Small-animal PET Imaging of human epidermal growth factor receptor type 2 expression with site-specific F-18-labeled protein scaffold molecules
ABSTRACT Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of (18)F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment.
In this study, anti-HER2 monomeric and dimeric protein scaffold molecules [Z(HER2:477) and (Z(HER2:477))(2), respectively] were radiofluorinated at a reasonable radiochemical yield (13%-18%) by use of site-specific oxime chemistry. The resulting radiofluorinated protein scaffold molecules were then evaluated as potential molecular probes for small-animal HER2 PET by use of a SKOV3 tumor-bearing mouse model.
The 4-(18)F-fluorobenzaldehyde conjugated aminooxy-protein scaffolds [(18)F-N-(4-fluorobenzylidene)oxime (FBO)-Z(HER2:477) and (18)F-FBO-(Z(HER2:477))(2)] both displayed specific HER2-binding ability in vitro. Biodistribution and small-animal PET imaging studies further revealed that (18)F-FBO-Z(HER2:477) showed rapid and high SKOV3 tumor accumulation and quick clearance from normal tissues, whereas (18)F-FBO-(Z(HER2:477))(2) showed poor in vivo performance (low tumor uptake and tumor-to-normal tissue ratios). The specificity of (18)F-FBO-Z(HER2:477) for SKOV3 tumors was confirmed by its lower uptake on pretreatment of tumor-bearing mice with the HER2-targeting agents Z(HER2) and trastuzumab. Moreover, small-animal PET imaging studies revealed that (18)F-FBO-Z(HER2:477) produced higher-quality tumor imaging than (18)F-FBO-(Z(HER2:477))(2). (18)F-FBO-Z(HER2:477) could clearly identify HER2-positive tumors with good contrast.
Overall, these data demonstrate that (18)F-FBO-Z(HER2:477) is a promising PET probe for imaging HER2 expression in living mice. It has a high potential for translation to clinical applications. The radiofluorination method developed can also be used as a general strategy for the site-specific labeling of other proteins with (18)F. The protein scaffold molecules used here are attractive for the further development of PET probes for other molecular targets.
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ABSTRACT: The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous PET studies showed that the EGFR-targeting Affibody molecules 64Cu-DOTA-ZEGFR:1907 and 18F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors, and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled Affibody probes (Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. Methods: The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al18F to produce Al18F-NOTA-ZEGFR:1907. In a second approach the prosthetic group 18F-labeled-2- cyanobenzothiazole (18F-CBT) was conjugated to Cys-ZEGFR:1907 to produce 18F-CBT-ZEGFR:1907. Binding affinity and specificity of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al18F-NOTA-ZEGFR:1907 or 18F-CBT-ZEGFR:1907 with or without co-injection of unlabeled Affibody proteins. Results: The radiosyntheses of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al18F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on co-injection of unlabeled Affibody. PET studies showed that Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 could clearly identify EGFR-positive tumors with good contrast. Conclusion: Two strategies for 18F-labeling of Affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to Affibody molecules that contain an N-terminal cysteine. Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al18F-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.Molecular Pharmaceutics 06/2014; 11(11). DOI:10.1021/mp5003043 · 4.79 Impact Factor
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ABSTRACT: Fluorescent affinity reagents are essential for dynamic live cell imaging studies. Antibodies are the conventional reagents in endogenous protein labeling, but the utility of the conjugate is limited by the conjugated dye and new conjugates must be prepared for different purposes. Recently, we developed a new technology based on noncovalent binding and almost instantaneous activation of a series of fluorogenic dyes by a single genetically encoded protein tag. Here we present the characterization of a new platform for affinity probes, in which a protein domain (FAP dL5**) capable of binding to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the EGFR binding affibody ZEGFR:1907, resulting in a recombinant and fluorogenic labeling reagent. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP-affibody fusions produced thousands-fold fluorescence enhancements, with high binding affinity and fast association rate. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nM surface Kd values were observed with the double-ZEGFR:1907 constructs. The application of light-harvesting fluorogens (dyedrons) significantly improved the detected fluorescence signal. We have shown that this probe can not only be used in tracing surface receptors trafficking under stimulation, but also the sequential addition of the probe and dyes allowed the differentiation between surface and endocytotic pools of receptors to reveal the dynamics of endocytic trafficking. Therefore, FAP/affibody coupling provides a new approach to construct compact and modular affinity probes for labeling of endogenous proteins on living cells.Bioconjugate Chemistry 12/2014; DOI:10.1021/bc500525b · 4.82 Impact Factor
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ABSTRACT: Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane tyrosine kinase receptor involved for example in angiogenesis. Overexpression and excessive signaling of PDGFRβ has been observed in multiple malignant tumors and fibrotic diseases, making this receptor a pharmaceutical target for monoclonal antibodies and tyrosine kinase inhibitors. Successful targeted therapy requires identification of responding patients. Radionuclide molecular imaging would enable determination of the PDGFRβ status in all lesions using a single non-invasive repeatable procedure. Recently, we have demonstrated that the affibody molecule Z09195 labeled with (111)In can specifically target PDGFR-expressing tumors in vivo. The use of PET as an imaging technique would provide superior resolution, sensitivity and quantitation accuracy. In this study, a DOTA-conjugated Z09195 was labeled with the generator-produced positron emitting radionuclide 68Ga (T1/2=67.6 min, Eβ+max=1899 keV, 89% β+). (68)Ga-DOTA-Z09591 retained the capacity to specifically bind to PDGFRβ-expressing U-87 MG glioma cells. The half maximum inhibition concentration (IC50) of 68Ga-DOTA-Z09591(6.6 ± 1.4 nM) was somewhat higher than that of (111)In-DOTA-Z09195 (1.4 ± 1.2 nM). (68)Ga-DOTA-Z09591demonstrated specific targeting of U-87 MG xenografts in immunodeficient mice. The tumor uptake at 2 h after injection was 3.7±1.7 %IA/g, which provided a tumor-to-blood ratio of 8.0±3.1. The only organ with higher accumulation of radioactivity was the kidney. MicroPET imaging provided high-contrast imaging of U-87 MG xenografts. In conclusion, the (68)Ga-labeled affibody molecule Z09195 is a promising candidate for further development as a probe for imaging PDGFRβ expression in vivo using PET.Molecular Pharmaceutics 06/2014; 11(11). DOI:10.1021/mp500284t · 4.79 Impact Factor