99mTc-maESE-ZHER2:342, 99mTc-maEES-ZHER2:342 and 99mTc-maSEE-ZHER2:342, respectively, were significantly reduced in comparison with 99mTc-maEEE-ZHER2:342 (102 ± 13%IA/g). For 99mTc-maESE-ZHER2:342, a tumour uptake of 9.6 ± 1.8%IA/g and a tumour-to-blood ratio of 58 ± 6 were reached at 4 h p.i. Conclusions  A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of 99mTc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.

Keyword
Affibody molecule, HER2, Renal uptake, Technetium-99m, Tumour targeting
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-17054 (URN)10.1007/s00259-008-0845-7 (DOI)000261654000012 ()18594815 (PubMedID)
Available from: 2008-06-16 Created: 2009-03-25 Last updated: 2017-12-08Bibliographically approved
7. Synthesis and chemoselective intramolecular crosslinking of a HER2-binding Affibody
Open this publication in new window or tab >>Synthesis and chemoselective intramolecular crosslinking of a HER2-binding Affibody
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2009 (English)In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 92, no 2, 116-123 p.Article in journal (Refereed) Published
Abstract [en]

The human epidermal growth factor receptor HER2 has emerged as an important target for molecular imaging of breast cancer. This article presents the design and synthesis of a HER2-targeting affibody molecule with improved stability and tumor targeting capacity and with potential use as an imaging agent. The 58 aa three-helix bundle protein was assembled using solid-phase peptide synthesis, and a chemoselective ligation strategy was used to establish an intramolecular thioether bond between the side chain thiol group of a cysteine residue, positioned in the loop between helices I and II, and a chloroacetyl group on the side chain amino group of the C-terminal lysine residue. The tethered protein offered an increased thermal stability, with a melting temperature of 64 degrees C, compared to 54 degrees C for the linear control. The ligation did not have a major influence on the HER2 binding affinity, which was 320 and 380 pM for the crosslinked and linear molecules, respectively. Biodistribution studies were performed both in normal and tumor-bearing mice to evaluate the impact of the crosslinking on the in vivo behavior and on the tumor targeting performance. The distribution pattern was characterized by a low uptake in all organs except kidney, and rapid clearance from blood and normal tissue. Crosslinking of the protein resulted in a significantly increased tumor accumulation, rendering the tethered HER2-binding affibody molecule a valuable lead in the development of superior HER2 imaging agents.

Keyword
affibody molecules; chemoselective ligation; thioalkylation; HER2; molecular imaging; tumor targeting
National Category
Biochemistry and Molecular Biology Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-9725 (URN)10.1002/bip.21142 (DOI)000264691400005 ()2-s2.0-67449106599 (Scopus ID)
Note
QC 20100716. Uppdaterad från submitted till published (20100716)Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved

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