title: Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules.
abstract: 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.
authors.affiliation: Division of Nuclear Medicine, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
authors.forename: Sara
authors.initials: S
authors.lastname: Ahlgren
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.forename: Daniel
authors.initials: D
authors.lastname: Rosik
authors.forename: Mattias
authors.initials: M
authors.lastname: Sandström
authors.forename: Anna
authors.initials: A
authors.lastname: Sjöberg
authors.forename: Barbro
authors.initials: B
authors.lastname: Baastrup
authors.forename: Olof
authors.initials: O
authors.lastname: Widmark
authors.forename: Gunilla
authors.initials: G
authors.lastname: Fant
authors.forename: Joachim
authors.initials: J
authors.lastname: Feldwisch
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
mesh_terms: D000818:Animals; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006639:Histidine / Q000378:metabolism; D007205:Indium Radioisotopes; D008301:Maleimides / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D009369:Neoplasms / Q000000981:diagnostic imaging; D010455:Peptides / Q000737:chemistry*; D011877:Radionuclide Imaging; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D014183:Transplantation, Heterologous
chemical_list: D002614:Chelating Agents; D003545:Cysteine; D006573:Heterocyclic Compounds, 1-Ring; D006639:Histidine; D007205:Indium Radioisotopes; D008301:Maleimides; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C520644:1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; C043592:maleimide