a b s t r a c tAffibody molecules are a class of engineered affinity proteins with proven potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. Fifteen years after its discovery, the Affibody technology is gaining use in many groups as a tool for creating molecular specificity wherever a small, engineering compatible tool is warranted. Here we summarize recent results using this technology, propose an Affibody nomenclature and give an overview of different HER2-specific Affibody molecules. Cumulative evidence suggests that the three helical scaffold domain used as basis for these molecules is highly suited to create a molecular affinity handle for vastly different applications.
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)
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.
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