Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Construction, Bacterial Expression and Characterization of a Bifunctional Single–Chain Antibody–Phosphatase Fusion Protein Targeted to the Human ERBB–2 Receptor

Abstract

We have constructed genes expressing single–chain antigen binding proteins (scFv) which recognize the human erbB–2 receptor. These genes encode the heavy and light chain variable domains of an erbB–2 receptor specific monoclonal antibody, MAb FRP5, connected by a peptide linker. In order to express a bifunctional molecule, a bacterial alkaline phosphatase gene was fused 3′ to the scFv gene. The scFv(FRP5) and scFv(FRP5)–alkaline phosphatase fusion protein (scFv(FRP5)–PhoA) expressed in E. coli specifically recognize the human erbB–2 protein and compete with MAb FRP5 for binding to the receptor. The bound scFv(FRP5)–PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

References

  1. Coussens, L., Yang-Feng, T.L., Liao, Y.-C., Chen, E., Gray, A., McGrath, J., Seeburg, P.H., Libermann, T.A., Schlessinger, J., Franke, U., Levinson, A. and Ullrich, A. 1985. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 230: 1132–1139.

    CAS  Article  PubMed  Google Scholar 

  2. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N., Miyajima, N., Saito, T. and Toyoshima, K. 1986. Similarity of protein encoded by the human c-erB-2 gene epidermal growth factor receptor. Nature 319: 230–234.

    CAS  Article  PubMed  Google Scholar 

  3. Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A. and McGuire, W.L. 1987. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235: 177–182.

    CAS  Article  PubMed  Google Scholar 

  4. Van de Vijver, M., van de Bersselaar, R., Devilee, P., Cornelisse, C., Peterse, J. and Nusse, R. 1987. Amplification of the neu (c-erbB-2) oncogene in human mammary tumors is relatively frequent and is often accompanied by amplification of the linked c-erbA oncogene. Mol. Cell. Biol. 7: 2019–2023.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Varley, J.M., Swallow, J.E., Brammer, W.J., Whittaker, J.L. and Walker, R. 1987. Alterations in either c-erbB-2(neu) or c-myc proto-oncogenes in breast carcinomas correlate with poor short-term prognosis. Oncogene 1: 423–430.

    CAS  PubMed  Google Scholar 

  6. Berger, M.S., Locher, G.W., Saurer, S., Gullick, W.J., Waterfield, M.D., Groner, B. and Hynes, N.E. 1988. Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading. Cancer Res. 48: 1238–1243.

    CAS  PubMed  Google Scholar 

  7. Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A., Wong, S.G., Kieth, D.E., Levin, W.J., Stuart, S.G., Udove, J., Ullrich, A. and Press, M.F. 1989. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244: 707–712.

    CAS  Article  PubMed  Google Scholar 

  8. Wright, C., Angus, B., Nicholson, S., Sainsbury, J.R.C., Cairns, J., Gullick, W.J., Kelly, P., Harris, A.L. and Horne, C.H.W. 1989. Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer. Cancer Res. 49: 2087–2090.

    CAS  PubMed  Google Scholar 

  9. Richner, J., Gerber, H.A., Locher, G.W., Goldhirsch, A., Gelber, R.D., Gullick, W.J., Berger, M.S., Groner, B. and Hynes, N.E. 1990. c-erbB-2 protein expression in node negative breast cancer. Annals of Oncology 1: 263–268.

    CAS  Article  PubMed  Google Scholar 

  10. Harwerth, I.-M., Wels, W., Marte, B. and Hynes, N.E. 1992. Monoclonal antibodies against the extracellular domain of the erbB-2 receptor function as partial ligand agonists. J. Biol. Chem. In press.

  11. Orlandi, R., Guessow, D.H., Jones, P.T. and Winter, G. 1989. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86: 3833–3837.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Prickett, K.S., Amberg, D.C. and Hopp, T.P. 1989. A calcium-dependent antibody for identification and purification of recombinant proteins. Biotechniques 7: 580–589.

    CAS  PubMed  Google Scholar 

  13. Chang, C.N., Kuang, W.-J. and Chen, E.Y. 1986. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli . Gene 44: 121–125.

    CAS  Article  PubMed  Google Scholar 

  14. Manoil, C. and Beckwith, J. 1985. TnphoA: A transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82: 8129–8133.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Hynes, N.E., Gerber, H.A., Saurer, S. and Groner, B. 1989. Overexpression of the c-erbB-2 protein in human breast tumor cell lines. J. Cell. Biochem. 39: 167–173.

    CAS  Article  PubMed  Google Scholar 

  16. Murdoch, A., Jenkinson, E.J., Johnson, G.D. and Owen, J.J.Y. 1990. Alkaline phosphatase-Fast Red, a new fluorescent label. J. Immunolog. Meth. 132: 45–49.

    CAS  Article  Google Scholar 

  17. Barton, R.L., Briggs, S.L. and Koppel, G.A. 1991. Monoclonal antibody drug targeting. Drug News & Persp. 4: 73–88.

    Google Scholar 

  18. Waldmann, T.A. 1991. Monoclonal antibodies in diagnosis and therapy. Science 252: 1657–1662.

    CAS  Article  PubMed  Google Scholar 

  19. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.-M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. 1988. Single-chain antigen-binding proteins. Science 242: 423–426.

    CAS  Article  PubMed  Google Scholar 

  20. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, S.-S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. and Oppermann, H. 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli . Proc. Natl. Acad. Sci. USA 85: 5879–5883.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Brinkmann, U., Pai, L.H., FitzGerald, D.J., Willingham, M. and Pastan, I. 1991. B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Sci. USA 88: 8616–8620.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Chaudary, V.K., Queen, C., Junghans, R.P., Waldmann, T.A., FitzGerald, D.J. and Pastan, I. 1989. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 339: 394–397.

    Article  Google Scholar 

  23. Colcher, D., Bird, R., Roselli, M., Hardman, K.D., Johnson, S., Pope, S., Dodd, S.W., Pantoliano, M.W., Milenic, D.E. and Schlom, J. 1990. In vivo tumor targeting of a recombinant single-chain antigen-binding protein. J. Natl. Cancer Inst. 82: 1191–1197.

    CAS  Article  PubMed  Google Scholar 

  24. Glockshuber, R., Malia, M., Pfitzinger, I. and Plückthun, A. 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29: 1362–1367.

    CAS  Article  PubMed  Google Scholar 

  25. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  26. Gueltekin, H. and Heermann, K.H. 1988. The use of polyvinylidene-difluoride membranes as a general blotting matrix. Anal. Biochem. 172: 320–329.

    CAS  Article  Google Scholar 

  27. Munson, P.J. and Rodbard, D. 1980. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107: 220–239.

    CAS  Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wels, W., Harwerth, IM., Zwickl, M. et al. Construction, Bacterial Expression and Characterization of a Bifunctional Single–Chain Antibody–Phosphatase Fusion Protein Targeted to the Human ERBB–2 Receptor. Nat Biotechnol 10, 1128–1132 (1992). https://doi.org/10.1038/nbt1092-1128

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1092-1128

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing