Amino Acids

, Volume 41, Issue 5, pp 1037–1047 | Cite as

Protein scaffold-based molecular probes for cancer molecular imaging

Review Article


Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.


Protein scaffolds Peptide, molecular imaging Cancer PET 


  1. Ahlgren S, Orlova A, Rosik D, Sandström M, Sjöberg A, Baastrup B, Widmark O, Fant G, Feldwisch J, Tolmachev V (2008) Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules. Bioconjug Chem 19:235–243PubMedCrossRefGoogle Scholar
  2. Ahlgren S, Wållberg H, Tran TA, Widström C, Hjertman M, Abrahmsén L, Berndorff D, Dinkelborg LM, Cyr JE, Feldwisch J, Orlova A, Tolmachev V (2009) Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine. J Nucl Med 50:781–789PubMedCrossRefGoogle Scholar
  3. Amstutz P, Binz HK, Parizek P, Stumpp MT, Kohl A, Grutter MG, Forrer P, Plückthun A (2005) Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. J Biol Chem 280:24715–24722PubMedCrossRefGoogle Scholar
  4. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521–526PubMedCrossRefGoogle Scholar
  5. Austin J, Wang W, Puttamadappa S, Shekhtman A, Camarero JA (2009) Biosynthesis and biological screening of a genetically encoded library based on the cyclotide MCoTI-I. ChemBioChem 10:2663–2670PubMedCrossRefGoogle Scholar
  6. Baum RP, Orlova A, Tolmachev V, Feldwisch J (2006) Receptor PET/CT and SPECT using an Affibody molecule for targeting and molecular imaging of HER2-positive cancer in animal xenografts and human breast cancer patients [abstract]. J Nucl Med 47(suppl):108PGoogle Scholar
  7. Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter MG, Plückthun A (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 22:575–582PubMedCrossRefGoogle Scholar
  8. Chen W, Zhu Z, Feng Y, Xiao X, Dimitrov DS (2008) Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain. J Mol Biol 382:779–789PubMedCrossRefGoogle Scholar
  9. Cheng Z, Padilla De Jesus O, Namavari M, De A, Levi J, Webster JM, Zhang R, Lee B, Syud FA, Gambhirl SS (2008) Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules. J Nucl Med 49:804–813PubMedCrossRefGoogle Scholar
  10. Cheng Z, De Jesus OP, Kramer DJ, De A, Webster JM, Gheysens O, Levi J, Namavari M, Wang S, Park JM, Zhang R, Liu H, Lee B, Syud FA, Gambhir SS (2009) 64Cu-labeled Affibody molecules for imaging of HER2 expressing tumors. Mol Imaging Biol (Epub ahead of print)Google Scholar
  11. Cortez-Retamozo V, Lahoutte T, Caveliers V, Gainkam LO, Hernot S, Packeu A, De Vos F, Vanhove C, Muyldermans S, Baetselier PD, Revets H (2008) 99mTc-labeled nanobodies: a new type of targeted probes for imaging antigen expression. Curr Radiopharm 1:37–41Google Scholar
  12. Di Carlo A, Mariano A, D’Alessandro V, Belli G, Romano G, Macchia V (2001) Evaluation of epidermal growth factor receptor, carcinoembryonic antigen and Lewis carbohydrate antigens in human colorectal and liver neoplasias. Oncol Rep 8:387–392PubMedGoogle Scholar
  13. Ekblad T, Tran T, Orlova A, Widström C, Feldwisch J, Abrahmsén L, Wennborg A, Karlström AE, Tolmachev V (2008) Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake. Eur J Nucl Med Mol Imaging. 35:2245–2255PubMedCrossRefGoogle Scholar
  14. Engfeldt T, Renberg B, Brumer H, Nygren PA, Karlström AE (2005) Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein. ChemBioChem 6:1043–1050PubMedCrossRefGoogle Scholar
  15. Gainkam LO, Huang L, Caveliers V, Keyaerts M, Hernot S, Vaneycken I, Vanhove C, Revets H, De Baetselier P, Lahoutte T (2008) Comparison of the biodistribution and tumor targeting of two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole SPECT/micro-CT. J Nucl Med 49:788–795PubMedCrossRefGoogle Scholar
  16. Gambhir SS (2002) Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2:683–693PubMedCrossRefGoogle Scholar
  17. Getmanova EV, Chen Y, Bloom L, Gokemeijer J, Shamah S, Warikoo V, Wang J, Ling V, Sun L (2006) Antagonists to human and mouse vascular endothelial growth factor receptor 2 generated by directed protein evolution in vitro. Chem. Biol. 13:549–556PubMedCrossRefGoogle Scholar
  18. Gill DS, Damle NK (2006) Biopharmaceutical drug discovery using novel protein scaffolds. Curr Opin Biotechnol 17:653–658PubMedCrossRefGoogle Scholar
  19. Goldman ER, Anderson GP, Liu JL, Delehanty JB, Sherwood LJ, Osborn LE, Cummins LB, Hayhurst A (2006) Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama library. Anal Chem 78:8245–8255PubMedCrossRefGoogle Scholar
  20. Harmsen MM, De Haard HJ (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77:13–22PubMedCrossRefGoogle Scholar
  21. Heyd B, Pecorari F, Collinet B, Adjadj E, Desmadril M, Minard P (2003) In vitro evolution of the binding specificity of neocarzinostatin, an enediyne-binding chromoprotein. Biochemistry 42:5674–5683PubMedCrossRefGoogle Scholar
  22. Hosse RJ, Rothe A, Power BE (2006) A new generation of protein display scaffolds for molecular recognition. Protein Sci 15:14–27PubMedCrossRefGoogle Scholar
  23. Huang L, Gainkam LO, Caveliers V, Vanhove C, Keyaerts M, De Baetselier P, Bossuyt A, Revets H, Lahoutte T (2008) SPECT imaging with 99mTc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expression. Mol Imaging Biol 10:167–175PubMedCrossRefGoogle Scholar
  24. Jiang L, Kimura RH, Miao Z, Silverman AP, Ren G, Liu HG, Li PY, Gambhir SS, Cochran JR, Cheng Z (2010) Evaluation of a 64Cu-labeled cystine-knot peptide based on agouti related protein for PET imaging of tumors expressing αvβ3 integrin. J Nucl Med 51(2):251–258Google Scholar
  25. Jonsson A, Dogan J, Herne N, Abrahmsén L, Nygren PA (2008) Engineering of a femtomolar affinity binding protein to human serum albumin. Protein Eng Des Sel 21:515–527PubMedCrossRefGoogle Scholar
  26. Jonsson A, Wållberg H, Herne N, Ståhl S, Frejd FY (2009) Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro. Biotechnol Appl Biochem 54:93–103PubMedCrossRefGoogle Scholar
  27. Kimura RH, Cheng Z, Gambhir SS, Cochran JR (2009a) Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects. Cancer Res 69:2435–2442PubMedCrossRefGoogle Scholar
  28. Kimura RH, Levin AM, Cochran FV, Cochran JR (2009b) Engineered cystine knot peptides that bind alphavbeta3, alphavbeta5, and alpha5beta1 integrins with low-nanomolar affinity. Proteins [Epub ahead of print]Google Scholar
  29. Kimura RH, Miao Z, Cheng Z, Gambhir SS, Cochran JR (2009c) A dual-labeled knottin peptide for PET and near-infrared fluorescence imaging of integrin expression in living subjects. World Molecular Imaging Congress (abstract)Google Scholar
  30. Kramer-Marek G, Kiesewetter DO, Martiniova L, Jagoda E, Lee SB, Capala J (2008) [18F]FBEM-Z(HER2:342)-Affibody molecule-a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography. Eur J Nucl Med Mol Imaging. 35:1008–1018PubMedCrossRefGoogle Scholar
  31. Kramer-Marek G, Kiesewetter DO, Capala J (2009) Changes in HER2 expression in breast cancer xenografts after therapy can be quantified using PET and 18F-labeled affibody molecules. J Nucl Med 50:1131–1139PubMedCrossRefGoogle Scholar
  32. Ku J, Schultz PG (1995) Alternate protein frameworks for molecular recognition. Proc Natl Acad Sci 92:6552–6556PubMedCrossRefGoogle Scholar
  33. Ladner RC (1999) Polypeptides from phage display: a superior source of in vivo imaging agents. Q J Nucl Med 43:119–124PubMedGoogle Scholar
  34. Lamla T, Erdmann VA (2003) Searching sequence space for high affinity binding peptides using ribosome display. J Mol Biol 329:381–388PubMedCrossRefGoogle Scholar
  35. Lamla T, Erdmann VA (2004) The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins. Protein Expr Purif 33:39–47PubMedCrossRefGoogle Scholar
  36. Lee SB, Hassan M, Fisher R, Chertov O, Chernomordik V, Kramer-Marek G, Gandjbakhche A, Capala J (2008) Affibody molecules for in vivo characterization of HER2-positive tumors by near-infrared imaging. Clin Cancer Res 14:3840–3849PubMedCrossRefGoogle Scholar
  37. Legendre D, Vucic B, Hougardy V, Girboux AL, Henrioul C, Van Haute J, Soumillion P, Fastrez J (2002) TEM-1 beta-lactamase as a scaffold for protein recognition and assay. Protein Sci 11:1506–1518PubMedCrossRefGoogle Scholar
  38. Lehtio J, Teeri TT, Nygren PA (2000) α-Amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold. Proteins 41:316–322PubMedCrossRefGoogle Scholar
  39. Li R, Hoess RH, Bennett JS, DeGrado WF (2003) Use of phage display to probe the evolution of binding specificity and affinity in integrins. Protein Eng 16:65–72PubMedCrossRefGoogle Scholar
  40. Massoud TF, Gambhir SS (2003) Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17:545–580PubMedCrossRefGoogle Scholar
  41. Miao Z, Ren G, Liu H, Kimura RH, Jiang L, Gambhir SS, Cochran J, Cheng Z (2009a) An engineered knottin peptide labeled with 18F for PET imaging of integrin expression. Bioconjug Chem 20(12):2342–2347Google Scholar
  42. Miao Z, Ren G, Liu H, Jiang L, Wang YH, Gambhir SS, Cheng Z (2009b) Affibody based molecular probes for EGFR PET and optical imaging. World Molecular Imaging Congress Meeting (abstract)Google Scholar
  43. Nygren PA, Skerra A (2004) Binding proteins from alternative scaffolds. J Immunol Methods 290:3–28PubMedCrossRefGoogle Scholar
  44. Orlova A, Magnusson M, Eriksson TL, Nilsson M, Larsson B, Höidén-Guthenberg I, Widström C, Carlsson J, Tolmachev V, Ståhl S, Nilsson FY (2006a) Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res 66:4339–4448PubMedCrossRefGoogle Scholar
  45. Orlova A, Nilsson FY, Wikman M, Widström C, Ståhl S, Carlsson J, Tolmachev V (2006b) Comparative in vivo evaluation of technetium and iodine labels on an anti-HER2 Affibody for single-photon imaging of HER2 expression in tumors. J Nucl Med 47:512–519PubMedGoogle Scholar
  46. Orlova A, Wållberg H, Stone-Elander S, Tolmachev V (2009) On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of a 124I-labeled affibody molecule and trastuzumab in a murine xenograft model. J Nucl Med 50:417–425PubMedCrossRefGoogle Scholar
  47. Ren G, Zhang R, Liu Z, Webster JM, Miao Z, Gambhir SS, Syud FA, Cheng Z (2009) A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression. J Nucl Med 50:1492–1499PubMedCrossRefGoogle Scholar
  48. Roberts BL, Markland W, Ley AC, Kent RB, White DW, Guterman SK, Ladner RC (1992) Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage. Proc Natl Acad Sci USA 89:2429–2433PubMedCrossRefGoogle Scholar
  49. Rothe A, Hosse RJ, Power BE (2006) In vitro display technologies reveal novel biopharmaceutics. FASEB J 20:1599–1610PubMedCrossRefGoogle Scholar
  50. Schneider S, Buchert M, Georgiev O, Catimel B, Halford M, Stacker SA, Baechi T, Moelling K, Hovens CM (1999) Mutagenesis and selection of PDZ domains that bind new protein targets. Nat Biotechnol 17(2):170–175PubMedCrossRefGoogle Scholar
  51. Signore A, Annovazzi A, Chianelli M, Corsetti F, Van de Wiele C, Watherhouse RN (2001) Peptide radiopharmaceuticals for diagnosis and therapy. Eur J Nucl Med 28:1556–1565Google Scholar
  52. Silverman AP, Levin AM, Lahti JL, Cochran JR (2009) Engineered cystine-knot peptides that bind alpha(v)beta (3) integrin with antibody-like affinities. J Mol Biol 385(4):1064–1075Google Scholar
  53. Thongyoo P, Bonomelli C, Leatherbarrow RJ, Tate EW (2009) Potent inhibitors of beta-tryptase and human leukocyte elastase based on the MCoTI-II scaffold. J Med Chem 52:6197–6200PubMedCrossRefGoogle Scholar
  54. Tolmachev V, Nilsson FY, Widström C, Andersson K, Rosik D, Gedda L, Wennborg A, Orlova A (2006) 111In-benzyl-DTPA-ZHER2:342, an Affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors. J Nucl Med 47:846–853PubMedGoogle Scholar
  55. Tolmachev V, Xu H, Wållberg H, Ahlgren S, Hjertman M, Sjöberg A, Sandström M, Abrahmsén L, Brechbiel MW, Orlova A (2008) Evaluation of a maleimido derivative of CHX-A″-DTPA for site-specific labeling of Affibody molecules. Bioconjug Chem 9:1579–1587CrossRefGoogle Scholar
  56. Tolmachev V, Friedman M, Sandström M, Eriksson TL, Rosik D, Hodik M, Ståhl S, Frejd FY, Orlova A (2009) Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry. J Nucl Med 50:274–283PubMedCrossRefGoogle Scholar
  57. Tran T, Engfeldt T, Orlova A, Sandström M, Feldwisch J, Abrahmsén L, Wennborg A, Tolmachev V, Karlström AE (2007a) 99mTc-maEEE-Z(HER2:342), an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors. Bioconjug Chem 18:1956–1964PubMedCrossRefGoogle Scholar
  58. Tran T, Engfeldt T, Orlova A, Widström C, Bruskin A, Tolmachev V, Karlström AE (2007b) In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumor-targeting Affibody molecules. Bioconjug Chem 18:549–558PubMedCrossRefGoogle Scholar
  59. Uchiyama F, Tanaka Y, Minari Y, Tokui N (2005) Designing scaffolds of peptides for phage display libraries. J Biosci Bioeng 99:448–456PubMedCrossRefGoogle Scholar
  60. Vogt M, Skerra A (2004) Construction of an artificial receptor protein (“anticalin”) based on the human apolipoprotein D. ChemBioChem 5:191–199PubMedCrossRefGoogle Scholar
  61. Webster JM, Zhang R, Gambhir SS, Cheng Z, Syud FA (2009) Engineered two-helix small proteins for molecular recognition. Chembiochem 10:1293–1296PubMedCrossRefGoogle Scholar
  62. Wu AM, Senter PD (2005) Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 23(9):1137–1146PubMedCrossRefGoogle Scholar
  63. Xu L, Aha P, Gu K, Kuimelis RG, Kurz M, Lam T, Lim AC, Liu H, Lohse PA, Sun L, Weng S, Wagner RW, Lipovsek D (2002) Directed evolution of high affinity antibody mimics using mRNA display. Chem Biol 9:933–942PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Department of Radiology, Molecular Imaging Program at Stanford (MIPS)Stanford UniversityStanfordUSA
  2. 2.Bio-X ProgramStanford UniversityStanfordUSA
  3. 3.Canary Center at StanfordStanford UniversityStanfordUSA
  4. 4.Department of Radiology, Molecular Imaging Program at Stanford, Stanford Cancer CenterStanford UniversityStanfordUSA

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