title: Tumor targeting using affibody molecules: interplay of affinity, target expression level, and binding site composition.
abstract: Radionuclide imaging of cancer-associated molecular alterations may contribute to patient stratification for targeting therapy. Scaffold high-affinity proteins, such as Affibody molecules, are a new, promising class of probes for in vivo imaging.
  
  
  METHODS
  The effects of human epidermal growth factor receptor 2 (HER2) affinity and binding site composition of HER2-binding Affibody molecules, and of the HER2 density on the tumor targeting, were studied in vivo. The tumor uptake and tumor-to-organ ratios of Affibody molecules with moderate (dissociation constant [K(D)] = 10(-9) M) or high (K(D) = 10(-10) M) affinity were compared between tumor xenografts with a high (SKOV-3) and low (LS174T) HER2 expression level in BALB/C nu/nu mice. Two Affibody molecules with similar affinity (K(D) = 10(-10) M) but having alternative amino acids in the binding site were compared.
  
  
  RESULTS
  In SKOV-3 xenografts, uptake was independent of affinity at 4 h after injection, but high-affinity binders provided 2-fold-higher tumor radioactivity retention at 24 h. In LS174T xenografts, uptake of high-affinity probes was already severalfold higher at 4 h after injection, and the difference was increased at 24 h. The clearance rate and tumor-to-organ ratios were influenced by the amino acid composition of the binding surface of the tracer protein.
  
  
  CONCLUSIONS
  The optimal affinity of HER2-binding Affibody molecules depends on the expression of a molecular target. At a high expression level (>10(6) receptors per cell), an affinity in the low-nanomolar range is sufficient. At moderate expression, subnanomolar affinity is desirable. The binding site composition can influence the imaging contrast. This information may be useful for development of imaging agents based on scaffold affinity proteins.
authors.affiliation: Department of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden. vladimir.tolmachev@bms.uu.se
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
authors.forename: Thuy A
authors.initials: TA
authors.lastname: Tran
authors.forename: Daniel
authors.initials: D
authors.lastname: Rosik
authors.forename: Anna
authors.initials: A
authors.lastname: Sjöberg
authors.forename: Lars
authors.initials: L
authors.lastname: Abrahmsén
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005260:Female; D007205:Indium Radioisotopes; D007553:Isotope Labeling; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000000981:diagnostic imaging*; D011877:Radionuclide Imaging; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*
chemical_list: D007205:Indium Radioisotopes; D018719:Erb-b2 Receptor Tyrosine Kinases