title: A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression.
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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University, Stanford, California, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Ren
authors.forename: Rong
authors.initials: R
authors.lastname: Zhang
authors.forename: Zhe
authors.initials: Z
authors.lastname: Liu
authors.forename: Jack M
authors.initials: JM
authors.lastname: Webster
authors.forename: Zheng
authors.initials: Z
authors.lastname: Miao
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
authors.forename: Faisal A
authors.initials: FA
authors.lastname: Syud
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000493:pharmacokinetics*; D045744:Cell Line, Tumor; D005260:Female; D005710:Gallium Radioisotopes / Q000737:chemistry / Q000493:pharmacokinetics*; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009928:Organ Specificity; D010051:Ovarian Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism*; D049268:Positron-Emission Tomography / Q000379:methods*; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D002352:Carrier Proteins; D005710:Gallium Radioisotopes; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases
references.citation: J Nucl Med. 2007 Oct;48(10):1741-8
references.pmid: 17873136
references.citation: Oncol Rep. 2008 Apr;19(4):853-7
references.pmid: 18357367
references.citation: Clin Cancer Res. 2008 Jun 15;14(12):3840-9
references.pmid: 18559604
references.citation: FEBS J. 2008 Jun;275(11):2668-76
references.pmid: 18435759
references.citation: Nat Biotechnol. 2005 Sep;23(9):1126-36
references.pmid: 16151406
references.citation: J Biotechnol. 2007 Jan 30;128(1):162-83
references.pmid: 17088007
references.citation: Oncol Rep. 2007 May;17(5):1141-7
references.pmid: 17390057
references.citation: Bioconjug Chem. 2008 Jan;19(1):235-43
references.pmid: 18163536
references.citation: J Nucl Med. 2009 Feb;50(2):274-83
references.pmid: 19164241
references.citation: Biotechnol Appl Biochem. 2009 Jan;52(Pt 1):17-27
references.pmid: 18260830
references.citation: Cancer Res. 2007 Mar 1;67(5):2178-86
references.pmid: 17332348
references.citation: Biotechnol Appl Biochem. 2008 Jun;50(Pt 2):97-112
references.pmid: 18284375
references.citation: Cancer Res. 2007 Mar 15;67(6):2773-82
references.pmid: 17363599
references.citation: Curr Opin Drug Discov Devel. 2007 Mar;10(2):167-75
references.pmid: 17436552
references.citation: Int J Mol Med. 2007 Feb;19(2):285-91
references.pmid: 17203203
references.citation: Eur J Nucl Med Mol Imaging. 2008 May;35(5):1008-18
references.pmid: 18157531
references.citation: J Nucl Med. 2006 May;47(5):846-53
references.pmid: 16644755
references.citation: J Nucl Med. 2007 Apr;48(4):596-601
references.pmid: 17401097
references.citation: Cancer Res. 2006 Apr 15;66(8):4339-48
references.pmid: 16618759
references.citation: Nucl Med Biol. 2005 Jan;32(1):51-8
references.pmid: 15691661
references.citation: J Nucl Med. 2006 Mar;47(3):512-9
references.pmid: 16513621
references.citation: J Nucl Med. 2006 May;47(5):793-6
references.pmid: 16644749
references.citation: Radiographics. 2007 Oct;27 Suppl 1:S215-29
references.pmid: 18180228
references.citation: Mol Imaging Biol. 2008 Jul-Aug;10(4):177-81
references.pmid: 18481153
references.citation: Nat Biotechnol. 2004 Jun;22(6):701-6
references.pmid: 15133471
references.citation: Bioconjug Chem. 2007 Mar-Apr;18(2):549-58
references.pmid: 17330952
references.citation: Breast Cancer. 2007;14(3):260-8
references.pmid: 17690502
references.citation: J Immunol Methods. 2004 Jul;290(1-2):3-28
references.pmid: 15261569
references.citation: J Biosci Bioeng. 2005 May;99(5):448-56
references.pmid: 16233816
references.citation: J Nucl Med. 2008 May;49(5):804-13
references.pmid: 18413392
references.citation: Chembiochem. 2009 May 25;10(8):1293-6
references.pmid: 19422008
references.citation: Eur J Nucl Med Mol Imaging. 2008 Jun;35(6):1100-8
references.pmid: 18204838
references.citation: FASEB J. 2006 Aug;20(10):1599-610
references.pmid: 16873883
references.citation: Q J Nucl Med Mol Imaging. 2007 Dec;51(4):314-23
references.pmid: 17464277
references.citation: Cancer J. 2008 May-Jun;14(3):149-53
references.pmid: 18536554
references.citation: Biotechnol Appl Biochem. 2006 Sep;45(Pt 2):93-105
references.pmid: 16712522
references.citation: Protein Eng Des Sel. 2008 Aug;21(8):515-27
references.pmid: 18499681