Development and evaluation of peptidic ligands targeting tumour-associated urokinase plasminogen activator receptor (uPAR) for use in α-emitter therapy for disseminated ovarian cancer

  • Sebastian Knör
  • Sumito Sato
  • Timo Huber
  • Alfred Morgenstern
  • Frank Bruchertseifer
  • Manfred Schmitt
  • Horst Kessler
  • Reingard Senekowitsch-Schmidtke
  • Viktor Magdolen
  • Christof Seidl
Original article



Among gynecologic malignancies, ovarian cancer has the highest mortality due to rapid peritoneal dissemination. Treatment failure particularly arises from failure to eliminate disseminated cells. Our aim was to develop peptidic radioligands targeting tumour cell-associated urokinase receptor (uPAR, CD87) for α-emitter therapy for advanced ovarian cancer.


DOTA-conjugated, uPAR-directed ligands were synthesised on solid-phase. Binding of peptides to human cells expressing uPAR was assayed by flow cytofluorometry or, in case of 213Bi-labelled peptides, by measuring cell-bound radioactivity. Bio-distribution of the 213Bi-labelled peptide P-P4D was analysed in nude mice 28 days after intraperitoneal inoculation of OV-MZ-6 ovarian cancer cells in the absence or presence of the plasma expander gelofusine.


uPAR-selective ligands were developed based on published high-affinity uPAR-binding peptides. For preparation of N-terminally cross-linked divalent ligands, a novel solid-phase procedure was developed. Specific binding of 213Bi-labelled peptides to monocytoid U937 and OV-MZ-6 cells was demonstrated using the natural ligand of uPAR, pro-uPA, or a soluble form of uPAR, suPAR, as competitors. The pseudo-symmetrical covalent dimer 213Bi-P-P4D displayed superior binding to OV-MZ-6 cells in vitro. Accumulation of 213Bi-P-P4D in tumour tissue was demonstrated by bio-distribution analysis in nude mice bearing intraperitoneal OV-MZ-6-derived tumours. Gelofusine reduced kidney uptake of 213Bi-P-P4D by half.


Ovarian cancer cells overexpressing uPAR were specifically targeted in vitro and in vivo by 213Bi-P-P4D. Kidney uptake of 213Bi-P-P4D was distinctly reduced using gelofusine. Thus, this radiopeptide may represent a promising option for therapy for disseminated ovarian cancer.


N-terminally cross-linked peptide dimer N-terminal dimerization on solid-phase OV-MZ-6 ovarian cancer cells Peritoneal carcinomatosis model α-emitter 213Bi 



We thank Birgit Pfost and Christel Schnelldorfer for excellent technical assistance in conducting the animal experiments and flow cytofluorometric analyses, respectively, and Nathalie Beaufort for helpful discussions. This study was supported by Deutsche Krebshilfe e.V., Germany (grant no. 106 185, V.M.) and by Deutsche Forschungsgemeinschaft (grants SE 962/2-4, R.S.-S.). H.K. thanks the Fonds der Chemischen Industrie for financial support.


  1. 1.
    Sadeghi B, Arvieux C, Glehen O, Beaujard AC, Rivoire M, Baulieux J, et al. Peritoneal carcinomatosis from non-gynecologic malignancies – Results of the EVOCAPE 1 multicentric prospective study. Cancer 2000;88:358–63.PubMedCrossRefGoogle Scholar
  2. 2.
    Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74–108.PubMedCrossRefGoogle Scholar
  3. 3.
    Bolis G, Villa A, Guarnerio P, Ferraris C, Gavoni N, Giardina G, et al. Survival of women with advanced ovarian cancer and complete pathologic response at second-look laparotomy. Cancer 1996;77:128–31.PubMedCrossRefGoogle Scholar
  4. 4.
    Couturier O, Supiot S, Degraef-Mougin M, Faivre-Chauvet A, Carlier T, Chatal JF, et al. Cancer radioimmunotherapy with alpha-emitting nuclides. Eur J Nucl Med Mol Imaging 2005;32:601–14.PubMedCrossRefGoogle Scholar
  5. 5.
    Borchardt PE, Yuan RR, Miederer M, McDevitt MR, Scheinberg DA. Targeted actinium-225 in vivo generators for therapy of ovarian cancer. Cancer Res 2003;63:5084–90.PubMedGoogle Scholar
  6. 6.
    Huber R, Seidl C, Schmid E, Seidenschwang S, Becker KF, Schuhmacher C, et al. Locoregional alpha-radioimmunotherapy of intraperitoneal tumor cell dissemination using a tumor-specific monoclonal antibody. Clin Cancer Res 2003;9:3922s–8s.PubMedGoogle Scholar
  7. 7.
    Allen BJ, Raja C, Rizvi S, Li Y, Tsui W, Graham P, et al. Intralesional targeted alpha therapy for metastatic melanoma. Cancer Biol Ther 2005;4:1318–24.PubMedCrossRefGoogle Scholar
  8. 8.
    Buchhorn HM, Seidl C, Beck R, Saur D, Apostolidis C, Morgenstern A, et al. Non-invasive visualisation of the development of peritoneal carcinomatosis and tumour regression after 213Bi-radioimmunotherapy using bioluminescence imaging. Eur J Nucl Med Mol Imaging 2007;34:841–9.PubMedCrossRefGoogle Scholar
  9. 9.
    Milenic D, Garmestani K, Dadachova E, Chappell L, Albert P, Hill D, et al. Radioimmunotherapy of human colon carcinoma xenografts using a 213Bi-labeled domain-deleted humanized monoclonal antibody. Cancer Biother Radiopharm 2004;19:135–47.PubMedCrossRefGoogle Scholar
  10. 10.
    Elgqvist J, Andersson H, Back T, Claesson I, Hultborn R, Jensen H, et al. Alpha-radioimmunotherapy of intraperitoneally growing OVCAR-3 tumors of variable dimensions: Outcome related to measured tumor size and mean absorbed dose. J Nucl Med 2006;47:1342–50.PubMedGoogle Scholar
  11. 11.
    Jurcic JG, Larson SM, Sgouros G, McDevitt MR, Finn RD, Divgi CR, et al. Targeted alpha particle immunotherapy for myeloid leukemia. Blood 2002;100:1233–9.PubMedGoogle Scholar
  12. 12.
    Zalutsky MR, Pozzi OR. Radioimmunotherapy with alpha-particle emitting radionuclides. Q J Nucl Med Mol Imaging 2004;48:289–96.PubMedGoogle Scholar
  13. 13.
    Heppeler A, Froidevaux S, Eberle AN, Maecke HR. Receptor targeting for tumor localisation and therapy with radiopeptides. Curr Med Chem 2000;7:971–94.PubMedGoogle Scholar
  14. 14.
    Reubi JC, Mäcke HR, Krenning EP. Candidates for peptide receptor radiotherapy today and in the future. J Nucl Med 2005;46 Suppl 1:67S–75S.PubMedGoogle Scholar
  15. 15.
    Ginj M, Zhang H, Waser B, Cescato R, Wild D, Wang X, et al. Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors. Proc Natl Acad Sci U S A 2006;103:16436–41.PubMedCrossRefGoogle Scholar
  16. 16.
    Kneifel S, Cordier D, Good S, Ionescu MCS, Ghaffari A, Hofer S, et al. Local targeting of malignant gliomas by the diffusible peptidic vector 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid-substance P. Clin Cancer Res 2006;12:3843–50.PubMedCrossRefGoogle Scholar
  17. 17.
    Nayak T, Norenberg J, Anderson T, Atcher R. A comparison of high- versus low-linear energy transfer somatostatin receptor targeted radionuclide therapy in vitro. Cancer Biother Radiopharm 2005;20:52–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Norenberg JP, Krenning BJ, Konings IR, Kusewitt DF, Nayak TK, Anderson TL, et al. 213Bi-[DOTA0, Tyr3]octreotide peptide receptor radionuclide therapy of pancreatic tumors in a preclinical animal model. Clin Cancer Res 2006;12:897–903.PubMedCrossRefGoogle Scholar
  19. 19.
    Sperl S, Mueller MM, Wilhelm OG, Schmitt M, Magdolen V, Moroder L. The uPA/uPA receptor system as a target for tumor therapy. Drug News Perspect 2001;14:401–11.PubMedCrossRefGoogle Scholar
  20. 20.
    Rao JS, Gondi C, Chetty C, Chittivelu S, Joseph PA, Lakka SS. Inhibition of invasion, angiogenesis, tumor growth, and metastasis by adenovirus-mediated transfer of antisense uPAR and MMP-9 in non-small cell lung cancer cells. Mol Cancer Ther 2005;4:1399–408.PubMedCrossRefGoogle Scholar
  21. 21.
    Setyono-Han B, Stürzebecher J, Schmalix WA, Muehlenweg B, Sieuwerts AM, Timmermans M, et al. Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1. Thromb Haemost 2005;93:779–86.PubMedGoogle Scholar
  22. 22.
    Rømer J, Nielsen BS, Ploug M. The urokinase receptor as a potential target in cancer therapy. Curr Pharm Des 2004;10:2359–76.PubMedCrossRefGoogle Scholar
  23. 23.
    Reuning U, Sperl S, Kopitz C, Kessler H, Krüger A, Schmitt M, et al. Urokinase-type plasminogen activator (uPA) and its receptor (uPAR): development of antagonists of uPA/uPAR interaction and their effects in vitro and in vivo. Curr Pharm Des 2003;9:1529–43.PubMedCrossRefGoogle Scholar
  24. 24.
    Stutchbury TK, Al-Ejeh F, Stillfried GE, Croucher DR, Andrews J, Irving D, et al. Preclinical evaluation of 213Bi-labeled plasminogen activator inhibitor type 2 in an orthotopic murine xenogenic model of human breast carcinoma. Mol Cancer Ther 2007;6:203–12.PubMedCrossRefGoogle Scholar
  25. 25.
    Qu CF, Song EY, Li Y, Rizvi SM, Raja C, Smith R, et al. Pre-clinical study of 213Bi labeled PAI2 for the control of micrometastatic pancreatic cancer. Clin Exp Metastasis 2005;22:575–86.PubMedCrossRefGoogle Scholar
  26. 26.
    Song YJ, Qu CF, Rizvi SM, Li Y, Robertson G, Raja C, et al. Cytotoxicity of PAI2, C595 and Herceptin vectors labeled with the alpha-emitting radioisotope Bismuth-213 for ovarian cancer cell monolayers and clusters. Cancer Lett 2006;234:176–83.PubMedCrossRefGoogle Scholar
  27. 27.
    Rizvi SMA, Li Y, Song EYJ, Qu CF, Raja C, Morgenstern A, et al. Preclinical studies of Bismuth-213 labeled plasminogen activator inhibitor type 2 (PAI2) in a prostate cancer nude mouse xenograft model. Cancer Biol Ther 2006;5:386–93.CrossRefGoogle Scholar
  28. 28.
    Schmiedeberg N, Schmitt M, Rol C, Truffault V, Sukopp M, Bürgle M, et al. Synthesis, solution structure, and biological evaluation of urokinase type plasminogen activator (uPA)-derived receptor binding domain mimetics. J Med Chem 2002;45:4984–94.PubMedCrossRefGoogle Scholar
  29. 29.
    Ploug M, Ostergaard S, Gårdsvoll H, Kovalski K, Holst-Hansen C, Holm A, et al. Peptide-derived antagonists of the urokinase receptor. Affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation. Biochemistry-US 2001;40:12157–68.CrossRefGoogle Scholar
  30. 30.
    Magdolen V, Bürgle M, de Prada NA, Schmiedeberg N, Riemer C, Schroeck F, et al. Cyclo(19,31)[d-Cys(19)]-uPA(19-31) is a potent competitive antagonist of the interaction of urokinase-type plasminogen activator with its receptor (CD87). Biol Chem 2001;382:1197–205.PubMedCrossRefGoogle Scholar
  31. 31.
    Bürgle M, Koppitz M, Riemer C, Kessler H, König B, Weidle UH, et al. Inhibition of the interaction of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) by synthetic peptides. Biol Chem 1997;378:231–7.PubMedCrossRefGoogle Scholar
  32. 32.
    Barlos K, Gatos D, Kallitsis J, Papaphotiu G, Sotiriu P, Yao WQ, et al. Synthesis of protected peptide-fragments using substituted triphenylmethyl resins. Tetrahedron Lett 1989;30:3943–6.CrossRefGoogle Scholar
  33. 33.
    Carpino LA, Han GY. 9-Fluorenylmethoxycarbonyl amino-protecting group. J Org Chem 1972;37:3404–9.CrossRefGoogle Scholar
  34. 34.
    Fields GB, Noble RL. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res 1990;35:161–214.PubMedCrossRefGoogle Scholar
  35. 35.
    Thumshirn G, Hersel U, Goodman SL, Kessler H. Multimeric cyclic RGD peptides as potential tools for tumor targeting: Solid-phase peptide synthesis and chemoselective oxime ligation. Chem-Eur J 2003;9:2717–25.CrossRefGoogle Scholar
  36. 36.
    Carpino LA, Sadat-Aalaee D, Chao HG, DeSelm RH. [(9-Fluorenylmethyl)oxy]carbonyl (FMOC) amino acid fluorides. Convienient new peptide coupling reagents applicable to the FMOC/tert-butyl strategy for solution and solid-phase syntheses. J Am Chem Soc 1990;112:9651–2.CrossRefGoogle Scholar
  37. 37.
    Paquet A. Introduction of 9-fluorenylmethyloxycarbonyl, trichloroethoxycarbonyl, and benzylcarbonyl amine protecting groups into O-unprotected hydroxamino acids using succinimidyl carbonates. Can J Chem 1981;60:976–80.CrossRefGoogle Scholar
  38. 38.
    Möbus VJ, Moll R, Gerharz CD, Kieback DG, Weikel W, Hoffmann G, et al. Establishment of new ovarian and colon-carcinoma cell-lines – Differentiation is only possible by cytokeratin analysis. Br J Cancer 1994;69:422–8.PubMedGoogle Scholar
  39. 39.
    Apostolidis C, Molinet R, Rasmussen G, Morgenstern A. Production of Ac-225 from Th-229 for targeted alpha therapy. Anal Chem 2005;77:6288–91.PubMedCrossRefGoogle Scholar
  40. 40.
    Nikula TK, Curcio MJ, Brechbiel MW, Gansow OA, Finn RD, Scheinberg DA. A rapid, single vessel method for preparation of clinical grade ligand conjugated monoclonal antibodies. Nucl Med Biol 1995;22:387–90.PubMedCrossRefGoogle Scholar
  41. 41.
    Magdolen V, Krüger A, Sato S, Nagel J, Sperl S, Reuning U, et al. Inhibition of the tumor-associated urokinase-type plasminogen activation system: effects of high-level synthesis of soluble urokinase receptor in ovarian and breast cancer cells in vitro and in vivo. Recent Results Cancer Res 2003;162:43–63.PubMedGoogle Scholar
  42. 42.
    Stoppelli MP, Corti A, Soffientini A, Cassani G, Blasi F, Assoian RK. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen-activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci U S A 1985;82:4939–43.PubMedCrossRefGoogle Scholar
  43. 43.
    Wester HJ, Kessler H. Molecular targeting with peptides or peptide-polymer conjugates: just a question of size? J Nucl Med 2005;46:1940–5.PubMedGoogle Scholar
  44. 44.
    Mammen M, Choi SK, Whitesides GM. Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed 1998;37:2755–94.CrossRefGoogle Scholar
  45. 45.
    Owen RM, Carlson CB, Xu JW, Mowery P, Fasella E, Kiessling LL. Bifunctional ligands that target cells displaying the alpha(v)beta(3) integrin. Chembiochem 2007;8:68–82.PubMedCrossRefGoogle Scholar
  46. 46.
    Heppeler A, Froidevaux S, Mäcke HR, Jermann E, Behe M, Powell P, et al. Radiometal-labelled macrocyclic chelator-derivatized somatostatin analogue with superb tumour-targeting properties and potential for receptor-mediated internal radiotherapy. Chem Eur J 1999;5:1974–81.CrossRefGoogle Scholar
  47. 47.
    Will C, Wilhelm O, Hohl S, Möbus V, Weidle U, Kreienberg R, et al. Expression of urokinase-type plasminogen-activator (uPA) and Its receptor (uPAR) in human ovarian-cancer cells and in-vitro invasion capacity. Int J Oncol 1994;5:753–61.Google Scholar
  48. 48.
    Vegt E, Wetzels JF, Russel FG, Masereeuw R, Boerman OC, van Eerd JE, et al. Renal uptake of radiolabeled octreotide in human subjects is efficiently inhibited by succinylated gelatin. J Nucl Med 2006;47:432–6.PubMedGoogle Scholar
  49. 49.
    van Eerd JE, Vegt E, Wetzels JF, Russel FG, Masereeuw R, Corstens FH, et al. Gelatin-based plasma expander effectively reduces renal uptake of 111In-octreotide in mice and rats. J Nucl Med 2006;47:528–33.PubMedGoogle Scholar
  50. 50.
    Milenic DE, Brady ED, Brechbiel MW. Antibody-targeted radiation cancer therapy. Nat Rev Drug Discov 2004;3:488–99.PubMedCrossRefGoogle Scholar
  51. 51.
    Goldenberg DM, Sharkey RM. Advances in cancer therapy with radiolabeled monoclonal antibodies. Q J Nucl Med Mol Imaging 2006;50:248–64.PubMedGoogle Scholar
  52. 52.
    Behe M, Kluge G, Becker W, Gotthardt M, Behr TM. Use of polyglutamic acids to reduce uptake of radiometal-labeled minigastrin in the kidneys. J Nucl Med 2005;46:1012–5.PubMedGoogle Scholar
  53. 53.
    Panigone S, Nunn AD. Lutetium-177-labeled gastrin releasing peptide receptor binding analogs: A novel approach to radionuclide therapy. Q J Nucl Med Mol Imaging 2006;50:310–21.PubMedGoogle Scholar
  54. 54.
    Wild D, Behe M, Wicki A, Storch D, Waser B, Gotthardt M, et al. [Lys40(Ahx-DTPA-111In)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting. J Nucl Med 2006;47:2025–33.PubMedGoogle Scholar
  55. 55.
    de Jong M, Breeman WA, Valkema R, Bernard BF, Krenning EP. Combination radionuclide therapy using 177Lu- and 90Y-labeled somatostatin analogs. J Nucl Med 2005;46 Suppl 1:13S–7S.PubMedGoogle Scholar
  56. 56.
    Kwekkeboom DJ, Mueller-Brand J, Paganelli G, Anthony LB, Pauwels S, Kvols LK, et al. Overview of results of peptide receptor radionuclide therapy with 3 radiolabeled somatostatin analogs. J Nucl Med 2005;46 Suppl 1:62S–6S.PubMedGoogle Scholar
  57. 57.
    Seidl C, Schröck H, Seidenschwang S, Beck R, Schmid E, Abend M, et al. Cell death triggered by alpha-emitting 213Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death. Eur J Nucl Med Mol Imaging 2005;32:274–85.PubMedCrossRefGoogle Scholar
  58. 58.
    Senekowitsch-Schmidtke R, Schuhmacher C, Becker KF, Nikula TK, Seidl C, Becker I, et al. Highly specific tumor binding of a 213Bi-labeled monoclonal antibody against mutant E-cadherin suggests its usefulness for locoregional alpha-radioimmunotherapy of diffuse-type gastric cancer. Cancer Res 2001;61:2804–8.PubMedGoogle Scholar
  59. 59.
    Beck R, Seidl C, Pfost B, Morgenstern A, Bruchertseifer F, Baum H, et al. 213Bi-radioimmunotherapy defeats early-stage disseminated gastric cancer in nude mice. Cancer Sci 2007;98:1215–22PubMedCrossRefGoogle Scholar
  60. 60.
    Danø K, Behrendt N, Høyer-Hansen G, Johnsen M, Lund LR, Ploug M, et al. Plasminogen activation and cancer. Thromb Haemost 2005;93:676–81.PubMedGoogle Scholar
  61. 61.
    Harbeck N, Kates RE, Gauger K, Willems A, Kiechle M, Magdolen V, et al. Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I: novel tumor-derived factors with a high prognostic and predictive impact in breast cancer. Thromb Haemost 2004;91:450–6.PubMedGoogle Scholar
  62. 62.
    Pillay V, Dass CR, Choong PF. The urokinase plasminogen activator receptor as a gene therapy target for cancer. Trends Biotechnol 2007;25:33–9.PubMedCrossRefGoogle Scholar
  63. 63.
    Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev 2003;24:389–427.PubMedCrossRefGoogle Scholar
  64. 64.
    Lambert B, Cybulla M, Weiner SM, Van De Wiele C, Ham H, Dierckx RA, et al. Renal toxicity after radionuclide therapy. Radiat Res 2004;161:607–11.PubMedCrossRefGoogle Scholar
  65. 65.
    Rolleman EJ, Valkema R, de Jong M, Kooij PP, Krenning EP. Safe and effective inhibition of renal uptake of radiolabelled octreotide by a combination of lysine and arginine. Eur J Nucl Med Mol Imaging 2003;30:9–15.PubMedCrossRefGoogle Scholar
  66. 66.
    Bodei L, Cremonesi M, Zoboli S, Grana C, Bartolomei M, Rocca P, et al. Receptor mediated radionuclide therapy with 90Y DOTATOC in association with amino acid infusion: A phase I study. Eur J Nucl Med Mol Imaging 2003;30:207–16.PubMedCrossRefGoogle Scholar
  67. 67.
    Jaggi JS, Seshan SV, McDevitt MR, Sgouros G, Hyjek E, Scheinberg DA. Mitigation of radiation nephropathy after internal alpha-particle irradiation of kidneys. Int J Radiat Oncol Biol Phys 2006;64:1503–12.PubMedGoogle Scholar
  68. 68.
    Kakizaki T, Yokoyama Y, Natsuhori M, Yamada N, Hashimoto M, Sato K, et al. Quantitative analysis of the effect of probenecid on pharmacokinetics of 99mTc-mercaptoacetyltriglycine in dogs. J Vet Pharmacol Ther 2005;28:559–64.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Sebastian Knör
    • 1
  • Sumito Sato
    • 2
  • Timo Huber
    • 1
  • Alfred Morgenstern
    • 3
  • Frank Bruchertseifer
    • 3
  • Manfred Schmitt
    • 2
  • Horst Kessler
    • 1
  • Reingard Senekowitsch-Schmidtke
    • 4
  • Viktor Magdolen
    • 2
  • Christof Seidl
    • 4
  1. 1.Department Chemie, Lehrstuhl II für Organische ChemieTechnische Universität MünchenGarchingGermany
  2. 2.Klinische Forschergruppe der FrauenklinikKlinikum rechts der Isar der TU MünchenMunichGermany
  3. 3.European Commission, Joint Research CentreInstitute for Transuranium ElementsKarlsruheGermany
  4. 4.Nuklearmedizinische Klinik und PoliklinikKlinikum rechts der Isar der TU MünchenMunichGermany

Personalised recommendations