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. 1990 Jun;87(12):4697–4701. doi: 10.1073/pnas.87.12.4697

Transforming growth factor alpha-Pseudomonas exotoxin fusion protein prolongs survival of nude mice bearing tumor xenografts.

D C Heimbrook 1, S M Stirdivant 1, J D Ahern 1, N L Balishin 1, D R Patrick 1, G M Edwards 1, D Defeo-Jones 1, D J FitzGerald 1, I Pastan 1, A Oliff 1
PMCID: PMC54184  PMID: 2352944

Abstract

Transforming growth factor alpha (TGF alpha)-Pseudomonas exotoxin 40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the Pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor-binding activity of TGF alpha and the cell-killing activity of PE40. These properties make TGF alpha-PE40 an effective cytotoxic agent for cells that possess epidermal growth factor receptors (EGFR). However, the utility of this protein as an anticancer agent has been unclear because many normal tissues express EGFR and may be damaged by exposure to TGF alpha-PE40. To address this issue, we injected nude mice with a lethal inoculum of either A431 or HT29 human tumor cells that possess EGFR or with Chinese hamster ovary (CHO) tumor cells that lack EGFR. Animals were treated with a derivative of TGF alpha-PE40 in which the cysteine residues are replaced by alanine, termed "TGF alpha-PE40 delta cys," or with saline once a day for 5 days. Mice bearing EGFR+ tumor cells lived significantly (P less than 0.001) longer when treated with TGF alpha-PE40 delta cys compared with saline-treated controls (median survival: A431 cells, 51.5 vs. 25.5 days; HT29 cells, 101 vs. 47.5 days). TGF alpha-PE40 delta cys did not prolong the survival of mice bearing tumor cells that lack EGFR (median survival: CHO cells, 15.5 vs. 19.5 days). The only toxicity to normal tissues was mild periportal hepatic necrosis. These studies indicate that a therapeutic window exists in vivo for the use of some growth factor-toxin fusion proteins as anticancer agents.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akiyama S., Gottesman M. M., Hanover J. A., Fitzgerald D. J., Willingham M. C., Pastan I. Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin. J Cell Physiol. 1984 Sep;120(3):271–279. doi: 10.1002/jcp.1041200303. [DOI] [PubMed] [Google Scholar]
  2. Allured V. S., Collier R. J., Carroll S. F., McKay D. B. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1320–1324. doi: 10.1073/pnas.83.5.1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chaudhary V. K., FitzGerald D. J., Adhya S., Pastan I. Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4538–4542. doi: 10.1073/pnas.84.13.4538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chung D. W., Collier R. J. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect Immun. 1977 Jun;16(3):832–841. doi: 10.1128/iai.16.3.832-841.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen S., Ushiro H., Stoscheck C., Chinkers M. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem. 1982 Feb 10;257(3):1523–1531. [PubMed] [Google Scholar]
  6. Defeo-Jones D., Tai J. Y., Wegrzyn R. J., Vuocolo G. A., Baker A. E., Payne L. S., Garsky V. M., Oliff A., Riemen M. W. Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha. Mol Cell Biol. 1988 Aug;8(8):2999–3007. doi: 10.1128/mcb.8.8.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edwards G. M., DeFeo-Jones D., Tai J. Y., Vuocolo G. A., Patrick D. R., Heimbrook D. C., Oliff A. Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins. Mol Cell Biol. 1989 Jul;9(7):2860–2867. doi: 10.1128/mcb.9.7.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fabricant R. N., De Larco J. E., Todaro G. J. Nerve growth factor receptors on human melanoma cells in culture. Proc Natl Acad Sci U S A. 1977 Feb;74(2):565–569. doi: 10.1073/pnas.74.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. FitzGerald D. J., Padmanabhan R., Pastan I., Willingham M. C. Adenovirus-induced release of epidermal growth factor and pseudomonas toxin into the cytosol of KB cells during receptor-mediated endocytosis. Cell. 1983 Feb;32(2):607–617. doi: 10.1016/0092-8674(83)90480-4. [DOI] [PubMed] [Google Scholar]
  10. Giard D. J., Aaronson S. A., Todaro G. J., Arnstein P., Kersey J. H., Dosik H., Parks W. P. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst. 1973 Nov;51(5):1417–1423. doi: 10.1093/jnci/51.5.1417. [DOI] [PubMed] [Google Scholar]
  11. Giard D. J., Aaronson S. A., Todaro G. J., Arnstein P., Kersey J. H., Dosik H., Parks W. P. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst. 1973 Nov;51(5):1417–1423. doi: 10.1093/jnci/51.5.1417. [DOI] [PubMed] [Google Scholar]
  12. Gray G. L., Smith D. H., Baldridge J. S., Harkins R. N., Vasil M. L., Chen E. Y., Heyneker H. L. Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1984 May;81(9):2645–2649. doi: 10.1073/pnas.81.9.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gusterson B., Cowley G., McIlhinney J., Ozanne B., Fisher C., Reeves B. Evidence for increased epidermal growth factor receptors in human sarcomas. Int J Cancer. 1985 Dec 15;36(6):689–693. doi: 10.1002/ijc.2910360612. [DOI] [PubMed] [Google Scholar]
  14. Haigler H., Ash J. F., Singer S. J., Cohen S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3317–3321. doi: 10.1073/pnas.75.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hwang J., Fitzgerald D. J., Adhya S., Pastan I. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell. 1987 Jan 16;48(1):129–136. doi: 10.1016/0092-8674(87)90363-1. [DOI] [PubMed] [Google Scholar]
  16. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim D. C., Sugiyama Y., Satoh H., Fuwa T., Iga T., Hanano M. Kinetic analysis of in vivo receptor-dependent binding of human epidermal growth factor by rat tissues. J Pharm Sci. 1988 Mar;77(3):200–207. doi: 10.1002/jps.2600770304. [DOI] [PubMed] [Google Scholar]
  19. Kondo T., FitzGerald D., Chaudhary V. K., Adhya S., Pastan I. Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain. J Biol Chem. 1988 Jul 5;263(19):9470–9475. [PubMed] [Google Scholar]
  20. Libermann T. A., Nusbaum H. R., Razon N., Kris R., Lax I., Soreq H., Whittle N., Waterfield M. D., Ullrich A., Schlessinger J. Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature. 1985 Jan 10;313(5998):144–147. doi: 10.1038/313144a0. [DOI] [PubMed] [Google Scholar]
  21. Lorberboum-Galski H., Barrett L. V., Kirkman R. L., Ogata M., Willingham M. C., FitzGerald D. J., Pastan I. Cardiac allograft survival in mice treated with IL-2-PE40. Proc Natl Acad Sci U S A. 1989 Feb;86(3):1008–1012. doi: 10.1073/pnas.86.3.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lorberboum-Galski H., FitzGerald D., Chaudhary V., Adhya S., Pastan I. Cytotoxic activity of an interleukin 2-Pseudomonas exotoxin chimeric protein produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1922–1926. doi: 10.1073/pnas.85.6.1922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mateo de Acosta C., Justiz E., Skoog L., Lage A. Biodistribution of radioactive epidermal growth factor in normal and tumor bearing mice. Anticancer Res. 1989 Jan-Feb;9(1):87–92. [PubMed] [Google Scholar]
  24. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  25. Ogata M., Chaudhary V. K., FitzGerald D. J., Pastan I. Cytotoxic activity of a recombinant fusion protein between interleukin 4 and Pseudomonas exotoxin. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4215–4219. doi: 10.1073/pnas.86.11.4215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ozawa S., Ueda M., Ando N., Abe O., Shimizu N. High incidence of EGF receptor hyperproduction in esophageal squamous-cell carcinomas. Int J Cancer. 1987 Mar 15;39(3):333–337. doi: 10.1002/ijc.2910390311. [DOI] [PubMed] [Google Scholar]
  27. Pérez R., Pascual M., Macías A., Lage A. Epidermal growth factor receptors in human breast cancer. Breast Cancer Res Treat. 1984;4(3):189–193. doi: 10.1007/BF01806484. [DOI] [PubMed] [Google Scholar]
  28. Riemen M. W., Wegrzyn R. J., Baker A. E., Hurni W. M., Bennett C. D., Oliff A., Stein R. B. Isolation of multiple biologically and chemically diverse species of epidermal growth factor. Peptides. 1987 Sep-Oct;8(5):877–885. doi: 10.1016/0196-9781(87)90075-1. [DOI] [PubMed] [Google Scholar]
  29. Siegall C. B., Chaudhary V. K., FitzGerald D. J., Pastan I. Cytotoxic activity of an interleukin 6-Pseudomonas exotoxin fusion protein on human myeloma cells. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9738–9742. doi: 10.1073/pnas.85.24.9738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Siegall C. B., Chaudhary V. K., FitzGerald D. J., Pastan I. Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin. J Biol Chem. 1989 Aug 25;264(24):14256–14261. [PubMed] [Google Scholar]
  31. Siegall C. B., Xu Y. H., Chaudhary V. K., Adhya S., Fitzgerald D., Pastan I. Cytotoxic activities of a fusion protein comprised of TGF alpha and Pseudomonas exotoxin. FASEB J. 1989 Dec;3(14):2647–2652. doi: 10.1096/fasebj.3.14.2556314. [DOI] [PubMed] [Google Scholar]
  32. Wong A. J., Bigner S. H., Bigner D. D., Kinzler K. W., Hamilton S. R., Vogelstein B. Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6899–6903. doi: 10.1073/pnas.84.19.6899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yanai S., Sugiyama Y., Kim D. C., Sato H., Fuwa T., Iga T., Hanano M. Binding of human epidermal growth factor to tissue homogenates of the rat. Chem Pharm Bull (Tokyo) 1987 Dec;35(12):4891–4897. doi: 10.1248/cpb.35.4891. [DOI] [PubMed] [Google Scholar]
  34. von Kleist S., Chany E., Burtin P., King M., Fogh J. Immunohistology of the antigenic pattern of a continuous cell line from a human colon tumor. J Natl Cancer Inst. 1975 Sep;55(3):555–560. doi: 10.1093/jnci/55.3.555. [DOI] [PubMed] [Google Scholar]

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