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Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
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2011 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 22, no 5, 894-902 p.Article in journal (Refereed) Published
Abstract [en]

Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:239S) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:239S) with In-111 gave a yield of >95% after 20 min at 60 degrees C. In vitro cell tests demonstrated specific binding of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) was 8.2 +/- 0.9% IA/g and the tumor-to-blood ratio was 31 +/- 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) and [In-111-MMA-DOTA-Cys(61)]-Z(HER2:239S) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.

Place, publisher, year, edition, pages
2011. Vol. 22, no 5, 894-902 p.
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology Organic Chemistry
URN: urn:nbn:se:kth:diva-34392DOI: 10.1021/bc100470xISI: 000290691600007ScopusID: 2-s2.0-79959574104OAI: diva2:420996
QC 20110607Available from: 2011-06-07 Created: 2011-06-07 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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