Abstract
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.
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References
Cesano, A. and Gayko, U. (2003) CD22 as a target of passive immunotherapy. Semin. Oncol. 30, 253–257.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947–950.
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.
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.
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.
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.
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.
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.
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.
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Krauss, J., Exner, E., Mavratzas, A., Seeber, S., Arndt, M.A. (2009). High-Level Production of a Humanized ImmunoRNase Fusion Protein from Stably Transfected Myeloma Cells. In: Dimitrov, A. (eds) Therapeutic Antibodies. Methods in Molecular Biology™, vol 525. Humana Press. https://doi.org/10.1007/978-1-59745-554-1_24
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DOI: https://doi.org/10.1007/978-1-59745-554-1_24
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