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.Experimental design: Sixteen women with known metastatic breast cancer and on-going treatment were included and underwent FDG PET/CT to identify viable metastases. After iv injection of 212±46 MBq [68Ga]ABY-025 whole-body PET was performed at 1, 2 and 4 h. In the first 10 patients (6 with HER2-positive and 4 with HER2-negative primary tumors), [68Ga]ABY-025 PET/CT with two different doses of injected peptide was performed one week apart. In the last six patients (5 HER2-positive and 1 HER2-negative primary tumors), repeated [68Ga]ABY-025 PET were performed one week apart as a test-retest of uptake in individual lesions. Biopsies from 16 metastases in 12 patients were collected for verification of HER2 expression by immunohistochemistry and in-situ hybridization.Results: Imaging 4h after injection with high peptide content discriminated HER2-positive metastases best (p<0.01). PET SUV correlated with biopsy HER2-scores (r=0.91, p<0.001). Uptake was five times higher in HER2-positive than in HER2-negative lesions with no overlap (p=0.005). The test-retest intra-class correlation was r=0.996. [68Ga]ABY-025 PET correctly identified conversion and mixed expression of HER2 and targeted treatment was changed in 3 of the 16 patients.Conclusion: [68Ga]ABY-025 PET accurately quantifies whole-body HER2-receptor status in metastatic breast cancer.

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 111 In-or 68 Ga-labeled DOTA 0 -Z HER2:342-pep2 (ABY-002). g-Camera, SPECT, or PET/CT images were compared with earlier 18 F-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 18 F-FDG-positive metastases as early as 2-3 h after injection. Conclusion: Molecular imaging using 111 In-or 68 Ga-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.

The expression status of human epidermal growth factor receptor type 2 (HER2) predicts the response of HER2-targeted therapy in breast cancer. ABY-025 is a small reengineered Affibody molecule targeting a unique epitope of the HER2 receptor, not occupied by current therapeutic agents. This study evaluated the distribution, safety, dosimetry, and efficacy of 111 In-ABY-025 for determining the HER2 status in metastatic breast cancer. Methods: Seven patients with metastatic breast cancer and HER2-positive (n 5 5) or -negative (n 5 2) primary tumors received an intravenous injection of approximately 100 μg (∼140 MBq) of 111 In-ABY-025. Planar γ-camera imaging was performed after 30 min, followed by SPECT/CT after 4, 24, and 48 h. Blood levels of radioactivity, antibodies, shed serum HER2, and toxicity markers were evaluated. Lesional HER2 status was verified by biopsies. The metastases were located by 18 F-FDG PET/CT 5 d before 111 In-ABY-025 imaging. Results: Injection of 111 In-ABY-025 yielded a mean effective dose of 0.15 mSv/MBq and was safe, well tolerated, and without drug-related adverse events. Fast blood clearance allowed high-contrast HER2 images within 4-24 h. No anti-ABY-025 antibodies were observed. When metastatic uptake at 24 h was normalized to uptake at 4 h, the ratio increased in HER2-positive metastases and decreased in negative ones (P , 0.05), with no overlap and confirmation by biopsies. In 1 patient, with HER2-positive primary tumor, 111 In-ABY-025 imaging correctly suggested a HER2-negative status of the metastases. The highest normal-tissue uptake was in the kidneys, followed by the liver and spleen. Conclusion: 111 In-ABY-025 appears safe for use in humans and is a promising noninvasive tool for discriminating HER2 status in metastatic breast cancer, regardless of ongoing HER2-targeted antibody treatment. Today,t reatment of breast cancer is based on the biologic profile of the individual tumor. Knowledge of the human epidermal growth factor receptor type 2 (HER2) status is crucial to predict the response of HER2-targeted therapy (1). Patients with breast cancer overexpressing HER2 have improved survival when treated with HER2-targeting agents such as trastuzumab, pertuzumab, and trastuzumab emtansine (2-10).The analysis of HER2 expression is usually based on a surgical specimen of the primary tumor or, in case of neoadjuvant therapy or inoperable disease, on a biopsy sample from the tumor (11). The pathologic analysis includes immunohistochemistry and in some cases fluorescence in situ hybridization (FISH). Therapy for patients with disseminated disease is often based on histopathologic classification of the primary tumor and not of the metastases. Disparities in HER2 expression of primary breast cancer and metastases have been reported. Metaanalysis of 26 studies including 2,520 patients revealed discordance in HER2 expression between the primary tumor and local lymph node metastases in the range of 2.4%-7.2% and discordance with distant metastases in the range of 6.9%-18.6%, with an abs...

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 (f7 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 Affibody molecule Z HER2:342-pep2 , site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N, N ¶,N 00 ,N Ø-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-Z HER2:342-pep2 folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of 111 In was achieved within 30 min at low (room temperature) and high temperatures (up to 90°C). Tumor uptake of 111 In-DOTA-Z HER2:342-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-allylaminogeldanamycin before administration of 111In-DOTA-Z HER2:342-pep2 obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-Z HER2:342-pep2 has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas.