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Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. (Affinity-protein Based Blocking of Cellular Proteins and their Delivery)
2013 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.

In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2013. , s. xi, 65
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2013:2
Emneord [en]
Affibody molecules, affinity proteins, radionuclide molecular imaging, intracellular targeting, EGFR, HER2, IGF-1R
HSV kategori
Forskningsprogram
SRA - Molekylär biovetenskap
Identifikatorer
URN: urn:nbn:se:kth:diva-116884ISBN: 978-91-7501-613-9 (tryckt)OAI: oai:DiVA.org:kth-116884DiVA, id: diva2:601387
Disputas
2013-02-20, FR4, AlbaNova University Center Roslagstullsbacken 21, Stockholm, 10:30 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Merknad

QC 20130129

Tilgjengelig fra: 2013-01-29 Laget: 2013-01-29 Sist oppdatert: 2022-06-24bibliografisk kontrollert
Delarbeid
1. HEHEHE-Tagged Affibody Molecule May Be Purified by IMAC, Is Conveniently Labeled with [Tc-99m(CO)(3)](+), and Shows Improved Biodistribution with Reduced Hepatic Radioactivity Accumulation
Åpne denne publikasjonen i ny fane eller vindu >>HEHEHE-Tagged Affibody Molecule May Be Purified by IMAC, Is Conveniently Labeled with [Tc-99m(CO)(3)](+), and Shows Improved Biodistribution with Reduced Hepatic Radioactivity Accumulation
Vise andre…
2010 (engelsk)Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, nr 11, s. 2013-2022Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [Tc-99m(CO)(3)](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER2:342) were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [Tc-99m(CO)(3)](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER2:342)-H-6 and (HE)(3)-Z(HER2:342), respectively, could be efficiently purified using IMAC and stably labeled with [Tc-99m(CO)(3)](+) and were subsequently compared with the parental H-6-Z(HER2:342) having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [Tc-99m(CO)(3)](+)-Z(HER2:342)-H-6 compared to [Tc-99m(CO)(3)](+)-H-6-Z(HER2:342), and more than 10-fold lower with [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342). These differences translated into appreciably superior tumor-to-liver ratio for [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342) compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.

Emneord
anti-her2 affibody, her2 expression, tumors, therapy, tc-99m, proteins, cancer, affinity
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-27384 (URN)10.1021/bc1002357 (DOI)000284203200010 ()20964447 (PubMedID)2-s2.0-78649309723 (Scopus ID)
Merknad
QC 20101213Tilgjengelig fra: 2010-12-13 Laget: 2010-12-13 Sist oppdatert: 2024-03-15bibliografisk kontrollert
2. Use of a HEHEHE Purification Tag Instead of a Hexahistidine Tag Improves Biodistribution of Affibody Molecules Site-Specifically Labeled with Tc-99m, In-111 and I-125
Åpne denne publikasjonen i ny fane eller vindu >>Use of a HEHEHE Purification Tag Instead of a Hexahistidine Tag Improves Biodistribution of Affibody Molecules Site-Specifically Labeled with Tc-99m, In-111 and I-125
Vise andre…
2011 (engelsk)Inngår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, nr 11, s. 3817-3826Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Affibody molecules are a class of small (similar to 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His(6))-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have demonstrated that replacement of the His(6)- tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of Affibody molecules by IMAC, enables labeling with [Tc-99m(CO)(3)](+), and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with In-111, Tc-99m, and I-125 at the C-terminus, having a His(6)-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHE-tag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents.

Emneord
ANTI-HER2 AFFIBODY, HER2 EXPRESSION, HER2-EXPRESSING TUMORS, RECOMBINANT PROTEINS, MALIGNANT-TUMORS, PEPTIDE, AFFINITY, TECHNETIUM, SCAFFOLD, THERAPY
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-34657 (URN)10.1021/jm200065e (DOI)000291082500010 ()21524142 (PubMedID)2-s2.0-79958148943 (Scopus ID)
Forskningsfinansiär
Swedish Research Council
Merknad
QC 20110627Tilgjengelig fra: 2011-06-27 Laget: 2011-06-13 Sist oppdatert: 2024-03-15bibliografisk kontrollert
3. HAHAHA, HEHEHE,HIHIHI or HKHKHK: influence of position and composition of histidine containing tags on biodistribution of [99mTc(CO)3]+-labeled Affibody molecules
Åpne denne publikasjonen i ny fane eller vindu >>HAHAHA, HEHEHE,HIHIHI or HKHKHK: influence of position and composition of histidine containing tags on biodistribution of [99mTc(CO)3]+-labeled Affibody molecules
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-116889 (URN)
Merknad

QS 2013

Tilgjengelig fra: 2013-01-29 Laget: 2013-01-29 Sist oppdatert: 2022-06-24bibliografisk kontrollert
4. [99mTc(CO)3]+-(HE)3-Z IGF1R45514551: a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours.
Åpne denne publikasjonen i ny fane eller vindu >>[99mTc(CO)3]+-(HE)3-Z IGF1R45514551: a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours.
Vise andre…
2013 (engelsk)Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, nr 3, s. 439-449Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

PURPOSE: Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule (111)In-DOTA-His(6)-Z(IGF1R:4551). The use of (99m)Tc instead of (111)In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His(6) tag in Z(IGF1R:4551) would permit its convenient purification using IMAC, enable labelling with [(99m)Tc(CO)(3)](+), and improve its biodistribution. METHODS: Z(IGF1R:4551) was expressed with a HEHEHE tag in the N terminus. The resulting (HE)(3)-Z(IGF1R:4551) construct was labelled with [(99m)Tc(CO)(3)](+). Targeting of IGF-1R-expressing cells using [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was evaluated in vitro and in vivo. RESULTS: (HE)(3)-Z(IGF1R:4551) was stably labelled with (99m)Tc with preserved specific binding to IGF-1R-expressing DU-145 prostate cancer cells in vitro. In mice, [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung and salivary gland). [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) demonstrated 3.6-fold lower accumulation in the liver and spleen than (111)In-DOTA-Z(IGF1R:4551). In NMRI nu/nu mice with DU-145 prostate cancer xenografts, the tumour uptake was 1.32 ± 0.11 %ID/g and the tumour-to-blood ratio was 4.4 ± 0.3 at 8 h after injection. The xenografts were visualized using a gamma camera 6 h after injection. CONCLUSION: (99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) is a promising candidate for visualization of IGF-1R expression in malignant tumours.

Emneord
Affibody molecules, affinity proteins, radionuclide molecular imaging, intracellular targeting, EGFR, HER2, IGF-1R
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-116887 (URN)10.1007/s00259-012-2284-8 (DOI)000314676900015 ()23179942 (PubMedID)2-s2.0-84873997284 (Scopus ID)
Forskningsfinansiär
Swedish Research Council
Merknad

QC 20130214

Tilgjengelig fra: 2013-01-29 Laget: 2013-01-29 Sist oppdatert: 2024-03-15bibliografisk kontrollert
5. Step-wise down regulationof the epidermal growth factor receptor by affinity-based intracellular redirection
Åpne denne publikasjonen i ny fane eller vindu >>Step-wise down regulationof the epidermal growth factor receptor by affinity-based intracellular redirection
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-116890 (URN)
Merknad

QS 2013

Tilgjengelig fra: 2013-01-29 Laget: 2013-01-29 Sist oppdatert: 2022-06-24bibliografisk kontrollert

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