Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules

Sara Ahlgren, Anna Orlova, Daniel Rosik, Mattias Sandström, Anna Sjöberg, Barbro Baastrup, Olof Widmark, Gunilla Fant, Joachim Feldwisch, Vladimir Tolmachev

Research output: Contribution to journalArticlepeer-review

85 Citations (Scopus)


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 111in-labeling of anti-HER2 Affibody molecules HisS6-ZHER2.342-Cys and ZHER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His6-ZHER2:342-Cys and 27 pM for ZHER2:2395-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 111In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111In-[MMA-DOTA-Cys 61]-ZHER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111In-[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.

Original languageEnglish
Pages (from-to)235-243
Number of pages9
JournalBioconjugate Chemistry
Issue number1
Publication statusPublished - Jan 2008
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Fingerprint Dive into the research topics of 'Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules'. Together they form a unique fingerprint.

Cite this