Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine.
The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics.


METHODS
A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts.


RESULTS
(99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys.


CONCLUSIONS
The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.
Ahlgren
Sara
S
Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
Wållberg
Helena
H
Tran
Thuy A
TA
Widström
Charles
C
Hjertman
Magnus
M
Abrahmsén
Lars
L
Berndorff
Dietmar
D
Dinkelborg
Ludger M
LM
Cyr
John E
JE
Feldwisch
Joachim
J
Orlova
Anna
A
Tolmachev
Vladimir
V
D000230:Adenocarcinoma / Q000000981:diagnostic imaging* / Q000378:metabolism*; D000818:Animals; D003545:Cysteine / Q000493:pharmacokinetics; D016503:Drug Delivery Systems / Q000379:methods; D005260:Female; D008657:Metabolic Clearance Rate; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D016513:Mice, SCID; D015336:Molecular Probe Techniques; D009928:Organ Specificity; D015609:Organotechnetium Compounds / Q000493:pharmacokinetics*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D014018:Tissue Distribution; D015899:Tomography, Emission-Computed, Single-Photon / Q000379:methods*
D003545:Cysteine; D015609:Organotechnetium Compounds; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C542077:(99m)Tc-Z(HER2:2395)-Cys