Change search
ReferencesLink to record
Permanent link

Direct link
Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of In-111-Labeled Synthetic Anti-HER2 Affibody Molecules
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Show others and affiliations
2012 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 8, 1661-1670 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with Tc-99m at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K-D values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1) K50-DOTA showed a slightly lower melting point (57 degrees C) compared to DOTA-A1-Z(HER2:S1) (64 degrees C) and DOTA-K5S-Z(HER2:S1) (62 degrees C), but all variants showed good refolding properties after heat treatment All conjugates were successfully labeled with In-III resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in L5174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1) and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

Place, publisher, year, edition, pages
2012. Vol. 23, no 8, 1661-1670 p.
Keyword [en]
Prostate-Cancer Xenografts, Metastatic Breast-Cancer, Monoclonal-Antibody, Binding-Proteins, Imaging Agents, Ovarian-Cancer, In-Vivo, Receptor, Her2, Overexpression
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-102343DOI: 10.1021/bc3002369ISI: 000307487300016ScopusID: 2-s2.0-84865145661OAI: diva2:552448
Swedish Research Council

QC 20120914

Available from: 2012-09-14 Created: 2012-09-14 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

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Search in DiVA

By author/editor
Perols, AnnaEriksson Karlström, Amelie
By organisation
Molecular Biotechnology
In the same journal
Bioconjugate chemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 42 hits
ReferencesLink to record
Permanent link

Direct link