title: Evaluation of a maleimido derivative of CHX-A'' DTPA for site-specific labeling of affibody molecules.
abstract: Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A'' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A'' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A'' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A'' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.
authors.affiliation: Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
authors.forename: Heng
authors.initials: H
authors.lastname: Xu
authors.forename: Helena
authors.initials: H
authors.lastname: Wållberg
authors.forename: Sara
authors.initials: S
authors.lastname: Ahlgren
authors.forename: Magnus
authors.initials: M
authors.lastname: Hjertman
authors.forename: Anna
authors.initials: A
authors.lastname: Sjöberg
authors.forename: Mattias
authors.initials: M
authors.lastname: Sandström
authors.forename: Lars
authors.initials: L
authors.lastname: Abrahmsén
authors.forename: Martin W
authors.initials: MW
authors.lastname: Brechbiel
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006573:Heterocyclic Compounds, 1-Ring / Q000378:metabolism; D006801:Humans; D007205:Indium Radioisotopes; D017879:Isothiocyanates / Q000737:chemistry* / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry*; D051379:Mice; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D014018:Tissue Distribution
chemical_list: D000906:Antibodies; D002614:Chelating Agents; D006573:Heterocyclic Compounds, 1-Ring; D007205:Indium Radioisotopes; D017879:Isothiocyanates; D008301:Maleimides; D004369:Pentetic Acid; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C515865:Z(HER2.4)2 affibody; C085678:N-(2-amino-3-(4-isothiocyanatophenyl)propyl)cyclohexane-1,2-diamine-N,N',N',N'',N''-pentaacetic acid; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; C043592:maleimide; C524056:Erbb2 protein, mouse