The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z HER2:342 showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging. (Cancer Res 2006; 66(8): 4339-48)

The organs at risk in radionuclide therapy with 177 Lu-octreotate are the bone marrow and the kidneys. The primary aim of this study was to develop an individualized dosimetry protocol for the bone marrow. The secondary aim was to identify those patients, undergoing fractionated therapy with 7.4 GBq/cycle, who first reached an accumulated dose of either 2 Gy to the bone marrow or 23 Gy to the kidneys. Methods: Two hundred patients with metastatic neuroendocrine tumors with high somatostatin receptor expression were included. After the administration of 7.4 GBq of 177 Lu-octreotate, blood samples were drawn 6 times within the first 24 h. In 50 patients, additional blood samples were obtained at 96 and 168 h. Moreover, urine was collected from 30 patients during the first 24 h. Planar whole-body and SPECT/CT images over the abdomen were acquired at 24, 96, and 168 h after the infusion. Calculation of the absorbed radiation dose to the bone marrow was based on blood and urinary activity curves combined with organ-based analysis of the whole-body images. The absorbed dose to the kidney was calculated from the pharmacokinetic data obtained from SPECT/CT. Results: For a single cycle of 7.4 GBq, the absorbed dose to the bone marrow and the kidney ranged from 0.05 to 0.4 Gy and from 2 to 10 Gy, respectively. In 197 of 200 patients, the kidneys accumulated an absorbed dose of 23 Gy before the bone marrow reached 2 Gy. Between 2 and 10 cycles of 177 Lu-octreotate could be administered before the upper dose limit for the individual patient was reached. Conclusion: A method based on repeated whole-body imaging in combination with blood and urinary activity data over time was developed to determine the absorbed dose to the bone marrow. The dose-limiting organ was the kidney in 197 of 200 patients. In 50% of the patients, more than 4 cycles of 7.4 GBq of 177 Lu-octreotate could be administered, whereas 20% of the subjects were treated with fewer than 4 cycles. Individualized absorbed dose calculation is essential to optimize the therapy.

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 sitespecific 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 g-camera imaging studies were performed in mice bearing HER2-expressing xenografts. Results: 99m TcHis 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 6 2.5 (mean 6 SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 6 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 6 24 and 121 6 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical g-camera 1 h after injection of 99m Tc-Z HER2:2395 -Cys. Conclusion: The Affibody molecule 99m Tc-Z HER2:2395 -Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled site-specific (99m)Tc labelling of the Z(HER2:342) Affibody molecule with preserved targeting capacity.

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 6 4.0 and 7.7 6 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. Conclusion: 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.