High-Level Production of a Humanized ImmunoRNase Fusion Protein from Stably Transfected Myeloma Cells

  • Jürgen Krauss
  • Evelyn Exner
  • Athanasios Mavratzas
  • Siegfried Seeber
  • Michaela A.E. Arndt
Part of the Methods in Molecular Biology™ book series (MIMB, volume 525)


ImmunoRNases represent a highly attractive alternative to conventional immunotoxins for cancer therapy. Quantitative production of immunoRNases in appropriate expression systems, however, remains a major challenge for further clinical development of these novel compounds. Here we describe a method for high-level production and purification of a fully functional immunoRNase fusion protein from supernatants of stably transfected mammalian cells.

Key words

ImmunoRNase fusion protein angiogenin humanized single-chain Fv eukaryotic expression ribonuclease zymogram tRNA assay cytotoxicity 


  1. 1.
    Cesano, A. and Gayko, U. (2003) CD22 as a target of passive immunotherapy. Semin. Oncol. 30, 253–257.PubMedCrossRefGoogle Scholar
  2. 2.
    Senderowicz, A. M., Vitetta, E., Headlee, D., Ghetie, V., Uhr, J. W., Figg, W. D., Lush, R. M., Stetler-Stevenson, M., Kershaw, G., Kingma, D. W., Jaffe, E. S., and Sausville, E. A. (1997) Complete sustained response of a refractory, post-transplantation, large B-cell lymphoma to an anti-CD22 immunotoxin. Ann. Intern. Med. 126, 882–885.PubMedGoogle Scholar
  3. 3.
    Kreitman, R. J., Wilson, W. H., Bergeron, K., Raggio, M., Stetler-Stevenson, M., FitzGerald, D. J., and Pastan, I. (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N. Engl. J. Med. 345, 241–247.PubMedCrossRefGoogle Scholar
  4. 4.
    Amlot, P. L., Stone, M. J., Cunningham, D., Fay, J., Newman, J., Collins, R., May, R., McCarthy, M., Richardson, J., and Ghetie, V. (1993) A phase I study of an anti-CD22-deglycosylated ricin A chain immunotoxin in the treatment of B-cell lymphomas resistant to conventional therapy. Blood 82, 2624–2633.PubMedGoogle Scholar
  5. 5.
    Sausville, E. A., Headlee, D., Stetler-Stevenson, M., Jaffe, E. S., Solomon, D., Figg, W. D., Herdt, J., Kopp, W. C., Rager, H., and Steinberg, S. M. (1995) Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a phase I study. Blood 85, 3457–3465.PubMedGoogle Scholar
  6. 6.
    Vitetta, E. S., Stone, M., Amlot, P., Fay, J., May, R., Till, M., Newman, J., Clark, P., Collins, R., Cunningham, D., et al. (1991) Phase I immunotoxin trial in patients with B-cell lymphoma. Cancer Res 51, 4052–4058.PubMedGoogle Scholar
  7. 7.
    Rybak, S. M., and Newton, D. L. (2001) Antibody targeted therapeutics for lymphoma: new focus on the CD22 antigen and RNA. Expert Opin. Biol. Ther. 1, 995–1003.CrossRefGoogle Scholar
  8. 8.
    St. Clair, D. K., Rybak, S. M., Riordan, J. F., and Vallee, B. L. (1987) Angiogenin abolishes cell-free protein synthesis by specific ribonucleolytic inactivation of ribosomes. Proc. Natl. Acad. Sci. USA 84, 8330–8334.CrossRefGoogle Scholar
  9. 9.
    Saxena, S. K., Rybak, S. M., Winkler, G., Meade, H. M., McGray, P., Youle, R. J., and Ackerman, E. J. (1991) Comparison of RNases and toxins upon injection into Xenopus oocytes. J. Biol. Chem. 266, 21208–21214.PubMedGoogle Scholar
  10. 10.
    Saxena, S. K., Rybak, S. M., Davey, R. T., Jr., Youle, R. J., and Ackerman, E. J. (1992) Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily. J. Biol. Chem. 267, 21982–21986.PubMedGoogle Scholar
  11. 11.
    Rybak, S. M., Hoogenboom, H. R., Meade, H. M., Raus, J. C., Schwartz, D., and Youle, R. J. (1992) Humanization of immunotoxins. Proc. Natl. Acad. Sci. USA 89, 3165–3169.PubMedCrossRefGoogle Scholar
  12. 12.
    Newton, D. L., Xue, Y., Olson, K. A., Fett, J. W., and Rybak, S. M. (1996) Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains. Biochemistry 35, 545–553.PubMedCrossRefGoogle Scholar
  13. 13.
    Stocker, M., Tur, M. K., Sasse, S., Krussmann, A., Barth, S., and Engert, A. (2003) Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. Protein Expr. Purif. 28, 211–219.PubMedCrossRefGoogle Scholar
  14. 14.
    Krauss, J., Arndt, M. A., Vu, B. K., Newton, D. L., and Rybak, S. M. (2005) Targeting malignant B-cell lymphoma with a humanized anti-CD22 scFv-angiogenin immunoenzyme. Br. J. Haematol. 128, 602–609.PubMedCrossRefGoogle Scholar
  15. 15.
    Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947–950.PubMedCrossRefGoogle Scholar
  16. 16.
    Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.PubMedCrossRefGoogle Scholar
  17. 17.
    Benedict, C. A., MacKrell, A. J., and Anderson, W. F. (1997) Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay. J. Immunol. Methods 201, 223–231.PubMedCrossRefGoogle Scholar
  18. 18.
    Nielsen, U. B., Adams, G. P., Weiner, L. M., and Marks, J. D. (2000) Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity. Cancer Res. 60, 6434–6440.PubMedGoogle Scholar
  19. 19.
    Bebbington, C. R., Renner, G., Thomson, S., King, D., Abrams, D., and Yarranton, G. T. (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (NY) 10, 169–175.CrossRefGoogle Scholar
  20. 20.
    Bravo, J., Fernandez, E., Ribo, M., de Llorens, R., and Cuchillo, C. M. (1994) A versatile negative-staining ribonuclease zymogram. Anal. Biochem. 219, 82–86.PubMedCrossRefGoogle Scholar
  21. 21.
    Korn, K., Foerster, H. H., and Hahn, U. (2000) Phage display of RNase A and an improved method for purification of phages displaying RNases. Biol. Chem. 381, 179–181.PubMedCrossRefGoogle Scholar
  22. 22.
    Arndt, M. A., Krauss, J., Vu, B. K., Newton, D. L., and Rybak, S. M. (2005) A dimeric angiogenin immunofusion protein mediates selective toxicity toward CD22+ tumor cells. J. Immunother. 28, 245–251.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jürgen Krauss
    • 1
  • Evelyn Exner
    • 1
  • Athanasios Mavratzas
    • 1
  • Siegfried Seeber
    • 2
  • Michaela A.E. Arndt
    • 1
  1. 1.University of HeidelbergGermany
  2. 2.University of Duisburg-EssenGermany

Personalised recommendations