title: Molecular design and optimization of 99mTc-labeled recombinant affibody molecules improves their biodistribution and imaging properties.
abstract: Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). (99m)Tc as a label offers advantages in clinical practice, and earlier studies demonstrated that (99m)Tc-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting.
  
  
  METHODS
  A series of recombinant derivatives of the HER2-binding Z(HER2)(:342) Affibody molecule with a C-terminal chelating sequence, -GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with (99m)Tc and evaluated in vitro and in vivo.
  
  
  RESULTS
  All variants were stably labeled with (99m)Tc, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, (99m)Tc-Z(HER2)(:V2), with the C-terminal chelating sequence -GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. (99m)Tc-Z(HER2)(:V2) displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 ± 4.0 and 7.7 ± 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake.
  
  
  CONCLUSIONS
  These results demonstrate that the biodistribution properties of recombinant (99m)Tc-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. (99m)Tc-Z(HER2)(:V2) is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.
authors.affiliation: School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Wållberg
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.forename: Mohammed
authors.initials: M
authors.lastname: Altai
authors.forename: Seyed Jalal
authors.initials: SJ
authors.lastname: Hosseinimehr
authors.forename: Charles
authors.initials: C
authors.lastname: Widström
authors.forename: Jennie
authors.initials: J
authors.lastname: Malmberg
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
mesh_terms: D000818:Animals; D015195:Drug Design; D007089:Image Enhancement / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D008657:Metabolic Clearance Rate; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D009374:Neoplasms, Experimental / Q000000981:diagnostic imaging* / Q000378:metabolism*; D009928:Organ Specificity; D011877:Radionuclide Imaging; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013667:Technetium / Q000493:pharmacokinetics*; D014018:Tissue Distribution
chemical_list: D011993:Recombinant Fusion Proteins; C533351:affibody (ZHER2-342)2; D013667:Technetium