Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake.
OBJECTIVE
Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, (99m)Tc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering.


METHODS
Anti-HER2 Z(HER2:342) Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with (99m)Tc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, (99m)Tc-maEEE-Z(HER2:342,) in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, (99m)Tc-maESE-Z(HER2:342,) was compared with radioiodinated Z(HER2:342).


RESULTS
All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 +/- 5, 68 +/- 21 and 71 +/- 10%IA/g, for(99m)Tc-maESE-Z(HER2:342), (99m)Tc-maEES-Z(HER2:342) and (99m)Tc-maSEE-Z(HER2:342), respectively, were significantly reduced in comparison with (99m)Tc-maEEE-Z(HER2:342) (102 +/- 13%IA/g). For (99m)Tc-maESE-Z(HER2:342), a tumour uptake of 9.6 +/- 1.8%IA/g and a tumour-to-blood ratio of 58 +/- 6 were reached at 4 h p.i.


CONCLUSIONS
A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of (99m)Tc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.
Ekblad
Torun
T
School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Tran
Thuy
T
Orlova
Anna
A
Widström
Charles
C
Feldwisch
Joachim
J
Abrahmsén
Lars
L
Wennborg
Anders
A
Karlström
Amelie Eriksson
AE
Tolmachev
Vladimir
V
D000818:Animals; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D018698:Glutamic Acid; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D015609:Organotechnetium Compounds / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000493:pharmacokinetics*; D012694:Serine; D013194:Staining and Labeling; D014018:Tissue Distribution; D014183:Transplantation, Heterologous
D002614:Chelating Agents; D015609:Organotechnetium Compounds; D010455:Peptides; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D012694:Serine