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- Hofström, Camilla, 1979-
(författare)
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Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Hofström, Camilla, et al.
(författare)
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HAHAHA, HEHEHE, HIHIHI, or HKHKHK : Influence of Position and Composition of Histidine Containing Tags on Biodistribution of [Tc-99m(CO)(3)](+)-Labeled Affibody Molecules
- 2013
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 56:12, s. 4966-4974
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Tidskriftsartikel (refereegranskat)abstract
- Engineered affibody molecules can be used for high contrast in vivo molecular imaging. Extending a recombinantly produced HER2 binding affibody molecule with a hexa-histidine tag allows for convenient purification by immobilized metal-ion affinity chromatography and labeling with [Tc-99m(CO)(3)](+) but increases radioactivity uptake in the liver. To investigate the impact of charge, lipophilicity, and position on biodistribution, 10 variants of a histidine-based tag was attached to a HER2 binding affibody molecule. The biochemical properties and the HER2 binding affinity appeared to be similar for all variants. In vivo, positive charge promoted liver uptake. For N-terminally placed tags, promoted liver uptake and decreased kidney uptake. Kidney uptake was higher for C-terminally placed tags compared to their N-terminal counterparts. The variant with the amino acid composition HEHEHE placed in the N-terminus gave the lowest nonspecific uptake.
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| 5. |
- Hofström, Camilla, et al.
(författare)
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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
- 2011
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 54:11, s. 3817-3826
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Hueting, David A., et al.
(författare)
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Design, structure and plasma binding of ancestral β-CoV scaffold antigens
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- We report the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interact with plasma of patients recovered from COVID-19 but do not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yield high resolution structures (2.6–2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. The structures reveal an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together. We show that AnSA-5 can induce and boost a broad-spectrum immune response against the wild-type RBD as well as circulating variants of concern in an immune organoid model derived from tonsils. Finally, we highlight how AnSAs are potent scaffolds by replacing the ancestral RBD with the wild-type sequence, which restores ACE2 binding and increases the interaction with convalescent plasma.
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| 7. |
- Larsson, Karin, et al.
(författare)
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Novel antigen design for the generation of antibodies to G-protein-coupled receptors
- 2011
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 370:1-2, s. 14-23
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.
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| 9. |
- Lindberg, Hanna, et al.
(författare)
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Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus
- 2012
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Ingår i: Tumor Biology. - : Springer. - 1010-4283 .- 1423-0380. ; 33:3, s. 641-651
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide Tc-99m. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been 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 immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with Tc-99m. Tc-99m-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, Tc-99m-Z(HER2:2395)-VDC and Tc-99m-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of Tc-99m-Z(HER2:2395)-VDC, but it was substantially higher than uptake of Tc-99m-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of Tc-99m was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of Tc-99m labelling due to a partial loss of site-specificity of nuclide chelation.
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