Journal of Neuro-Oncology

, Volume 103, Issue 2, pp 255–266 | Cite as

A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature

  • Alexander K. Tsai
  • Seunguk Oh
  • Hua Chen
  • Yanqun Shu
  • John R. Ohlfest
  • Daniel A. Vallera
Laboratory Investigation - Human/Animal Tissue


A bispecific ligand-directed toxin (BLT), called EGFATFKDEL, consisting of human epidermal growth factor, a fragment of urokinase, and truncated pseudomonas exotoxin (PE38) was assembled in order to target human glioblastoma. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGFATFKDEL 7mut. In vitro, the drug selectively killed several human glioblastoma cell lines. EGFATFKDEL is our first BLT designed to simultaneously target EGFR on solid tumors and uPAR on the tumor neovasculature. In vitro assays revealed that the agent is effective against glioblastoma cell lines as well as human umbilical vein endothelial cells (HUVEC). Additionally, the bispecific drug displayed enhanced binding to overexpressed epidermal growth factor receptor and urokinase receptor when compared to similar monospecific drugs, EGFKDEL and ATFKDEL. In vivo, an aggressive human glioblastoma cell line was genetically marked with a firefly luciferase reporter gene and administered to the flanks of nude mice. Treatment with intratumoral injections of EGFATFKDEL 7mut eradicated small tumors in over half of the treated mice, which survived with tumor free status at least 100 days post tumor inoculation. ATFKDEL, which primarily targets the tumor neovasculature, prevented tumor growth but did not result in tumor-free mice in most cases. Specificity was shown by treating with an irrelevant BLT control which did not protect mice. Finally, immunization experiments in immunocompetent mice revealed significantly reduced anti-toxin production in EGFATFKDEL 7mut treated groups. Thus, EGFATFKDEL 7mut is an effective drug for glioblastoma therapy in this murine model and warrants further study.


Immunotoxin Pseudomonas exotoxin Glioblastoma Xenograft model EGF 



This work was supported in part by the US Public Health Service Grants RO1-CA36725 and RO1-CA082154 awarded by the NCI and the NIAID, DHHS and the Martha L. Kramer Fund. We thank Travis M. Spangler and Zintis Inde for assistance. This manuscript partially fulfilled requirements for the Master of Science degree for A. Tsai, CLS program, University of Minnesota.


  1. 1.
    Lowe S, Schmidt U, Unterberg A, Halatsch ME (2009) The epidermal growth factor receptor as a therapeutic target in glioblastoma multiforme and other malignant neoplasms. Anticancer Agents Med Chem 9:703–715Google Scholar
  2. 2.
    Kioi M, Husain SR, Croteau D, Kunwar S, Puri RK (2006) Convection-enhanced delivery of interleukin-14 receptor-directed cytotoxin for malignant glioma therapy. Technol Cancer Res Treat 5:239–250PubMedGoogle Scholar
  3. 3.
    Laske DW, Youle RJ, Oldfield EH (1997) Tumor regression with regional distribution of the targeted toxin TF-CRM 107 in patients with malignant brain tumors. Nat Med 3:1362–1368PubMedCrossRefGoogle Scholar
  4. 4.
    Hall WA, Vallera DA (2006) Efficacy of antiangiogenic targeted toxins against glioblastoma multiforme. Neurosurg Focus 20:E23PubMedCrossRefGoogle Scholar
  5. 5.
    Fuchs H, Bachran C (2009) Targeted tumor therapies at a glance. Curr Drug Targets 10:89–93PubMedCrossRefGoogle Scholar
  6. 6.
    Stish BJ, Oh S, Chen H, Dudek AZ, Kratzke RA, Vallera DA (2009) Design and modification of EGF4KDEL 7mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity. Br J Cancer 101:1114–1123PubMedCrossRefGoogle Scholar
  7. 7.
    Vallera DA, Chen H, Sicheneder AR, Panoskaltsis-Mortari A, Taras EP (2009) Genetic alteration of a bispecific ligand-directed toxin targeting human CD19 and CD22 receptors resulting in improved efficacy against systemic B cell malignancy. Leuk Res 33:1233–1242PubMedCrossRefGoogle Scholar
  8. 8.
    Stish BJ, Chen H, Shu Y, Panoskaltsis-Mortarti A, Vallera DA (2007) A bispecific recombinant cytotoxin (DTEGF13) targeting human IL-13 and EGF receptors in a mouse xenograft model of prostate cancer. Clin Cancer Res 13:6486–6493PubMedCrossRefGoogle Scholar
  9. 9.
    Stish BJ, Chen H, Shu Y, Panoskaltsis-Mortari A, Vallera DA (2007) Increasing anticarcinoma activity of an anti-erbB2 recombinant immunotoxin by the addition of an anti-EpCAM sFv. Clin Cancer Res 15:3058–3067CrossRefGoogle Scholar
  10. 10.
    Pastan I, Chaudhary V, FitzGerald DJ (1992) Recombinant toxins as novel therapeutic agents. Annu Rev Biochem 61:331–354PubMedCrossRefGoogle Scholar
  11. 11.
    Kreitman RJ, Pastan I (1998) Accumulation of a recombinant immunotoxin in a tumor in vivo: fewer than 1000 molecules per cell are sufficient for complete responses. Cancer Res 58:968–975PubMedGoogle Scholar
  12. 12.
    Kreitman RJ, Pastan I (1995) Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. Biochem J 307:29–37PubMedGoogle Scholar
  13. 13.
    Yarden Y, Sliwkowski MX (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127–137PubMedCrossRefGoogle Scholar
  14. 14.
    Pytel P, Lukas RV (2009) Update on diagnostic practice: tumors of the nervous system. Arch Pathol Lab Med 133:1062–1077PubMedGoogle Scholar
  15. 15.
    Wang MY, Lu KV, Zhu S et al (2006) Mammalian target of rapamycin inhibition promotes response to epidermal growth factor receptor kinase inhibitors in PTEN-deficient and PTEN-intact glioblastoma cells. Cancer Res 66:7864–7869PubMedCrossRefGoogle Scholar
  16. 16.
    Haas-Kogan DA, Prados MD, Lamborn KR, Tihan T, Berger MS, Stokoe D (2005) Biomarkers to predict response to epidermal growth factor receptor inhibitors. Cell Cycle 4:1369–1372PubMedCrossRefGoogle Scholar
  17. 17.
    Rich JN, Reardon DA, Peery T et al (2004) Phase II trial of gefitinib in recurrent glioblastoma. J Clin Oncol 22:133–142PubMedCrossRefGoogle Scholar
  18. 18.
    Mori T, Abe T, Wakabayashi Y et al (2000) Up-regulation of urokinase-type plasminogen activator and its receptor correlates with enhanced invasion activity of human glioma cells mediated by transforming growth factor-α or basic fibroblast growth factor. J Neurooncol 46:115–123PubMedCrossRefGoogle Scholar
  19. 19.
    Grondahl-Hansen J, Peters HA, van Putten WL et al (1995) Prognostic significance of the receptor for urokinase plasminogen activator in breast cancer. Clin Cancer Res 1:1079–1087PubMedGoogle Scholar
  20. 20.
    Verspaget HW, Sier CF, Ganesh S, Griffoen G, Lamers CB (1995) Prognostic value of plasminogen activators and their inhibitors in colorectal cancer. Eur J Cancer 31:1105–1109CrossRefGoogle Scholar
  21. 21.
    Prager GW, Breuss JM, Steurer S, Mihaly J, Binder BR (2004) Vascular endothelial growth factor (VEGF) induces rapid prourokinase (pro-uPA) activation on the surface of endothelial cells. Blood 103:955–962PubMedCrossRefGoogle Scholar
  22. 22.
    Choong PF, Nadesapillai AP (2003) Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis. Clin Orthop Relat Res 415(Supple):S46–S58PubMedCrossRefGoogle Scholar
  23. 23.
    Rustamzadeh E, Hall WA, Todhunter DA et al (2006) Intracranial therapy of glioblastoma with the fusion protein DTAT in immunodeficient mice. Int J Cancer 120:411–419CrossRefGoogle Scholar
  24. 24.
    Hassan R, Bullock S, Premkumar A et al (2007) Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. Clin Cancer Res 13:5144–5149PubMedCrossRefGoogle Scholar
  25. 25.
    Onda M, Nagata S, FitzGerald DJ et al (2006) Characterization of the B cell epitopes associated with a truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins for the treatment of cancer patients. J Immunol 177:8822–8834PubMedGoogle Scholar
  26. 26.
    Vogelbaum MA, Sampson JH, Kunwar S et al (2007) Convection-enhanced delivery of cintredekin besudotox (interleukin-13-PE38QQR) followed by radiation therapy with and without temozolomide in newly diagnosed malignant gliomas: phase 1 study of final safety results. Neurosurgery 61:1031–1037PubMedCrossRefGoogle Scholar
  27. 27.
    Vallera DA, Todhunter DA, Kuroki DW, Shu Y, Sicheneder A, Chen H (2005) A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma. Clin Cancer Res 11:3879–3888PubMedCrossRefGoogle Scholar
  28. 28.
    Vallera DA, Li C, Jin N, Panoskaltsis-Mortari A, Hall WA (2002) Targeting urokinase-type plasminogen activator receptor on human glioblastoma tumors with diphtheria toxin fusion protein DTAT. J Nat Cancer Inst 94:597–605PubMedGoogle Scholar
  29. 29.
    Vallera DA, Todhunter D, Kuroki DW, Shu Y, Sicheneder A, Panoskaltsis-Mortari A, Vallera VD, Chen H (2005) Molecular modification of a recombinant, bivalent anti-human CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice. Leuk Res 29:331–341PubMedCrossRefGoogle Scholar
  30. 30.
    Vallera DA, Shu Y, Chen H et al (2008) Genetically designing a more potent anti-pancreatic cancer agent by simultaneously cotargeting human IL-13 and EGF receptors in a mouse xenograft model. Gut 57:634–641PubMedCrossRefGoogle Scholar
  31. 31.
    Vallera DA, Ash RC, Zanjani ED, Kersey JH, LeBien TW, Beverley PC, Neville DM Jr, Youle RJ (1983) Anti-T-cell reagents for human bone marrow transplantation: ricin linked to three monoclonal antibodies. Science 222:512–515PubMedCrossRefGoogle Scholar
  32. 32.
    Vallera DA, Taylor PA, Sprent J, Blazar BR (1994) The role of host T cell subsets in bone marrow rejection directed to isolated major histocompatability complex class I versus class II differences of bm1 and bm12 mutant mice. Transplantation 57:249–256PubMedCrossRefGoogle Scholar
  33. 33.
    Nagata S, Pastan I (2009) Removal of B cell epitopes as a practical approach for reducing the immunogenicity of foreign protein-based therapeutics. Adv Drug Deliv Rev 61:977–985PubMedCrossRefGoogle Scholar
  34. 34.
    Onda M, Beers R, Xiang L, Nagata S, Wang Q, Pastan I (2008) An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes. PNAS 105:11311–11316PubMedCrossRefGoogle Scholar
  35. 35.
    Oh S, Stish BJ, Sachdev D, Chen H, Dudek AZ, Vallera DA (2009) A novel reduced immunogenicity bispecific targeted toxin simultaneously recognizing human epidermal growth factor and interleukin-4 receptors in a mouse model of metastatic breast carcinoma. Clin Cancer Res 15:6137–6147PubMedCrossRefGoogle Scholar
  36. 36.
    Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407:249–257PubMedCrossRefGoogle Scholar
  37. 37.
    Kreitman RJ, Stetler-Stevenson M, Margulies I et al (2009) Phase II trial of recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) in patients with hairy cell leukemia. J Clin Oncol 27:2983–2990PubMedCrossRefGoogle Scholar
  38. 38.
    Oh S, Ohlfest JR, Todhunter DA et al (2009) Intracranial elimination of glioblastoma brain tumors in nude rats using the bispecific ligand-directed toxin, DTEGF13 and convection enhanced delivery. J Neurooncol 95:331–342PubMedCrossRefGoogle Scholar
  39. 39.
    Ho M, Nagata S, Pastan I (2006) Isolation of anti-CD22 Fv with high affinity by Fv display on human cells. Proc Natl Acad Sci USA 103:9637–9642PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Alexander K. Tsai
    • 1
  • Seunguk Oh
    • 1
  • Hua Chen
    • 1
  • Yanqun Shu
    • 1
  • John R. Ohlfest
    • 2
  • Daniel A. Vallera
    • 1
  1. 1.Department of Therapeutic Radiology-Radiation OncologySection on Molecular Cancer Therapeutics, University of Minnesota Masonic Cancer CenterMinneapolisUSA
  2. 2.Department of PediatricsUniversity of Minnesota Masonic Cancer CenterMinneapolisUSA

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