| 1. |
- Ahlgren, Sara, et al.
(författare)
-
Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules
- 2008
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 19:1, s. 235-243
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.
|
|
| 2. |
- Ahlgren, Sara, 1979-
(författare)
-
Molecular Radionuclide Imaging Using Site-specifically Labelled Recombinant Affibody Molecules : Preparation and Preclinical Evaluation
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Radionuclide molecular imaging is an emerging multidisciplinary technique that is used in modern medicine to visualise diseases at cellular and molecular levels. This thesis is based on five papers (I-V) and focuses on the development of site-specific radiolabelled recombinant anti-HER2 Affibody molecules and preclinical evaluations in vitro and in vivo of the labelled conjugates. This work is part of a preclinical development of an Affibody molecule-based tracer for molecular imaging of HER2 expressing tumours. Papers I and II report the evaluation of the Affibody molecule ZHER2:2395-C, site-specifically labelled with the radiometals 111In (for SPECT) and 57Co (as a surrogate for 55Co, suitable for PET applications) using a thiol reactive DOTA derivative as a chelator. Both conjugates demonstrated very suitable biodistribution properties, enabling high contrast imaging just a few hours after injection. Papers III and IV report the development and optimization of a technique for site-specific labelling of ZHER2:2395-C with 99mTc using an N3S chelating peptide sequence. 99mTc-ZHER2:2395-C demonstrated high and specific tumour uptake and rapid clearance of non-bound tracer from the blood, resulting in high tumour-to-non-tumour ratios shortly after injection, enabling high contrast imaging. In addition, in the study described in paper IV, freeze-dried kits previously developed for 99mTc-labelling were optimised, resulting in the development of a kit in which all the reagents and protein needed for labelling of ZHER2:2395-C with 99mTc were contained in a single vial. Paper V reports the evaluation of an anti-HER2 Affibody molecule, ABY-025, with a fundamentally re-engineered scaffold. Despite the profound re-engineering, the biodistribution pattern of 111In-ABY-025 was very similar to that of two variants of the parental molecule. It seems reasonable to believe that these results will also be applicable to Affibody molecules towards other targets. Hopefully, this work will also be helpful in the development of other small proteinaceous tracers.
|
|
| 3. |
- Ahlgren, Sara, et al.
(författare)
-
Targeting of HER2-Expressing Tumors Using 111In-ABY-025, a Second-Generation Affibody Molecule with a Fundamentally Reengineered Scaffold
- 2010
-
Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 51:7, s. 1131-1138
-
Tidskriftsartikel (refereegranskat)abstract
- Overexpression of HER2 in breast carcinomas predicts response to trastuzumab therapy. Affibody molecules based on a non-immunoglobulin scaffold have demon-strated high potential for in vivo molecular imaging of HER2-expressing tumors. Re-engineering of the molecular scaffold has led to a second generation of optimized Affibody molecules, having a surface distinctly different from the parental protein domain from staphylococcal protein A. The new tracer showed further increased melting point, stability and overall hydrophilicity compared to the parental molecule, and was shown to be more amenable for chemical peptide synthesis. The goal of this study was to assess potential effects of this extensive re-engineering on HER2 targeting, using ABY-025, a DOTA conjugated variant of the novel tracer. Methods: 111In-ABY-025 was compared with previously evaluated parent HER2-binding Affibody tracers in vitro and in vivo. The in vivo behavior was further evaluated in mice bearing SKOV-3 xenografts, in rats and in cynomolgus macaques. Results: 111In-ABY-025 bound specifically to HER2 in vitro and in vivo. Direct comparison with the previous generation of HER2-binding tracers showed that ABY-025 retained excellent targeting properties. Rapid blood clearance was shown in mice, rats and macaques. A highly specific tumor uptake of 16.7 ± 2.5 %IA/g was seen at 4 h after injection. The tumor-to-blood ratio was 6.3 at 0.5 h, 88 at 4 h, and increased up to 3 days after injection. Gamma camera imaging of tumors was already possible 0.5 h after injection. Furthermore, repeated i.v. administration of ABY-025 did not induce antibody formation in rats. Conclusions: The biodistribution of 111In-ABY-025 was in remarkably good agreement with the parent tracers, despite profound re-engineering of the non-binding surface. The molecule displayed rapid blood clearance in all species investigated and excellent targeting capacity in tumor bearing mice, leading to high tumor-to-organ-ratios and high contrast imaging shortly after injection.
|
|
| 4. |
- Ahlgren, Sara, et al.
(författare)
-
Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine
- 2009
-
Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 50:5, s. 781-789
-
Tidskriftsartikel (refereegranskat)abstract
- 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. CONCLUSION: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.
|
|
| 5. |
|
|
| 6. |
- Alhuseinalkhudhur, Ali, et al.
(författare)
-
Human Epidermal Growth Factor Receptor 2-Targeting [68Ga]Ga-ABY-025 PET/CT Predicts Early Metabolic Response in Metastatic Breast Cancer.
- 2023
-
Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667. ; 64:9, s. 1364-1370
-
Tidskriftsartikel (refereegranskat)abstract
- Imaging using the human epidermal growth factor receptor 2 (HER2)-binding tracer 68Ga-labeled ZHER2:2891-Cys-MMA-DOTA ([68Ga]Ga-ABY-025) was shown to reflect HER2 status determined by immunohistochemistry and in situ hybridization in metastatic breast cancer (MBC). This single-center open-label phase II study investigated how [68Ga]Ga-ABY-025 uptake corresponds to biopsy results and early treatment response in both primary breast cancer (PBC) planned for neoadjuvant chemotherapy and MBC. Methods: Forty patients with known positive HER2 status were included: 19 with PBC and 21 with MBC (median, 3 previous treatments). [68Ga]Ga-ABY-025 PET/CT, [18F]F-FDG PET/CT, and core-needle biopsies from targeted lesions were performed at baseline. [18F]F-FDG PET/CT was repeated after 2 cycles of therapy to calculate the directional change in tumor lesion glycolysis (Δ-TLG). The largest lesions (up to 5) were evaluated in all 3 scans per patient. SUVs from [68Ga]Ga-ABY-025 PET/CT were compared with the biopsied HER2 status and Δ-TLG by receiver operating characteristic analyses. Results: Trial biopsies were HER2-positive in 31 patients, HER2-negative in 6 patients, and borderline HER2-positive in 3 patients. The [68Ga]Ga-ABY-025 PET/CT cutoff SUVmax of 6.0 predicted a Δ-TLG lower than -25% with 86% sensitivity and 67% specificity in soft-tissue lesions (area under the curve, 0.74 [95% CI, 0.67-0.82]; P = 0.01). Compared with the HER2 status, this cutoff resulted in clinically relevant discordant findings in 12 of 40 patients. Metabolic response (Δ-TLG) was more pronounced in PBC (-71% [95% CI, -58% to -83%]; P < 0.0001) than in MBC (-27% [95% CI, -16% to -38%]; P < 0.0001), but [68Ga]Ga-ABY-025 SUVmax was similar in both with a mean SUVmax of 9.8 (95% CI, 6.3-13.3) and 13.9 (95% CI, 10.5-17.2), respectively (P = 0.10). In multivariate analysis, global Δ-TLG was positively associated with the number of previous treatments (P = 0.0004) and negatively associated with [68Ga]Ga-ABY-025 PET/CT SUVmax (P = 0.018) but not with HER2 status (P = 0.09). Conclusion: [68Ga]Ga-ABY-025 PET/CT predicted early metabolic response to HER2-targeted therapy in HER2-positive breast cancer. Metabolic response was attenuated in recurrent disease. [68Ga]Ga-ABY-025 PET/CT appears to provide an estimate of the HER2 expression required to induce tumor metabolic remission by targeted therapies and might be useful as an adjunct diagnostic tool.
|
|
| 7. |
- Alhuseinalkhudhur, Ali, et al.
(författare)
-
Kinetic analysis of HER2-binding ABY-025 Affibody molecule using dynamic PET in patients with metastatic breast cancer
- 2020
-
Ingår i: EJNMMI Research. - : SPRINGEROPEN. - 2191-219X. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: High expression of human epidermal growth factor receptor type 2 (HER2) represents an aggressive subtype of breast cancer. Anti-HER2 treatment requires a theragnostic approach wherein sufficiently high receptor expression in biopsy material is mandatory. Heterogeneity and discordance of HER2 expression between primary tumour and metastases, as well as within a lesion, present a complication for the treatment and require multiple biopsies. Molecular imaging using the HER2-targeting Affibody peptide ABY-025 radiolabelled with Ga-68-gallium for PET/CT is currently under investigation as a non-invasive tool for whole-body evaluation of metastatic HER2 expression. Initial studies demonstrated a high correlation between Ga-68-ABY-025 standardized uptake values (SUVs) and histopathology. However, detecting small liver lesions might be compromised by high background uptake. This study aimed to explore the applicability of kinetic modelling and parametric image analysis for absolute quantification of Ga-68-ABY-025 uptake and HER2-receptor expression and how that relates to static SUVs.Methods: Dynamic Ga-68-ABY-025 PET of the upper abdomen was performed 0-45 min post-injection in 16 patients with metastatic breast cancer. Five patients underwent two examinations to test reproducibility. Parametric images of tracer delivery (K-1) and irreversible binding (K-i) were created with an irreversible two-tissue compartment model and Patlak graphical analysis using an image-derived input function from the descending aorta. A volume of interest (VOI)-based analysis was performed to validate parametric images. SUVs were calculated from 2 h and 4 h post-injection static whole-body images and compared to K-i.Results: Characterization of HER2 expression in smaller liver metastases was improved using parametric images. K-i values from parametric images agreed very well with VOI-based gold standard (R-2 > 0.99, p < 0.001). SUVs of metastases at 2 h and 4 h post-injection were highly correlated with K-i values from both the two-tissue compartment model and Patlak method (R-2 = 0.87 and 0.95, both p < 0.001). Ga-68-ABY-025 PET yielded high test-retest reliability (relative repeatability coefficient for Patlak 30% and for the two-tissue compartment model 47%).Conclusion: Ga-68-ABY-025 binding in HER2-positive metastases was well characterized by irreversible two-tissue compartment model wherein K-i highly correlated with SUVs at 2 and 4 h. Dynamic scanning with parametric image formation can be used to evaluate metastatic HER2 expression accurately.
|
|
| 8. |
|
|
| 9. |
- Baum, Richard P, et al.
(författare)
-
Molecular imaging of HER2-expressing malignant tumors in breast cancer patients using synthetic 111In- or 68Ga-labeled affibody molecules
- 2010
-
Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 51:6, s. 892-897
-
Tidskriftsartikel (refereegranskat)abstract
- 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). gamma-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.
|
|
| 10. |
- Eissler, Nina, et al.
(författare)
-
Affibody PET Imaging of HER2-Expressing Cancers as a Key to Guide HER2-Targeted Therapy
- 2024
-
Ingår i: Biomedicines. - : MDPI. - 2227-9059. ; 12:5
-
Forskningsöversikt (refereegranskat)abstract
- Human epidermal growth factor receptor 2 (HER2) is a major prognostic and predictive marker overexpressed in 15-20% of breast cancers. The diagnostic reference standard for selecting patients for HER2-targeted therapy is based on the analysis of tumor biopsies. Previously patients were defined as HER2-positive or -negative; however, with the approval of novel treatment options, specifically the antibody-drug conjugate trastuzumab deruxtecan, many breast cancer patients with tumors expressing low levels of HER2 have become eligible for HER2-targeted therapy. Such patients will need to be reliably identified by suitable diagnostic methods. Biopsy-based diagnostics are invasive, and repeat biopsies are not always feasible. They cannot visualize the heterogeneity of HER2 expression, leading to a substantial number of misdiagnosed patients. An alternative and highly accurate diagnostic method is molecular imaging with radiotracers. In the case of HER2, various studies demonstrate the clinical utility and feasibility of such approaches. Radiotracers based on Affibody((R)) molecules, small, engineered affinity proteins with a size of similar to 6.5 kDa, are clinically validated molecules with favorable characteristics for imaging. In this article, we summarize the HER2-targeted therapeutic landscape, describe our experience with imaging diagnostics for HER2, and review the currently available clinical data on HER2-Affibody-based molecular imaging as a novel diagnostic tool in breast cancer and beyond.
|
|
