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, ZHER2:342 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 clinical utility of a human epidermal growth factor receptor 2 (HER2)–targeting Affibody molecule for detection and characterization of HER2-positive lesions was investigated in patients with recurrent metastatic breast cancer. Methods: Three patients received ¹¹¹In- or ⁶⁸Ga-labeled DOTA⁰-Z_{HER2:342-pep2} (ABY-002). γ-Camera, SPECT, or PET/CT images were compared with earlier ¹⁸F-FDG PET/CT results. Results: Administration of radiolabeled ABY-002 was well tolerated. Blood kinetics of radiolabeled ABY-002 showed a first half-life of 4–14 min, second half-life of 1–4 h, and third half-life of 12–18 h. Radiolabeled ABY-002 detected 9 of 11 ¹⁸F-FDG–positive metastases as early as 2–3 h after injection. Conclusion: Molecular imaging using ¹¹¹In- or ⁶⁸Ga-labeled ABY-002 has the potential to localize metastatic lesions in vivo, adds qualitative information not available today by conventional imaging techniques, and may allow the HER2 status to be determined for metastases not amenable to biopsy. To our knowledge, this is the first report on clinical imaging data obtained with a non–immunoglobulin-based scaffold protein.

Purpose: Positron Emission Tomography (PET) imaging of HER2 expression could potentially be used to select patients for HER2-targed therapy, predict response based on uptake and be used for monitoring. In this phase I/II study the HER2-binding Affibody molecule ABY-025 was labeled with ⁶⁸Ga-gallium ([⁶⁸Ga]ABY-025) for PET to study effect of peptide mass, test-retest variability and correlation of quantified uptake in tumors to histopathology.

A radiolabeled anti-HER2 Affibody molecule (Z_HER2:342) targets HER2-expressing xenografts with high selectivity and gives good imaging contrast. However, the small size (∼7 kDa) results in rapid glomerular filtration and high renal accumulation of radiometals, thus excluding targeted therapy. Here, we report that reversible binding to albumin efficiently reduces the renal excretion and uptake, enabling radiometal-based nuclide therapy. The dimeric Affibody molecule (Z_HER2:342)₂ was fused with an albumin-binding domain (ABD) conjugated with the isothiocyanate derivative of CHX-A″-DTPA and labeled with the low-energy β-emitter ¹⁷⁷Lu. The obtained conjugate [CHX-A″-DTPA-ABD-(Z_HER2:342)₂] had a dissociation constant of 18 pmol/L to HER2 and 8.2 and 31 nmol/L for human and murine albumin, respectively. The radiolabeled conjugate displayed specific binding to HER2-expressing cells and good cellular retention in vitro. In vivo, fusion with ABD enabled a 25-fold reduction of renal uptake in comparison with the nonfused dimer molecule (Z_HER2:342)₂. Furthermore, the biodistribution showed high and specific uptake of the conjugate in HER2-expressing tumors. Treatment of SKOV-3 microxenografts (high HER2 expression) with 17 or 22 MBq ¹⁷⁷Lu-CHX-A″-DTPA-ABD-(Z_HER2:342)₂ completely prevented formation of tumors, in contrast to mice given PBS or 22 MBq of a radiolabeled non–HER2-binding Affibody molecule. In LS174T xenografts (low HER2 expression), this treatment resulted in a small but significant increase of the survival time. Thus, fusion with ABD improved the in vivo biodistribution, and the results highlight ¹⁷⁷Lu-CHX-A″-DTPA-ABD-(Z_HER2:342)₂ as a candidate for treatment of disseminated tumors with a high level of HER2 expression. [Cancer Res 2007;67(6):2773–82]

The Affibody molecule ZHER2:342-pep2, site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-ZHER2:342-pep2 folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of 111In was achieved within 30 min at low (room temperature) and high temperatures (up to 90°C). Tumor uptake of 111In-DOTA-ZHER2:342-pep2 was specific for HER2-positive xenografts. A high tumor uptake of 23% injected activity per gram tissue, a tumor-to-blood ratio of >7.5, and high-contrast gamma camera images were obtained already 1 h after injection. Pretreatment with Herceptin did not interfere with tumor targeting, whereas degradation of HER2 using the heat shock protein 90 inhibitor 17-allylamino-geldanamycin before administration of 111In-DOTA-ZHER2:342-pep2 obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-ZHER2:342-pep2 has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas. [Cancer Res 2007;67(5):2178–86]