A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression.

Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University, Stanford, California, USA.
Journal of Nuclear Medicine (Impact Factor: 5.56). 09/2009; 50(9):1492-9. DOI: 10.2967/jnumed.109.064287
Source: PubMed

ABSTRACT Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation.
In this study, a 2-helix small protein, MUT-DS, was chemically modified with a metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA-MUT-DS was then site-specifically radiolabeled with an important PET radionuclide, (68)Ga. The resulting radiolabeled anti-HER2 2-helix molecule was further evaluated as a potential molecular probe for small-animal PET HER2 imaging in a SKOV3 tumor mouse model.
The 2-helix DOTA-MUT-DS showed high HER2-binding affinity (dissociation constant, 4.76 nM). The radiolabeled probe displayed high stability in mouse serum and specificity toward HER2 in cell cultures. Biodistribution and small-animal PET studies further showed that (68)Ga-DOTA-MUT-DS had rapid and high SKOV3 tumor accumulation and quick clearance from normal organs. The specificity of (68)Ga-DOTA-MUT-DS for SKOV3 tumors was confirmed by monitoring modulation of HER2 protein on treatment of tumor mice with heat shock protein 90 inhibitor 17-N,N-dimethyl ethylene diamine-geldanamycin in vivo.
This proof-of-concept research clearly demonstrated that synthetic 2-helix (68)Ga-DOTA-MUT-DS is a promising PET probe for imaging HER2 expression in vivo. The Affibody-derived small 2-helix protein scaffold has great potential for developing targeting agents for a variety of tumor-associated biomarkers.

  • [Show abstract] [Hide abstract]
    ABSTRACT: We aimed to develop a gallium-68 (Ga-68)-labeled single-chain variable fragment (scFv) targeting the human epidermal growth factor receptor 2 (HER2) to rapidly and noninvasively evaluate the status of HER2 expression.
    Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 07/2014; 17(1). DOI:10.1007/s11307-014-0769-5 · 2.87 Impact Factor
  • [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: While [(11) C]palmitate continues to be a promising tracer for cardiovascular Positron Emission Tomography (PET) imaging, unfavourable logistics due to the short half-life of (11) C (20 min) and cumbersome labeling methodologies are the major impediments that limit its widespread use. In order to circumvent such limitations, an attempt has been made to explore the potential of (68) Ga-labeled fatty acid analogs for metabolic imaging owing to the availability of (68) Ga through a (68) Ge/(68) Ga generator on an on-demand basis. In this study, two fatty acid conjugates were synthesized by conjugation of p-SCN-benzyl NOTA with the ω-amino group of 11-amino undecanoic acid and 12-amino dodecanoic acid, respectively, under alkaline conditions. Both derivatives were radiolabeled in high yields with (68) Ga obtained from an in-house (68) Ge/(68) Ga generator. Biodistribution studies in Swiss mice showed reasonable myocardial uptake at 2 min for both derivatives (7.4 ± 2.8% ID/g for 11-carbon fatty acid-NOTA conjugate and 6.4 ± 2.1% ID/g for 12-carbon fatty acid-NOTA conjugate), which cleared rapidly over 30 min. However, significant activity was found in blood for both tracers, with heart/blood ratios observed to be below 0.5 at all time points, diminishing the potential of the synthesized complexes for cardiac imaging. Copyright © 2014 John Wiley & Sons, Ltd.
    Journal of Labelled Compounds and Radiopharmaceuticals 06/2014; 57(7):463-9. DOI:10.1002/jlcr.3205 · 1.19 Impact Factor