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Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with In-111 using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
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2012 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no 3, 481-492 p.Article in journal (Refereed) Published
Abstract [en]

Purpose In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer. Methods A synthetic variant of the anti-HER2 Z(HER2:342) Affibody molecule, Z(HER2:S1), was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with In-111, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts. Results The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1), respectively. A comparative study of In-111-labelled DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1) in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. In-111-NODAGA-Z(HER2:S1) had the most rapid clearance from blood and healthy tissues. In-111-NOTA-Z(HER2:S1) showed high hepatic uptake and was excluded from further evaluation. In-111-DOTA-Z(HER2:S1) and In-111-NODAGAZHER2: S1 demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of In-111-NODAGA-Z(HER2:S1), 5.6 +/- 0.4% ID/g, was significantly lower than the uptake of In-111-DOTA-Z(HER2:S1), 7.4 +/- 0.5% ID/g, presumably because of lower bioavailability due to more rapid clearance. In-111-NODAGA-Z(HER2:S1) provided higher tumour-to-blood ratio, but somewhat lower tumour-to-liver, tumour-to-spleen and tumour-to-bone ratios. Conclusion Since distant prostate cancer metastases are situated in bone or bone marrow, the higher tumour-to-bone ratio is the most important. This renders In-111-DOTA-Z(HER2:S1) a preferable agent for imaging of HER2 expression in disseminated prostate cancer.

Place, publisher, year, edition, pages
2012. Vol. 39, no 3, 481-492 p.
Keyword [en]
HER2 expression, Affibody molecule, Radionuclide molecular imaging, Prostate cancer, Macrocyclic chelators
National Category
Radiology, Nuclear Medicine and Medical Imaging
URN: urn:nbn:se:kth:diva-93940DOI: 10.1007/s00259-011-1992-9ISI: 000302287200015ScopusID: 2-s2.0-84859635393OAI: diva2:524748
Swedish Research Council
QC 20120503Available from: 2012-05-03 Created: 2012-05-03 Last updated: 2014-09-29Bibliographically approved
In thesis
1. Site-specific labeling of affinity molecules for in vitro and in vivo studies
Open this publication in new window or tab >>Site-specific labeling of affinity molecules for in vitro and in vivo studies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant.

In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast.

Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. 83 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:14
Affibody molecules, molecular imaging, site-specific labeling, solid phase peptide synthesis, IgG-binding domains, photoconjugation.
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-152349 (URN)978-91-7595-252-9 (ISBN)
Public defence
2014-10-17, FR4, Oscar Klein, AlbaNova Universitetscenter, Roslagstullsbacken 21,, Stockholm, 10:00 (English)

QC 20140929

Available from: 2014-09-29 Created: 2014-09-25 Last updated: 2014-10-28Bibliographically approved

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