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Molecular Design and Optimization of Tc-99m-Labeled Recombinant Affibody Molecules Improves Their Biodistribution and Imaging Properties
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
Vise andre og tillknytning
2011 (engelsk)Inngår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 52, nr 3, s. 461-469Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). Tc-99m as a label offers advantages in clinical practice, and earlier studies demonstrated that Tc-99m-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting. Methods: A series of recombinant derivatives of the HER2-binding Z(HER2:342) Affibody molecule with a C-terminal chelating sequence, -GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with Tc-99m and evaluated in vitro and in vivo. Results: All variants were stably labeled with Tc-99m, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, Tc-99m-Z(HER2:V2), with the C-terminal chelating sequence -GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. Tc-99m-Z(HER2:V2) displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 +/- 4.0 and 7.7 +/- 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake. Conclusion: These results demonstrate that the biodistribution properties of recombinant Tc-99m-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. Tc-99m-Z(HER2:V2) is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.

sted, utgiver, år, opplag, sider
2011. Vol. 52, nr 3, s. 461-469
Emneord [en]
Affibody molecule, Tc-99m, molecular imaging, HER2, C-terminal cysteine
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-31327DOI: 10.2967/jnumed.110.083592ISI: 000287578200029Scopus ID: 2-s2.0-79952798198OAI: oai:DiVA.org:kth-31327DiVA, id: diva2:404680
Merknad
QC 20110318Tilgjengelig fra: 2011-03-18 Laget: 2011-03-14 Sist oppdatert: 2017-12-11bibliografisk kontrollert
Inngår i avhandling
1. Design and Evaluation of Radiolabeled Affibody Tracers for Imaging of HER2-expressing Tumors
Åpne denne publikasjonen i ny fane eller vindu >>Design and Evaluation of Radiolabeled Affibody Tracers for Imaging of HER2-expressing Tumors
2011 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The growing understanding of tumor biology and the identification of tumor specificgenetic and molecular alterations, such as the overexpression of human epidermal growthfactor receptor 2 (HER2), opens up for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools usedto identify patients that are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising non-invasive method to visualize and characterize the expression of such targets. This thesis, based on five papers, is focused on the development of radiolabeled Affibody molecules for imaging of HER2-expression in malignant tumors. Affibody molecules, which represent a rather novel class of affinity proteins developed by combinatorial protein engineering of the protein A derived Z-domain, display manyfeatures that make them promising tracers for molecular imaging applications. The aim of the work presented here was to further develop the tracer format for improved in vivo properties and flexibility in the choice of radionuclide.

In paper I, the development of an assay that enables quantitative studies of the internalization rate and cellular processing of high affinity Affibody molecules is described. The assay was applied to a HER2-binding Affibody variant that was efficiently retained by HER2-expressing cells, although characterized by a slow internalization rate. This may have implications for the choice of label for Affibody molecules since high affinity to the target may be equally, or more, important for good imaging quality than residualizing properties of the radiolabel. In paper II, a HER2-binding Affibody molecule and the monoclonal antibody trastuzumab were labeled with positron emitting 124I, for a head-to-head in vivocomparison of the two tracer formats. The effects of tracer size and presence of an Fc region on the biodistribution profile were investigated. In paper III, a HER2-binding Affibody molecule was site-specifically labeled with radiocobalt and evaluated in vitro and in vivo.A head-to-head in vivo comparison with the well-studied 111In-labeled counterpart was performed, revealing promising potential for the cobalt-labeled molecule as a PET-tracerfor visualization of HER2. Paper IV describes the in vitro and in vivo evaluation of a panel of Affibody molecules with different C-terminal peptide-based chelators for the coordination of 99mTc. Even small changes in the C-terminal sequence had appreciable impact on the biodistribution of the Affibody molecules and by optimizing the design of the chelator, the kidney uptake of 99mTc could be significantly reduced. Finally, in paper V we describe the development of a HER2-targeting Affibody variant equipped with a Sel-tag for site-specific labeling with the short-lived positron emitter 11C. This novel Affibody tracer could be used to image HER2-expressing tumors in vivo within one hour after injection.

Taken together, Affibody molecules show great promise as targeting tracers for radionuclide molecular imaging of HER2. Careful design and optimization of the tracer protein is important and can be used to improve the biodistribution and targeting properties of Affibody molecules.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2011. s. xi, 79
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011:21
Emneord
Affibody molecule, radionuclide molecular imaging, HER2, radiotracer, SPECT, PET, biodistribution, protein engineering, radiolabeling
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-40890 (URN)978-91-7501-092-2 (ISBN)
Disputas
2011-10-21, FD5, AlbaNova University center, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad
QC 20110922Tilgjengelig fra: 2011-09-22 Laget: 2011-09-22 Sist oppdatert: 2011-09-22bibliografisk kontrollert

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