Cancer Immunology, Immunotherapy

, Volume 55, Issue 12, pp 1590–1600

Target-selective activation of a TNF prodrug by urokinase-type plasminogen activator (uPA) mediated proteolytic processing at the cell surface

  • Jeannette Gerspach
  • Julia Németh
  • Sabine Münkel
  • Harald Wajant
  • Klaus Pfizenmaier
Original Article


We have previously developed TNF prodrugs comprised of a N-terminal scFv targeting, a TNF effector and a C-terminal TNFR1-derived inhibitor module linked to TNF via a MMP-2 motif containing peptide, allowing activation by MMP-2-expressing tumor cells. To overcome the known heterogeneity of matrix metalloprotease expression, we developed TNF prodrugs that become processed by other tumor and/or stroma-associated proteases. These TNF prodrugs comprise either an uPA-selective or a dual uPA-MMP-2-specific linker which displayed efficient, target-dependent and cleavage sequence-specific activation by the corresponding tumor cell-expressed proteases. Selective pharmacologic inhibition of endogenous uPA and MMP-2 confirm independent prodrug processing by these two model proteases and indicate the functional superiority of a prodrug containing a multi-specific protease linker. Processing optimised TNF prodrugs should increase the proportion of active therapeutic within the targeted tissue and thus potentially enhance tumor response rate.


Targeted TNF activation Prodrug processing uPA MMP-2 





Fibroblast activation protein




Tumor necrosis factor


TNF receptor

scFv 36

FAP-specific single chain variable fragment


Urokinase-type plasminogen activator


  1. 1.
    Andreasen PA, Kjoller L, Christensen L, Duffy MJ (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 72:1PubMedCrossRefGoogle Scholar
  2. 2.
    Andreasen PA, Egelund R, Petersen HH (2000) The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 57:25PubMedCrossRefGoogle Scholar
  3. 3.
    Bauer S, Adrian N, Williamson B, Panousis C, Fadle N, Smerd J, Fettah I, Scott AM, Pfreundschuh M, Renner C (2004) Targeted bioactivity of membrane-anchored TNF by an antibody-derived TNF fusion protein. J Immunol 172:3930PubMedGoogle Scholar
  4. 4.
    Blasi F, Carmeliet P (2002) uPAR: a versatile signalling orchestrator. Nat Rev Mol Cell Biol 3:932PubMedCrossRefGoogle Scholar
  5. 5.
    Borsi L, Balza E, Carnemolla B, Sassi F, Castellani P, Berndt A, Kosmehl H, Biro A, Siri A, Orecchia P, Grassi J, Neri D, Zardi L (2003) Selective targeted delivery of TNFalpha to tumor blood vessels. Blood 102:4384PubMedCrossRefGoogle Scholar
  6. 6.
    Bremer E, Samplonius DF, van Genne L, Dijkstra MH, Kroesen BJ, de Leij LF, Helfrich W (2005) Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR. J Biol Chem 280:10025PubMedCrossRefGoogle Scholar
  7. 7.
    Bryde S, Grunwald I, Hammer A, Krippner-Heidenreich A, Schiestel T, Brunner H, Tovar GE, Pfizenmaier K, Scheurich P (2005) Tumor necrosis factor (TNF)-functionalized nanostructured particles for the stimulation of membrane TNF-specific cell responses. Bioconjug Chem 16:1459PubMedCrossRefGoogle Scholar
  8. 8.
    Cha SS, Kim JS, Cho HS, Shin NK, Jeong W, Shin HC, Kim YJ, Hahn JH, Oh BH (1998) High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity. J Biol Chem 273:2153PubMedCrossRefGoogle Scholar
  9. 9.
    Corti A (2004) Strategies for improving the anti-neoplastic activity of TNF by tumor targeting. Methods Mol Med 98:247PubMedGoogle Scholar
  10. 10.
    Curnis F, Sacchi A, Borgna L, Magni F, Gasparri A, Corti A (2000) Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nat Biotechnol 18:1185PubMedCrossRefGoogle Scholar
  11. 11.
    Curnis F, Sacchi A, Corti A (2002) Improving chemotherapeutic drug penetration in tumors by vascular targeting and barrier alteration. J Clin Invest 110:475PubMedGoogle Scholar
  12. 12.
    Curnis F, Gasparri A, Sacchi A, Longhi R, Corti A (2004) Coupling tumor necrosis factor-alpha with alphaV integrin ligands improves its antineoplastic activity. Cancer Res 64:565PubMedCrossRefGoogle Scholar
  13. 13.
    Duffy MJ, Maguire TM, McDermott EW, O’Higgins N (1999) Urokinase plasminogen activator: a prognostic marker in multiple types of cancer. J Surg Oncol 71:130PubMedCrossRefGoogle Scholar
  14. 14.
    Duffy MJ, Maguire TM, Hill A, McDermott E, O’Higgins N (2000) Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res 2:252PubMedCrossRefGoogle Scholar
  15. 15.
    Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2:161PubMedCrossRefGoogle Scholar
  16. 16.
    Eggermont AM, ten Hagen TL (2001) Isolated limb perfusion for extremity soft-tissue sarcomas, in-transit metastases, and other unresectable tumors: credits, debits, and future perspectives. Curr Oncol Rep 3:359PubMedCrossRefGoogle Scholar
  17. 17.
    Fesik SW (2005) Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer 5:876PubMedCrossRefGoogle Scholar
  18. 18.
    Garin-Chesa P, Old LJ, Rettig WJ (1990) Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc Natl Acad Sci USA 87:7235PubMedCrossRefGoogle Scholar
  19. 19.
    Gerspach J, Muller D, Munkel S, Selchow O, Nemeth J, Noack M, Petrul H, Menrad A, Wajant H, Pfizenmaier K (2006) Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug. Cell Death Differ 13:273PubMedCrossRefGoogle Scholar
  20. 20.
    Ginestra A, Monea S, Seghezzi G, Dolo V, Nagase H, Mignatti P, Vittorelli ML (1997) Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells. J Biol Chem 272:17216PubMedCrossRefGoogle Scholar
  21. 21.
    Grell M, Douni E, Wajant H, Lohden M, Clauss M, Maxeiner B, Georgopoulos S, Lesslauer W, Kollias G, Pfizenmaier K, Scheurich P (1995) The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 83:793PubMedCrossRefGoogle Scholar
  22. 22.
    Heppner KJ, Matrisian LM, Jensen RA, Rodgers WH (1996) Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response. Am J Pathol 149:273PubMedGoogle Scholar
  23. 23.
    Hofmann UB, Westphal JR, van Muijen GN, Ruiter DJ (2000) Matrix metalloproteinases in human melanoma. J Invest Dermatol 115:337PubMedCrossRefGoogle Scholar
  24. 24.
    Kamada H, Tsutsumi Y, Yamamoto Y, Kihira T, Kaneda Y, Mu Y, Kodaira H, Tsunoda SI, Nakagawa S, Mayumi T (2000) Antitumor activity of tumor necrosis factor-alpha conjugated with polyvinylpyrrolidone on solid tumors in mice. Cancer Res 60:6416PubMedGoogle Scholar
  25. 25.
    Ke SH, Coombs GS, Tachias K, Corey DR, Madison EL (1997) Optimal subsite occupancy and design of a selective inhibitor of urokinase. J Biol Chem 272:20456PubMedCrossRefGoogle Scholar
  26. 26.
    Kuroda K, Miyata K, Fujita F, Koike M, Fujita M, Nomura M, Nakagawa S, Tsutsumi Y, Kawagoe T, Mitsuishi Y, Mayumi T (2000) Human tumor necrosis factor-alpha mutant RGD-V29 (F4614) shows potent antitumor activity and reduced toxicity against human tumor xenografted nude mice. Cancer Lett 159:33PubMedCrossRefGoogle Scholar
  27. 27.
    Lafleur MA, Tester AM, Thompson EW (2003) Selective involvement of TIMP-2 in the second activational cleavage of pro-MMP-2: refinement of the pro-MMP-2 activation mechanism. FEBS Lett 553:457PubMedCrossRefGoogle Scholar
  28. 28.
    Laug WE, Wang K, Mundi R, Rideout W III, Kruithof EK, Bogenmann E (1992) Clonal variation of expression of the genes coding for plasminogen activators, their inhibitors and the urokinase receptor in HT1080 sarcoma cells. Int J Cancer 52:298PubMedCrossRefGoogle Scholar
  29. 29.
    Liabakk NB, Talbot I, Smith RA, Wilkinson K, Balkwill F (1996) Matrix metalloprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Res 56:190PubMedGoogle Scholar
  30. 30.
    Lim YT, Sugiura Y, Laug WE, Sun B, Garcia A, DeClerck YA (1996) Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells. J Cell Physiol 167:333PubMedCrossRefGoogle Scholar
  31. 31.
    Liu S, Bugge TH, Leppla SH (2001) Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin. J Biol Chem 276:17976PubMedCrossRefGoogle Scholar
  32. 32.
    Look MP, van Putten WL, Duffy MJ, Harbeck N, Christensen IJ, Thomssen C, Kates R, Spyratos F, Ferno M, Eppenberger-Castori S, Sweep CG, Ulm K, Peyrat JP, Martin PM, Magdelenat H, Brunner N, Duggan C, Lisboa BW, Bendahl PO, Quillien V, Daver A, Ricolleau G, Meijer-van Gelder ME, Manders P, Fiets WE, Blankenstein MA, Broet P, Romain S, Daxenbichler G, Windbichler G, Cufer T, Borstnar S, Kueng W, Beex LV, Klijn JG, O’Higgins N, Eppenberger U, Janicke F, Schmitt M, Foekens JA (2002) Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst 94:116PubMedGoogle Scholar
  33. 33.
    Lu F, Chen ZM, Liu QH, Chen CQ (2001) A novel human tumor necrosis factor alpha mutant showed potent antitumor activity and reduced toxicity in vivo. Acta Pharmacol Sin 22:619PubMedGoogle Scholar
  34. 34.
    Maquoi E, Frankenne F, Noel A, Krell HW, Grams F, Foidart JM (2000) Type IV collagen induces matrix metalloproteinase 2 activation in HT1080 fibrosarcoma cells. Exp Cell Res 261:348PubMedCrossRefGoogle Scholar
  35. 35.
    Mueller H (1998) Tumor necrosis factor as an antineoplastic agent: pitfalls and promises. Cell Mol Life Sci 54:1291PubMedCrossRefGoogle Scholar
  36. 36.
    Ohba K, Miyata Y, Kanda S, Koga S, Hayashi T, Kanetake H (2005) Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and plasminogen activator inhibitors in patients with renal cell carcinoma: correlation with tumor associated macrophage and prognosis. J Urol 174:461PubMedCrossRefGoogle Scholar
  37. 37.
    Pyke C, Kristensen P, Ralfkiaer E, Grondahl-Hansen J, Eriksen J, Blasi F, Dano K (1991) Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas. Am J Pathol 138:1059PubMedGoogle Scholar
  38. 38.
    Samel D, Muller D, Gerspach J, Assohou-Luty C, Sass G, Tiegs G, Pfizenmaier K, Wajant H (2003) Generation of a FasL-based proapoptotic fusion protein devoid of systemic toxicity due to cell-surface antigen-restricted activation. J Biol Chem 278:32077PubMedCrossRefGoogle Scholar
  39. 39.
    Schmalfeldt B, Prechtel D, Harting K, Spathe K, Rutke S, Konik E, Fridman R, Berger U, Schmitt M, Kuhn W, Lengyel E (2001) Increased expression of matrix metalloproteinases (MMP)-2, MMP-9, and the urokinase-type plasminogen activator is associated with progression from benign to advanced ovarian cancer. Clin Cancer Res 7:2396PubMedGoogle Scholar
  40. 40.
    Stanton H, Gavrilovic J, Atkinson SJ, d’Ortho MP, Yamada KM, Zardi L, Murphy G (1998) The activation of ProMMP-2 (gelatinase A) by HT1080 fibrosarcoma cells is promoted by culture on a fibronectin substrate and is concomitant with an increase in processing of MT1-MMP (MMP-14) to a 45 kDa form. J Cell Sci 111(Pt 18):2789PubMedGoogle Scholar
  41. 41.
    Torng PL, Mao TL, Chan WY, Huang SC, Lin CT (2004) Prognostic significance of stromal metalloproteinase-2 in ovarian adenocarcinoma and its relation to carcinoma progression. Gynecol Oncol 92:559PubMedCrossRefGoogle Scholar
  42. 42.
    Turk BE, Huang LL, Piro ET, Cantley LC (2001) Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat Biotechnol 19:661PubMedCrossRefGoogle Scholar
  43. 43.
    Wajant H, Moosmayer D, Wuest T, Bartke T, Gerlach E, Schonherr U, Peters N, Scheurich P, Pfizenmaier K (2001) Differential activation of TRAIL-R1 and -2 by soluble and membrane TRAIL allows selective surface antigen-directed activation of TRAIL-R2 by a soluble TRAIL derivative. Oncogene 20:4101PubMedCrossRefGoogle Scholar
  44. 44.
    Wajant H, Gerspach J, Pfizenmaier K (2005) Tumor therapeutics by design: targeting and activation of death receptors. Cytokine Growth Factor Rev 16:55PubMedCrossRefGoogle Scholar
  45. 45.
    Wuest T, Gerlach E, Banerjee D, Gerspach J, Moosmayer D, Pfizenmaier K (2002) TNF-Selectokine: a novel prodrug generated for tumor targeting and site-specific activation of tumor necrosis factor. Oncogene 21:4257PubMedCrossRefGoogle Scholar
  46. 46.
    Yamamoto Y, Tsutsumi Y, Yoshioka Y, Nishibata T, Kobayashi K, Okamoto T, Mukai Y, Shimizu T, Nakagawa S, Nagata S, Mayumi T (2003) Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity. Nat Biotechnol 21:546PubMedCrossRefGoogle Scholar
  47. 47.
    Yamashita K, Tanaka Y, Mimori K, Inoue H, Mori M (2004) Differential expression of MMP and uPA systems and prognostic relevance of their expression in esophageal squamous cell carcinoma. Int J Cancer 110:201PubMedCrossRefGoogle Scholar
  48. 48.
    Yoshioka Y, Tsutsumi Y, Ikemizu S, Yamamoto Y, Shibata H, Nishibata T, Mukai Y, Okamoto T, Taniai M, Kawamura M, Abe Y, Nakagawa S, Nagata S, Yamagata Y, Mayumi T (2004) Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency. Biochem Biophys Res Commun 315:808PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Jeannette Gerspach
    • 1
  • Julia Németh
    • 1
  • Sabine Münkel
    • 1
  • Harald Wajant
    • 2
  • Klaus Pfizenmaier
    • 1
  1. 1.Institute of Cell Biology and Immunology University of StuttgartStuttgartGermany
  2. 2.Department of Molecular Internal Medicine, Medical Clinic and Polyclinic IIUniversity of WürzburgWurzburgGermany

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