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Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi, Enheten för biomedicinsk strålningsvetenskap.ORCID-id: 0000-0001-6120-2683
Affibody AB, Stockholm, Sweden.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi, Enheten för biomedicinsk strålningsvetenskap.
Vise andre og tillknytning
2008 (engelsk)Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, nr 1, s. 235-243Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.

sted, utgiver, år, opplag, sider
2008. Vol. 19, nr 1, s. 235-243
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-104530DOI: 10.1021/bc700307yISI: 000252520300030PubMedID: 18163536OAI: oai:DiVA.org:uu-104530DiVA, id: diva2:219999
Tilgjengelig fra: 2009-05-29 Laget: 2009-05-28 Sist oppdatert: 2017-12-13bibliografisk kontrollert
Inngår i avhandling
1. Molecular Radionuclide Imaging Using Site-specifically Labelled Recombinant Affibody Molecules: Preparation and Preclinical Evaluation
Åpne denne publikasjonen i ny fane eller vindu >>Molecular Radionuclide Imaging Using Site-specifically Labelled Recombinant Affibody Molecules: Preparation and Preclinical Evaluation
2010 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Radionuclide molecular imaging is an emerging multidisciplinary technique that is used in modern medicine to visualise diseases at cellular and molecular levels. This thesis is based on five papers (I-V) and focuses on the development of site-specific radiolabelled recombinant anti-HER2 Affibody molecules and preclinical evaluations in vitro and in vivo of the labelled conjugates. This work is part of a preclinical development of an Affibody molecule-based tracer for molecular imaging of HER2 expressing tumours.

Papers I and II report the evaluation of the Affibody molecule ZHER2:2395-C, site-specifically labelled with the radiometals 111In (for SPECT) and 57Co (as a surrogate for 55Co, suitable for PET applications) using a thiol reactive DOTA derivative as a chelator. Both conjugates demonstrated very suitable biodistribution properties, enabling high contrast imaging just a few hours after injection.

Papers III and IV report the development and optimization of a technique for site-specific labelling of ZHER2:2395-C with 99mTc using an N3S chelating peptide sequence. 99mTc-ZHER2:2395-C demonstrated high and specific tumour uptake and rapid clearance of non-bound tracer from the blood, resulting in high tumour-to-non-tumour ratios shortly after injection, enabling high contrast imaging. In addition, in the study described in paper IV, freeze-dried kits previously developed for 99mTc-labelling were optimised, resulting in the development of a kit in which all the reagents and protein needed for labelling of ZHER2:2395-C with 99mTc were contained in a single vial.

Paper V reports the evaluation of an anti-HER2 Affibody molecule, ABY-025, with a fundamentally re-engineered scaffold. Despite the profound re-engineering, the biodistribution pattern of 111In-ABY-025 was very similar to that of two variants of the parental molecule.

It seems reasonable to believe that these results will also be applicable to Affibody molecules towards other targets. Hopefully, this work will also be helpful in the development of other small proteinaceous tracers.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2010. s. 70
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 553
Emneord
molecular radionuclide imaging, Affibody molecules, HER2, cancer detection, radiolabelling, technetium, indium, cobalt, SPECT, PET
HSV kategori
Forskningsprogram
Biomedicinsk strålningsvetenskap; Medicinsk vetenskap
Identifikatorer
urn:nbn:se:uu:diva-122177 (URN)978-91-554-7787-5 (ISBN)
Disputas
2010-05-22, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2010-04-29 Laget: 2010-04-07 Sist oppdatert: 2010-04-29bibliografisk kontrollert

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